Research Article Niacin Inhibits Vascular Inflammation via...

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Research Article Niacin Inhibits Vascular Inflammation via Downregulating Nuclear Transcription Factor-B Signaling Pathway Yanhong Si, 1,2 Ying Zhang, 1 Jilong Zhao, 3 Shoudong Guo, 1 Lei Zhai, 1 Shutong Yao, 1,2 Hui Sang, 1,2 Nana Yang, 1 Guohua Song, 1 Jue Gu, 4 and Shucun Qin 1 1 Key Laboratory of Atherosclerosis in Universities of Shandong and Institute of Atherosclerosis, Taishan Medical University, Shandong 271000, China 2 College of Basic Medical Sciences, Taishan Medical University, Shandong 271000, China 3 Shandong Agricultural University, Shandong 271000, China 4 Department of Geriatrics, PLA Navy General Hospital, Beijing 100048, China Correspondence should be addressed to Jue Gu; [email protected] and Shucun Qin; [email protected] Received 9 January 2014; Revised 1 April 2014; Accepted 16 April 2014; Published 27 May 2014 Academic Editor: Clara Di Filippo Copyright © 2014 Yanhong Si et al. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. e study aimed to investigate the effect of niacin on vascular inflammatory lesions in vivo and in vitro as well as its lipid-regulating mechanism. In vivo study revealed that niacin downregulated the levels of inflammatory factors (IL-6 and TNF-) in plasma, suppressed protein expression of CD68 and NF-B p65 in arterial wall, and attenuated oxidative stress in guinea pigs that have been fed high fat diet. In vitro study further confirmed that niacin decreased the secretion of IL-6 and TNF- and inhibited NF-B p65 and notch1 protein expression in oxLDL-stimulated HUVECs and THP-1 macrophages. Moreover, niacin attenuated oxLDL- induced apoptosis of HUVECs as well. In addition, niacin significantly lessened lipid deposition in arterial wall, increased HDL- C and apoA levels and decreased TG and non-HDL-C levels in plasma, and upregulated the mRNA amount of cholesterol 7- hydroxylase A1 in liver of guinea pigs. ese data suggest for the first time that niacin inhibits vascular inflammation in vivo and in vitro via downregulating NF-B signaling pathway. Furthermore, niacin also modulates plasma lipid by upregulating the expression of factors involved in the process of reverse cholesterol transport. 1. Introduction Niacin (nicotinic acid, vitamin B3) is a water-soluble vitamin. In 1955, Altschul et al. reported for the first time that pharma- cological doses of niacin can lower plasma cholesterol level in normal persons as well as hypercholesterolemic patients [1]. Several subsequent clinical studies established the usage of niacin as a broad-spectrum lipid-regulating medication. Niacin alone or in combination can slow or reverse the pro- gression of atherosclerosis (AS) and reduce cardiovascular event rates and total mortality in patients with hypercholes- terolemia and atherosclerotic cardiovascular disease [2, 3]. ese effects are generally attributed to favorable actions on the lipoprotein profile, which includes LDL-C reduction and HDL-C elevation. However, it is not clear whether the beneficial effects of niacin on cardiovascular disease can be completely explained by alterations of plasma lipids. Recent studies indicated niacin also has the anti- inflammatory and antioxidant properties. In human aortic endothelial cells in vitro, niacin significantly suppressed the adhesion and accumulation of monocytes/macrophages and inhibited angiotensin II-induced reactive oxygen species (ROS) production and LDL oxidation [4], but it is unknown whether niacin has the same effects or not in vivo. In addi- tion, Kuvin et al. confirmed that extended-release niaspan not only had a beneficial lipid-regulating effect but also decreased C-reactive protein (CRP) by 15% in patients with stable coronary artery disease [5]. CRP is one of the most important inflammatory markers. erefore, we hypothesize that niacin has vascular anti-inflammatory and potentially vascular-protective property independent of its effect on lipid regulation. Guinea pigs have been successfully used to study the damage of hyperlipidemia [6]. Like human, guinea pig is Hindawi Publishing Corporation Mediators of Inflammation Volume 2014, Article ID 263786, 12 pages http://dx.doi.org/10.1155/2014/263786

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Research ArticleNiacin Inhibits Vascular Inflammation via DownregulatingNuclear Transcription Factor-120581B Signaling Pathway

Yanhong Si12 Ying Zhang1 Jilong Zhao3 Shoudong Guo1 Lei Zhai1 Shutong Yao12

Hui Sang12 Nana Yang1 Guohua Song1 Jue Gu4 and Shucun Qin1

1 Key Laboratory of Atherosclerosis in Universities of Shandong and Institute of Atherosclerosis Taishan Medical UniversityShandong 271000 China

2 College of Basic Medical Sciences Taishan Medical University Shandong 271000 China3 Shandong Agricultural University Shandong 271000 China4Department of Geriatrics PLA Navy General Hospital Beijing 100048 China

Correspondence should be addressed to Jue Gu gujue0705163com and Shucun Qin shucunqinhotmailcom

Received 9 January 2014 Revised 1 April 2014 Accepted 16 April 2014 Published 27 May 2014

Academic Editor Clara Di Filippo

Copyright copy 2014 Yanhong Si et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The study aimed to investigate the effect of niacin on vascular inflammatory lesions in vivo and in vitro as well as its lipid-regulatingmechanism In vivo study revealed that niacin downregulated the levels of inflammatory factors (IL-6 and TNF-120572) in plasmasuppressed protein expression of CD68 and NF-120581B p65 in arterial wall and attenuated oxidative stress in guinea pigs that havebeen fed high fat diet In vitro study further confirmed that niacin decreased the secretion of IL-6 and TNF-120572 and inhibited NF-120581Bp65 and notch1 protein expression in oxLDL-stimulated HUVECs and THP-1 macrophages Moreover niacin attenuated oxLDL-induced apoptosis of HUVECs as well In addition niacin significantly lessened lipid deposition in arterial wall increased HDL-C and apoA levels and decreased TG and non-HDL-C levels in plasma and upregulated the mRNA amount of cholesterol 7120572-hydroxylase A1 in liver of guinea pigsThese data suggest for the first time that niacin inhibits vascular inflammation in vivo and invitro via downregulatingNF-120581B signaling pathway Furthermore niacin alsomodulates plasma lipid by upregulating the expressionof factors involved in the process of reverse cholesterol transport

1 Introduction

Niacin (nicotinic acid vitamin B3) is a water-soluble vitaminIn 1955 Altschul et al reported for the first time that pharma-cological doses of niacin can lower plasma cholesterol levelin normal persons as well as hypercholesterolemic patients[1] Several subsequent clinical studies established the usageof niacin as a broad-spectrum lipid-regulating medicationNiacin alone or in combination can slow or reverse the pro-gression of atherosclerosis (AS) and reduce cardiovascularevent rates and total mortality in patients with hypercholes-terolemia and atherosclerotic cardiovascular disease [2 3]These effects are generally attributed to favorable actionson the lipoprotein profile which includes LDL-C reductionand HDL-C elevation However it is not clear whether thebeneficial effects of niacin on cardiovascular disease can becompletely explained by alterations of plasma lipids

Recent studies indicated niacin also has the anti-inflammatory and antioxidant properties In human aorticendothelial cells in vitro niacin significantly suppressed theadhesion and accumulation of monocytesmacrophages andinhibited angiotensin II-induced reactive oxygen species(ROS) production and LDL oxidation [4] but it is unknownwhether niacin has the same effects or not in vivo In addi-tion Kuvin et al confirmed that extended-release niaspannot only had a beneficial lipid-regulating effect but alsodecreased C-reactive protein (CRP) by 15 in patients withstable coronary artery disease [5] CRP is one of the mostimportant inflammatory markers Therefore we hypothesizethat niacin has vascular anti-inflammatory and potentiallyvascular-protective property independent of its effect on lipidregulation

Guinea pigs have been successfully used to study thedamage of hyperlipidemia [6] Like human guinea pig is

Hindawi Publishing CorporationMediators of InflammationVolume 2014 Article ID 263786 12 pageshttpdxdoiorg1011552014263786

2 Mediators of Inflammation

one of the few species that carry the majority of cholesterolin LDL [7] It is possible to induce the initial stages ofAS quickly by feeding them high fat diet [8] In additionsignificant atherogenic inflammation with an increase ofaortic cytokines has been documented in guinea pigs [7] Inthe study we select guinea pigs as animal model to study theprotective effect of niacin on vascular inflammatory lesionsinduced by high fat diet in vivo Meanwhile to furtherconfirm the direct anti-inflammatory property of niacin itseffect on oxLDL-induced inflammation of endothelial cellsand macrophages is also explored

2 Materials and Methods

21 Animal and Experimental Design Thirty-two male Eng-land short-hair guinea pigs (260sim310 g 5 months old) werebought from the Animal Laboratory Center of Taibang Bio-logical Products Co Shandong China All experiments wereapproved by the Laboratory Animalsrsquo Ethical Committee ofTaishanMedical University and abided by national guidelinesfor the care and use of animals All guinea pigswere randomlydivided into 4 groups regular chow diet group (CD) highfat diet group (HFD 10 lard + 10 yolk power + 030cholesterol + 797 grass) HFD with niacin group (HFD-NHFD+ 100mgkgd niacin) andHFDwith simvastatin group(HFD-S HFD + 20mgkgd simvastatin) Each group isassigned 8 guinea pigsThedrugs (niacin or simvastatin)werefed by oral gavage once daily for 8 weeks

22 Cell Culture Human umbilical vein endothelial cells(HUVECs EAhy926) and THP-1 (human monocyte) wereall purchased from Shanghai Institute of Biochemistry Theywere maintained in RPMI 1640 medium (HyClone China)supplementedwith 10 fetal bovine serum (HyClone China)in a 5 CO2 incubator at 37∘C To induce monocytesdifferentiation into macrophages THP-1 cells were culturedwith 50 ngmL phorbol 12-myristate 13-acetate (PMA Sigma)for 24 hours as described previously [9]

23 Immunohistochemistry Examination Tissue sections(5 120583m) from formalin-fixed paraffin-embedded specimenswere stained with specific antibodies against NF-120581B p65 andCD68 proteins (Zhongshan Biotechnology Co Ltd BeijingChina) respectively The sections were developed with 331015840-diaminobenzidine (Vector Laboratories) and counterstainedwith Mayerrsquos hematoxylin (Saturatedard Allen) Images werecaptured using microscope (Olympus) All quantificationswere examined by calculating the percentage of integrateoptical density (IOD) of the antigen positive staining to theentire cross-sectional vessel wall by Image-Pro Plus software

24 Analysis of Lipid Deposition in the Arterial Wall Theproximal aorta attached to the heart was applied to preparecryosections Cryosections (8120583m) were cut gathered andstained with oil redOThe quantification of stained lipids wasexamined by calculating the percentage of the positive area tothe total cross-sectional vessel wall area by using Image-ProPlus software 45 (Media Cybernetics) The percentage wascalculated from five sections from an animal

25 Measurement of Inflammatory Factors Plasma concen-trations of IL-6 TNF-120572 and CRP which can be secreted dur-ing the process of inflammation were determined by ELISAkit (American RampD) in accordance with the manufacturerrsquosinstructions

ELISA kits were also used for the measurement of TNF-120572 and IL-6 protein levels in the medium of HUVECs andTHP-1 macrophages Cells were incubated in the absenceor presence of niacin (025ndash1mM) for 24 h in serum-freemedia and then stimulated by 150 120583gmL ox-LDL (BeijingXiesheng Biotechnology Co Ltd China) for 6 h TNF-120572 andIL-6 protein levels were assessed respectively Results werecalculated as ngL and expressed as a percentage of thoseobtained with blanks

26 Measurement of Oxidative Stress Reaction in PlasmaPlasma level of malondialdehyde (MDA) amarker for oxida-tive stress was determined by a spectrophotometric mea-surement of thiobarbituric acid-reactive substances (TBARS)in accordance with the manufacturerrsquos instructions (NanjingJiancheng Bioengineering Institute China)

27 Plasma Lipid Analysis After being treated by niacin orsimvastatin for 8 weeks blood was collected by cardiac punc-ture from guinea pigs without dietary exposure for 12 hoursConcentrations of plasma total cholesterol (TC) triglyceride(TG) and HDL cholesterol (HDL-C) were determined byenzymatic methods (BioSino Beijing China) Non-HDL-Cwas calculated as TC minus HDL-C

28 Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophore-sis (SDS-PAGE)Analysis of Apolipoproteins inHDL Lipopro-tein isolation was carried out by sequential ultracentrifu-gation in a LE-80K ultracentrifuge (Beckman Coulter IncBrea CA USA) as described before Separation was doneaccording to the following density fractionation 119889 lt1019 gmL for VLDL and IDL d 1019ndash109 gmL for LDLand d 109ndash124 gmL for HDL [10] The isolated specimenswere dialyzed in 150mmolL NaCl and 03mmolL EDTAat 4∘C HDL containing equal amounts of cholesterol wasloaded on a 15 sodium dodecyl sulfate (SDS) polyacry-lamide gradient gel and apolipoprotein samples were stainedwith coomassie brilliant blue as described by Jiang et al [11]Meanwhile the marker (Invitrogen LC5800) was put in lane1 and HDL from human was put in the last lane for contrastStained gels were scanned and analyzed by Quantity One(Bio-Rad Hercules CA USA) software program

29 Analysis of Cell Apoptosis by Flow Cytometry AnnexinV-FITCPI double-staining assay was applied to measureapoptosis according to the manufacturerrsquos instructions AfterHUVECs were stimulated by ox-LDL for 24 h cells werecentrifuged washed twice with PBS resuspended in 500 uLbinding buffer and incubated with 5 uL fluorescein isoth-iocyanate (FITC)-labeled Annexin V and 5 uL propidiumiodide (PI) for 10 minutes at room temperature in the darkThe scatter parameters of cells were analyzed by FAC Scanflow cytometer and Cell Quest analysis software (Becton-Dickinson CA USA) Four cell populations were discerned

Mediators of Inflammation 3

according to the following status live cells in the lower-leftquadrant (low-PI and FITC signals) early apoptotic cells inthe lower-right quadrant (low-PI and high-FITC signals) lateapoptotic or necrotic cells in the upper-right quadrant (high-PI and high-FITC signals) and necrotic cells in the upper-leftquadrant (high-PI and low-FITC signals)

210 Western Blot The entire proteins from fresh aorticwalls or treated cells were extracted using RIPA lysis bufferThen the nuclear protein fraction was prepared by a nuclearprotein extraction kit (BestBio China) in accordance withthe manufacturerrsquos instructions Equal amounts of proteinwere subjected to 8 to 15 SDS-PAGE and transferredonto PVDF membranes by electroblotting After blockingin Tris-buffered saline (TBS) containing 01 Tween 20and 10 nonfat dry milk for 2 h at room temperature themembranes were incubated with primary antibodies for3 h at room temperature or overnight at 4∘C After beingwashed four times with TBS containing 01 Tween 20 themembranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperatureImmunoblots were revealed by ECL reaction and visualizedusing a high-performance chemiluminescence filmThe IODvalue of immunoreactive bands was measured by Image-ProPlus software and normalized by house-keeping protein (120573-actin or histone H3)

211 Quantitative Real-Time PCR RT-PCR assay was appliedto detect the expression of LDL receptor (LDL-R) scav-enger receptor Class B Type 1 (SR-B1) 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR) and cholesterol 7120572-hydroxylase A1 (CYP7A1) in liver The first two are recep-tors of plasma cholesterol and the rest are key enzymesof cholesterol metabolism Primer Design 41 Softwarewas used to design the following primers 120573-actin for-ward primer 51015840-TTACTACTTTGCTGCGTTACACC-31015840reverse primer 51015840-CATGCCAATCTCATCTCGTTT-31015840(length of 78 bp) LDL-R forward primer 51015840-GACGTGTCC-CAGAGGAAGAT-31015840 reverse primer 51015840-CGAGTCGGT-CCAGTAGATGTT-31015840 (length of 144 bp) SR-B1 forwardprimer 51015840-TCTCCCACCCGCATTTCT-31015840 reverse primer51015840-CGCATACTGCACGTAGCACA-31015840 (length of 317 bp)CYP7A1 forward primer 51015840-CAGTATGCTGCTGTTTAT-G-31015840 reverse primer 51015840-GTTCTCGGTGGTGTTTCC-31015840(length of 335 bp) HMGR forward primer 51015840-TGATAG-CACCAGCAGATT-31015840 reverse primer 51015840-TATAAAGGT-TGCGTCCAG-31015840 (length of 68 bp) The primers were syn-thesized by TaKaRa Total liver RNA was separated by TRI-ZOL Reagent (Invitrogen) cDNA synthesis was performedusing MuLV Reverse Transcriptase (Applied Biosystems)Real-time PCR was performed using a SYBR-green PCRmaster mix kit (TianGen Biotech) The data was analyzed byusingRotor-geneQ software ver17 (Qiagen) RelativemRNAlevels were calculated by the 2minusΔΔCt method and normalizedagainst 120573-actin Each experiment was repeated three times

212 Statistical Analysis Results are shown as the mean plusmn SDfor at least three independent experiments Statistical analysiswas performed using one-way analysis of variance (ANOVA)

Table 1 Effect of niacin and simvastatin on plasma inflammatorymarkers (CRP IL-6 and TNF-120572) of guinea pig fed high fat diet

Group CRP (ngL) IL-6 (ngL) TNF-120572 (ngL)CD 31699 plusmn 2197 2588 plusmn 252 1308 plusmn 92

HFD 32113 plusmn 1538 2658 plusmn 242 1430 plusmn 157

HFD-N 30231 plusmn 1806 2152 plusmn 385lowastlowast

1179 plusmn 179lowastlowast

HFD-S 29557 plusmn 2578lowast

2367 plusmn 216lowast

1144 plusmn 193lowastlowast

The contents of CRP IL-6 and TNF-120572 in guinea pig plasma were measuredby ELISAmethod after 8 weeksrsquo treatment Data are presented as mean plusmn SD(119899 = 8) lowast119875 lt 005 lowastlowast119875 lt 001 versus HFD group

followed by Student-Newmann-Keuls multiple comparisontests with the SPSS 130 software for Windows 119875 values lessthan 005 were considered statistically significant

3 Results

At the beginning of the experiment 32 guinea pigs weredivided into 4 groups randomly andmean initial body weightwas 30227 plusmn 2367 g All guinea pigs survived for 8 weeksin the experiment and mean final body weight was 38489 plusmn2618 g No significant differences were observed among thesegroups for both the initial and final mean body weights

31 Niacin Attenuated the Systemic and Aortic Inflammationin Guinea Pigs Fed High Fat Diet

311 Niacin Significantly Downregulated IL-6 and TNF-120572Levels in Plasma of Guinea Pigs Fed High Fat Diet Inflam-matory process within the vessel wall can lead to vasculardysfunction and cause cardiovascular disease In this processinflammatory factors play a key role In this study the levelsof three major inflammatory factors (CRP IL-6 and TNF-120572) in plasma were measured Compared with CD grouphigh fat diet for 8 weeks lightly increased the levels ofCRP IL-6 and TNF-120572 in plasma but the increase was notstatistically significant (119875 gt 005) Compared with HFDgroup niacin decreased IL-6 level by 19 and decreasedTNF-120572 level by 18 whereas its effect on CRP had nostatistical difference Simvastatin decreased the levels of threeinflammatory markers in plasma compared with HFD group(Table 1)

312 Niacin Suppressed Protein Expression of CD68 and NF-120581B p65 in the Arterial Wall of Guinea Pigs Fed High FatDiet The invasion of monocyte into arterial intima and itsdifferentiation into resident macrophages may contribute toarterial inflammation in experimental atherosclerosis andhypertension In the study the immunohistochemical exam-ination of CD68 protein in the vessel wall was done tomark monocytesmacrophages Densitometric quantitativeimmunohistochemistry imaging revealed that comparedwith CD group the positive staining of CD68 was signifi-cantly increased in HFD group (119875 lt 001) compared withHFD group niacin and simvastatin significantly decreasedthe densitometric value of positive staining (Figures 1(a)

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CD HFD

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Figure 1Niacin and simvastatin reduced the expressions of CD68 andNF-120581Bp65 in the arterial wall of guinea pigs by immunohistochemistryanalysis after treatment for 8 weeks (a) and (c) show the representative immunostained aortic sections of CD68 and NF-120581B p65 respectively(20x magnification blue = nuclei and brown = target protein) (b) and (d) show the relative levels of CD68 and NF-120581B positive cells of perview field by densitometric quantitation respectively Data are presented as mean plusmn SD (119899 = 8)

119875 lt 001 versus CD group lowastlowast119875 lt 001versus HFD group

and 1(b)) These results indicated that niacin weakened theadhesion and invasion of monocytes in the arterial wall

NF-120581B is a transcription factor associatedwith the expres-sion of various proinflammatory mediators [12] When notstimulated it is found in the cytoplasm connected to itsinhibiting protein inhibitor 120581B kinase (I120581B) Stimulationby inflammatory factors causes degradation of I120581B proteinand then translocates NF-120581B to the nucleus In nucleus NF-120581B upregulates gene expressions of inflammatory moleculesincluding chemokines cytokines adhesion molecules andproteases [13] In order to further explore the mechanismthrough which niacin inhibited inflammatory progress wedetermined the expression of nuclear protein NF-120581B p65in the arterial wall by immunohistochemistry analysis andwestern blot The results all indicated that compared with

CD group high fat diet promoted the expression of activeNF-120581B p65 in the arterial wall (119875 lt 001) compared withHFD group niacin and simvastatin significantly decreasedthe expression (Figures 1(c) 1(d) 2(a) and 2(b))

313 Niacin Attenuated Oxidative Stress in Guinea Pigs FedHigh Fat Diet Oxidative stress plays an important role inthe inflammatory process [14] MDA is one of the mostreliable and widely used indices of oxidative stress [15]In our study we determined MDA level in plasma As shownin Figure 7 compared with that of CD group the level ofMDA in plasma was significantly increased in HFD group(119875 lt 001) Compared with that of HFD group niacin andsimvastatin significantly lowered the MDA level by 38 and43 respectively (Figure 3)

Mediators of Inflammation 5

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Figure 2 Niacin and simvastatin suppressed the expression ofnuclear proteinNF-120581Bp65 in the arterial wall of guinea pigs fed highfat diet The protein expression was analyzed by western blot andnormalized to histone H3 level (a) shows the representative imageby western blot (b) shows the IOD ratio of NF-120581B p65 to HistoneH3 Data are presented as mean plusmn SD of at least three independentexperiments

119875 lt 001 versus CD group lowastlowast119875 lt 001 versus HFDgroup

32 Niacin Inhibited oxLDL-Stimulated Apoptosis and Inflam-mation in HUVECs

321 Niacin Attenuated oxLDL-Stimulated Apoptosis ofHUVECs The inflammation and further damage includingapoptosis of arterial ECs are considered as early and neces-sary steps of AS as well as thrombosis In this study HUVECsapoptosis was analyzed via flow cytometry After HUVECswere stained with Annexin V-FITCPI flow cytometry anal-ysis revealed that apoptosis was induced by 150 120583gmL ox-LDL for 24 h which was significantly improved by niacin(Figure 4(a))

322 Niacin Decreased the Secretion of Inflammatory Cytok-ines TNF-120572 and IL-6 in oxLDL-Stimulated HUVECs Asshown in Figures 4(b) and 4(c) ox-LDL (150 120583gmL)markedly increased TNF-120572 and IL-6 protein levels by 76and 31 respectively in the medium of HUVECs Preincu-bation of cells with niacin (025ndash1mM) significantly inhibitedTNF-120572 expression by 12 21 and 27 respectively Mean-while 1mM niacin lowered IL-6 level by 15 in the medium

323 Niacin Suppressed NF-120581B p65 and Notch1 Expression inoxLDL-Stimulated HUVECs Notch signaling pathway playsa key role in the inflammatory progress It can activateNF-120581B and promote inflammatory factors secretion fromendothelial cells and macrophages [16] To further explorethe mechanism through which niacin inhibited cell injury

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Figure 3 Niacin and simvastatin decreased the level of plasmaMDA in guinea pigs after treatment for 8 weeks MDA wasdetermined by a spectrophotometricmeasurement of thiobarbituricacid-reactive substances (TBARS) according to the manufacturerrsquosinstruction Data are presented as mean plusmn SD (119899 = 8)

119875 lt 001

versus CD group lowastlowast119875 lt 001 versus HFD group

we determined the expressions of nuclear protein NF-120581B p65and notch1 by western blot The results showed that oxLDLmarkedly increased the protein levels of active NF-120581B p65and notch1 in HUVECs which were suppressed by prein-cubation of cells with niacin in a dose-dependent manner(Figures 4(d) 4(e) 4(f) and 4(g))

33 Niacin Suppressed Inflammatory Response Stimulated byoxLDL in THP-1 Macrophages

331 Niacin Decreased TNF-120572 and IL-6 Protein Secretionin the Medium of THP-1 Macrophages Next we assessedanti-inflammatory property of niacin in THP-1macrophagesAs shown in Figures 5(a) and 5(b) ox-LDL significantlypromoted TNF-120572 and IL-6 secretion by 89 and 23respectively in THP-1 macrophages Niacin (025ndash1mM)remarkably inhibited TNF-120572 expression by 11ndash30 and IL-6expression by 8ndash22 in the medium

332 Niacin Inhibited NF-120581B p65 and Notch1 Protein Expres-sion in oxLDL-Induced THP-1 Macrophages The effect ofniacin on the protein expressions of NF-120581B p65 in nuclei andnotch1 stimulated by ox-LDLwere examined Results showedthat niacin (025ndash1mM) significantly decreased NF-120581B levelby 75ndash83 and niacin (1mM) decreased notch1 level by 20(Figures 5(c) 5(d) 5(e) and 5(f))

34 Niacin Significantly Lessened Lipid Deposition in the Arte-rial Wall and Modified Lipoprotein Profile in Plasma viaModulatingCholesterolMetabolism in Liver of Guinea PigsFed High Fat Diet

341 Niacin Significantly Lessened Lipid Deposition in theArterial Wall of Guinea Pigs Fed High Fat Diet Oil red Ostaining in the aorta was found in HFD group but not inCD group because of chow diet without high fat Compared

6 Mediators of Inflammation

BlankoxLDL

0mM niacin 025mM niacin 05mM niacin 1mM niacin961 plusmn 063

702 plusmn 085

2551 plusmn 462

2132 plusmn 194

1844 plusmn 340

1463 plusmn 268

1371 plusmn 232

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750 plusmn 132

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Annexin-Y104103102101100

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(g)

Figure 4 Niacin inhibited oxLDL-stimulated apoptosis and inflammation in HUVECs (a) shows the ratio of apoptotic HUVECs stainedwith annexin V-FITC and PI Representative data of flow cytometry analysis are presented (b) and (c) show the relative levels of TNF-120572 andIL-6 in the medium of HUVECs The concentrations of IL-6 and TNF-120572 were determined by ELISA kit (d) and (e) show the representativeimages of NF-120581B p65 and notch1 protein expression in HUVECs by western blot (f) and (g) show the IOD ratios of NF-120581B p65 and notch1expression respectively Data are presented as mean plusmn SD

119875 lt 001 versus blank group lowast119875 lt 005 lowastlowast119875 lt 001 versus oxLDL-treatedgroup without niacin

Mediators of Inflammation 7

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oxLDL ++++minus

Niacin (mM) 0 0 025 05 1oxLDL ++++minus

Niacin (mM) 0 0 025 05 1

Figure 5 Niacin decreased the secretion of TNF-120572 and IL-6 and inhibited NF-120581B and notch1 expression in oxLDL-stimulated THP-1macrophages (a) and (b) show the relative levels of TNF-120572 and IL-6 secretion in the medium of THP-1 macrophages The concentrations ofIL-6 and TNF-120572 were determined by ELISA kit (c) and (d) show the representative images of NF-120581B p65 and notch1 protein expression inTHP-1 macrophages by western blot (e) and (f) show the IOD ratios of NF-120581B p65 and notch1 expression respectively Data are presented asmean plusmn SD

119875 lt 001 versus blank group lowast119875 lt 005 lowastlowast119875 lt 001 versus oxLDL-treated group without niacin

with that of HFD group niacin and simvastatin significantlydecreased the percentages of stained area to the total cross-sectional vessel wall by 56 and 67 respectively (Figure 6)The effect of simvastatin was superior to that of niacin

342 Niacin Increased HDL-C and ApoA I Levels andDecreased TG and Non-HDL-C Levels in Plasma of GuineaPigs Fed High Fat Diet As shown in Figure 7 after highfat diet for 8 weeks the levels of plasma TC TG HDL-Cand non-HDL-C were significantly increased in HFD groupcompared with CD group (119875 lt 001) which indicated asuccessful hyperlipidemic model in guinea pigs Comparedwith HFD group niacin decreased the levels of TG and

non-HDL-C by 27 and 12 respectively and increasedHDL level by 21 Niacin had no statistical influence onTC level in plasma Compared with HFD group simvastatindecreased the levels of TG non-HDL-C and TC by 1853 and 51 respectively Simvastatin had no significantinfluence on HDL-C level

The level of apoA I in plasma was also detected by SDS-PAGE in this study Comparedwith that ofHFDgroup niacinsignificantly promoted the level of apoA I by 42 whereassimvastatin had no significant influence on apoA I (Figure 8)

343 Niacin Significantly Upregulated the mRNA Amountof CYP7A1 in Liver Cholesterol metabolism in liver is a

8 Mediators of Inflammation

CD HFD

HFD-N HFD-S

(a)

0

05

1

15

2

CD HFD HFD-N HFD-S

Oil

red

O st

aine

d ar

ea (

)

lowastlowast

lowastlowast

(b)

Figure 6 Niacin and simvastatin significantly lessened lipid depo-sition in the arterial wall of guinea pigs fed high fat diet Lipiddeposition in the aorta wall was analyzed by oil red O staining aftertreatment for 8 weeksThe quantification of stained lipids was deter-mined by calculating the percentage of the positive area to the totalcross-sectional vessel wall area by Image-Pro Plus software Data arepresented as mean plusmn SD (119899 = 8)

119875 lt 001 versus CD grouplowastlowast

119875 lt 001 versus HFD group

complicated homeostasis involving multiple steps includingcholesterol ingression synthesis and conversion SR-B1 andLDL-R in liver play a critical role in cholesterol ingressionSR-B1 is the HDL receptor on the hepatocyte surface LDL-R can bind to LDL and VLDL and internalize them intohepatocytes [17] In the study we determined the effect ofniacin on the expressions of SR-B1 and LDL-R mRNA inliver As shown in Figures 9(a) and 9(b) after treatment withhigh fat diet for 8 weeks the LDL-R mRNA level was down-regulated (119875 lt 001) and the SR-B1 mRNA level wasnot significantly changed in HFD group Compared withHFD group niacin had no significant effect on SR-B1 and

LDL-R mRNA levels but simvastatin upregulated LDL-RmRNA level by 71

Cholesterol in liver can be converted into bile acidthrough cytochrome P450-meidiated oxidation The rate-limiting enzyme for the dominant pathway of bile acidsynthesis is cytochrome P450 7A1 (CYP7A1) As shown inFigure 9(c) compared with HFD group niacin significantlyupregulated the CYP7A1 mRNA level by 59 whereassimvastatin had no significant influence on its level HMGCRis the rate-limiting enzyme in the process of cholesterolsynthesis Compared with that of CD group the mRNA levelof HMGCR was significantly decreased in HFD group (119875 lt001) Compared with HFD group simvastatin upregulatedthe HMGCR mRNA levels by 46 whereas niacin had nosignificant influence on its level (Figure 9(d))

4 Discussion

For the first time to our knowledge this report demonstratesniacin inhibited vascular inflammation in guinea pig fedhigh fat diet and suppressed oxLDL-stimulated inflammatoryresponse even injury of endothelial cells and macrophagesin vitro The result indicates a new mechanism for niacinrsquosprotective action on cardiovascular disease in addition to itsestablished effects on lipid metabolism

The augmentation of inflammatory response has beenclearly documented in pathogenesis of vascular impairmentThe chronic inflammatory pathogenesis in the arterial wall isas follows Harmful substances in blood such as hypercholes-terolemia can induce endothelial dysfunction This causesthe production of ROS and the secretion of cellular adhesionmolecules (CAMs) cytokines and chemokines which facili-tate adherence and endothelial transmigration of leukocytes(monocytes and T helper lymphocytes) Monocytes in thearterial wall will be activated by proinflammatory cytokinesand differentiated into macrophages Activated macrophagesincrease the expression of CAMs and cytokines which resultsin recruitment of more leukocytes into the arterial wall acti-vates the complement pathways of immune system and theacute phase response stimulates proliferation and migrationof smooth muscle cells (SMCs) and promotes fibrous tissuedeposition [18] In progress the signaling molecule NF-120581Bis a proinflammatory major switch that can upregulate theexpression of lots of cytokines [19] Activated NF-120581B canresult in the enhanced efflux of TNF-120572 and IL-6 in serum[20] Early events in AS are usually driven by NF-120581B and thedisruption of NF-120581B signaling pathway has been shown toslow down the vascular impairment [21]

In the present study we demonstrate that niacin attenu-ated vascular inflammation induced by high fat diet in vivoThe involved evidences are as follows (1) Niacin lowered thenumber of macrophages (CD68 positive cells) in the arterialwall and significantly downregulated the inflammatory fac-tors (IL-6 and TNF-120572) levels in plasma of guinea pigs fed highfat diet (2) Both immunohistochemistry and western blotanalysis indicated niacin suppressed the expression of activeNF-120581B p65 in nuclei of the arterial wall The activated NF-120581Bis reported to form a heterodimer which usually consists oftwo proteins p65 and p50 subunitsThe p65 subunit has been

Mediators of Inflammation 9

0

50

100

150

200

250

CD HFD HFD-N HFD-S

TG (m

gdL

)

lowastlowastlowastlowast

(a)

0

500

1000

1500

2000

CD HFD HFD-N HFD-S

TC (m

gdL

)

lowastlowast

(b)

0

20

40

60

80

100

120

CD HFD HFD-N HFD-S

HD

L-C

(mg

dL)

lowast

(c)

0

500

1000

1500

2000

CD HFD HFD-N HFD-S

Non

-HD

L-C

(mg

dL)

lowastlowast

lowast

(d)

Figure 7 Effect of niacin and simvastatin on plasma lipid of guinea pigs fed high fat diet The levels of TG (a) TC (b) HDL-C (c) andnon-HDL-C (d) in plasma of guinea pigs were determined by enzyme method after 8 weeksrsquo treatment Data are presented as mean plusmn SD(119899 = 8)

119875 lt 001 versus CD group lowast119875 lt 005 lowastlowast119875 lt 001 versus HFD group

apoAI20

30

40

50

6080

160110

Mar

ker

CD HFD

HFD

-N

HFD

-S

Hum

an

(a)

0

05

1

15

2

CD HFD HFD-N HFD-S

Fold

indu

ctio

n of

apoA

I in

HD

L

lowastlowast

(b)

Figure 8 Niacin upregulated apoA I level in plasma of guinea pigs fed high fat diet HDL (density = 109ndash124 gmL) was separatedfrom plasma of guinea pigs by sequential ultracentrifugation The SDS-PAGE was performed on 15 SDS polyacrylamide gel and theapolipoproteins were stained with coomassie brilliant blue Densitometric quantitation of SDS-PAGE image was analyzed by Image-Pro Plussoftware (a) shows the representative SDS-PAGE image ofHDL (b) shows the relative level of apoA I in plasma by densitometric quantitationData are presented as mean plusmn SD of at least three independent experiments

119875 lt 005 versus CD group lowastlowast119875 lt 001 versus HFD group

10 Mediators of Inflammation

0

02

04

06

08

1

12

14

16

CD HFD HFD-N HFD-S

Relat

ive m

RNA

leve

l of S

R-B1

(a)

0

02

04

06

08

1

12

CD HFD HFD-N HFD-S

Relat

ive m

RNA

leve

l of L

DL-

R

lowastlowast

(b)

0

05

1

15

2

CD HFD HFD-N HFD-S

Relat

ive m

RNA

leve

l of C

YP7A

1 lowastlowast

(c)

0

02

04

06

08

1

12

14

CD HFD HFD-N HFD-S

Relat

ive m

RNA

leve

l of H

MG

CR

lowastlowast

(d)

Figure 9 Effect of niacin and simvastatin on the mRNA abundance of SR-B1 LDL-R CYP7A1 and HMGCR in liver of guinea pigs fedhigh fat dietThe mRNA levels which were analyzed by quantitative real-time PCR were calculated after being adjusted for 120573-actin usingthe 2minusΔΔCt method Data are presented as mean plusmn SD of at least three independent experiments

119875 lt 005 119875 lt 001 versus CD group

lowastlowast

119875 lt 001 versus HFD group

demonstrated to exert critical activity on the transcriptionof many inflammatory genes including adhesion moleculescytokines and chemokines [22] (3) CRP an early acute phasereactant is closely relevant to inflammation Baseline level ofCRP is a strong independent predictor of the risk of futuremyocardial infarction peripheral vascular disease strokeand vascular death amonghealthy individualswithout knownvascular disease [23] Kuvin et al have shown that niacindecreased CRP level by 15 in patients with stable coronaryartery disease [5] In patients with metabolic syndromeafter treatment with extended-release niacin (1 gday) for 52weeks their endothelial function was improved by 22 andthere was a decrease in CRP level by 20 [24] Our resultsalso showed niacin slightly lowered CRP level but hadno statistical difference (Table 1) (4) Oxidative stress wassuppressed by niacin Oxidative stress is closely related to theinflammation in the arterial wall Increased ROS productioncan initiate a cascade of signal transduction which results inendothelial dysfunction changes in vascular tone vascularremodeling and vascular inflammatory responses [25]

To further confirm the direct anti-inflammatory prop-erty of niacin its effect on oxLDL-induced inflammatoryresponse of endothelial cells and macrophages was studiedoxLDL is pivotal in the development of AS and representsa crucial proinflammatory stimulus [26] Upon enteringinto the intima of arteries oxLDL activates endothelial

cells and upregulates adhesion molecule expression andinflammatory factors secretion all of which contribute tothe recruitment of circulating leukocytes Monocytes andormacrophages infiltrating the arterial wall take up oxLDL andform ldquofoam cellsrdquo which in turn promote further secretionof inflammatory mediators [27] Our data indicated niacinremarkably downregulated the secretion of TNF-120572 and IL-6stimulated by oxLDL in HUVECs and THP-1 macrophagesNotch1 signal pathway is an evolutionarily highly conservedmechanism for communication which can improve NF-120581Bactivity and have a positive correlation with the developmentof inflammation [28] A previous study demonstrates thatnotch knockout in macrophage markedly downregulates theexpression of proinflammatory factors Instead when thenotch signal pathway is activatedNF-120581B expression increasesand inflammatory factors secretion is also promoted [29]Our findings indicated niacin decreased the expressions ofNF-120581B and notch1 in oxLDL-induced inflammation Vas-cular endothelial apoptosis plays an important role in theinitiation of AS It may compromise vessel wall permeabilityto cytokines growth factors lipids and immune cells andincrease coagulation [30] In this study we indicated niacinattenuated oxLDL-induced apoptosis of HUVECs

Taken together these observations lend initial but com-pelling evidence that niacin has protective effects on car-diovascular disease through its vascular anti-inflammatory

Mediators of Inflammation 11

properties in addition to its established effects on lipidmetabolism

Reverse cholesterol transport (RCT) is a potent defensemechanism against AS It involves transporting cholesterolfromperipheral tissues and cells to liver transforming choles-terol into bile acid and finally eliminating it from the body[31] HDL and apoA play a key role in RCT They can drivecholesterol efflux from macrophage foam cells that residein vessel intima [32 33] Similar to previous studies ourfindings also indicated that niacin increased the levels ofHDL and apoA I in plasma significantly In the process ofRCT CYP7A1 is an important regulatory factor which canconvert cholesterol to bile acid in liver and be further excretedinto intestine for elimination from body with feces Here weshowed that the mRNA abundance of CYP7A1 in liver wassignificantly upregulated by niacin treatment These findingssuggest that niacin may modulate plasma lipid and exertantiatherosclerotic effects by promoting RCT progress

In clinical practice the oral dose of niacin for lipid-lowering purpose is usually 1ndash3 g per day equivalently 14ndash43mgkg per day According to the formula of Meeh-Rubnerequation we calculate the dose for guinea pig to be about80ndash250mgkg per day In addition a recent report showedthat up to 400mgkg per day of niacin treatment for 7 daysand 100mgkg per day of niacin treatment for 4 weeks aresafe and do not cause toxicity in rodent models [34] In thisstudy the applied dose of niacin for guinea pig is 100mgkgdwhich has pharmacologic effects and does not have varioustoxicities based on the changes in food intake body weightand behaviors In human plasma level of niacin was foundto be about 03mM after oral ingestion of 1 g niacin [3536] Since the reported plasma concentrations are one timemeasurement it is unclear whether these levels reflect troughor peak values Furthermore there is no reported clinicaldata available regarding plasma concentration of niacin afteringestion of 3 g niacin Extrapolation of the available datausing oral dose of 1 g niacin would indicate that the 3 g niacinoral administration may lead to the plasma concentrations ofniacin in the range of 08ndash1mM In our in-vitro studies weused 025 to 1mM niacin

Statins are the first-line therapy in the treatment ofcholesterol-induced atherosclerotic cardiovascular diseases[37] They suppress the conversion of HMG-CoA to meval-onate by competitively blocking HMGCR therefore theendogenous cholesterol synthesis in the liver is remarkablyreduced Consequently it results in the increase of LDL-Rexpression on hepatic cell surfaces and the decrease of LDL-Cin plasma [38]Meanwhile inhibition of cholesterol synthesiscauses an overexpression of HMGCR due to a feedback-regulatory mechanism [39] Similar to previous studies ourfindings also revealed that simvastatin significantly decreasedthe level of non-HDL-C in plasma followed by upregulatingLDL-R expression in liver and the HMGCR mRNA level inliver was increased in a compensatory way

5 Conclusion

In summary our data presented herein support the novelconcept that niacin has vascular anti-inflammatory and

potentially vascular-protective property which is indepen-dent of its effect on lipid regulation The anti-inflammatoryproperty of niacin is realized by downregulating nucleartranscription factor-120581B signaling pathway Meanwhile ourfinding also suggests that niacin modulates plasma lipid bystimulating the expression of factors involved in the processof RCT and promoting RCT

Conflict of Interests

The authors declare that they have no conflict of interest

Authorsrsquo Contribution

Yanhong Si and Ying Zhang have equally contributed to thisstudy

Acknowledgments

This research was supported by the financial supportsfrom the Taishan scholar foundation of Shandong Province(200867) and the Natural Science Foundations of China(81170785 and 81371234)

References

[1] R Altschul A Hoffer and J D Stephen ldquoInfluence of nicotinicacid on serum cholesterol in manrdquoArchives of Biochemistry andBiophysics vol 54 no 2 pp 558ndash559 1955

[2] C D Meyers V S Kamanna and M L Kashyap ldquoNiacin ther-apy in atherosclerosisrdquo Current Opinion in Lipidology vol 15no 6 pp 659ndash665 2004

[3] L A Carlson ldquoNicotinic acid the broad-spectrum lipid drug A50th anniversary reviewrdquo Journal of Internal Medicine vol 258no 2 pp 94ndash114 2005

[4] S H Ganji S Qin L Zhang V S Kamanna andM L KashyapldquoNiacin inhibits vascular oxidative stress redox-sensitive genesand monocyte adhesion to human aortic endothelial cellsrdquoAtherosclerosis vol 202 no 1 pp 68ndash75 2009

[5] J T Kuvin D M Dave K A Sliney et al ldquoEffects of extended-release niacin on lipoprotein particle size distribution andinflammatorymarkers in patients with coronary artery diseaserdquoAmerican Journal of Cardiology vol 98 no 6 pp 743ndash745 2006

[6] R Yang P Guo X Song F Liu and N Gao ldquoHyperlipidemicguinea pig model mechanisms of triglyceride metabolismdisorder and comparison to ratrdquo Biological and PharmaceuticalBulletin vol 34 no 7 pp 1046ndash1051 2011

[7] X Li Y Liu H Zhang L Ren Q Li and N Li ldquoAnimal modelsfor the atherosclerosis research a reviewrdquo Protein and Cell vol2 no 3 pp 189ndash201 2011

[8] J O Leite U Vaishnav M Puglisi H Fraser J Trias and M LFernandez ldquoA-002 (Varespladib) a phospholipase A2 inhibitorreduces atherosclerosis in guinea pigsrdquo BMC CardiovascularDisorders vol 9 article 7 2009

[9] J Auwerx ldquoThe human leukemia cell line THP-1 a multi-facetted model for the study of monocyte-macrophage differ-entiationrdquo Experientia vol 47 no 1 pp 22ndash31 1991

[10] M J Chapman G L Mills and J H Ledford ldquoThe distributionand partial characterization of the serum apolipoproteins in the

12 Mediators of Inflammation

guinea pigrdquo Biochemical Journal vol 149 no 2 pp 423ndash4361975

[11] X C Jiang C Bruce J Mar et al ldquoTargetedmutation of plasmaphospholipid transfer protein gene markedly reduces high-density lipoprotein levelsrdquo Journal of Clinical Investigation vol103 no 6 pp 907ndash914 1999

[12] T Collins and M I Cybulsky ldquoNF-120581B pivotal mediator orinnocent bystander in atherogenesisrdquo Journal of Clinical Inves-tigation vol 107 no 3 pp 255ndash264 2001

[13] Y Tsubosaka T Murata K Yamada D Uemura M Horiand H Ozaki ldquoHalichlorine reduces monocyte adhesion toendothelium through the suppression of nuclear factor-120581Bactivationrdquo Journal of Pharmacological Sciences vol 113 no 3pp 208ndash213 2010

[14] H Tsutsui ldquoMitochondrial oxidative stress and heart failurerdquoInternal Medicine vol 45 no 13 pp 809ndash813 2006

[15] M Seddon Y H Looi and A M Shah ldquoOxidative stressand redox signalling in cardiac hypertrophy and heart failurerdquoHeart vol 93 no 8 pp 903ndash907 2007

[16] WWongchana and T Palaga ldquoDirect regulation of interleukin-6 expression by Notch signaling in macrophagesrdquo Cellular andMolecular Immunology vol 9 no 2 pp 155ndash162 2012

[17] J Chen H Zhao X Ma et al ldquoThe effects of jiang-zhi-ningand itsmain components on cholesterol metabolismrdquo Evidence-Based Complementary and Alternative Medicine vol 2012Article ID 928234 10 pages 2012

[18] G Francisco C Hernandez and R Simo ldquoSerum markers ofvascular inflammation in dyslipemiardquo Clinica Chimica Actavol 369 no 2 pp 1ndash16 2006

[19] J Andersson P Libby and G K Hansson ldquoAdaptive immunityand atherosclerosisrdquoClinical Immunology vol 134 no 1 pp 33ndash46 2010

[20] W J Oh U Jung H S Eom H J Shin and H R Park ldquoInhibi-tion of lipopolysaccharide-induced proinflammatory responsesby Buddleja officinalis extract in BV-2 microglial cells vianegative regulation of NF-kB and ERK12 signalingrdquoMoleculesvol 18 no 8 pp 9195ndash9206 2013

[21] T M Wang C J Chen T S Lee et al ldquoDocosahexaenoic acidattenuates VCAM-1 expression and NF-120581B activation in TNF-120572-treated human aortic endothelial cellsrdquo Journal of NutritionalBiochemistry vol 22 no 2 pp 187ndash194 2011

[22] M S Islam H Yoshida N Matsuki et al ldquoAntioxidant freeradical-scavenging and NF-120581B-inhibitory activities of phy-tosteryl ferulates structure-activity studiesrdquo Journal of Pharma-cological Sciences vol 111 no 4 pp 328ndash337 2009

[23] P M Ridker M J Stampfer and N Rifai ldquoNovel risk factorsfor systemic atherosclerosis a comparison ofC-reactive proteinfibrinogen homocysteine lipoprotein(a) and standard choles-terol screening as predictors of peripheral arterial diseaserdquoJournal of the AmericanMedical Association vol 285 no 19 pp2481ndash2485 2001

[24] M Thoenes A Oguchi S Nagamia et al ldquoThe effects ofextended-release niacin on carotid intimal media thicknessendothelial function and inflammatory markers in patientswith the metabolic syndromerdquo International Journal of ClinicalPractice vol 61 no 11 pp 1942ndash1948 2007

[25] N R Madamanchi A Vendrov and M S Runge ldquoOxidativestress and vascular diseaserdquo Arteriosclerosis Thrombosis andVascular Biology vol 25 no 1 pp 29ndash38 2005

[26] E Matsuura F Atzeni P Sarzi-Puttini M Turiel L R Lopezand M T Nurmohamed ldquoIs atherosclerosis an autoimmunediseaserdquo BMCMedicine vol 12 no 1 article 47 2014

[27] A Pirillo G D Norata and A L Catapano ldquoLOX-1 OxLDLand atherosclerosisrdquo Mediators of Inflammation vol 2013Article ID 152786 12 pages 2013

[28] C L Wang M Meng S B Liu L R Wang L H Hou and XH Cao ldquochemically sulfated polysaccharide from Grifola fron-dos induces HepG2 cell apoptosis by notch1-NF-120581B pathwayrdquoCarbohydrate Polymers vol 95 no 1 pp 282ndash287 2013

[29] WWongchana and T Palaga ldquoDirect regulation of interleukin-6 expression by Notch signaling in macrophagesrdquo Cellular andMolecular Immunology vol 9 no 2 pp 155ndash162 2012

[30] J C Choy D J Granville D W C Hunt and B M McManusldquoEndothelial cell apoptosis biochemical characteristics andpotential implications for atherosclerosisrdquo Journal of Molecularand Cellular Cardiology vol 33 no 9 pp 1673ndash1690 2001

[31] M Cuchel and D J Rader ldquoMacrophage reverse cholesteroltransport key to the regression of atherosclerosisrdquo Circulationvol 113 no 21 pp 2548ndash2555 2006

[32] A Yazdanyar C Yeang and X C Jiang ldquoRole of phospholipidtransfer protein in high-density lipoprotein-mediated reversecholesterol transportrdquo Current Atherosclerosis Reports vol 13no 3 pp 242ndash248 2011

[33] X Wang and D J Rader ldquoMolecular regulation of macrophagereverse cholesterol transportrdquo Current Opinion in Cardiologyvol 22 no 4 pp 368ndash372 2007

[34] S J Yang J M Choi L Kim et al ldquoNicotinamide improvesglucose metabolism and affects the hepatic NAD-sirtuin path-way in a rodent model of obesity and type 2 diabetesrdquo Journalof Nutritional Biochemistry vol 25 no 1 pp 66ndash72 2014

[35] L A Carlson L Oro and J Ostman ldquoEffect of a single dose ofnicotinic acid on plasma lipids in patients with hyperlipopro-teinemiardquo Acta Medica Scandinavica vol 183 no 5 pp 457ndash465 1968

[36] R H Stern J D Spence D J Freeman and A Parbtani ldquoTol-erance to nicotinic acid flushingrdquo Clinical Pharmacology andTherapeutics vol 50 no 1 pp 66ndash70 1991

[37] S M Grundy J I Cleeman C N Bairey Merz et al ldquoImpli-cations of recent clinical trials for the National CholesterolEducation Program Adult Treatment Panel III guidelinesrdquoCirculation vol 110 no 2 pp 227ndash239 2004

[38] M S Brown and J L Goldstein ldquoLowering plasma cholesterolby raising LDL receptorsrdquoNewEngland Journal ofMedicine vol305 no 9 pp 515ndash517 1981

[39] R Hampton D Dimster-Denk and J Rine ldquoThe biology ofHMG-CoA reductase the pros of contra-regulationrdquo Trends inBiochemical Sciences vol 21 no 4 pp 140ndash145 1996

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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 2: Research Article Niacin Inhibits Vascular Inflammation via ...downloads.hindawi.com/journals/mi/2014/263786.pdf · Research Article Niacin Inhibits Vascular Inflammation via Downregulating

2 Mediators of Inflammation

one of the few species that carry the majority of cholesterolin LDL [7] It is possible to induce the initial stages ofAS quickly by feeding them high fat diet [8] In additionsignificant atherogenic inflammation with an increase ofaortic cytokines has been documented in guinea pigs [7] Inthe study we select guinea pigs as animal model to study theprotective effect of niacin on vascular inflammatory lesionsinduced by high fat diet in vivo Meanwhile to furtherconfirm the direct anti-inflammatory property of niacin itseffect on oxLDL-induced inflammation of endothelial cellsand macrophages is also explored

2 Materials and Methods

21 Animal and Experimental Design Thirty-two male Eng-land short-hair guinea pigs (260sim310 g 5 months old) werebought from the Animal Laboratory Center of Taibang Bio-logical Products Co Shandong China All experiments wereapproved by the Laboratory Animalsrsquo Ethical Committee ofTaishanMedical University and abided by national guidelinesfor the care and use of animals All guinea pigswere randomlydivided into 4 groups regular chow diet group (CD) highfat diet group (HFD 10 lard + 10 yolk power + 030cholesterol + 797 grass) HFD with niacin group (HFD-NHFD+ 100mgkgd niacin) andHFDwith simvastatin group(HFD-S HFD + 20mgkgd simvastatin) Each group isassigned 8 guinea pigsThedrugs (niacin or simvastatin)werefed by oral gavage once daily for 8 weeks

22 Cell Culture Human umbilical vein endothelial cells(HUVECs EAhy926) and THP-1 (human monocyte) wereall purchased from Shanghai Institute of Biochemistry Theywere maintained in RPMI 1640 medium (HyClone China)supplementedwith 10 fetal bovine serum (HyClone China)in a 5 CO2 incubator at 37∘C To induce monocytesdifferentiation into macrophages THP-1 cells were culturedwith 50 ngmL phorbol 12-myristate 13-acetate (PMA Sigma)for 24 hours as described previously [9]

23 Immunohistochemistry Examination Tissue sections(5 120583m) from formalin-fixed paraffin-embedded specimenswere stained with specific antibodies against NF-120581B p65 andCD68 proteins (Zhongshan Biotechnology Co Ltd BeijingChina) respectively The sections were developed with 331015840-diaminobenzidine (Vector Laboratories) and counterstainedwith Mayerrsquos hematoxylin (Saturatedard Allen) Images werecaptured using microscope (Olympus) All quantificationswere examined by calculating the percentage of integrateoptical density (IOD) of the antigen positive staining to theentire cross-sectional vessel wall by Image-Pro Plus software

24 Analysis of Lipid Deposition in the Arterial Wall Theproximal aorta attached to the heart was applied to preparecryosections Cryosections (8120583m) were cut gathered andstained with oil redOThe quantification of stained lipids wasexamined by calculating the percentage of the positive area tothe total cross-sectional vessel wall area by using Image-ProPlus software 45 (Media Cybernetics) The percentage wascalculated from five sections from an animal

25 Measurement of Inflammatory Factors Plasma concen-trations of IL-6 TNF-120572 and CRP which can be secreted dur-ing the process of inflammation were determined by ELISAkit (American RampD) in accordance with the manufacturerrsquosinstructions

ELISA kits were also used for the measurement of TNF-120572 and IL-6 protein levels in the medium of HUVECs andTHP-1 macrophages Cells were incubated in the absenceor presence of niacin (025ndash1mM) for 24 h in serum-freemedia and then stimulated by 150 120583gmL ox-LDL (BeijingXiesheng Biotechnology Co Ltd China) for 6 h TNF-120572 andIL-6 protein levels were assessed respectively Results werecalculated as ngL and expressed as a percentage of thoseobtained with blanks

26 Measurement of Oxidative Stress Reaction in PlasmaPlasma level of malondialdehyde (MDA) amarker for oxida-tive stress was determined by a spectrophotometric mea-surement of thiobarbituric acid-reactive substances (TBARS)in accordance with the manufacturerrsquos instructions (NanjingJiancheng Bioengineering Institute China)

27 Plasma Lipid Analysis After being treated by niacin orsimvastatin for 8 weeks blood was collected by cardiac punc-ture from guinea pigs without dietary exposure for 12 hoursConcentrations of plasma total cholesterol (TC) triglyceride(TG) and HDL cholesterol (HDL-C) were determined byenzymatic methods (BioSino Beijing China) Non-HDL-Cwas calculated as TC minus HDL-C

28 Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophore-sis (SDS-PAGE)Analysis of Apolipoproteins inHDL Lipopro-tein isolation was carried out by sequential ultracentrifu-gation in a LE-80K ultracentrifuge (Beckman Coulter IncBrea CA USA) as described before Separation was doneaccording to the following density fractionation 119889 lt1019 gmL for VLDL and IDL d 1019ndash109 gmL for LDLand d 109ndash124 gmL for HDL [10] The isolated specimenswere dialyzed in 150mmolL NaCl and 03mmolL EDTAat 4∘C HDL containing equal amounts of cholesterol wasloaded on a 15 sodium dodecyl sulfate (SDS) polyacry-lamide gradient gel and apolipoprotein samples were stainedwith coomassie brilliant blue as described by Jiang et al [11]Meanwhile the marker (Invitrogen LC5800) was put in lane1 and HDL from human was put in the last lane for contrastStained gels were scanned and analyzed by Quantity One(Bio-Rad Hercules CA USA) software program

29 Analysis of Cell Apoptosis by Flow Cytometry AnnexinV-FITCPI double-staining assay was applied to measureapoptosis according to the manufacturerrsquos instructions AfterHUVECs were stimulated by ox-LDL for 24 h cells werecentrifuged washed twice with PBS resuspended in 500 uLbinding buffer and incubated with 5 uL fluorescein isoth-iocyanate (FITC)-labeled Annexin V and 5 uL propidiumiodide (PI) for 10 minutes at room temperature in the darkThe scatter parameters of cells were analyzed by FAC Scanflow cytometer and Cell Quest analysis software (Becton-Dickinson CA USA) Four cell populations were discerned

Mediators of Inflammation 3

according to the following status live cells in the lower-leftquadrant (low-PI and FITC signals) early apoptotic cells inthe lower-right quadrant (low-PI and high-FITC signals) lateapoptotic or necrotic cells in the upper-right quadrant (high-PI and high-FITC signals) and necrotic cells in the upper-leftquadrant (high-PI and low-FITC signals)

210 Western Blot The entire proteins from fresh aorticwalls or treated cells were extracted using RIPA lysis bufferThen the nuclear protein fraction was prepared by a nuclearprotein extraction kit (BestBio China) in accordance withthe manufacturerrsquos instructions Equal amounts of proteinwere subjected to 8 to 15 SDS-PAGE and transferredonto PVDF membranes by electroblotting After blockingin Tris-buffered saline (TBS) containing 01 Tween 20and 10 nonfat dry milk for 2 h at room temperature themembranes were incubated with primary antibodies for3 h at room temperature or overnight at 4∘C After beingwashed four times with TBS containing 01 Tween 20 themembranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperatureImmunoblots were revealed by ECL reaction and visualizedusing a high-performance chemiluminescence filmThe IODvalue of immunoreactive bands was measured by Image-ProPlus software and normalized by house-keeping protein (120573-actin or histone H3)

211 Quantitative Real-Time PCR RT-PCR assay was appliedto detect the expression of LDL receptor (LDL-R) scav-enger receptor Class B Type 1 (SR-B1) 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR) and cholesterol 7120572-hydroxylase A1 (CYP7A1) in liver The first two are recep-tors of plasma cholesterol and the rest are key enzymesof cholesterol metabolism Primer Design 41 Softwarewas used to design the following primers 120573-actin for-ward primer 51015840-TTACTACTTTGCTGCGTTACACC-31015840reverse primer 51015840-CATGCCAATCTCATCTCGTTT-31015840(length of 78 bp) LDL-R forward primer 51015840-GACGTGTCC-CAGAGGAAGAT-31015840 reverse primer 51015840-CGAGTCGGT-CCAGTAGATGTT-31015840 (length of 144 bp) SR-B1 forwardprimer 51015840-TCTCCCACCCGCATTTCT-31015840 reverse primer51015840-CGCATACTGCACGTAGCACA-31015840 (length of 317 bp)CYP7A1 forward primer 51015840-CAGTATGCTGCTGTTTAT-G-31015840 reverse primer 51015840-GTTCTCGGTGGTGTTTCC-31015840(length of 335 bp) HMGR forward primer 51015840-TGATAG-CACCAGCAGATT-31015840 reverse primer 51015840-TATAAAGGT-TGCGTCCAG-31015840 (length of 68 bp) The primers were syn-thesized by TaKaRa Total liver RNA was separated by TRI-ZOL Reagent (Invitrogen) cDNA synthesis was performedusing MuLV Reverse Transcriptase (Applied Biosystems)Real-time PCR was performed using a SYBR-green PCRmaster mix kit (TianGen Biotech) The data was analyzed byusingRotor-geneQ software ver17 (Qiagen) RelativemRNAlevels were calculated by the 2minusΔΔCt method and normalizedagainst 120573-actin Each experiment was repeated three times

212 Statistical Analysis Results are shown as the mean plusmn SDfor at least three independent experiments Statistical analysiswas performed using one-way analysis of variance (ANOVA)

Table 1 Effect of niacin and simvastatin on plasma inflammatorymarkers (CRP IL-6 and TNF-120572) of guinea pig fed high fat diet

Group CRP (ngL) IL-6 (ngL) TNF-120572 (ngL)CD 31699 plusmn 2197 2588 plusmn 252 1308 plusmn 92

HFD 32113 plusmn 1538 2658 plusmn 242 1430 plusmn 157

HFD-N 30231 plusmn 1806 2152 plusmn 385lowastlowast

1179 plusmn 179lowastlowast

HFD-S 29557 plusmn 2578lowast

2367 plusmn 216lowast

1144 plusmn 193lowastlowast

The contents of CRP IL-6 and TNF-120572 in guinea pig plasma were measuredby ELISAmethod after 8 weeksrsquo treatment Data are presented as mean plusmn SD(119899 = 8) lowast119875 lt 005 lowastlowast119875 lt 001 versus HFD group

followed by Student-Newmann-Keuls multiple comparisontests with the SPSS 130 software for Windows 119875 values lessthan 005 were considered statistically significant

3 Results

At the beginning of the experiment 32 guinea pigs weredivided into 4 groups randomly andmean initial body weightwas 30227 plusmn 2367 g All guinea pigs survived for 8 weeksin the experiment and mean final body weight was 38489 plusmn2618 g No significant differences were observed among thesegroups for both the initial and final mean body weights

31 Niacin Attenuated the Systemic and Aortic Inflammationin Guinea Pigs Fed High Fat Diet

311 Niacin Significantly Downregulated IL-6 and TNF-120572Levels in Plasma of Guinea Pigs Fed High Fat Diet Inflam-matory process within the vessel wall can lead to vasculardysfunction and cause cardiovascular disease In this processinflammatory factors play a key role In this study the levelsof three major inflammatory factors (CRP IL-6 and TNF-120572) in plasma were measured Compared with CD grouphigh fat diet for 8 weeks lightly increased the levels ofCRP IL-6 and TNF-120572 in plasma but the increase was notstatistically significant (119875 gt 005) Compared with HFDgroup niacin decreased IL-6 level by 19 and decreasedTNF-120572 level by 18 whereas its effect on CRP had nostatistical difference Simvastatin decreased the levels of threeinflammatory markers in plasma compared with HFD group(Table 1)

312 Niacin Suppressed Protein Expression of CD68 and NF-120581B p65 in the Arterial Wall of Guinea Pigs Fed High FatDiet The invasion of monocyte into arterial intima and itsdifferentiation into resident macrophages may contribute toarterial inflammation in experimental atherosclerosis andhypertension In the study the immunohistochemical exam-ination of CD68 protein in the vessel wall was done tomark monocytesmacrophages Densitometric quantitativeimmunohistochemistry imaging revealed that comparedwith CD group the positive staining of CD68 was signifi-cantly increased in HFD group (119875 lt 001) compared withHFD group niacin and simvastatin significantly decreasedthe densitometric value of positive staining (Figures 1(a)

4 Mediators of Inflammation

CD HFD

HFD-N HFD-S

CD HFD

HFD-N HFD-S

0

10

20

30

40

50

CD HFD HFD-N HFD-S

Posit

ive s

tain

ing

(CD

68) (

)

0

10

20

30

40

50

60

CD HFD HFD-N HFD-S

(a)

(b)

(c)

(d)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

Posit

ive s

tain

ing

(NF-120581

B) (

)

Figure 1Niacin and simvastatin reduced the expressions of CD68 andNF-120581Bp65 in the arterial wall of guinea pigs by immunohistochemistryanalysis after treatment for 8 weeks (a) and (c) show the representative immunostained aortic sections of CD68 and NF-120581B p65 respectively(20x magnification blue = nuclei and brown = target protein) (b) and (d) show the relative levels of CD68 and NF-120581B positive cells of perview field by densitometric quantitation respectively Data are presented as mean plusmn SD (119899 = 8)

119875 lt 001 versus CD group lowastlowast119875 lt 001versus HFD group

and 1(b)) These results indicated that niacin weakened theadhesion and invasion of monocytes in the arterial wall

NF-120581B is a transcription factor associatedwith the expres-sion of various proinflammatory mediators [12] When notstimulated it is found in the cytoplasm connected to itsinhibiting protein inhibitor 120581B kinase (I120581B) Stimulationby inflammatory factors causes degradation of I120581B proteinand then translocates NF-120581B to the nucleus In nucleus NF-120581B upregulates gene expressions of inflammatory moleculesincluding chemokines cytokines adhesion molecules andproteases [13] In order to further explore the mechanismthrough which niacin inhibited inflammatory progress wedetermined the expression of nuclear protein NF-120581B p65in the arterial wall by immunohistochemistry analysis andwestern blot The results all indicated that compared with

CD group high fat diet promoted the expression of activeNF-120581B p65 in the arterial wall (119875 lt 001) compared withHFD group niacin and simvastatin significantly decreasedthe expression (Figures 1(c) 1(d) 2(a) and 2(b))

313 Niacin Attenuated Oxidative Stress in Guinea Pigs FedHigh Fat Diet Oxidative stress plays an important role inthe inflammatory process [14] MDA is one of the mostreliable and widely used indices of oxidative stress [15]In our study we determined MDA level in plasma As shownin Figure 7 compared with that of CD group the level ofMDA in plasma was significantly increased in HFD group(119875 lt 001) Compared with that of HFD group niacin andsimvastatin significantly lowered the MDA level by 38 and43 respectively (Figure 3)

Mediators of Inflammation 5

CD HFD HFD-N HFD-S

Histone H3

NF-120581Bp65

(a)

0

02

04

06

08

1

CD HFD HFD-N HFD-S

lowastlowast

lowastlowast

Relat

ive p

rote

in le

vel o

f nuc

lear

NF-120581

B in

the a

rter

ial w

all

(b)

Figure 2 Niacin and simvastatin suppressed the expression ofnuclear proteinNF-120581Bp65 in the arterial wall of guinea pigs fed highfat diet The protein expression was analyzed by western blot andnormalized to histone H3 level (a) shows the representative imageby western blot (b) shows the IOD ratio of NF-120581B p65 to HistoneH3 Data are presented as mean plusmn SD of at least three independentexperiments

119875 lt 001 versus CD group lowastlowast119875 lt 001 versus HFDgroup

32 Niacin Inhibited oxLDL-Stimulated Apoptosis and Inflam-mation in HUVECs

321 Niacin Attenuated oxLDL-Stimulated Apoptosis ofHUVECs The inflammation and further damage includingapoptosis of arterial ECs are considered as early and neces-sary steps of AS as well as thrombosis In this study HUVECsapoptosis was analyzed via flow cytometry After HUVECswere stained with Annexin V-FITCPI flow cytometry anal-ysis revealed that apoptosis was induced by 150 120583gmL ox-LDL for 24 h which was significantly improved by niacin(Figure 4(a))

322 Niacin Decreased the Secretion of Inflammatory Cytok-ines TNF-120572 and IL-6 in oxLDL-Stimulated HUVECs Asshown in Figures 4(b) and 4(c) ox-LDL (150 120583gmL)markedly increased TNF-120572 and IL-6 protein levels by 76and 31 respectively in the medium of HUVECs Preincu-bation of cells with niacin (025ndash1mM) significantly inhibitedTNF-120572 expression by 12 21 and 27 respectively Mean-while 1mM niacin lowered IL-6 level by 15 in the medium

323 Niacin Suppressed NF-120581B p65 and Notch1 Expression inoxLDL-Stimulated HUVECs Notch signaling pathway playsa key role in the inflammatory progress It can activateNF-120581B and promote inflammatory factors secretion fromendothelial cells and macrophages [16] To further explorethe mechanism through which niacin inhibited cell injury

0

2

4

6

8

10

CD HFD HFD-N HFD-S

MD

A (n

mol

mL)

in p

lasm

a

lowastlowast

lowastlowast

Figure 3 Niacin and simvastatin decreased the level of plasmaMDA in guinea pigs after treatment for 8 weeks MDA wasdetermined by a spectrophotometricmeasurement of thiobarbituricacid-reactive substances (TBARS) according to the manufacturerrsquosinstruction Data are presented as mean plusmn SD (119899 = 8)

119875 lt 001

versus CD group lowastlowast119875 lt 001 versus HFD group

we determined the expressions of nuclear protein NF-120581B p65and notch1 by western blot The results showed that oxLDLmarkedly increased the protein levels of active NF-120581B p65and notch1 in HUVECs which were suppressed by prein-cubation of cells with niacin in a dose-dependent manner(Figures 4(d) 4(e) 4(f) and 4(g))

33 Niacin Suppressed Inflammatory Response Stimulated byoxLDL in THP-1 Macrophages

331 Niacin Decreased TNF-120572 and IL-6 Protein Secretionin the Medium of THP-1 Macrophages Next we assessedanti-inflammatory property of niacin in THP-1macrophagesAs shown in Figures 5(a) and 5(b) ox-LDL significantlypromoted TNF-120572 and IL-6 secretion by 89 and 23respectively in THP-1 macrophages Niacin (025ndash1mM)remarkably inhibited TNF-120572 expression by 11ndash30 and IL-6expression by 8ndash22 in the medium

332 Niacin Inhibited NF-120581B p65 and Notch1 Protein Expres-sion in oxLDL-Induced THP-1 Macrophages The effect ofniacin on the protein expressions of NF-120581B p65 in nuclei andnotch1 stimulated by ox-LDLwere examined Results showedthat niacin (025ndash1mM) significantly decreased NF-120581B levelby 75ndash83 and niacin (1mM) decreased notch1 level by 20(Figures 5(c) 5(d) 5(e) and 5(f))

34 Niacin Significantly Lessened Lipid Deposition in the Arte-rial Wall and Modified Lipoprotein Profile in Plasma viaModulatingCholesterolMetabolism in Liver of Guinea PigsFed High Fat Diet

341 Niacin Significantly Lessened Lipid Deposition in theArterial Wall of Guinea Pigs Fed High Fat Diet Oil red Ostaining in the aorta was found in HFD group but not inCD group because of chow diet without high fat Compared

6 Mediators of Inflammation

BlankoxLDL

0mM niacin 025mM niacin 05mM niacin 1mM niacin961 plusmn 063

702 plusmn 085

2551 plusmn 462

2132 plusmn 194

1844 plusmn 340

1463 plusmn 268

1371 plusmn 232

902 plusmn 084

1429 plusmn 282

750 plusmn 132

104

103

102

101

100

104103102101100

PI

Annexin-Y104103102101100

Annexin-Y104103102101100

Annexin-Y104103102101100

Annexin-Y104103102101100

Annexin-Y

(a)

002040608

112141618

2

oxLDL

lowast

lowastlowastlowastlowast

++++minus

Niacin (mM) 0 0 025 05 1

Relat

ive l

evel

of T

NF-120572

secr

etio

n

(b)

002040608

1121416

Relat

ive l

evel

of I

L-6

secr

etio

n

lowastlowast

oxLDL ++++minus

Niacin (mM) 0 0 025 05 1

(c)

Histone H3

NF-120581B p65

oxLDL ++++minus

Niacin (mM) 0 0 025 05 1

(d)

Notch1

120573-Actin

oxLDL ++++minus

Niacin (mM) 0 0 025 05 1

(e)

0

05

1

15

2

25

3

35

Relat

ive p

rote

in le

vel o

fN

F-120581

lowastlowast

lowastlowastlowastlowast

oxLDL ++++minus

Niacin (mM) 0 0 025 05 1

B p6

5in

nuc

lei o

f HU

VEC

s

(f)

0

05

1

15

2

Rela

tive p

rote

in le

vel o

fno

tch1

in H

UV

ECs

lowastlowastlowastlowast

lowastlowast

oxLDL ++++minus

Niacin (mM) 0 0 025 05 1

(g)

Figure 4 Niacin inhibited oxLDL-stimulated apoptosis and inflammation in HUVECs (a) shows the ratio of apoptotic HUVECs stainedwith annexin V-FITC and PI Representative data of flow cytometry analysis are presented (b) and (c) show the relative levels of TNF-120572 andIL-6 in the medium of HUVECs The concentrations of IL-6 and TNF-120572 were determined by ELISA kit (d) and (e) show the representativeimages of NF-120581B p65 and notch1 protein expression in HUVECs by western blot (f) and (g) show the IOD ratios of NF-120581B p65 and notch1expression respectively Data are presented as mean plusmn SD

119875 lt 001 versus blank group lowast119875 lt 005 lowastlowast119875 lt 001 versus oxLDL-treatedgroup without niacin

Mediators of Inflammation 7

0

05

1

15

2

25

0

02

04

06

08

1

12

14

Relat

ive l

evel

of I

L-6

secr

etio

n

0

02

04

06

08

1

12

14

Relat

ive p

rote

in le

vel o

f not

ch1

0

02

04

06

08

1

12

14

(a) (b)

(c)

(e)

(d)

(f)

oxLDL

lowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

++++minus

Niacin (mM) 0 0 025 05 1oxLDL ++++minus

Niacin (mM) 0 0 025 05 1

Relat

ive l

evel

of T

NF-120572

secr

etio

n

Histone H3

Notch1

Relat

ive p

rote

in le

vel o

fN

F-120581

Bp65

in n

ucle

i

NF-120581B p65

120573-Actin

oxLDL ++++minus

Niacin (mM) 0 0 025 05 1

oxLDL ++++minus

Niacin (mM) 0 0 025 05 1

oxLDL ++++minus

Niacin (mM) 0 0 025 05 1oxLDL ++++minus

Niacin (mM) 0 0 025 05 1

Figure 5 Niacin decreased the secretion of TNF-120572 and IL-6 and inhibited NF-120581B and notch1 expression in oxLDL-stimulated THP-1macrophages (a) and (b) show the relative levels of TNF-120572 and IL-6 secretion in the medium of THP-1 macrophages The concentrations ofIL-6 and TNF-120572 were determined by ELISA kit (c) and (d) show the representative images of NF-120581B p65 and notch1 protein expression inTHP-1 macrophages by western blot (e) and (f) show the IOD ratios of NF-120581B p65 and notch1 expression respectively Data are presented asmean plusmn SD

119875 lt 001 versus blank group lowast119875 lt 005 lowastlowast119875 lt 001 versus oxLDL-treated group without niacin

with that of HFD group niacin and simvastatin significantlydecreased the percentages of stained area to the total cross-sectional vessel wall by 56 and 67 respectively (Figure 6)The effect of simvastatin was superior to that of niacin

342 Niacin Increased HDL-C and ApoA I Levels andDecreased TG and Non-HDL-C Levels in Plasma of GuineaPigs Fed High Fat Diet As shown in Figure 7 after highfat diet for 8 weeks the levels of plasma TC TG HDL-Cand non-HDL-C were significantly increased in HFD groupcompared with CD group (119875 lt 001) which indicated asuccessful hyperlipidemic model in guinea pigs Comparedwith HFD group niacin decreased the levels of TG and

non-HDL-C by 27 and 12 respectively and increasedHDL level by 21 Niacin had no statistical influence onTC level in plasma Compared with HFD group simvastatindecreased the levels of TG non-HDL-C and TC by 1853 and 51 respectively Simvastatin had no significantinfluence on HDL-C level

The level of apoA I in plasma was also detected by SDS-PAGE in this study Comparedwith that ofHFDgroup niacinsignificantly promoted the level of apoA I by 42 whereassimvastatin had no significant influence on apoA I (Figure 8)

343 Niacin Significantly Upregulated the mRNA Amountof CYP7A1 in Liver Cholesterol metabolism in liver is a

8 Mediators of Inflammation

CD HFD

HFD-N HFD-S

(a)

0

05

1

15

2

CD HFD HFD-N HFD-S

Oil

red

O st

aine

d ar

ea (

)

lowastlowast

lowastlowast

(b)

Figure 6 Niacin and simvastatin significantly lessened lipid depo-sition in the arterial wall of guinea pigs fed high fat diet Lipiddeposition in the aorta wall was analyzed by oil red O staining aftertreatment for 8 weeksThe quantification of stained lipids was deter-mined by calculating the percentage of the positive area to the totalcross-sectional vessel wall area by Image-Pro Plus software Data arepresented as mean plusmn SD (119899 = 8)

119875 lt 001 versus CD grouplowastlowast

119875 lt 001 versus HFD group

complicated homeostasis involving multiple steps includingcholesterol ingression synthesis and conversion SR-B1 andLDL-R in liver play a critical role in cholesterol ingressionSR-B1 is the HDL receptor on the hepatocyte surface LDL-R can bind to LDL and VLDL and internalize them intohepatocytes [17] In the study we determined the effect ofniacin on the expressions of SR-B1 and LDL-R mRNA inliver As shown in Figures 9(a) and 9(b) after treatment withhigh fat diet for 8 weeks the LDL-R mRNA level was down-regulated (119875 lt 001) and the SR-B1 mRNA level wasnot significantly changed in HFD group Compared withHFD group niacin had no significant effect on SR-B1 and

LDL-R mRNA levels but simvastatin upregulated LDL-RmRNA level by 71

Cholesterol in liver can be converted into bile acidthrough cytochrome P450-meidiated oxidation The rate-limiting enzyme for the dominant pathway of bile acidsynthesis is cytochrome P450 7A1 (CYP7A1) As shown inFigure 9(c) compared with HFD group niacin significantlyupregulated the CYP7A1 mRNA level by 59 whereassimvastatin had no significant influence on its level HMGCRis the rate-limiting enzyme in the process of cholesterolsynthesis Compared with that of CD group the mRNA levelof HMGCR was significantly decreased in HFD group (119875 lt001) Compared with HFD group simvastatin upregulatedthe HMGCR mRNA levels by 46 whereas niacin had nosignificant influence on its level (Figure 9(d))

4 Discussion

For the first time to our knowledge this report demonstratesniacin inhibited vascular inflammation in guinea pig fedhigh fat diet and suppressed oxLDL-stimulated inflammatoryresponse even injury of endothelial cells and macrophagesin vitro The result indicates a new mechanism for niacinrsquosprotective action on cardiovascular disease in addition to itsestablished effects on lipid metabolism

The augmentation of inflammatory response has beenclearly documented in pathogenesis of vascular impairmentThe chronic inflammatory pathogenesis in the arterial wall isas follows Harmful substances in blood such as hypercholes-terolemia can induce endothelial dysfunction This causesthe production of ROS and the secretion of cellular adhesionmolecules (CAMs) cytokines and chemokines which facili-tate adherence and endothelial transmigration of leukocytes(monocytes and T helper lymphocytes) Monocytes in thearterial wall will be activated by proinflammatory cytokinesand differentiated into macrophages Activated macrophagesincrease the expression of CAMs and cytokines which resultsin recruitment of more leukocytes into the arterial wall acti-vates the complement pathways of immune system and theacute phase response stimulates proliferation and migrationof smooth muscle cells (SMCs) and promotes fibrous tissuedeposition [18] In progress the signaling molecule NF-120581Bis a proinflammatory major switch that can upregulate theexpression of lots of cytokines [19] Activated NF-120581B canresult in the enhanced efflux of TNF-120572 and IL-6 in serum[20] Early events in AS are usually driven by NF-120581B and thedisruption of NF-120581B signaling pathway has been shown toslow down the vascular impairment [21]

In the present study we demonstrate that niacin attenu-ated vascular inflammation induced by high fat diet in vivoThe involved evidences are as follows (1) Niacin lowered thenumber of macrophages (CD68 positive cells) in the arterialwall and significantly downregulated the inflammatory fac-tors (IL-6 and TNF-120572) levels in plasma of guinea pigs fed highfat diet (2) Both immunohistochemistry and western blotanalysis indicated niacin suppressed the expression of activeNF-120581B p65 in nuclei of the arterial wall The activated NF-120581Bis reported to form a heterodimer which usually consists oftwo proteins p65 and p50 subunitsThe p65 subunit has been

Mediators of Inflammation 9

0

50

100

150

200

250

CD HFD HFD-N HFD-S

TG (m

gdL

)

lowastlowastlowastlowast

(a)

0

500

1000

1500

2000

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TC (m

gdL

)

lowastlowast

(b)

0

20

40

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120

CD HFD HFD-N HFD-S

HD

L-C

(mg

dL)

lowast

(c)

0

500

1000

1500

2000

CD HFD HFD-N HFD-S

Non

-HD

L-C

(mg

dL)

lowastlowast

lowast

(d)

Figure 7 Effect of niacin and simvastatin on plasma lipid of guinea pigs fed high fat diet The levels of TG (a) TC (b) HDL-C (c) andnon-HDL-C (d) in plasma of guinea pigs were determined by enzyme method after 8 weeksrsquo treatment Data are presented as mean plusmn SD(119899 = 8)

119875 lt 001 versus CD group lowast119875 lt 005 lowastlowast119875 lt 001 versus HFD group

apoAI20

30

40

50

6080

160110

Mar

ker

CD HFD

HFD

-N

HFD

-S

Hum

an

(a)

0

05

1

15

2

CD HFD HFD-N HFD-S

Fold

indu

ctio

n of

apoA

I in

HD

L

lowastlowast

(b)

Figure 8 Niacin upregulated apoA I level in plasma of guinea pigs fed high fat diet HDL (density = 109ndash124 gmL) was separatedfrom plasma of guinea pigs by sequential ultracentrifugation The SDS-PAGE was performed on 15 SDS polyacrylamide gel and theapolipoproteins were stained with coomassie brilliant blue Densitometric quantitation of SDS-PAGE image was analyzed by Image-Pro Plussoftware (a) shows the representative SDS-PAGE image ofHDL (b) shows the relative level of apoA I in plasma by densitometric quantitationData are presented as mean plusmn SD of at least three independent experiments

119875 lt 005 versus CD group lowastlowast119875 lt 001 versus HFD group

10 Mediators of Inflammation

0

02

04

06

08

1

12

14

16

CD HFD HFD-N HFD-S

Relat

ive m

RNA

leve

l of S

R-B1

(a)

0

02

04

06

08

1

12

CD HFD HFD-N HFD-S

Relat

ive m

RNA

leve

l of L

DL-

R

lowastlowast

(b)

0

05

1

15

2

CD HFD HFD-N HFD-S

Relat

ive m

RNA

leve

l of C

YP7A

1 lowastlowast

(c)

0

02

04

06

08

1

12

14

CD HFD HFD-N HFD-S

Relat

ive m

RNA

leve

l of H

MG

CR

lowastlowast

(d)

Figure 9 Effect of niacin and simvastatin on the mRNA abundance of SR-B1 LDL-R CYP7A1 and HMGCR in liver of guinea pigs fedhigh fat dietThe mRNA levels which were analyzed by quantitative real-time PCR were calculated after being adjusted for 120573-actin usingthe 2minusΔΔCt method Data are presented as mean plusmn SD of at least three independent experiments

119875 lt 005 119875 lt 001 versus CD group

lowastlowast

119875 lt 001 versus HFD group

demonstrated to exert critical activity on the transcriptionof many inflammatory genes including adhesion moleculescytokines and chemokines [22] (3) CRP an early acute phasereactant is closely relevant to inflammation Baseline level ofCRP is a strong independent predictor of the risk of futuremyocardial infarction peripheral vascular disease strokeand vascular death amonghealthy individualswithout knownvascular disease [23] Kuvin et al have shown that niacindecreased CRP level by 15 in patients with stable coronaryartery disease [5] In patients with metabolic syndromeafter treatment with extended-release niacin (1 gday) for 52weeks their endothelial function was improved by 22 andthere was a decrease in CRP level by 20 [24] Our resultsalso showed niacin slightly lowered CRP level but hadno statistical difference (Table 1) (4) Oxidative stress wassuppressed by niacin Oxidative stress is closely related to theinflammation in the arterial wall Increased ROS productioncan initiate a cascade of signal transduction which results inendothelial dysfunction changes in vascular tone vascularremodeling and vascular inflammatory responses [25]

To further confirm the direct anti-inflammatory prop-erty of niacin its effect on oxLDL-induced inflammatoryresponse of endothelial cells and macrophages was studiedoxLDL is pivotal in the development of AS and representsa crucial proinflammatory stimulus [26] Upon enteringinto the intima of arteries oxLDL activates endothelial

cells and upregulates adhesion molecule expression andinflammatory factors secretion all of which contribute tothe recruitment of circulating leukocytes Monocytes andormacrophages infiltrating the arterial wall take up oxLDL andform ldquofoam cellsrdquo which in turn promote further secretionof inflammatory mediators [27] Our data indicated niacinremarkably downregulated the secretion of TNF-120572 and IL-6stimulated by oxLDL in HUVECs and THP-1 macrophagesNotch1 signal pathway is an evolutionarily highly conservedmechanism for communication which can improve NF-120581Bactivity and have a positive correlation with the developmentof inflammation [28] A previous study demonstrates thatnotch knockout in macrophage markedly downregulates theexpression of proinflammatory factors Instead when thenotch signal pathway is activatedNF-120581B expression increasesand inflammatory factors secretion is also promoted [29]Our findings indicated niacin decreased the expressions ofNF-120581B and notch1 in oxLDL-induced inflammation Vas-cular endothelial apoptosis plays an important role in theinitiation of AS It may compromise vessel wall permeabilityto cytokines growth factors lipids and immune cells andincrease coagulation [30] In this study we indicated niacinattenuated oxLDL-induced apoptosis of HUVECs

Taken together these observations lend initial but com-pelling evidence that niacin has protective effects on car-diovascular disease through its vascular anti-inflammatory

Mediators of Inflammation 11

properties in addition to its established effects on lipidmetabolism

Reverse cholesterol transport (RCT) is a potent defensemechanism against AS It involves transporting cholesterolfromperipheral tissues and cells to liver transforming choles-terol into bile acid and finally eliminating it from the body[31] HDL and apoA play a key role in RCT They can drivecholesterol efflux from macrophage foam cells that residein vessel intima [32 33] Similar to previous studies ourfindings also indicated that niacin increased the levels ofHDL and apoA I in plasma significantly In the process ofRCT CYP7A1 is an important regulatory factor which canconvert cholesterol to bile acid in liver and be further excretedinto intestine for elimination from body with feces Here weshowed that the mRNA abundance of CYP7A1 in liver wassignificantly upregulated by niacin treatment These findingssuggest that niacin may modulate plasma lipid and exertantiatherosclerotic effects by promoting RCT progress

In clinical practice the oral dose of niacin for lipid-lowering purpose is usually 1ndash3 g per day equivalently 14ndash43mgkg per day According to the formula of Meeh-Rubnerequation we calculate the dose for guinea pig to be about80ndash250mgkg per day In addition a recent report showedthat up to 400mgkg per day of niacin treatment for 7 daysand 100mgkg per day of niacin treatment for 4 weeks aresafe and do not cause toxicity in rodent models [34] In thisstudy the applied dose of niacin for guinea pig is 100mgkgdwhich has pharmacologic effects and does not have varioustoxicities based on the changes in food intake body weightand behaviors In human plasma level of niacin was foundto be about 03mM after oral ingestion of 1 g niacin [3536] Since the reported plasma concentrations are one timemeasurement it is unclear whether these levels reflect troughor peak values Furthermore there is no reported clinicaldata available regarding plasma concentration of niacin afteringestion of 3 g niacin Extrapolation of the available datausing oral dose of 1 g niacin would indicate that the 3 g niacinoral administration may lead to the plasma concentrations ofniacin in the range of 08ndash1mM In our in-vitro studies weused 025 to 1mM niacin

Statins are the first-line therapy in the treatment ofcholesterol-induced atherosclerotic cardiovascular diseases[37] They suppress the conversion of HMG-CoA to meval-onate by competitively blocking HMGCR therefore theendogenous cholesterol synthesis in the liver is remarkablyreduced Consequently it results in the increase of LDL-Rexpression on hepatic cell surfaces and the decrease of LDL-Cin plasma [38]Meanwhile inhibition of cholesterol synthesiscauses an overexpression of HMGCR due to a feedback-regulatory mechanism [39] Similar to previous studies ourfindings also revealed that simvastatin significantly decreasedthe level of non-HDL-C in plasma followed by upregulatingLDL-R expression in liver and the HMGCR mRNA level inliver was increased in a compensatory way

5 Conclusion

In summary our data presented herein support the novelconcept that niacin has vascular anti-inflammatory and

potentially vascular-protective property which is indepen-dent of its effect on lipid regulation The anti-inflammatoryproperty of niacin is realized by downregulating nucleartranscription factor-120581B signaling pathway Meanwhile ourfinding also suggests that niacin modulates plasma lipid bystimulating the expression of factors involved in the processof RCT and promoting RCT

Conflict of Interests

The authors declare that they have no conflict of interest

Authorsrsquo Contribution

Yanhong Si and Ying Zhang have equally contributed to thisstudy

Acknowledgments

This research was supported by the financial supportsfrom the Taishan scholar foundation of Shandong Province(200867) and the Natural Science Foundations of China(81170785 and 81371234)

References

[1] R Altschul A Hoffer and J D Stephen ldquoInfluence of nicotinicacid on serum cholesterol in manrdquoArchives of Biochemistry andBiophysics vol 54 no 2 pp 558ndash559 1955

[2] C D Meyers V S Kamanna and M L Kashyap ldquoNiacin ther-apy in atherosclerosisrdquo Current Opinion in Lipidology vol 15no 6 pp 659ndash665 2004

[3] L A Carlson ldquoNicotinic acid the broad-spectrum lipid drug A50th anniversary reviewrdquo Journal of Internal Medicine vol 258no 2 pp 94ndash114 2005

[4] S H Ganji S Qin L Zhang V S Kamanna andM L KashyapldquoNiacin inhibits vascular oxidative stress redox-sensitive genesand monocyte adhesion to human aortic endothelial cellsrdquoAtherosclerosis vol 202 no 1 pp 68ndash75 2009

[5] J T Kuvin D M Dave K A Sliney et al ldquoEffects of extended-release niacin on lipoprotein particle size distribution andinflammatorymarkers in patients with coronary artery diseaserdquoAmerican Journal of Cardiology vol 98 no 6 pp 743ndash745 2006

[6] R Yang P Guo X Song F Liu and N Gao ldquoHyperlipidemicguinea pig model mechanisms of triglyceride metabolismdisorder and comparison to ratrdquo Biological and PharmaceuticalBulletin vol 34 no 7 pp 1046ndash1051 2011

[7] X Li Y Liu H Zhang L Ren Q Li and N Li ldquoAnimal modelsfor the atherosclerosis research a reviewrdquo Protein and Cell vol2 no 3 pp 189ndash201 2011

[8] J O Leite U Vaishnav M Puglisi H Fraser J Trias and M LFernandez ldquoA-002 (Varespladib) a phospholipase A2 inhibitorreduces atherosclerosis in guinea pigsrdquo BMC CardiovascularDisorders vol 9 article 7 2009

[9] J Auwerx ldquoThe human leukemia cell line THP-1 a multi-facetted model for the study of monocyte-macrophage differ-entiationrdquo Experientia vol 47 no 1 pp 22ndash31 1991

[10] M J Chapman G L Mills and J H Ledford ldquoThe distributionand partial characterization of the serum apolipoproteins in the

12 Mediators of Inflammation

guinea pigrdquo Biochemical Journal vol 149 no 2 pp 423ndash4361975

[11] X C Jiang C Bruce J Mar et al ldquoTargetedmutation of plasmaphospholipid transfer protein gene markedly reduces high-density lipoprotein levelsrdquo Journal of Clinical Investigation vol103 no 6 pp 907ndash914 1999

[12] T Collins and M I Cybulsky ldquoNF-120581B pivotal mediator orinnocent bystander in atherogenesisrdquo Journal of Clinical Inves-tigation vol 107 no 3 pp 255ndash264 2001

[13] Y Tsubosaka T Murata K Yamada D Uemura M Horiand H Ozaki ldquoHalichlorine reduces monocyte adhesion toendothelium through the suppression of nuclear factor-120581Bactivationrdquo Journal of Pharmacological Sciences vol 113 no 3pp 208ndash213 2010

[14] H Tsutsui ldquoMitochondrial oxidative stress and heart failurerdquoInternal Medicine vol 45 no 13 pp 809ndash813 2006

[15] M Seddon Y H Looi and A M Shah ldquoOxidative stressand redox signalling in cardiac hypertrophy and heart failurerdquoHeart vol 93 no 8 pp 903ndash907 2007

[16] WWongchana and T Palaga ldquoDirect regulation of interleukin-6 expression by Notch signaling in macrophagesrdquo Cellular andMolecular Immunology vol 9 no 2 pp 155ndash162 2012

[17] J Chen H Zhao X Ma et al ldquoThe effects of jiang-zhi-ningand itsmain components on cholesterol metabolismrdquo Evidence-Based Complementary and Alternative Medicine vol 2012Article ID 928234 10 pages 2012

[18] G Francisco C Hernandez and R Simo ldquoSerum markers ofvascular inflammation in dyslipemiardquo Clinica Chimica Actavol 369 no 2 pp 1ndash16 2006

[19] J Andersson P Libby and G K Hansson ldquoAdaptive immunityand atherosclerosisrdquoClinical Immunology vol 134 no 1 pp 33ndash46 2010

[20] W J Oh U Jung H S Eom H J Shin and H R Park ldquoInhibi-tion of lipopolysaccharide-induced proinflammatory responsesby Buddleja officinalis extract in BV-2 microglial cells vianegative regulation of NF-kB and ERK12 signalingrdquoMoleculesvol 18 no 8 pp 9195ndash9206 2013

[21] T M Wang C J Chen T S Lee et al ldquoDocosahexaenoic acidattenuates VCAM-1 expression and NF-120581B activation in TNF-120572-treated human aortic endothelial cellsrdquo Journal of NutritionalBiochemistry vol 22 no 2 pp 187ndash194 2011

[22] M S Islam H Yoshida N Matsuki et al ldquoAntioxidant freeradical-scavenging and NF-120581B-inhibitory activities of phy-tosteryl ferulates structure-activity studiesrdquo Journal of Pharma-cological Sciences vol 111 no 4 pp 328ndash337 2009

[23] P M Ridker M J Stampfer and N Rifai ldquoNovel risk factorsfor systemic atherosclerosis a comparison ofC-reactive proteinfibrinogen homocysteine lipoprotein(a) and standard choles-terol screening as predictors of peripheral arterial diseaserdquoJournal of the AmericanMedical Association vol 285 no 19 pp2481ndash2485 2001

[24] M Thoenes A Oguchi S Nagamia et al ldquoThe effects ofextended-release niacin on carotid intimal media thicknessendothelial function and inflammatory markers in patientswith the metabolic syndromerdquo International Journal of ClinicalPractice vol 61 no 11 pp 1942ndash1948 2007

[25] N R Madamanchi A Vendrov and M S Runge ldquoOxidativestress and vascular diseaserdquo Arteriosclerosis Thrombosis andVascular Biology vol 25 no 1 pp 29ndash38 2005

[26] E Matsuura F Atzeni P Sarzi-Puttini M Turiel L R Lopezand M T Nurmohamed ldquoIs atherosclerosis an autoimmunediseaserdquo BMCMedicine vol 12 no 1 article 47 2014

[27] A Pirillo G D Norata and A L Catapano ldquoLOX-1 OxLDLand atherosclerosisrdquo Mediators of Inflammation vol 2013Article ID 152786 12 pages 2013

[28] C L Wang M Meng S B Liu L R Wang L H Hou and XH Cao ldquochemically sulfated polysaccharide from Grifola fron-dos induces HepG2 cell apoptosis by notch1-NF-120581B pathwayrdquoCarbohydrate Polymers vol 95 no 1 pp 282ndash287 2013

[29] WWongchana and T Palaga ldquoDirect regulation of interleukin-6 expression by Notch signaling in macrophagesrdquo Cellular andMolecular Immunology vol 9 no 2 pp 155ndash162 2012

[30] J C Choy D J Granville D W C Hunt and B M McManusldquoEndothelial cell apoptosis biochemical characteristics andpotential implications for atherosclerosisrdquo Journal of Molecularand Cellular Cardiology vol 33 no 9 pp 1673ndash1690 2001

[31] M Cuchel and D J Rader ldquoMacrophage reverse cholesteroltransport key to the regression of atherosclerosisrdquo Circulationvol 113 no 21 pp 2548ndash2555 2006

[32] A Yazdanyar C Yeang and X C Jiang ldquoRole of phospholipidtransfer protein in high-density lipoprotein-mediated reversecholesterol transportrdquo Current Atherosclerosis Reports vol 13no 3 pp 242ndash248 2011

[33] X Wang and D J Rader ldquoMolecular regulation of macrophagereverse cholesterol transportrdquo Current Opinion in Cardiologyvol 22 no 4 pp 368ndash372 2007

[34] S J Yang J M Choi L Kim et al ldquoNicotinamide improvesglucose metabolism and affects the hepatic NAD-sirtuin path-way in a rodent model of obesity and type 2 diabetesrdquo Journalof Nutritional Biochemistry vol 25 no 1 pp 66ndash72 2014

[35] L A Carlson L Oro and J Ostman ldquoEffect of a single dose ofnicotinic acid on plasma lipids in patients with hyperlipopro-teinemiardquo Acta Medica Scandinavica vol 183 no 5 pp 457ndash465 1968

[36] R H Stern J D Spence D J Freeman and A Parbtani ldquoTol-erance to nicotinic acid flushingrdquo Clinical Pharmacology andTherapeutics vol 50 no 1 pp 66ndash70 1991

[37] S M Grundy J I Cleeman C N Bairey Merz et al ldquoImpli-cations of recent clinical trials for the National CholesterolEducation Program Adult Treatment Panel III guidelinesrdquoCirculation vol 110 no 2 pp 227ndash239 2004

[38] M S Brown and J L Goldstein ldquoLowering plasma cholesterolby raising LDL receptorsrdquoNewEngland Journal ofMedicine vol305 no 9 pp 515ndash517 1981

[39] R Hampton D Dimster-Denk and J Rine ldquoThe biology ofHMG-CoA reductase the pros of contra-regulationrdquo Trends inBiochemical Sciences vol 21 no 4 pp 140ndash145 1996

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

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OncologyJournal of

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Oxidative Medicine and Cellular Longevity

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PPAR Research

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Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 3: Research Article Niacin Inhibits Vascular Inflammation via ...downloads.hindawi.com/journals/mi/2014/263786.pdf · Research Article Niacin Inhibits Vascular Inflammation via Downregulating

Mediators of Inflammation 3

according to the following status live cells in the lower-leftquadrant (low-PI and FITC signals) early apoptotic cells inthe lower-right quadrant (low-PI and high-FITC signals) lateapoptotic or necrotic cells in the upper-right quadrant (high-PI and high-FITC signals) and necrotic cells in the upper-leftquadrant (high-PI and low-FITC signals)

210 Western Blot The entire proteins from fresh aorticwalls or treated cells were extracted using RIPA lysis bufferThen the nuclear protein fraction was prepared by a nuclearprotein extraction kit (BestBio China) in accordance withthe manufacturerrsquos instructions Equal amounts of proteinwere subjected to 8 to 15 SDS-PAGE and transferredonto PVDF membranes by electroblotting After blockingin Tris-buffered saline (TBS) containing 01 Tween 20and 10 nonfat dry milk for 2 h at room temperature themembranes were incubated with primary antibodies for3 h at room temperature or overnight at 4∘C After beingwashed four times with TBS containing 01 Tween 20 themembranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperatureImmunoblots were revealed by ECL reaction and visualizedusing a high-performance chemiluminescence filmThe IODvalue of immunoreactive bands was measured by Image-ProPlus software and normalized by house-keeping protein (120573-actin or histone H3)

211 Quantitative Real-Time PCR RT-PCR assay was appliedto detect the expression of LDL receptor (LDL-R) scav-enger receptor Class B Type 1 (SR-B1) 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR) and cholesterol 7120572-hydroxylase A1 (CYP7A1) in liver The first two are recep-tors of plasma cholesterol and the rest are key enzymesof cholesterol metabolism Primer Design 41 Softwarewas used to design the following primers 120573-actin for-ward primer 51015840-TTACTACTTTGCTGCGTTACACC-31015840reverse primer 51015840-CATGCCAATCTCATCTCGTTT-31015840(length of 78 bp) LDL-R forward primer 51015840-GACGTGTCC-CAGAGGAAGAT-31015840 reverse primer 51015840-CGAGTCGGT-CCAGTAGATGTT-31015840 (length of 144 bp) SR-B1 forwardprimer 51015840-TCTCCCACCCGCATTTCT-31015840 reverse primer51015840-CGCATACTGCACGTAGCACA-31015840 (length of 317 bp)CYP7A1 forward primer 51015840-CAGTATGCTGCTGTTTAT-G-31015840 reverse primer 51015840-GTTCTCGGTGGTGTTTCC-31015840(length of 335 bp) HMGR forward primer 51015840-TGATAG-CACCAGCAGATT-31015840 reverse primer 51015840-TATAAAGGT-TGCGTCCAG-31015840 (length of 68 bp) The primers were syn-thesized by TaKaRa Total liver RNA was separated by TRI-ZOL Reagent (Invitrogen) cDNA synthesis was performedusing MuLV Reverse Transcriptase (Applied Biosystems)Real-time PCR was performed using a SYBR-green PCRmaster mix kit (TianGen Biotech) The data was analyzed byusingRotor-geneQ software ver17 (Qiagen) RelativemRNAlevels were calculated by the 2minusΔΔCt method and normalizedagainst 120573-actin Each experiment was repeated three times

212 Statistical Analysis Results are shown as the mean plusmn SDfor at least three independent experiments Statistical analysiswas performed using one-way analysis of variance (ANOVA)

Table 1 Effect of niacin and simvastatin on plasma inflammatorymarkers (CRP IL-6 and TNF-120572) of guinea pig fed high fat diet

Group CRP (ngL) IL-6 (ngL) TNF-120572 (ngL)CD 31699 plusmn 2197 2588 plusmn 252 1308 plusmn 92

HFD 32113 plusmn 1538 2658 plusmn 242 1430 plusmn 157

HFD-N 30231 plusmn 1806 2152 plusmn 385lowastlowast

1179 plusmn 179lowastlowast

HFD-S 29557 plusmn 2578lowast

2367 plusmn 216lowast

1144 plusmn 193lowastlowast

The contents of CRP IL-6 and TNF-120572 in guinea pig plasma were measuredby ELISAmethod after 8 weeksrsquo treatment Data are presented as mean plusmn SD(119899 = 8) lowast119875 lt 005 lowastlowast119875 lt 001 versus HFD group

followed by Student-Newmann-Keuls multiple comparisontests with the SPSS 130 software for Windows 119875 values lessthan 005 were considered statistically significant

3 Results

At the beginning of the experiment 32 guinea pigs weredivided into 4 groups randomly andmean initial body weightwas 30227 plusmn 2367 g All guinea pigs survived for 8 weeksin the experiment and mean final body weight was 38489 plusmn2618 g No significant differences were observed among thesegroups for both the initial and final mean body weights

31 Niacin Attenuated the Systemic and Aortic Inflammationin Guinea Pigs Fed High Fat Diet

311 Niacin Significantly Downregulated IL-6 and TNF-120572Levels in Plasma of Guinea Pigs Fed High Fat Diet Inflam-matory process within the vessel wall can lead to vasculardysfunction and cause cardiovascular disease In this processinflammatory factors play a key role In this study the levelsof three major inflammatory factors (CRP IL-6 and TNF-120572) in plasma were measured Compared with CD grouphigh fat diet for 8 weeks lightly increased the levels ofCRP IL-6 and TNF-120572 in plasma but the increase was notstatistically significant (119875 gt 005) Compared with HFDgroup niacin decreased IL-6 level by 19 and decreasedTNF-120572 level by 18 whereas its effect on CRP had nostatistical difference Simvastatin decreased the levels of threeinflammatory markers in plasma compared with HFD group(Table 1)

312 Niacin Suppressed Protein Expression of CD68 and NF-120581B p65 in the Arterial Wall of Guinea Pigs Fed High FatDiet The invasion of monocyte into arterial intima and itsdifferentiation into resident macrophages may contribute toarterial inflammation in experimental atherosclerosis andhypertension In the study the immunohistochemical exam-ination of CD68 protein in the vessel wall was done tomark monocytesmacrophages Densitometric quantitativeimmunohistochemistry imaging revealed that comparedwith CD group the positive staining of CD68 was signifi-cantly increased in HFD group (119875 lt 001) compared withHFD group niacin and simvastatin significantly decreasedthe densitometric value of positive staining (Figures 1(a)

4 Mediators of Inflammation

CD HFD

HFD-N HFD-S

CD HFD

HFD-N HFD-S

0

10

20

30

40

50

CD HFD HFD-N HFD-S

Posit

ive s

tain

ing

(CD

68) (

)

0

10

20

30

40

50

60

CD HFD HFD-N HFD-S

(a)

(b)

(c)

(d)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

Posit

ive s

tain

ing

(NF-120581

B) (

)

Figure 1Niacin and simvastatin reduced the expressions of CD68 andNF-120581Bp65 in the arterial wall of guinea pigs by immunohistochemistryanalysis after treatment for 8 weeks (a) and (c) show the representative immunostained aortic sections of CD68 and NF-120581B p65 respectively(20x magnification blue = nuclei and brown = target protein) (b) and (d) show the relative levels of CD68 and NF-120581B positive cells of perview field by densitometric quantitation respectively Data are presented as mean plusmn SD (119899 = 8)

119875 lt 001 versus CD group lowastlowast119875 lt 001versus HFD group

and 1(b)) These results indicated that niacin weakened theadhesion and invasion of monocytes in the arterial wall

NF-120581B is a transcription factor associatedwith the expres-sion of various proinflammatory mediators [12] When notstimulated it is found in the cytoplasm connected to itsinhibiting protein inhibitor 120581B kinase (I120581B) Stimulationby inflammatory factors causes degradation of I120581B proteinand then translocates NF-120581B to the nucleus In nucleus NF-120581B upregulates gene expressions of inflammatory moleculesincluding chemokines cytokines adhesion molecules andproteases [13] In order to further explore the mechanismthrough which niacin inhibited inflammatory progress wedetermined the expression of nuclear protein NF-120581B p65in the arterial wall by immunohistochemistry analysis andwestern blot The results all indicated that compared with

CD group high fat diet promoted the expression of activeNF-120581B p65 in the arterial wall (119875 lt 001) compared withHFD group niacin and simvastatin significantly decreasedthe expression (Figures 1(c) 1(d) 2(a) and 2(b))

313 Niacin Attenuated Oxidative Stress in Guinea Pigs FedHigh Fat Diet Oxidative stress plays an important role inthe inflammatory process [14] MDA is one of the mostreliable and widely used indices of oxidative stress [15]In our study we determined MDA level in plasma As shownin Figure 7 compared with that of CD group the level ofMDA in plasma was significantly increased in HFD group(119875 lt 001) Compared with that of HFD group niacin andsimvastatin significantly lowered the MDA level by 38 and43 respectively (Figure 3)

Mediators of Inflammation 5

CD HFD HFD-N HFD-S

Histone H3

NF-120581Bp65

(a)

0

02

04

06

08

1

CD HFD HFD-N HFD-S

lowastlowast

lowastlowast

Relat

ive p

rote

in le

vel o

f nuc

lear

NF-120581

B in

the a

rter

ial w

all

(b)

Figure 2 Niacin and simvastatin suppressed the expression ofnuclear proteinNF-120581Bp65 in the arterial wall of guinea pigs fed highfat diet The protein expression was analyzed by western blot andnormalized to histone H3 level (a) shows the representative imageby western blot (b) shows the IOD ratio of NF-120581B p65 to HistoneH3 Data are presented as mean plusmn SD of at least three independentexperiments

119875 lt 001 versus CD group lowastlowast119875 lt 001 versus HFDgroup

32 Niacin Inhibited oxLDL-Stimulated Apoptosis and Inflam-mation in HUVECs

321 Niacin Attenuated oxLDL-Stimulated Apoptosis ofHUVECs The inflammation and further damage includingapoptosis of arterial ECs are considered as early and neces-sary steps of AS as well as thrombosis In this study HUVECsapoptosis was analyzed via flow cytometry After HUVECswere stained with Annexin V-FITCPI flow cytometry anal-ysis revealed that apoptosis was induced by 150 120583gmL ox-LDL for 24 h which was significantly improved by niacin(Figure 4(a))

322 Niacin Decreased the Secretion of Inflammatory Cytok-ines TNF-120572 and IL-6 in oxLDL-Stimulated HUVECs Asshown in Figures 4(b) and 4(c) ox-LDL (150 120583gmL)markedly increased TNF-120572 and IL-6 protein levels by 76and 31 respectively in the medium of HUVECs Preincu-bation of cells with niacin (025ndash1mM) significantly inhibitedTNF-120572 expression by 12 21 and 27 respectively Mean-while 1mM niacin lowered IL-6 level by 15 in the medium

323 Niacin Suppressed NF-120581B p65 and Notch1 Expression inoxLDL-Stimulated HUVECs Notch signaling pathway playsa key role in the inflammatory progress It can activateNF-120581B and promote inflammatory factors secretion fromendothelial cells and macrophages [16] To further explorethe mechanism through which niacin inhibited cell injury

0

2

4

6

8

10

CD HFD HFD-N HFD-S

MD

A (n

mol

mL)

in p

lasm

a

lowastlowast

lowastlowast

Figure 3 Niacin and simvastatin decreased the level of plasmaMDA in guinea pigs after treatment for 8 weeks MDA wasdetermined by a spectrophotometricmeasurement of thiobarbituricacid-reactive substances (TBARS) according to the manufacturerrsquosinstruction Data are presented as mean plusmn SD (119899 = 8)

119875 lt 001

versus CD group lowastlowast119875 lt 001 versus HFD group

we determined the expressions of nuclear protein NF-120581B p65and notch1 by western blot The results showed that oxLDLmarkedly increased the protein levels of active NF-120581B p65and notch1 in HUVECs which were suppressed by prein-cubation of cells with niacin in a dose-dependent manner(Figures 4(d) 4(e) 4(f) and 4(g))

33 Niacin Suppressed Inflammatory Response Stimulated byoxLDL in THP-1 Macrophages

331 Niacin Decreased TNF-120572 and IL-6 Protein Secretionin the Medium of THP-1 Macrophages Next we assessedanti-inflammatory property of niacin in THP-1macrophagesAs shown in Figures 5(a) and 5(b) ox-LDL significantlypromoted TNF-120572 and IL-6 secretion by 89 and 23respectively in THP-1 macrophages Niacin (025ndash1mM)remarkably inhibited TNF-120572 expression by 11ndash30 and IL-6expression by 8ndash22 in the medium

332 Niacin Inhibited NF-120581B p65 and Notch1 Protein Expres-sion in oxLDL-Induced THP-1 Macrophages The effect ofniacin on the protein expressions of NF-120581B p65 in nuclei andnotch1 stimulated by ox-LDLwere examined Results showedthat niacin (025ndash1mM) significantly decreased NF-120581B levelby 75ndash83 and niacin (1mM) decreased notch1 level by 20(Figures 5(c) 5(d) 5(e) and 5(f))

34 Niacin Significantly Lessened Lipid Deposition in the Arte-rial Wall and Modified Lipoprotein Profile in Plasma viaModulatingCholesterolMetabolism in Liver of Guinea PigsFed High Fat Diet

341 Niacin Significantly Lessened Lipid Deposition in theArterial Wall of Guinea Pigs Fed High Fat Diet Oil red Ostaining in the aorta was found in HFD group but not inCD group because of chow diet without high fat Compared

6 Mediators of Inflammation

BlankoxLDL

0mM niacin 025mM niacin 05mM niacin 1mM niacin961 plusmn 063

702 plusmn 085

2551 plusmn 462

2132 plusmn 194

1844 plusmn 340

1463 plusmn 268

1371 plusmn 232

902 plusmn 084

1429 plusmn 282

750 plusmn 132

104

103

102

101

100

104103102101100

PI

Annexin-Y104103102101100

Annexin-Y104103102101100

Annexin-Y104103102101100

Annexin-Y104103102101100

Annexin-Y

(a)

002040608

112141618

2

oxLDL

lowast

lowastlowastlowastlowast

++++minus

Niacin (mM) 0 0 025 05 1

Relat

ive l

evel

of T

NF-120572

secr

etio

n

(b)

002040608

1121416

Relat

ive l

evel

of I

L-6

secr

etio

n

lowastlowast

oxLDL ++++minus

Niacin (mM) 0 0 025 05 1

(c)

Histone H3

NF-120581B p65

oxLDL ++++minus

Niacin (mM) 0 0 025 05 1

(d)

Notch1

120573-Actin

oxLDL ++++minus

Niacin (mM) 0 0 025 05 1

(e)

0

05

1

15

2

25

3

35

Relat

ive p

rote

in le

vel o

fN

F-120581

lowastlowast

lowastlowastlowastlowast

oxLDL ++++minus

Niacin (mM) 0 0 025 05 1

B p6

5in

nuc

lei o

f HU

VEC

s

(f)

0

05

1

15

2

Rela

tive p

rote

in le

vel o

fno

tch1

in H

UV

ECs

lowastlowastlowastlowast

lowastlowast

oxLDL ++++minus

Niacin (mM) 0 0 025 05 1

(g)

Figure 4 Niacin inhibited oxLDL-stimulated apoptosis and inflammation in HUVECs (a) shows the ratio of apoptotic HUVECs stainedwith annexin V-FITC and PI Representative data of flow cytometry analysis are presented (b) and (c) show the relative levels of TNF-120572 andIL-6 in the medium of HUVECs The concentrations of IL-6 and TNF-120572 were determined by ELISA kit (d) and (e) show the representativeimages of NF-120581B p65 and notch1 protein expression in HUVECs by western blot (f) and (g) show the IOD ratios of NF-120581B p65 and notch1expression respectively Data are presented as mean plusmn SD

119875 lt 001 versus blank group lowast119875 lt 005 lowastlowast119875 lt 001 versus oxLDL-treatedgroup without niacin

Mediators of Inflammation 7

0

05

1

15

2

25

0

02

04

06

08

1

12

14

Relat

ive l

evel

of I

L-6

secr

etio

n

0

02

04

06

08

1

12

14

Relat

ive p

rote

in le

vel o

f not

ch1

0

02

04

06

08

1

12

14

(a) (b)

(c)

(e)

(d)

(f)

oxLDL

lowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

++++minus

Niacin (mM) 0 0 025 05 1oxLDL ++++minus

Niacin (mM) 0 0 025 05 1

Relat

ive l

evel

of T

NF-120572

secr

etio

n

Histone H3

Notch1

Relat

ive p

rote

in le

vel o

fN

F-120581

Bp65

in n

ucle

i

NF-120581B p65

120573-Actin

oxLDL ++++minus

Niacin (mM) 0 0 025 05 1

oxLDL ++++minus

Niacin (mM) 0 0 025 05 1

oxLDL ++++minus

Niacin (mM) 0 0 025 05 1oxLDL ++++minus

Niacin (mM) 0 0 025 05 1

Figure 5 Niacin decreased the secretion of TNF-120572 and IL-6 and inhibited NF-120581B and notch1 expression in oxLDL-stimulated THP-1macrophages (a) and (b) show the relative levels of TNF-120572 and IL-6 secretion in the medium of THP-1 macrophages The concentrations ofIL-6 and TNF-120572 were determined by ELISA kit (c) and (d) show the representative images of NF-120581B p65 and notch1 protein expression inTHP-1 macrophages by western blot (e) and (f) show the IOD ratios of NF-120581B p65 and notch1 expression respectively Data are presented asmean plusmn SD

119875 lt 001 versus blank group lowast119875 lt 005 lowastlowast119875 lt 001 versus oxLDL-treated group without niacin

with that of HFD group niacin and simvastatin significantlydecreased the percentages of stained area to the total cross-sectional vessel wall by 56 and 67 respectively (Figure 6)The effect of simvastatin was superior to that of niacin

342 Niacin Increased HDL-C and ApoA I Levels andDecreased TG and Non-HDL-C Levels in Plasma of GuineaPigs Fed High Fat Diet As shown in Figure 7 after highfat diet for 8 weeks the levels of plasma TC TG HDL-Cand non-HDL-C were significantly increased in HFD groupcompared with CD group (119875 lt 001) which indicated asuccessful hyperlipidemic model in guinea pigs Comparedwith HFD group niacin decreased the levels of TG and

non-HDL-C by 27 and 12 respectively and increasedHDL level by 21 Niacin had no statistical influence onTC level in plasma Compared with HFD group simvastatindecreased the levels of TG non-HDL-C and TC by 1853 and 51 respectively Simvastatin had no significantinfluence on HDL-C level

The level of apoA I in plasma was also detected by SDS-PAGE in this study Comparedwith that ofHFDgroup niacinsignificantly promoted the level of apoA I by 42 whereassimvastatin had no significant influence on apoA I (Figure 8)

343 Niacin Significantly Upregulated the mRNA Amountof CYP7A1 in Liver Cholesterol metabolism in liver is a

8 Mediators of Inflammation

CD HFD

HFD-N HFD-S

(a)

0

05

1

15

2

CD HFD HFD-N HFD-S

Oil

red

O st

aine

d ar

ea (

)

lowastlowast

lowastlowast

(b)

Figure 6 Niacin and simvastatin significantly lessened lipid depo-sition in the arterial wall of guinea pigs fed high fat diet Lipiddeposition in the aorta wall was analyzed by oil red O staining aftertreatment for 8 weeksThe quantification of stained lipids was deter-mined by calculating the percentage of the positive area to the totalcross-sectional vessel wall area by Image-Pro Plus software Data arepresented as mean plusmn SD (119899 = 8)

119875 lt 001 versus CD grouplowastlowast

119875 lt 001 versus HFD group

complicated homeostasis involving multiple steps includingcholesterol ingression synthesis and conversion SR-B1 andLDL-R in liver play a critical role in cholesterol ingressionSR-B1 is the HDL receptor on the hepatocyte surface LDL-R can bind to LDL and VLDL and internalize them intohepatocytes [17] In the study we determined the effect ofniacin on the expressions of SR-B1 and LDL-R mRNA inliver As shown in Figures 9(a) and 9(b) after treatment withhigh fat diet for 8 weeks the LDL-R mRNA level was down-regulated (119875 lt 001) and the SR-B1 mRNA level wasnot significantly changed in HFD group Compared withHFD group niacin had no significant effect on SR-B1 and

LDL-R mRNA levels but simvastatin upregulated LDL-RmRNA level by 71

Cholesterol in liver can be converted into bile acidthrough cytochrome P450-meidiated oxidation The rate-limiting enzyme for the dominant pathway of bile acidsynthesis is cytochrome P450 7A1 (CYP7A1) As shown inFigure 9(c) compared with HFD group niacin significantlyupregulated the CYP7A1 mRNA level by 59 whereassimvastatin had no significant influence on its level HMGCRis the rate-limiting enzyme in the process of cholesterolsynthesis Compared with that of CD group the mRNA levelof HMGCR was significantly decreased in HFD group (119875 lt001) Compared with HFD group simvastatin upregulatedthe HMGCR mRNA levels by 46 whereas niacin had nosignificant influence on its level (Figure 9(d))

4 Discussion

For the first time to our knowledge this report demonstratesniacin inhibited vascular inflammation in guinea pig fedhigh fat diet and suppressed oxLDL-stimulated inflammatoryresponse even injury of endothelial cells and macrophagesin vitro The result indicates a new mechanism for niacinrsquosprotective action on cardiovascular disease in addition to itsestablished effects on lipid metabolism

The augmentation of inflammatory response has beenclearly documented in pathogenesis of vascular impairmentThe chronic inflammatory pathogenesis in the arterial wall isas follows Harmful substances in blood such as hypercholes-terolemia can induce endothelial dysfunction This causesthe production of ROS and the secretion of cellular adhesionmolecules (CAMs) cytokines and chemokines which facili-tate adherence and endothelial transmigration of leukocytes(monocytes and T helper lymphocytes) Monocytes in thearterial wall will be activated by proinflammatory cytokinesand differentiated into macrophages Activated macrophagesincrease the expression of CAMs and cytokines which resultsin recruitment of more leukocytes into the arterial wall acti-vates the complement pathways of immune system and theacute phase response stimulates proliferation and migrationof smooth muscle cells (SMCs) and promotes fibrous tissuedeposition [18] In progress the signaling molecule NF-120581Bis a proinflammatory major switch that can upregulate theexpression of lots of cytokines [19] Activated NF-120581B canresult in the enhanced efflux of TNF-120572 and IL-6 in serum[20] Early events in AS are usually driven by NF-120581B and thedisruption of NF-120581B signaling pathway has been shown toslow down the vascular impairment [21]

In the present study we demonstrate that niacin attenu-ated vascular inflammation induced by high fat diet in vivoThe involved evidences are as follows (1) Niacin lowered thenumber of macrophages (CD68 positive cells) in the arterialwall and significantly downregulated the inflammatory fac-tors (IL-6 and TNF-120572) levels in plasma of guinea pigs fed highfat diet (2) Both immunohistochemistry and western blotanalysis indicated niacin suppressed the expression of activeNF-120581B p65 in nuclei of the arterial wall The activated NF-120581Bis reported to form a heterodimer which usually consists oftwo proteins p65 and p50 subunitsThe p65 subunit has been

Mediators of Inflammation 9

0

50

100

150

200

250

CD HFD HFD-N HFD-S

TG (m

gdL

)

lowastlowastlowastlowast

(a)

0

500

1000

1500

2000

CD HFD HFD-N HFD-S

TC (m

gdL

)

lowastlowast

(b)

0

20

40

60

80

100

120

CD HFD HFD-N HFD-S

HD

L-C

(mg

dL)

lowast

(c)

0

500

1000

1500

2000

CD HFD HFD-N HFD-S

Non

-HD

L-C

(mg

dL)

lowastlowast

lowast

(d)

Figure 7 Effect of niacin and simvastatin on plasma lipid of guinea pigs fed high fat diet The levels of TG (a) TC (b) HDL-C (c) andnon-HDL-C (d) in plasma of guinea pigs were determined by enzyme method after 8 weeksrsquo treatment Data are presented as mean plusmn SD(119899 = 8)

119875 lt 001 versus CD group lowast119875 lt 005 lowastlowast119875 lt 001 versus HFD group

apoAI20

30

40

50

6080

160110

Mar

ker

CD HFD

HFD

-N

HFD

-S

Hum

an

(a)

0

05

1

15

2

CD HFD HFD-N HFD-S

Fold

indu

ctio

n of

apoA

I in

HD

L

lowastlowast

(b)

Figure 8 Niacin upregulated apoA I level in plasma of guinea pigs fed high fat diet HDL (density = 109ndash124 gmL) was separatedfrom plasma of guinea pigs by sequential ultracentrifugation The SDS-PAGE was performed on 15 SDS polyacrylamide gel and theapolipoproteins were stained with coomassie brilliant blue Densitometric quantitation of SDS-PAGE image was analyzed by Image-Pro Plussoftware (a) shows the representative SDS-PAGE image ofHDL (b) shows the relative level of apoA I in plasma by densitometric quantitationData are presented as mean plusmn SD of at least three independent experiments

119875 lt 005 versus CD group lowastlowast119875 lt 001 versus HFD group

10 Mediators of Inflammation

0

02

04

06

08

1

12

14

16

CD HFD HFD-N HFD-S

Relat

ive m

RNA

leve

l of S

R-B1

(a)

0

02

04

06

08

1

12

CD HFD HFD-N HFD-S

Relat

ive m

RNA

leve

l of L

DL-

R

lowastlowast

(b)

0

05

1

15

2

CD HFD HFD-N HFD-S

Relat

ive m

RNA

leve

l of C

YP7A

1 lowastlowast

(c)

0

02

04

06

08

1

12

14

CD HFD HFD-N HFD-S

Relat

ive m

RNA

leve

l of H

MG

CR

lowastlowast

(d)

Figure 9 Effect of niacin and simvastatin on the mRNA abundance of SR-B1 LDL-R CYP7A1 and HMGCR in liver of guinea pigs fedhigh fat dietThe mRNA levels which were analyzed by quantitative real-time PCR were calculated after being adjusted for 120573-actin usingthe 2minusΔΔCt method Data are presented as mean plusmn SD of at least three independent experiments

119875 lt 005 119875 lt 001 versus CD group

lowastlowast

119875 lt 001 versus HFD group

demonstrated to exert critical activity on the transcriptionof many inflammatory genes including adhesion moleculescytokines and chemokines [22] (3) CRP an early acute phasereactant is closely relevant to inflammation Baseline level ofCRP is a strong independent predictor of the risk of futuremyocardial infarction peripheral vascular disease strokeand vascular death amonghealthy individualswithout knownvascular disease [23] Kuvin et al have shown that niacindecreased CRP level by 15 in patients with stable coronaryartery disease [5] In patients with metabolic syndromeafter treatment with extended-release niacin (1 gday) for 52weeks their endothelial function was improved by 22 andthere was a decrease in CRP level by 20 [24] Our resultsalso showed niacin slightly lowered CRP level but hadno statistical difference (Table 1) (4) Oxidative stress wassuppressed by niacin Oxidative stress is closely related to theinflammation in the arterial wall Increased ROS productioncan initiate a cascade of signal transduction which results inendothelial dysfunction changes in vascular tone vascularremodeling and vascular inflammatory responses [25]

To further confirm the direct anti-inflammatory prop-erty of niacin its effect on oxLDL-induced inflammatoryresponse of endothelial cells and macrophages was studiedoxLDL is pivotal in the development of AS and representsa crucial proinflammatory stimulus [26] Upon enteringinto the intima of arteries oxLDL activates endothelial

cells and upregulates adhesion molecule expression andinflammatory factors secretion all of which contribute tothe recruitment of circulating leukocytes Monocytes andormacrophages infiltrating the arterial wall take up oxLDL andform ldquofoam cellsrdquo which in turn promote further secretionof inflammatory mediators [27] Our data indicated niacinremarkably downregulated the secretion of TNF-120572 and IL-6stimulated by oxLDL in HUVECs and THP-1 macrophagesNotch1 signal pathway is an evolutionarily highly conservedmechanism for communication which can improve NF-120581Bactivity and have a positive correlation with the developmentof inflammation [28] A previous study demonstrates thatnotch knockout in macrophage markedly downregulates theexpression of proinflammatory factors Instead when thenotch signal pathway is activatedNF-120581B expression increasesand inflammatory factors secretion is also promoted [29]Our findings indicated niacin decreased the expressions ofNF-120581B and notch1 in oxLDL-induced inflammation Vas-cular endothelial apoptosis plays an important role in theinitiation of AS It may compromise vessel wall permeabilityto cytokines growth factors lipids and immune cells andincrease coagulation [30] In this study we indicated niacinattenuated oxLDL-induced apoptosis of HUVECs

Taken together these observations lend initial but com-pelling evidence that niacin has protective effects on car-diovascular disease through its vascular anti-inflammatory

Mediators of Inflammation 11

properties in addition to its established effects on lipidmetabolism

Reverse cholesterol transport (RCT) is a potent defensemechanism against AS It involves transporting cholesterolfromperipheral tissues and cells to liver transforming choles-terol into bile acid and finally eliminating it from the body[31] HDL and apoA play a key role in RCT They can drivecholesterol efflux from macrophage foam cells that residein vessel intima [32 33] Similar to previous studies ourfindings also indicated that niacin increased the levels ofHDL and apoA I in plasma significantly In the process ofRCT CYP7A1 is an important regulatory factor which canconvert cholesterol to bile acid in liver and be further excretedinto intestine for elimination from body with feces Here weshowed that the mRNA abundance of CYP7A1 in liver wassignificantly upregulated by niacin treatment These findingssuggest that niacin may modulate plasma lipid and exertantiatherosclerotic effects by promoting RCT progress

In clinical practice the oral dose of niacin for lipid-lowering purpose is usually 1ndash3 g per day equivalently 14ndash43mgkg per day According to the formula of Meeh-Rubnerequation we calculate the dose for guinea pig to be about80ndash250mgkg per day In addition a recent report showedthat up to 400mgkg per day of niacin treatment for 7 daysand 100mgkg per day of niacin treatment for 4 weeks aresafe and do not cause toxicity in rodent models [34] In thisstudy the applied dose of niacin for guinea pig is 100mgkgdwhich has pharmacologic effects and does not have varioustoxicities based on the changes in food intake body weightand behaviors In human plasma level of niacin was foundto be about 03mM after oral ingestion of 1 g niacin [3536] Since the reported plasma concentrations are one timemeasurement it is unclear whether these levels reflect troughor peak values Furthermore there is no reported clinicaldata available regarding plasma concentration of niacin afteringestion of 3 g niacin Extrapolation of the available datausing oral dose of 1 g niacin would indicate that the 3 g niacinoral administration may lead to the plasma concentrations ofniacin in the range of 08ndash1mM In our in-vitro studies weused 025 to 1mM niacin

Statins are the first-line therapy in the treatment ofcholesterol-induced atherosclerotic cardiovascular diseases[37] They suppress the conversion of HMG-CoA to meval-onate by competitively blocking HMGCR therefore theendogenous cholesterol synthesis in the liver is remarkablyreduced Consequently it results in the increase of LDL-Rexpression on hepatic cell surfaces and the decrease of LDL-Cin plasma [38]Meanwhile inhibition of cholesterol synthesiscauses an overexpression of HMGCR due to a feedback-regulatory mechanism [39] Similar to previous studies ourfindings also revealed that simvastatin significantly decreasedthe level of non-HDL-C in plasma followed by upregulatingLDL-R expression in liver and the HMGCR mRNA level inliver was increased in a compensatory way

5 Conclusion

In summary our data presented herein support the novelconcept that niacin has vascular anti-inflammatory and

potentially vascular-protective property which is indepen-dent of its effect on lipid regulation The anti-inflammatoryproperty of niacin is realized by downregulating nucleartranscription factor-120581B signaling pathway Meanwhile ourfinding also suggests that niacin modulates plasma lipid bystimulating the expression of factors involved in the processof RCT and promoting RCT

Conflict of Interests

The authors declare that they have no conflict of interest

Authorsrsquo Contribution

Yanhong Si and Ying Zhang have equally contributed to thisstudy

Acknowledgments

This research was supported by the financial supportsfrom the Taishan scholar foundation of Shandong Province(200867) and the Natural Science Foundations of China(81170785 and 81371234)

References

[1] R Altschul A Hoffer and J D Stephen ldquoInfluence of nicotinicacid on serum cholesterol in manrdquoArchives of Biochemistry andBiophysics vol 54 no 2 pp 558ndash559 1955

[2] C D Meyers V S Kamanna and M L Kashyap ldquoNiacin ther-apy in atherosclerosisrdquo Current Opinion in Lipidology vol 15no 6 pp 659ndash665 2004

[3] L A Carlson ldquoNicotinic acid the broad-spectrum lipid drug A50th anniversary reviewrdquo Journal of Internal Medicine vol 258no 2 pp 94ndash114 2005

[4] S H Ganji S Qin L Zhang V S Kamanna andM L KashyapldquoNiacin inhibits vascular oxidative stress redox-sensitive genesand monocyte adhesion to human aortic endothelial cellsrdquoAtherosclerosis vol 202 no 1 pp 68ndash75 2009

[5] J T Kuvin D M Dave K A Sliney et al ldquoEffects of extended-release niacin on lipoprotein particle size distribution andinflammatorymarkers in patients with coronary artery diseaserdquoAmerican Journal of Cardiology vol 98 no 6 pp 743ndash745 2006

[6] R Yang P Guo X Song F Liu and N Gao ldquoHyperlipidemicguinea pig model mechanisms of triglyceride metabolismdisorder and comparison to ratrdquo Biological and PharmaceuticalBulletin vol 34 no 7 pp 1046ndash1051 2011

[7] X Li Y Liu H Zhang L Ren Q Li and N Li ldquoAnimal modelsfor the atherosclerosis research a reviewrdquo Protein and Cell vol2 no 3 pp 189ndash201 2011

[8] J O Leite U Vaishnav M Puglisi H Fraser J Trias and M LFernandez ldquoA-002 (Varespladib) a phospholipase A2 inhibitorreduces atherosclerosis in guinea pigsrdquo BMC CardiovascularDisorders vol 9 article 7 2009

[9] J Auwerx ldquoThe human leukemia cell line THP-1 a multi-facetted model for the study of monocyte-macrophage differ-entiationrdquo Experientia vol 47 no 1 pp 22ndash31 1991

[10] M J Chapman G L Mills and J H Ledford ldquoThe distributionand partial characterization of the serum apolipoproteins in the

12 Mediators of Inflammation

guinea pigrdquo Biochemical Journal vol 149 no 2 pp 423ndash4361975

[11] X C Jiang C Bruce J Mar et al ldquoTargetedmutation of plasmaphospholipid transfer protein gene markedly reduces high-density lipoprotein levelsrdquo Journal of Clinical Investigation vol103 no 6 pp 907ndash914 1999

[12] T Collins and M I Cybulsky ldquoNF-120581B pivotal mediator orinnocent bystander in atherogenesisrdquo Journal of Clinical Inves-tigation vol 107 no 3 pp 255ndash264 2001

[13] Y Tsubosaka T Murata K Yamada D Uemura M Horiand H Ozaki ldquoHalichlorine reduces monocyte adhesion toendothelium through the suppression of nuclear factor-120581Bactivationrdquo Journal of Pharmacological Sciences vol 113 no 3pp 208ndash213 2010

[14] H Tsutsui ldquoMitochondrial oxidative stress and heart failurerdquoInternal Medicine vol 45 no 13 pp 809ndash813 2006

[15] M Seddon Y H Looi and A M Shah ldquoOxidative stressand redox signalling in cardiac hypertrophy and heart failurerdquoHeart vol 93 no 8 pp 903ndash907 2007

[16] WWongchana and T Palaga ldquoDirect regulation of interleukin-6 expression by Notch signaling in macrophagesrdquo Cellular andMolecular Immunology vol 9 no 2 pp 155ndash162 2012

[17] J Chen H Zhao X Ma et al ldquoThe effects of jiang-zhi-ningand itsmain components on cholesterol metabolismrdquo Evidence-Based Complementary and Alternative Medicine vol 2012Article ID 928234 10 pages 2012

[18] G Francisco C Hernandez and R Simo ldquoSerum markers ofvascular inflammation in dyslipemiardquo Clinica Chimica Actavol 369 no 2 pp 1ndash16 2006

[19] J Andersson P Libby and G K Hansson ldquoAdaptive immunityand atherosclerosisrdquoClinical Immunology vol 134 no 1 pp 33ndash46 2010

[20] W J Oh U Jung H S Eom H J Shin and H R Park ldquoInhibi-tion of lipopolysaccharide-induced proinflammatory responsesby Buddleja officinalis extract in BV-2 microglial cells vianegative regulation of NF-kB and ERK12 signalingrdquoMoleculesvol 18 no 8 pp 9195ndash9206 2013

[21] T M Wang C J Chen T S Lee et al ldquoDocosahexaenoic acidattenuates VCAM-1 expression and NF-120581B activation in TNF-120572-treated human aortic endothelial cellsrdquo Journal of NutritionalBiochemistry vol 22 no 2 pp 187ndash194 2011

[22] M S Islam H Yoshida N Matsuki et al ldquoAntioxidant freeradical-scavenging and NF-120581B-inhibitory activities of phy-tosteryl ferulates structure-activity studiesrdquo Journal of Pharma-cological Sciences vol 111 no 4 pp 328ndash337 2009

[23] P M Ridker M J Stampfer and N Rifai ldquoNovel risk factorsfor systemic atherosclerosis a comparison ofC-reactive proteinfibrinogen homocysteine lipoprotein(a) and standard choles-terol screening as predictors of peripheral arterial diseaserdquoJournal of the AmericanMedical Association vol 285 no 19 pp2481ndash2485 2001

[24] M Thoenes A Oguchi S Nagamia et al ldquoThe effects ofextended-release niacin on carotid intimal media thicknessendothelial function and inflammatory markers in patientswith the metabolic syndromerdquo International Journal of ClinicalPractice vol 61 no 11 pp 1942ndash1948 2007

[25] N R Madamanchi A Vendrov and M S Runge ldquoOxidativestress and vascular diseaserdquo Arteriosclerosis Thrombosis andVascular Biology vol 25 no 1 pp 29ndash38 2005

[26] E Matsuura F Atzeni P Sarzi-Puttini M Turiel L R Lopezand M T Nurmohamed ldquoIs atherosclerosis an autoimmunediseaserdquo BMCMedicine vol 12 no 1 article 47 2014

[27] A Pirillo G D Norata and A L Catapano ldquoLOX-1 OxLDLand atherosclerosisrdquo Mediators of Inflammation vol 2013Article ID 152786 12 pages 2013

[28] C L Wang M Meng S B Liu L R Wang L H Hou and XH Cao ldquochemically sulfated polysaccharide from Grifola fron-dos induces HepG2 cell apoptosis by notch1-NF-120581B pathwayrdquoCarbohydrate Polymers vol 95 no 1 pp 282ndash287 2013

[29] WWongchana and T Palaga ldquoDirect regulation of interleukin-6 expression by Notch signaling in macrophagesrdquo Cellular andMolecular Immunology vol 9 no 2 pp 155ndash162 2012

[30] J C Choy D J Granville D W C Hunt and B M McManusldquoEndothelial cell apoptosis biochemical characteristics andpotential implications for atherosclerosisrdquo Journal of Molecularand Cellular Cardiology vol 33 no 9 pp 1673ndash1690 2001

[31] M Cuchel and D J Rader ldquoMacrophage reverse cholesteroltransport key to the regression of atherosclerosisrdquo Circulationvol 113 no 21 pp 2548ndash2555 2006

[32] A Yazdanyar C Yeang and X C Jiang ldquoRole of phospholipidtransfer protein in high-density lipoprotein-mediated reversecholesterol transportrdquo Current Atherosclerosis Reports vol 13no 3 pp 242ndash248 2011

[33] X Wang and D J Rader ldquoMolecular regulation of macrophagereverse cholesterol transportrdquo Current Opinion in Cardiologyvol 22 no 4 pp 368ndash372 2007

[34] S J Yang J M Choi L Kim et al ldquoNicotinamide improvesglucose metabolism and affects the hepatic NAD-sirtuin path-way in a rodent model of obesity and type 2 diabetesrdquo Journalof Nutritional Biochemistry vol 25 no 1 pp 66ndash72 2014

[35] L A Carlson L Oro and J Ostman ldquoEffect of a single dose ofnicotinic acid on plasma lipids in patients with hyperlipopro-teinemiardquo Acta Medica Scandinavica vol 183 no 5 pp 457ndash465 1968

[36] R H Stern J D Spence D J Freeman and A Parbtani ldquoTol-erance to nicotinic acid flushingrdquo Clinical Pharmacology andTherapeutics vol 50 no 1 pp 66ndash70 1991

[37] S M Grundy J I Cleeman C N Bairey Merz et al ldquoImpli-cations of recent clinical trials for the National CholesterolEducation Program Adult Treatment Panel III guidelinesrdquoCirculation vol 110 no 2 pp 227ndash239 2004

[38] M S Brown and J L Goldstein ldquoLowering plasma cholesterolby raising LDL receptorsrdquoNewEngland Journal ofMedicine vol305 no 9 pp 515ndash517 1981

[39] R Hampton D Dimster-Denk and J Rine ldquoThe biology ofHMG-CoA reductase the pros of contra-regulationrdquo Trends inBiochemical Sciences vol 21 no 4 pp 140ndash145 1996

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Behavioural Neurology

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

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OncologyJournal of

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Oxidative Medicine and Cellular Longevity

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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 4: Research Article Niacin Inhibits Vascular Inflammation via ...downloads.hindawi.com/journals/mi/2014/263786.pdf · Research Article Niacin Inhibits Vascular Inflammation via Downregulating

4 Mediators of Inflammation

CD HFD

HFD-N HFD-S

CD HFD

HFD-N HFD-S

0

10

20

30

40

50

CD HFD HFD-N HFD-S

Posit

ive s

tain

ing

(CD

68) (

)

0

10

20

30

40

50

60

CD HFD HFD-N HFD-S

(a)

(b)

(c)

(d)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

Posit

ive s

tain

ing

(NF-120581

B) (

)

Figure 1Niacin and simvastatin reduced the expressions of CD68 andNF-120581Bp65 in the arterial wall of guinea pigs by immunohistochemistryanalysis after treatment for 8 weeks (a) and (c) show the representative immunostained aortic sections of CD68 and NF-120581B p65 respectively(20x magnification blue = nuclei and brown = target protein) (b) and (d) show the relative levels of CD68 and NF-120581B positive cells of perview field by densitometric quantitation respectively Data are presented as mean plusmn SD (119899 = 8)

119875 lt 001 versus CD group lowastlowast119875 lt 001versus HFD group

and 1(b)) These results indicated that niacin weakened theadhesion and invasion of monocytes in the arterial wall

NF-120581B is a transcription factor associatedwith the expres-sion of various proinflammatory mediators [12] When notstimulated it is found in the cytoplasm connected to itsinhibiting protein inhibitor 120581B kinase (I120581B) Stimulationby inflammatory factors causes degradation of I120581B proteinand then translocates NF-120581B to the nucleus In nucleus NF-120581B upregulates gene expressions of inflammatory moleculesincluding chemokines cytokines adhesion molecules andproteases [13] In order to further explore the mechanismthrough which niacin inhibited inflammatory progress wedetermined the expression of nuclear protein NF-120581B p65in the arterial wall by immunohistochemistry analysis andwestern blot The results all indicated that compared with

CD group high fat diet promoted the expression of activeNF-120581B p65 in the arterial wall (119875 lt 001) compared withHFD group niacin and simvastatin significantly decreasedthe expression (Figures 1(c) 1(d) 2(a) and 2(b))

313 Niacin Attenuated Oxidative Stress in Guinea Pigs FedHigh Fat Diet Oxidative stress plays an important role inthe inflammatory process [14] MDA is one of the mostreliable and widely used indices of oxidative stress [15]In our study we determined MDA level in plasma As shownin Figure 7 compared with that of CD group the level ofMDA in plasma was significantly increased in HFD group(119875 lt 001) Compared with that of HFD group niacin andsimvastatin significantly lowered the MDA level by 38 and43 respectively (Figure 3)

Mediators of Inflammation 5

CD HFD HFD-N HFD-S

Histone H3

NF-120581Bp65

(a)

0

02

04

06

08

1

CD HFD HFD-N HFD-S

lowastlowast

lowastlowast

Relat

ive p

rote

in le

vel o

f nuc

lear

NF-120581

B in

the a

rter

ial w

all

(b)

Figure 2 Niacin and simvastatin suppressed the expression ofnuclear proteinNF-120581Bp65 in the arterial wall of guinea pigs fed highfat diet The protein expression was analyzed by western blot andnormalized to histone H3 level (a) shows the representative imageby western blot (b) shows the IOD ratio of NF-120581B p65 to HistoneH3 Data are presented as mean plusmn SD of at least three independentexperiments

119875 lt 001 versus CD group lowastlowast119875 lt 001 versus HFDgroup

32 Niacin Inhibited oxLDL-Stimulated Apoptosis and Inflam-mation in HUVECs

321 Niacin Attenuated oxLDL-Stimulated Apoptosis ofHUVECs The inflammation and further damage includingapoptosis of arterial ECs are considered as early and neces-sary steps of AS as well as thrombosis In this study HUVECsapoptosis was analyzed via flow cytometry After HUVECswere stained with Annexin V-FITCPI flow cytometry anal-ysis revealed that apoptosis was induced by 150 120583gmL ox-LDL for 24 h which was significantly improved by niacin(Figure 4(a))

322 Niacin Decreased the Secretion of Inflammatory Cytok-ines TNF-120572 and IL-6 in oxLDL-Stimulated HUVECs Asshown in Figures 4(b) and 4(c) ox-LDL (150 120583gmL)markedly increased TNF-120572 and IL-6 protein levels by 76and 31 respectively in the medium of HUVECs Preincu-bation of cells with niacin (025ndash1mM) significantly inhibitedTNF-120572 expression by 12 21 and 27 respectively Mean-while 1mM niacin lowered IL-6 level by 15 in the medium

323 Niacin Suppressed NF-120581B p65 and Notch1 Expression inoxLDL-Stimulated HUVECs Notch signaling pathway playsa key role in the inflammatory progress It can activateNF-120581B and promote inflammatory factors secretion fromendothelial cells and macrophages [16] To further explorethe mechanism through which niacin inhibited cell injury

0

2

4

6

8

10

CD HFD HFD-N HFD-S

MD

A (n

mol

mL)

in p

lasm

a

lowastlowast

lowastlowast

Figure 3 Niacin and simvastatin decreased the level of plasmaMDA in guinea pigs after treatment for 8 weeks MDA wasdetermined by a spectrophotometricmeasurement of thiobarbituricacid-reactive substances (TBARS) according to the manufacturerrsquosinstruction Data are presented as mean plusmn SD (119899 = 8)

119875 lt 001

versus CD group lowastlowast119875 lt 001 versus HFD group

we determined the expressions of nuclear protein NF-120581B p65and notch1 by western blot The results showed that oxLDLmarkedly increased the protein levels of active NF-120581B p65and notch1 in HUVECs which were suppressed by prein-cubation of cells with niacin in a dose-dependent manner(Figures 4(d) 4(e) 4(f) and 4(g))

33 Niacin Suppressed Inflammatory Response Stimulated byoxLDL in THP-1 Macrophages

331 Niacin Decreased TNF-120572 and IL-6 Protein Secretionin the Medium of THP-1 Macrophages Next we assessedanti-inflammatory property of niacin in THP-1macrophagesAs shown in Figures 5(a) and 5(b) ox-LDL significantlypromoted TNF-120572 and IL-6 secretion by 89 and 23respectively in THP-1 macrophages Niacin (025ndash1mM)remarkably inhibited TNF-120572 expression by 11ndash30 and IL-6expression by 8ndash22 in the medium

332 Niacin Inhibited NF-120581B p65 and Notch1 Protein Expres-sion in oxLDL-Induced THP-1 Macrophages The effect ofniacin on the protein expressions of NF-120581B p65 in nuclei andnotch1 stimulated by ox-LDLwere examined Results showedthat niacin (025ndash1mM) significantly decreased NF-120581B levelby 75ndash83 and niacin (1mM) decreased notch1 level by 20(Figures 5(c) 5(d) 5(e) and 5(f))

34 Niacin Significantly Lessened Lipid Deposition in the Arte-rial Wall and Modified Lipoprotein Profile in Plasma viaModulatingCholesterolMetabolism in Liver of Guinea PigsFed High Fat Diet

341 Niacin Significantly Lessened Lipid Deposition in theArterial Wall of Guinea Pigs Fed High Fat Diet Oil red Ostaining in the aorta was found in HFD group but not inCD group because of chow diet without high fat Compared

6 Mediators of Inflammation

BlankoxLDL

0mM niacin 025mM niacin 05mM niacin 1mM niacin961 plusmn 063

702 plusmn 085

2551 plusmn 462

2132 plusmn 194

1844 plusmn 340

1463 plusmn 268

1371 plusmn 232

902 plusmn 084

1429 plusmn 282

750 plusmn 132

104

103

102

101

100

104103102101100

PI

Annexin-Y104103102101100

Annexin-Y104103102101100

Annexin-Y104103102101100

Annexin-Y104103102101100

Annexin-Y

(a)

002040608

112141618

2

oxLDL

lowast

lowastlowastlowastlowast

++++minus

Niacin (mM) 0 0 025 05 1

Relat

ive l

evel

of T

NF-120572

secr

etio

n

(b)

002040608

1121416

Relat

ive l

evel

of I

L-6

secr

etio

n

lowastlowast

oxLDL ++++minus

Niacin (mM) 0 0 025 05 1

(c)

Histone H3

NF-120581B p65

oxLDL ++++minus

Niacin (mM) 0 0 025 05 1

(d)

Notch1

120573-Actin

oxLDL ++++minus

Niacin (mM) 0 0 025 05 1

(e)

0

05

1

15

2

25

3

35

Relat

ive p

rote

in le

vel o

fN

F-120581

lowastlowast

lowastlowastlowastlowast

oxLDL ++++minus

Niacin (mM) 0 0 025 05 1

B p6

5in

nuc

lei o

f HU

VEC

s

(f)

0

05

1

15

2

Rela

tive p

rote

in le

vel o

fno

tch1

in H

UV

ECs

lowastlowastlowastlowast

lowastlowast

oxLDL ++++minus

Niacin (mM) 0 0 025 05 1

(g)

Figure 4 Niacin inhibited oxLDL-stimulated apoptosis and inflammation in HUVECs (a) shows the ratio of apoptotic HUVECs stainedwith annexin V-FITC and PI Representative data of flow cytometry analysis are presented (b) and (c) show the relative levels of TNF-120572 andIL-6 in the medium of HUVECs The concentrations of IL-6 and TNF-120572 were determined by ELISA kit (d) and (e) show the representativeimages of NF-120581B p65 and notch1 protein expression in HUVECs by western blot (f) and (g) show the IOD ratios of NF-120581B p65 and notch1expression respectively Data are presented as mean plusmn SD

119875 lt 001 versus blank group lowast119875 lt 005 lowastlowast119875 lt 001 versus oxLDL-treatedgroup without niacin

Mediators of Inflammation 7

0

05

1

15

2

25

0

02

04

06

08

1

12

14

Relat

ive l

evel

of I

L-6

secr

etio

n

0

02

04

06

08

1

12

14

Relat

ive p

rote

in le

vel o

f not

ch1

0

02

04

06

08

1

12

14

(a) (b)

(c)

(e)

(d)

(f)

oxLDL

lowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

++++minus

Niacin (mM) 0 0 025 05 1oxLDL ++++minus

Niacin (mM) 0 0 025 05 1

Relat

ive l

evel

of T

NF-120572

secr

etio

n

Histone H3

Notch1

Relat

ive p

rote

in le

vel o

fN

F-120581

Bp65

in n

ucle

i

NF-120581B p65

120573-Actin

oxLDL ++++minus

Niacin (mM) 0 0 025 05 1

oxLDL ++++minus

Niacin (mM) 0 0 025 05 1

oxLDL ++++minus

Niacin (mM) 0 0 025 05 1oxLDL ++++minus

Niacin (mM) 0 0 025 05 1

Figure 5 Niacin decreased the secretion of TNF-120572 and IL-6 and inhibited NF-120581B and notch1 expression in oxLDL-stimulated THP-1macrophages (a) and (b) show the relative levels of TNF-120572 and IL-6 secretion in the medium of THP-1 macrophages The concentrations ofIL-6 and TNF-120572 were determined by ELISA kit (c) and (d) show the representative images of NF-120581B p65 and notch1 protein expression inTHP-1 macrophages by western blot (e) and (f) show the IOD ratios of NF-120581B p65 and notch1 expression respectively Data are presented asmean plusmn SD

119875 lt 001 versus blank group lowast119875 lt 005 lowastlowast119875 lt 001 versus oxLDL-treated group without niacin

with that of HFD group niacin and simvastatin significantlydecreased the percentages of stained area to the total cross-sectional vessel wall by 56 and 67 respectively (Figure 6)The effect of simvastatin was superior to that of niacin

342 Niacin Increased HDL-C and ApoA I Levels andDecreased TG and Non-HDL-C Levels in Plasma of GuineaPigs Fed High Fat Diet As shown in Figure 7 after highfat diet for 8 weeks the levels of plasma TC TG HDL-Cand non-HDL-C were significantly increased in HFD groupcompared with CD group (119875 lt 001) which indicated asuccessful hyperlipidemic model in guinea pigs Comparedwith HFD group niacin decreased the levels of TG and

non-HDL-C by 27 and 12 respectively and increasedHDL level by 21 Niacin had no statistical influence onTC level in plasma Compared with HFD group simvastatindecreased the levels of TG non-HDL-C and TC by 1853 and 51 respectively Simvastatin had no significantinfluence on HDL-C level

The level of apoA I in plasma was also detected by SDS-PAGE in this study Comparedwith that ofHFDgroup niacinsignificantly promoted the level of apoA I by 42 whereassimvastatin had no significant influence on apoA I (Figure 8)

343 Niacin Significantly Upregulated the mRNA Amountof CYP7A1 in Liver Cholesterol metabolism in liver is a

8 Mediators of Inflammation

CD HFD

HFD-N HFD-S

(a)

0

05

1

15

2

CD HFD HFD-N HFD-S

Oil

red

O st

aine

d ar

ea (

)

lowastlowast

lowastlowast

(b)

Figure 6 Niacin and simvastatin significantly lessened lipid depo-sition in the arterial wall of guinea pigs fed high fat diet Lipiddeposition in the aorta wall was analyzed by oil red O staining aftertreatment for 8 weeksThe quantification of stained lipids was deter-mined by calculating the percentage of the positive area to the totalcross-sectional vessel wall area by Image-Pro Plus software Data arepresented as mean plusmn SD (119899 = 8)

119875 lt 001 versus CD grouplowastlowast

119875 lt 001 versus HFD group

complicated homeostasis involving multiple steps includingcholesterol ingression synthesis and conversion SR-B1 andLDL-R in liver play a critical role in cholesterol ingressionSR-B1 is the HDL receptor on the hepatocyte surface LDL-R can bind to LDL and VLDL and internalize them intohepatocytes [17] In the study we determined the effect ofniacin on the expressions of SR-B1 and LDL-R mRNA inliver As shown in Figures 9(a) and 9(b) after treatment withhigh fat diet for 8 weeks the LDL-R mRNA level was down-regulated (119875 lt 001) and the SR-B1 mRNA level wasnot significantly changed in HFD group Compared withHFD group niacin had no significant effect on SR-B1 and

LDL-R mRNA levels but simvastatin upregulated LDL-RmRNA level by 71

Cholesterol in liver can be converted into bile acidthrough cytochrome P450-meidiated oxidation The rate-limiting enzyme for the dominant pathway of bile acidsynthesis is cytochrome P450 7A1 (CYP7A1) As shown inFigure 9(c) compared with HFD group niacin significantlyupregulated the CYP7A1 mRNA level by 59 whereassimvastatin had no significant influence on its level HMGCRis the rate-limiting enzyme in the process of cholesterolsynthesis Compared with that of CD group the mRNA levelof HMGCR was significantly decreased in HFD group (119875 lt001) Compared with HFD group simvastatin upregulatedthe HMGCR mRNA levels by 46 whereas niacin had nosignificant influence on its level (Figure 9(d))

4 Discussion

For the first time to our knowledge this report demonstratesniacin inhibited vascular inflammation in guinea pig fedhigh fat diet and suppressed oxLDL-stimulated inflammatoryresponse even injury of endothelial cells and macrophagesin vitro The result indicates a new mechanism for niacinrsquosprotective action on cardiovascular disease in addition to itsestablished effects on lipid metabolism

The augmentation of inflammatory response has beenclearly documented in pathogenesis of vascular impairmentThe chronic inflammatory pathogenesis in the arterial wall isas follows Harmful substances in blood such as hypercholes-terolemia can induce endothelial dysfunction This causesthe production of ROS and the secretion of cellular adhesionmolecules (CAMs) cytokines and chemokines which facili-tate adherence and endothelial transmigration of leukocytes(monocytes and T helper lymphocytes) Monocytes in thearterial wall will be activated by proinflammatory cytokinesand differentiated into macrophages Activated macrophagesincrease the expression of CAMs and cytokines which resultsin recruitment of more leukocytes into the arterial wall acti-vates the complement pathways of immune system and theacute phase response stimulates proliferation and migrationof smooth muscle cells (SMCs) and promotes fibrous tissuedeposition [18] In progress the signaling molecule NF-120581Bis a proinflammatory major switch that can upregulate theexpression of lots of cytokines [19] Activated NF-120581B canresult in the enhanced efflux of TNF-120572 and IL-6 in serum[20] Early events in AS are usually driven by NF-120581B and thedisruption of NF-120581B signaling pathway has been shown toslow down the vascular impairment [21]

In the present study we demonstrate that niacin attenu-ated vascular inflammation induced by high fat diet in vivoThe involved evidences are as follows (1) Niacin lowered thenumber of macrophages (CD68 positive cells) in the arterialwall and significantly downregulated the inflammatory fac-tors (IL-6 and TNF-120572) levels in plasma of guinea pigs fed highfat diet (2) Both immunohistochemistry and western blotanalysis indicated niacin suppressed the expression of activeNF-120581B p65 in nuclei of the arterial wall The activated NF-120581Bis reported to form a heterodimer which usually consists oftwo proteins p65 and p50 subunitsThe p65 subunit has been

Mediators of Inflammation 9

0

50

100

150

200

250

CD HFD HFD-N HFD-S

TG (m

gdL

)

lowastlowastlowastlowast

(a)

0

500

1000

1500

2000

CD HFD HFD-N HFD-S

TC (m

gdL

)

lowastlowast

(b)

0

20

40

60

80

100

120

CD HFD HFD-N HFD-S

HD

L-C

(mg

dL)

lowast

(c)

0

500

1000

1500

2000

CD HFD HFD-N HFD-S

Non

-HD

L-C

(mg

dL)

lowastlowast

lowast

(d)

Figure 7 Effect of niacin and simvastatin on plasma lipid of guinea pigs fed high fat diet The levels of TG (a) TC (b) HDL-C (c) andnon-HDL-C (d) in plasma of guinea pigs were determined by enzyme method after 8 weeksrsquo treatment Data are presented as mean plusmn SD(119899 = 8)

119875 lt 001 versus CD group lowast119875 lt 005 lowastlowast119875 lt 001 versus HFD group

apoAI20

30

40

50

6080

160110

Mar

ker

CD HFD

HFD

-N

HFD

-S

Hum

an

(a)

0

05

1

15

2

CD HFD HFD-N HFD-S

Fold

indu

ctio

n of

apoA

I in

HD

L

lowastlowast

(b)

Figure 8 Niacin upregulated apoA I level in plasma of guinea pigs fed high fat diet HDL (density = 109ndash124 gmL) was separatedfrom plasma of guinea pigs by sequential ultracentrifugation The SDS-PAGE was performed on 15 SDS polyacrylamide gel and theapolipoproteins were stained with coomassie brilliant blue Densitometric quantitation of SDS-PAGE image was analyzed by Image-Pro Plussoftware (a) shows the representative SDS-PAGE image ofHDL (b) shows the relative level of apoA I in plasma by densitometric quantitationData are presented as mean plusmn SD of at least three independent experiments

119875 lt 005 versus CD group lowastlowast119875 lt 001 versus HFD group

10 Mediators of Inflammation

0

02

04

06

08

1

12

14

16

CD HFD HFD-N HFD-S

Relat

ive m

RNA

leve

l of S

R-B1

(a)

0

02

04

06

08

1

12

CD HFD HFD-N HFD-S

Relat

ive m

RNA

leve

l of L

DL-

R

lowastlowast

(b)

0

05

1

15

2

CD HFD HFD-N HFD-S

Relat

ive m

RNA

leve

l of C

YP7A

1 lowastlowast

(c)

0

02

04

06

08

1

12

14

CD HFD HFD-N HFD-S

Relat

ive m

RNA

leve

l of H

MG

CR

lowastlowast

(d)

Figure 9 Effect of niacin and simvastatin on the mRNA abundance of SR-B1 LDL-R CYP7A1 and HMGCR in liver of guinea pigs fedhigh fat dietThe mRNA levels which were analyzed by quantitative real-time PCR were calculated after being adjusted for 120573-actin usingthe 2minusΔΔCt method Data are presented as mean plusmn SD of at least three independent experiments

119875 lt 005 119875 lt 001 versus CD group

lowastlowast

119875 lt 001 versus HFD group

demonstrated to exert critical activity on the transcriptionof many inflammatory genes including adhesion moleculescytokines and chemokines [22] (3) CRP an early acute phasereactant is closely relevant to inflammation Baseline level ofCRP is a strong independent predictor of the risk of futuremyocardial infarction peripheral vascular disease strokeand vascular death amonghealthy individualswithout knownvascular disease [23] Kuvin et al have shown that niacindecreased CRP level by 15 in patients with stable coronaryartery disease [5] In patients with metabolic syndromeafter treatment with extended-release niacin (1 gday) for 52weeks their endothelial function was improved by 22 andthere was a decrease in CRP level by 20 [24] Our resultsalso showed niacin slightly lowered CRP level but hadno statistical difference (Table 1) (4) Oxidative stress wassuppressed by niacin Oxidative stress is closely related to theinflammation in the arterial wall Increased ROS productioncan initiate a cascade of signal transduction which results inendothelial dysfunction changes in vascular tone vascularremodeling and vascular inflammatory responses [25]

To further confirm the direct anti-inflammatory prop-erty of niacin its effect on oxLDL-induced inflammatoryresponse of endothelial cells and macrophages was studiedoxLDL is pivotal in the development of AS and representsa crucial proinflammatory stimulus [26] Upon enteringinto the intima of arteries oxLDL activates endothelial

cells and upregulates adhesion molecule expression andinflammatory factors secretion all of which contribute tothe recruitment of circulating leukocytes Monocytes andormacrophages infiltrating the arterial wall take up oxLDL andform ldquofoam cellsrdquo which in turn promote further secretionof inflammatory mediators [27] Our data indicated niacinremarkably downregulated the secretion of TNF-120572 and IL-6stimulated by oxLDL in HUVECs and THP-1 macrophagesNotch1 signal pathway is an evolutionarily highly conservedmechanism for communication which can improve NF-120581Bactivity and have a positive correlation with the developmentof inflammation [28] A previous study demonstrates thatnotch knockout in macrophage markedly downregulates theexpression of proinflammatory factors Instead when thenotch signal pathway is activatedNF-120581B expression increasesand inflammatory factors secretion is also promoted [29]Our findings indicated niacin decreased the expressions ofNF-120581B and notch1 in oxLDL-induced inflammation Vas-cular endothelial apoptosis plays an important role in theinitiation of AS It may compromise vessel wall permeabilityto cytokines growth factors lipids and immune cells andincrease coagulation [30] In this study we indicated niacinattenuated oxLDL-induced apoptosis of HUVECs

Taken together these observations lend initial but com-pelling evidence that niacin has protective effects on car-diovascular disease through its vascular anti-inflammatory

Mediators of Inflammation 11

properties in addition to its established effects on lipidmetabolism

Reverse cholesterol transport (RCT) is a potent defensemechanism against AS It involves transporting cholesterolfromperipheral tissues and cells to liver transforming choles-terol into bile acid and finally eliminating it from the body[31] HDL and apoA play a key role in RCT They can drivecholesterol efflux from macrophage foam cells that residein vessel intima [32 33] Similar to previous studies ourfindings also indicated that niacin increased the levels ofHDL and apoA I in plasma significantly In the process ofRCT CYP7A1 is an important regulatory factor which canconvert cholesterol to bile acid in liver and be further excretedinto intestine for elimination from body with feces Here weshowed that the mRNA abundance of CYP7A1 in liver wassignificantly upregulated by niacin treatment These findingssuggest that niacin may modulate plasma lipid and exertantiatherosclerotic effects by promoting RCT progress

In clinical practice the oral dose of niacin for lipid-lowering purpose is usually 1ndash3 g per day equivalently 14ndash43mgkg per day According to the formula of Meeh-Rubnerequation we calculate the dose for guinea pig to be about80ndash250mgkg per day In addition a recent report showedthat up to 400mgkg per day of niacin treatment for 7 daysand 100mgkg per day of niacin treatment for 4 weeks aresafe and do not cause toxicity in rodent models [34] In thisstudy the applied dose of niacin for guinea pig is 100mgkgdwhich has pharmacologic effects and does not have varioustoxicities based on the changes in food intake body weightand behaviors In human plasma level of niacin was foundto be about 03mM after oral ingestion of 1 g niacin [3536] Since the reported plasma concentrations are one timemeasurement it is unclear whether these levels reflect troughor peak values Furthermore there is no reported clinicaldata available regarding plasma concentration of niacin afteringestion of 3 g niacin Extrapolation of the available datausing oral dose of 1 g niacin would indicate that the 3 g niacinoral administration may lead to the plasma concentrations ofniacin in the range of 08ndash1mM In our in-vitro studies weused 025 to 1mM niacin

Statins are the first-line therapy in the treatment ofcholesterol-induced atherosclerotic cardiovascular diseases[37] They suppress the conversion of HMG-CoA to meval-onate by competitively blocking HMGCR therefore theendogenous cholesterol synthesis in the liver is remarkablyreduced Consequently it results in the increase of LDL-Rexpression on hepatic cell surfaces and the decrease of LDL-Cin plasma [38]Meanwhile inhibition of cholesterol synthesiscauses an overexpression of HMGCR due to a feedback-regulatory mechanism [39] Similar to previous studies ourfindings also revealed that simvastatin significantly decreasedthe level of non-HDL-C in plasma followed by upregulatingLDL-R expression in liver and the HMGCR mRNA level inliver was increased in a compensatory way

5 Conclusion

In summary our data presented herein support the novelconcept that niacin has vascular anti-inflammatory and

potentially vascular-protective property which is indepen-dent of its effect on lipid regulation The anti-inflammatoryproperty of niacin is realized by downregulating nucleartranscription factor-120581B signaling pathway Meanwhile ourfinding also suggests that niacin modulates plasma lipid bystimulating the expression of factors involved in the processof RCT and promoting RCT

Conflict of Interests

The authors declare that they have no conflict of interest

Authorsrsquo Contribution

Yanhong Si and Ying Zhang have equally contributed to thisstudy

Acknowledgments

This research was supported by the financial supportsfrom the Taishan scholar foundation of Shandong Province(200867) and the Natural Science Foundations of China(81170785 and 81371234)

References

[1] R Altschul A Hoffer and J D Stephen ldquoInfluence of nicotinicacid on serum cholesterol in manrdquoArchives of Biochemistry andBiophysics vol 54 no 2 pp 558ndash559 1955

[2] C D Meyers V S Kamanna and M L Kashyap ldquoNiacin ther-apy in atherosclerosisrdquo Current Opinion in Lipidology vol 15no 6 pp 659ndash665 2004

[3] L A Carlson ldquoNicotinic acid the broad-spectrum lipid drug A50th anniversary reviewrdquo Journal of Internal Medicine vol 258no 2 pp 94ndash114 2005

[4] S H Ganji S Qin L Zhang V S Kamanna andM L KashyapldquoNiacin inhibits vascular oxidative stress redox-sensitive genesand monocyte adhesion to human aortic endothelial cellsrdquoAtherosclerosis vol 202 no 1 pp 68ndash75 2009

[5] J T Kuvin D M Dave K A Sliney et al ldquoEffects of extended-release niacin on lipoprotein particle size distribution andinflammatorymarkers in patients with coronary artery diseaserdquoAmerican Journal of Cardiology vol 98 no 6 pp 743ndash745 2006

[6] R Yang P Guo X Song F Liu and N Gao ldquoHyperlipidemicguinea pig model mechanisms of triglyceride metabolismdisorder and comparison to ratrdquo Biological and PharmaceuticalBulletin vol 34 no 7 pp 1046ndash1051 2011

[7] X Li Y Liu H Zhang L Ren Q Li and N Li ldquoAnimal modelsfor the atherosclerosis research a reviewrdquo Protein and Cell vol2 no 3 pp 189ndash201 2011

[8] J O Leite U Vaishnav M Puglisi H Fraser J Trias and M LFernandez ldquoA-002 (Varespladib) a phospholipase A2 inhibitorreduces atherosclerosis in guinea pigsrdquo BMC CardiovascularDisorders vol 9 article 7 2009

[9] J Auwerx ldquoThe human leukemia cell line THP-1 a multi-facetted model for the study of monocyte-macrophage differ-entiationrdquo Experientia vol 47 no 1 pp 22ndash31 1991

[10] M J Chapman G L Mills and J H Ledford ldquoThe distributionand partial characterization of the serum apolipoproteins in the

12 Mediators of Inflammation

guinea pigrdquo Biochemical Journal vol 149 no 2 pp 423ndash4361975

[11] X C Jiang C Bruce J Mar et al ldquoTargetedmutation of plasmaphospholipid transfer protein gene markedly reduces high-density lipoprotein levelsrdquo Journal of Clinical Investigation vol103 no 6 pp 907ndash914 1999

[12] T Collins and M I Cybulsky ldquoNF-120581B pivotal mediator orinnocent bystander in atherogenesisrdquo Journal of Clinical Inves-tigation vol 107 no 3 pp 255ndash264 2001

[13] Y Tsubosaka T Murata K Yamada D Uemura M Horiand H Ozaki ldquoHalichlorine reduces monocyte adhesion toendothelium through the suppression of nuclear factor-120581Bactivationrdquo Journal of Pharmacological Sciences vol 113 no 3pp 208ndash213 2010

[14] H Tsutsui ldquoMitochondrial oxidative stress and heart failurerdquoInternal Medicine vol 45 no 13 pp 809ndash813 2006

[15] M Seddon Y H Looi and A M Shah ldquoOxidative stressand redox signalling in cardiac hypertrophy and heart failurerdquoHeart vol 93 no 8 pp 903ndash907 2007

[16] WWongchana and T Palaga ldquoDirect regulation of interleukin-6 expression by Notch signaling in macrophagesrdquo Cellular andMolecular Immunology vol 9 no 2 pp 155ndash162 2012

[17] J Chen H Zhao X Ma et al ldquoThe effects of jiang-zhi-ningand itsmain components on cholesterol metabolismrdquo Evidence-Based Complementary and Alternative Medicine vol 2012Article ID 928234 10 pages 2012

[18] G Francisco C Hernandez and R Simo ldquoSerum markers ofvascular inflammation in dyslipemiardquo Clinica Chimica Actavol 369 no 2 pp 1ndash16 2006

[19] J Andersson P Libby and G K Hansson ldquoAdaptive immunityand atherosclerosisrdquoClinical Immunology vol 134 no 1 pp 33ndash46 2010

[20] W J Oh U Jung H S Eom H J Shin and H R Park ldquoInhibi-tion of lipopolysaccharide-induced proinflammatory responsesby Buddleja officinalis extract in BV-2 microglial cells vianegative regulation of NF-kB and ERK12 signalingrdquoMoleculesvol 18 no 8 pp 9195ndash9206 2013

[21] T M Wang C J Chen T S Lee et al ldquoDocosahexaenoic acidattenuates VCAM-1 expression and NF-120581B activation in TNF-120572-treated human aortic endothelial cellsrdquo Journal of NutritionalBiochemistry vol 22 no 2 pp 187ndash194 2011

[22] M S Islam H Yoshida N Matsuki et al ldquoAntioxidant freeradical-scavenging and NF-120581B-inhibitory activities of phy-tosteryl ferulates structure-activity studiesrdquo Journal of Pharma-cological Sciences vol 111 no 4 pp 328ndash337 2009

[23] P M Ridker M J Stampfer and N Rifai ldquoNovel risk factorsfor systemic atherosclerosis a comparison ofC-reactive proteinfibrinogen homocysteine lipoprotein(a) and standard choles-terol screening as predictors of peripheral arterial diseaserdquoJournal of the AmericanMedical Association vol 285 no 19 pp2481ndash2485 2001

[24] M Thoenes A Oguchi S Nagamia et al ldquoThe effects ofextended-release niacin on carotid intimal media thicknessendothelial function and inflammatory markers in patientswith the metabolic syndromerdquo International Journal of ClinicalPractice vol 61 no 11 pp 1942ndash1948 2007

[25] N R Madamanchi A Vendrov and M S Runge ldquoOxidativestress and vascular diseaserdquo Arteriosclerosis Thrombosis andVascular Biology vol 25 no 1 pp 29ndash38 2005

[26] E Matsuura F Atzeni P Sarzi-Puttini M Turiel L R Lopezand M T Nurmohamed ldquoIs atherosclerosis an autoimmunediseaserdquo BMCMedicine vol 12 no 1 article 47 2014

[27] A Pirillo G D Norata and A L Catapano ldquoLOX-1 OxLDLand atherosclerosisrdquo Mediators of Inflammation vol 2013Article ID 152786 12 pages 2013

[28] C L Wang M Meng S B Liu L R Wang L H Hou and XH Cao ldquochemically sulfated polysaccharide from Grifola fron-dos induces HepG2 cell apoptosis by notch1-NF-120581B pathwayrdquoCarbohydrate Polymers vol 95 no 1 pp 282ndash287 2013

[29] WWongchana and T Palaga ldquoDirect regulation of interleukin-6 expression by Notch signaling in macrophagesrdquo Cellular andMolecular Immunology vol 9 no 2 pp 155ndash162 2012

[30] J C Choy D J Granville D W C Hunt and B M McManusldquoEndothelial cell apoptosis biochemical characteristics andpotential implications for atherosclerosisrdquo Journal of Molecularand Cellular Cardiology vol 33 no 9 pp 1673ndash1690 2001

[31] M Cuchel and D J Rader ldquoMacrophage reverse cholesteroltransport key to the regression of atherosclerosisrdquo Circulationvol 113 no 21 pp 2548ndash2555 2006

[32] A Yazdanyar C Yeang and X C Jiang ldquoRole of phospholipidtransfer protein in high-density lipoprotein-mediated reversecholesterol transportrdquo Current Atherosclerosis Reports vol 13no 3 pp 242ndash248 2011

[33] X Wang and D J Rader ldquoMolecular regulation of macrophagereverse cholesterol transportrdquo Current Opinion in Cardiologyvol 22 no 4 pp 368ndash372 2007

[34] S J Yang J M Choi L Kim et al ldquoNicotinamide improvesglucose metabolism and affects the hepatic NAD-sirtuin path-way in a rodent model of obesity and type 2 diabetesrdquo Journalof Nutritional Biochemistry vol 25 no 1 pp 66ndash72 2014

[35] L A Carlson L Oro and J Ostman ldquoEffect of a single dose ofnicotinic acid on plasma lipids in patients with hyperlipopro-teinemiardquo Acta Medica Scandinavica vol 183 no 5 pp 457ndash465 1968

[36] R H Stern J D Spence D J Freeman and A Parbtani ldquoTol-erance to nicotinic acid flushingrdquo Clinical Pharmacology andTherapeutics vol 50 no 1 pp 66ndash70 1991

[37] S M Grundy J I Cleeman C N Bairey Merz et al ldquoImpli-cations of recent clinical trials for the National CholesterolEducation Program Adult Treatment Panel III guidelinesrdquoCirculation vol 110 no 2 pp 227ndash239 2004

[38] M S Brown and J L Goldstein ldquoLowering plasma cholesterolby raising LDL receptorsrdquoNewEngland Journal ofMedicine vol305 no 9 pp 515ndash517 1981

[39] R Hampton D Dimster-Denk and J Rine ldquoThe biology ofHMG-CoA reductase the pros of contra-regulationrdquo Trends inBiochemical Sciences vol 21 no 4 pp 140ndash145 1996

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

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Page 5: Research Article Niacin Inhibits Vascular Inflammation via ...downloads.hindawi.com/journals/mi/2014/263786.pdf · Research Article Niacin Inhibits Vascular Inflammation via Downregulating

Mediators of Inflammation 5

CD HFD HFD-N HFD-S

Histone H3

NF-120581Bp65

(a)

0

02

04

06

08

1

CD HFD HFD-N HFD-S

lowastlowast

lowastlowast

Relat

ive p

rote

in le

vel o

f nuc

lear

NF-120581

B in

the a

rter

ial w

all

(b)

Figure 2 Niacin and simvastatin suppressed the expression ofnuclear proteinNF-120581Bp65 in the arterial wall of guinea pigs fed highfat diet The protein expression was analyzed by western blot andnormalized to histone H3 level (a) shows the representative imageby western blot (b) shows the IOD ratio of NF-120581B p65 to HistoneH3 Data are presented as mean plusmn SD of at least three independentexperiments

119875 lt 001 versus CD group lowastlowast119875 lt 001 versus HFDgroup

32 Niacin Inhibited oxLDL-Stimulated Apoptosis and Inflam-mation in HUVECs

321 Niacin Attenuated oxLDL-Stimulated Apoptosis ofHUVECs The inflammation and further damage includingapoptosis of arterial ECs are considered as early and neces-sary steps of AS as well as thrombosis In this study HUVECsapoptosis was analyzed via flow cytometry After HUVECswere stained with Annexin V-FITCPI flow cytometry anal-ysis revealed that apoptosis was induced by 150 120583gmL ox-LDL for 24 h which was significantly improved by niacin(Figure 4(a))

322 Niacin Decreased the Secretion of Inflammatory Cytok-ines TNF-120572 and IL-6 in oxLDL-Stimulated HUVECs Asshown in Figures 4(b) and 4(c) ox-LDL (150 120583gmL)markedly increased TNF-120572 and IL-6 protein levels by 76and 31 respectively in the medium of HUVECs Preincu-bation of cells with niacin (025ndash1mM) significantly inhibitedTNF-120572 expression by 12 21 and 27 respectively Mean-while 1mM niacin lowered IL-6 level by 15 in the medium

323 Niacin Suppressed NF-120581B p65 and Notch1 Expression inoxLDL-Stimulated HUVECs Notch signaling pathway playsa key role in the inflammatory progress It can activateNF-120581B and promote inflammatory factors secretion fromendothelial cells and macrophages [16] To further explorethe mechanism through which niacin inhibited cell injury

0

2

4

6

8

10

CD HFD HFD-N HFD-S

MD

A (n

mol

mL)

in p

lasm

a

lowastlowast

lowastlowast

Figure 3 Niacin and simvastatin decreased the level of plasmaMDA in guinea pigs after treatment for 8 weeks MDA wasdetermined by a spectrophotometricmeasurement of thiobarbituricacid-reactive substances (TBARS) according to the manufacturerrsquosinstruction Data are presented as mean plusmn SD (119899 = 8)

119875 lt 001

versus CD group lowastlowast119875 lt 001 versus HFD group

we determined the expressions of nuclear protein NF-120581B p65and notch1 by western blot The results showed that oxLDLmarkedly increased the protein levels of active NF-120581B p65and notch1 in HUVECs which were suppressed by prein-cubation of cells with niacin in a dose-dependent manner(Figures 4(d) 4(e) 4(f) and 4(g))

33 Niacin Suppressed Inflammatory Response Stimulated byoxLDL in THP-1 Macrophages

331 Niacin Decreased TNF-120572 and IL-6 Protein Secretionin the Medium of THP-1 Macrophages Next we assessedanti-inflammatory property of niacin in THP-1macrophagesAs shown in Figures 5(a) and 5(b) ox-LDL significantlypromoted TNF-120572 and IL-6 secretion by 89 and 23respectively in THP-1 macrophages Niacin (025ndash1mM)remarkably inhibited TNF-120572 expression by 11ndash30 and IL-6expression by 8ndash22 in the medium

332 Niacin Inhibited NF-120581B p65 and Notch1 Protein Expres-sion in oxLDL-Induced THP-1 Macrophages The effect ofniacin on the protein expressions of NF-120581B p65 in nuclei andnotch1 stimulated by ox-LDLwere examined Results showedthat niacin (025ndash1mM) significantly decreased NF-120581B levelby 75ndash83 and niacin (1mM) decreased notch1 level by 20(Figures 5(c) 5(d) 5(e) and 5(f))

34 Niacin Significantly Lessened Lipid Deposition in the Arte-rial Wall and Modified Lipoprotein Profile in Plasma viaModulatingCholesterolMetabolism in Liver of Guinea PigsFed High Fat Diet

341 Niacin Significantly Lessened Lipid Deposition in theArterial Wall of Guinea Pigs Fed High Fat Diet Oil red Ostaining in the aorta was found in HFD group but not inCD group because of chow diet without high fat Compared

6 Mediators of Inflammation

BlankoxLDL

0mM niacin 025mM niacin 05mM niacin 1mM niacin961 plusmn 063

702 plusmn 085

2551 plusmn 462

2132 plusmn 194

1844 plusmn 340

1463 plusmn 268

1371 plusmn 232

902 plusmn 084

1429 plusmn 282

750 plusmn 132

104

103

102

101

100

104103102101100

PI

Annexin-Y104103102101100

Annexin-Y104103102101100

Annexin-Y104103102101100

Annexin-Y104103102101100

Annexin-Y

(a)

002040608

112141618

2

oxLDL

lowast

lowastlowastlowastlowast

++++minus

Niacin (mM) 0 0 025 05 1

Relat

ive l

evel

of T

NF-120572

secr

etio

n

(b)

002040608

1121416

Relat

ive l

evel

of I

L-6

secr

etio

n

lowastlowast

oxLDL ++++minus

Niacin (mM) 0 0 025 05 1

(c)

Histone H3

NF-120581B p65

oxLDL ++++minus

Niacin (mM) 0 0 025 05 1

(d)

Notch1

120573-Actin

oxLDL ++++minus

Niacin (mM) 0 0 025 05 1

(e)

0

05

1

15

2

25

3

35

Relat

ive p

rote

in le

vel o

fN

F-120581

lowastlowast

lowastlowastlowastlowast

oxLDL ++++minus

Niacin (mM) 0 0 025 05 1

B p6

5in

nuc

lei o

f HU

VEC

s

(f)

0

05

1

15

2

Rela

tive p

rote

in le

vel o

fno

tch1

in H

UV

ECs

lowastlowastlowastlowast

lowastlowast

oxLDL ++++minus

Niacin (mM) 0 0 025 05 1

(g)

Figure 4 Niacin inhibited oxLDL-stimulated apoptosis and inflammation in HUVECs (a) shows the ratio of apoptotic HUVECs stainedwith annexin V-FITC and PI Representative data of flow cytometry analysis are presented (b) and (c) show the relative levels of TNF-120572 andIL-6 in the medium of HUVECs The concentrations of IL-6 and TNF-120572 were determined by ELISA kit (d) and (e) show the representativeimages of NF-120581B p65 and notch1 protein expression in HUVECs by western blot (f) and (g) show the IOD ratios of NF-120581B p65 and notch1expression respectively Data are presented as mean plusmn SD

119875 lt 001 versus blank group lowast119875 lt 005 lowastlowast119875 lt 001 versus oxLDL-treatedgroup without niacin

Mediators of Inflammation 7

0

05

1

15

2

25

0

02

04

06

08

1

12

14

Relat

ive l

evel

of I

L-6

secr

etio

n

0

02

04

06

08

1

12

14

Relat

ive p

rote

in le

vel o

f not

ch1

0

02

04

06

08

1

12

14

(a) (b)

(c)

(e)

(d)

(f)

oxLDL

lowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

++++minus

Niacin (mM) 0 0 025 05 1oxLDL ++++minus

Niacin (mM) 0 0 025 05 1

Relat

ive l

evel

of T

NF-120572

secr

etio

n

Histone H3

Notch1

Relat

ive p

rote

in le

vel o

fN

F-120581

Bp65

in n

ucle

i

NF-120581B p65

120573-Actin

oxLDL ++++minus

Niacin (mM) 0 0 025 05 1

oxLDL ++++minus

Niacin (mM) 0 0 025 05 1

oxLDL ++++minus

Niacin (mM) 0 0 025 05 1oxLDL ++++minus

Niacin (mM) 0 0 025 05 1

Figure 5 Niacin decreased the secretion of TNF-120572 and IL-6 and inhibited NF-120581B and notch1 expression in oxLDL-stimulated THP-1macrophages (a) and (b) show the relative levels of TNF-120572 and IL-6 secretion in the medium of THP-1 macrophages The concentrations ofIL-6 and TNF-120572 were determined by ELISA kit (c) and (d) show the representative images of NF-120581B p65 and notch1 protein expression inTHP-1 macrophages by western blot (e) and (f) show the IOD ratios of NF-120581B p65 and notch1 expression respectively Data are presented asmean plusmn SD

119875 lt 001 versus blank group lowast119875 lt 005 lowastlowast119875 lt 001 versus oxLDL-treated group without niacin

with that of HFD group niacin and simvastatin significantlydecreased the percentages of stained area to the total cross-sectional vessel wall by 56 and 67 respectively (Figure 6)The effect of simvastatin was superior to that of niacin

342 Niacin Increased HDL-C and ApoA I Levels andDecreased TG and Non-HDL-C Levels in Plasma of GuineaPigs Fed High Fat Diet As shown in Figure 7 after highfat diet for 8 weeks the levels of plasma TC TG HDL-Cand non-HDL-C were significantly increased in HFD groupcompared with CD group (119875 lt 001) which indicated asuccessful hyperlipidemic model in guinea pigs Comparedwith HFD group niacin decreased the levels of TG and

non-HDL-C by 27 and 12 respectively and increasedHDL level by 21 Niacin had no statistical influence onTC level in plasma Compared with HFD group simvastatindecreased the levels of TG non-HDL-C and TC by 1853 and 51 respectively Simvastatin had no significantinfluence on HDL-C level

The level of apoA I in plasma was also detected by SDS-PAGE in this study Comparedwith that ofHFDgroup niacinsignificantly promoted the level of apoA I by 42 whereassimvastatin had no significant influence on apoA I (Figure 8)

343 Niacin Significantly Upregulated the mRNA Amountof CYP7A1 in Liver Cholesterol metabolism in liver is a

8 Mediators of Inflammation

CD HFD

HFD-N HFD-S

(a)

0

05

1

15

2

CD HFD HFD-N HFD-S

Oil

red

O st

aine

d ar

ea (

)

lowastlowast

lowastlowast

(b)

Figure 6 Niacin and simvastatin significantly lessened lipid depo-sition in the arterial wall of guinea pigs fed high fat diet Lipiddeposition in the aorta wall was analyzed by oil red O staining aftertreatment for 8 weeksThe quantification of stained lipids was deter-mined by calculating the percentage of the positive area to the totalcross-sectional vessel wall area by Image-Pro Plus software Data arepresented as mean plusmn SD (119899 = 8)

119875 lt 001 versus CD grouplowastlowast

119875 lt 001 versus HFD group

complicated homeostasis involving multiple steps includingcholesterol ingression synthesis and conversion SR-B1 andLDL-R in liver play a critical role in cholesterol ingressionSR-B1 is the HDL receptor on the hepatocyte surface LDL-R can bind to LDL and VLDL and internalize them intohepatocytes [17] In the study we determined the effect ofniacin on the expressions of SR-B1 and LDL-R mRNA inliver As shown in Figures 9(a) and 9(b) after treatment withhigh fat diet for 8 weeks the LDL-R mRNA level was down-regulated (119875 lt 001) and the SR-B1 mRNA level wasnot significantly changed in HFD group Compared withHFD group niacin had no significant effect on SR-B1 and

LDL-R mRNA levels but simvastatin upregulated LDL-RmRNA level by 71

Cholesterol in liver can be converted into bile acidthrough cytochrome P450-meidiated oxidation The rate-limiting enzyme for the dominant pathway of bile acidsynthesis is cytochrome P450 7A1 (CYP7A1) As shown inFigure 9(c) compared with HFD group niacin significantlyupregulated the CYP7A1 mRNA level by 59 whereassimvastatin had no significant influence on its level HMGCRis the rate-limiting enzyme in the process of cholesterolsynthesis Compared with that of CD group the mRNA levelof HMGCR was significantly decreased in HFD group (119875 lt001) Compared with HFD group simvastatin upregulatedthe HMGCR mRNA levels by 46 whereas niacin had nosignificant influence on its level (Figure 9(d))

4 Discussion

For the first time to our knowledge this report demonstratesniacin inhibited vascular inflammation in guinea pig fedhigh fat diet and suppressed oxLDL-stimulated inflammatoryresponse even injury of endothelial cells and macrophagesin vitro The result indicates a new mechanism for niacinrsquosprotective action on cardiovascular disease in addition to itsestablished effects on lipid metabolism

The augmentation of inflammatory response has beenclearly documented in pathogenesis of vascular impairmentThe chronic inflammatory pathogenesis in the arterial wall isas follows Harmful substances in blood such as hypercholes-terolemia can induce endothelial dysfunction This causesthe production of ROS and the secretion of cellular adhesionmolecules (CAMs) cytokines and chemokines which facili-tate adherence and endothelial transmigration of leukocytes(monocytes and T helper lymphocytes) Monocytes in thearterial wall will be activated by proinflammatory cytokinesand differentiated into macrophages Activated macrophagesincrease the expression of CAMs and cytokines which resultsin recruitment of more leukocytes into the arterial wall acti-vates the complement pathways of immune system and theacute phase response stimulates proliferation and migrationof smooth muscle cells (SMCs) and promotes fibrous tissuedeposition [18] In progress the signaling molecule NF-120581Bis a proinflammatory major switch that can upregulate theexpression of lots of cytokines [19] Activated NF-120581B canresult in the enhanced efflux of TNF-120572 and IL-6 in serum[20] Early events in AS are usually driven by NF-120581B and thedisruption of NF-120581B signaling pathway has been shown toslow down the vascular impairment [21]

In the present study we demonstrate that niacin attenu-ated vascular inflammation induced by high fat diet in vivoThe involved evidences are as follows (1) Niacin lowered thenumber of macrophages (CD68 positive cells) in the arterialwall and significantly downregulated the inflammatory fac-tors (IL-6 and TNF-120572) levels in plasma of guinea pigs fed highfat diet (2) Both immunohistochemistry and western blotanalysis indicated niacin suppressed the expression of activeNF-120581B p65 in nuclei of the arterial wall The activated NF-120581Bis reported to form a heterodimer which usually consists oftwo proteins p65 and p50 subunitsThe p65 subunit has been

Mediators of Inflammation 9

0

50

100

150

200

250

CD HFD HFD-N HFD-S

TG (m

gdL

)

lowastlowastlowastlowast

(a)

0

500

1000

1500

2000

CD HFD HFD-N HFD-S

TC (m

gdL

)

lowastlowast

(b)

0

20

40

60

80

100

120

CD HFD HFD-N HFD-S

HD

L-C

(mg

dL)

lowast

(c)

0

500

1000

1500

2000

CD HFD HFD-N HFD-S

Non

-HD

L-C

(mg

dL)

lowastlowast

lowast

(d)

Figure 7 Effect of niacin and simvastatin on plasma lipid of guinea pigs fed high fat diet The levels of TG (a) TC (b) HDL-C (c) andnon-HDL-C (d) in plasma of guinea pigs were determined by enzyme method after 8 weeksrsquo treatment Data are presented as mean plusmn SD(119899 = 8)

119875 lt 001 versus CD group lowast119875 lt 005 lowastlowast119875 lt 001 versus HFD group

apoAI20

30

40

50

6080

160110

Mar

ker

CD HFD

HFD

-N

HFD

-S

Hum

an

(a)

0

05

1

15

2

CD HFD HFD-N HFD-S

Fold

indu

ctio

n of

apoA

I in

HD

L

lowastlowast

(b)

Figure 8 Niacin upregulated apoA I level in plasma of guinea pigs fed high fat diet HDL (density = 109ndash124 gmL) was separatedfrom plasma of guinea pigs by sequential ultracentrifugation The SDS-PAGE was performed on 15 SDS polyacrylamide gel and theapolipoproteins were stained with coomassie brilliant blue Densitometric quantitation of SDS-PAGE image was analyzed by Image-Pro Plussoftware (a) shows the representative SDS-PAGE image ofHDL (b) shows the relative level of apoA I in plasma by densitometric quantitationData are presented as mean plusmn SD of at least three independent experiments

119875 lt 005 versus CD group lowastlowast119875 lt 001 versus HFD group

10 Mediators of Inflammation

0

02

04

06

08

1

12

14

16

CD HFD HFD-N HFD-S

Relat

ive m

RNA

leve

l of S

R-B1

(a)

0

02

04

06

08

1

12

CD HFD HFD-N HFD-S

Relat

ive m

RNA

leve

l of L

DL-

R

lowastlowast

(b)

0

05

1

15

2

CD HFD HFD-N HFD-S

Relat

ive m

RNA

leve

l of C

YP7A

1 lowastlowast

(c)

0

02

04

06

08

1

12

14

CD HFD HFD-N HFD-S

Relat

ive m

RNA

leve

l of H

MG

CR

lowastlowast

(d)

Figure 9 Effect of niacin and simvastatin on the mRNA abundance of SR-B1 LDL-R CYP7A1 and HMGCR in liver of guinea pigs fedhigh fat dietThe mRNA levels which were analyzed by quantitative real-time PCR were calculated after being adjusted for 120573-actin usingthe 2minusΔΔCt method Data are presented as mean plusmn SD of at least three independent experiments

119875 lt 005 119875 lt 001 versus CD group

lowastlowast

119875 lt 001 versus HFD group

demonstrated to exert critical activity on the transcriptionof many inflammatory genes including adhesion moleculescytokines and chemokines [22] (3) CRP an early acute phasereactant is closely relevant to inflammation Baseline level ofCRP is a strong independent predictor of the risk of futuremyocardial infarction peripheral vascular disease strokeand vascular death amonghealthy individualswithout knownvascular disease [23] Kuvin et al have shown that niacindecreased CRP level by 15 in patients with stable coronaryartery disease [5] In patients with metabolic syndromeafter treatment with extended-release niacin (1 gday) for 52weeks their endothelial function was improved by 22 andthere was a decrease in CRP level by 20 [24] Our resultsalso showed niacin slightly lowered CRP level but hadno statistical difference (Table 1) (4) Oxidative stress wassuppressed by niacin Oxidative stress is closely related to theinflammation in the arterial wall Increased ROS productioncan initiate a cascade of signal transduction which results inendothelial dysfunction changes in vascular tone vascularremodeling and vascular inflammatory responses [25]

To further confirm the direct anti-inflammatory prop-erty of niacin its effect on oxLDL-induced inflammatoryresponse of endothelial cells and macrophages was studiedoxLDL is pivotal in the development of AS and representsa crucial proinflammatory stimulus [26] Upon enteringinto the intima of arteries oxLDL activates endothelial

cells and upregulates adhesion molecule expression andinflammatory factors secretion all of which contribute tothe recruitment of circulating leukocytes Monocytes andormacrophages infiltrating the arterial wall take up oxLDL andform ldquofoam cellsrdquo which in turn promote further secretionof inflammatory mediators [27] Our data indicated niacinremarkably downregulated the secretion of TNF-120572 and IL-6stimulated by oxLDL in HUVECs and THP-1 macrophagesNotch1 signal pathway is an evolutionarily highly conservedmechanism for communication which can improve NF-120581Bactivity and have a positive correlation with the developmentof inflammation [28] A previous study demonstrates thatnotch knockout in macrophage markedly downregulates theexpression of proinflammatory factors Instead when thenotch signal pathway is activatedNF-120581B expression increasesand inflammatory factors secretion is also promoted [29]Our findings indicated niacin decreased the expressions ofNF-120581B and notch1 in oxLDL-induced inflammation Vas-cular endothelial apoptosis plays an important role in theinitiation of AS It may compromise vessel wall permeabilityto cytokines growth factors lipids and immune cells andincrease coagulation [30] In this study we indicated niacinattenuated oxLDL-induced apoptosis of HUVECs

Taken together these observations lend initial but com-pelling evidence that niacin has protective effects on car-diovascular disease through its vascular anti-inflammatory

Mediators of Inflammation 11

properties in addition to its established effects on lipidmetabolism

Reverse cholesterol transport (RCT) is a potent defensemechanism against AS It involves transporting cholesterolfromperipheral tissues and cells to liver transforming choles-terol into bile acid and finally eliminating it from the body[31] HDL and apoA play a key role in RCT They can drivecholesterol efflux from macrophage foam cells that residein vessel intima [32 33] Similar to previous studies ourfindings also indicated that niacin increased the levels ofHDL and apoA I in plasma significantly In the process ofRCT CYP7A1 is an important regulatory factor which canconvert cholesterol to bile acid in liver and be further excretedinto intestine for elimination from body with feces Here weshowed that the mRNA abundance of CYP7A1 in liver wassignificantly upregulated by niacin treatment These findingssuggest that niacin may modulate plasma lipid and exertantiatherosclerotic effects by promoting RCT progress

In clinical practice the oral dose of niacin for lipid-lowering purpose is usually 1ndash3 g per day equivalently 14ndash43mgkg per day According to the formula of Meeh-Rubnerequation we calculate the dose for guinea pig to be about80ndash250mgkg per day In addition a recent report showedthat up to 400mgkg per day of niacin treatment for 7 daysand 100mgkg per day of niacin treatment for 4 weeks aresafe and do not cause toxicity in rodent models [34] In thisstudy the applied dose of niacin for guinea pig is 100mgkgdwhich has pharmacologic effects and does not have varioustoxicities based on the changes in food intake body weightand behaviors In human plasma level of niacin was foundto be about 03mM after oral ingestion of 1 g niacin [3536] Since the reported plasma concentrations are one timemeasurement it is unclear whether these levels reflect troughor peak values Furthermore there is no reported clinicaldata available regarding plasma concentration of niacin afteringestion of 3 g niacin Extrapolation of the available datausing oral dose of 1 g niacin would indicate that the 3 g niacinoral administration may lead to the plasma concentrations ofniacin in the range of 08ndash1mM In our in-vitro studies weused 025 to 1mM niacin

Statins are the first-line therapy in the treatment ofcholesterol-induced atherosclerotic cardiovascular diseases[37] They suppress the conversion of HMG-CoA to meval-onate by competitively blocking HMGCR therefore theendogenous cholesterol synthesis in the liver is remarkablyreduced Consequently it results in the increase of LDL-Rexpression on hepatic cell surfaces and the decrease of LDL-Cin plasma [38]Meanwhile inhibition of cholesterol synthesiscauses an overexpression of HMGCR due to a feedback-regulatory mechanism [39] Similar to previous studies ourfindings also revealed that simvastatin significantly decreasedthe level of non-HDL-C in plasma followed by upregulatingLDL-R expression in liver and the HMGCR mRNA level inliver was increased in a compensatory way

5 Conclusion

In summary our data presented herein support the novelconcept that niacin has vascular anti-inflammatory and

potentially vascular-protective property which is indepen-dent of its effect on lipid regulation The anti-inflammatoryproperty of niacin is realized by downregulating nucleartranscription factor-120581B signaling pathway Meanwhile ourfinding also suggests that niacin modulates plasma lipid bystimulating the expression of factors involved in the processof RCT and promoting RCT

Conflict of Interests

The authors declare that they have no conflict of interest

Authorsrsquo Contribution

Yanhong Si and Ying Zhang have equally contributed to thisstudy

Acknowledgments

This research was supported by the financial supportsfrom the Taishan scholar foundation of Shandong Province(200867) and the Natural Science Foundations of China(81170785 and 81371234)

References

[1] R Altschul A Hoffer and J D Stephen ldquoInfluence of nicotinicacid on serum cholesterol in manrdquoArchives of Biochemistry andBiophysics vol 54 no 2 pp 558ndash559 1955

[2] C D Meyers V S Kamanna and M L Kashyap ldquoNiacin ther-apy in atherosclerosisrdquo Current Opinion in Lipidology vol 15no 6 pp 659ndash665 2004

[3] L A Carlson ldquoNicotinic acid the broad-spectrum lipid drug A50th anniversary reviewrdquo Journal of Internal Medicine vol 258no 2 pp 94ndash114 2005

[4] S H Ganji S Qin L Zhang V S Kamanna andM L KashyapldquoNiacin inhibits vascular oxidative stress redox-sensitive genesand monocyte adhesion to human aortic endothelial cellsrdquoAtherosclerosis vol 202 no 1 pp 68ndash75 2009

[5] J T Kuvin D M Dave K A Sliney et al ldquoEffects of extended-release niacin on lipoprotein particle size distribution andinflammatorymarkers in patients with coronary artery diseaserdquoAmerican Journal of Cardiology vol 98 no 6 pp 743ndash745 2006

[6] R Yang P Guo X Song F Liu and N Gao ldquoHyperlipidemicguinea pig model mechanisms of triglyceride metabolismdisorder and comparison to ratrdquo Biological and PharmaceuticalBulletin vol 34 no 7 pp 1046ndash1051 2011

[7] X Li Y Liu H Zhang L Ren Q Li and N Li ldquoAnimal modelsfor the atherosclerosis research a reviewrdquo Protein and Cell vol2 no 3 pp 189ndash201 2011

[8] J O Leite U Vaishnav M Puglisi H Fraser J Trias and M LFernandez ldquoA-002 (Varespladib) a phospholipase A2 inhibitorreduces atherosclerosis in guinea pigsrdquo BMC CardiovascularDisorders vol 9 article 7 2009

[9] J Auwerx ldquoThe human leukemia cell line THP-1 a multi-facetted model for the study of monocyte-macrophage differ-entiationrdquo Experientia vol 47 no 1 pp 22ndash31 1991

[10] M J Chapman G L Mills and J H Ledford ldquoThe distributionand partial characterization of the serum apolipoproteins in the

12 Mediators of Inflammation

guinea pigrdquo Biochemical Journal vol 149 no 2 pp 423ndash4361975

[11] X C Jiang C Bruce J Mar et al ldquoTargetedmutation of plasmaphospholipid transfer protein gene markedly reduces high-density lipoprotein levelsrdquo Journal of Clinical Investigation vol103 no 6 pp 907ndash914 1999

[12] T Collins and M I Cybulsky ldquoNF-120581B pivotal mediator orinnocent bystander in atherogenesisrdquo Journal of Clinical Inves-tigation vol 107 no 3 pp 255ndash264 2001

[13] Y Tsubosaka T Murata K Yamada D Uemura M Horiand H Ozaki ldquoHalichlorine reduces monocyte adhesion toendothelium through the suppression of nuclear factor-120581Bactivationrdquo Journal of Pharmacological Sciences vol 113 no 3pp 208ndash213 2010

[14] H Tsutsui ldquoMitochondrial oxidative stress and heart failurerdquoInternal Medicine vol 45 no 13 pp 809ndash813 2006

[15] M Seddon Y H Looi and A M Shah ldquoOxidative stressand redox signalling in cardiac hypertrophy and heart failurerdquoHeart vol 93 no 8 pp 903ndash907 2007

[16] WWongchana and T Palaga ldquoDirect regulation of interleukin-6 expression by Notch signaling in macrophagesrdquo Cellular andMolecular Immunology vol 9 no 2 pp 155ndash162 2012

[17] J Chen H Zhao X Ma et al ldquoThe effects of jiang-zhi-ningand itsmain components on cholesterol metabolismrdquo Evidence-Based Complementary and Alternative Medicine vol 2012Article ID 928234 10 pages 2012

[18] G Francisco C Hernandez and R Simo ldquoSerum markers ofvascular inflammation in dyslipemiardquo Clinica Chimica Actavol 369 no 2 pp 1ndash16 2006

[19] J Andersson P Libby and G K Hansson ldquoAdaptive immunityand atherosclerosisrdquoClinical Immunology vol 134 no 1 pp 33ndash46 2010

[20] W J Oh U Jung H S Eom H J Shin and H R Park ldquoInhibi-tion of lipopolysaccharide-induced proinflammatory responsesby Buddleja officinalis extract in BV-2 microglial cells vianegative regulation of NF-kB and ERK12 signalingrdquoMoleculesvol 18 no 8 pp 9195ndash9206 2013

[21] T M Wang C J Chen T S Lee et al ldquoDocosahexaenoic acidattenuates VCAM-1 expression and NF-120581B activation in TNF-120572-treated human aortic endothelial cellsrdquo Journal of NutritionalBiochemistry vol 22 no 2 pp 187ndash194 2011

[22] M S Islam H Yoshida N Matsuki et al ldquoAntioxidant freeradical-scavenging and NF-120581B-inhibitory activities of phy-tosteryl ferulates structure-activity studiesrdquo Journal of Pharma-cological Sciences vol 111 no 4 pp 328ndash337 2009

[23] P M Ridker M J Stampfer and N Rifai ldquoNovel risk factorsfor systemic atherosclerosis a comparison ofC-reactive proteinfibrinogen homocysteine lipoprotein(a) and standard choles-terol screening as predictors of peripheral arterial diseaserdquoJournal of the AmericanMedical Association vol 285 no 19 pp2481ndash2485 2001

[24] M Thoenes A Oguchi S Nagamia et al ldquoThe effects ofextended-release niacin on carotid intimal media thicknessendothelial function and inflammatory markers in patientswith the metabolic syndromerdquo International Journal of ClinicalPractice vol 61 no 11 pp 1942ndash1948 2007

[25] N R Madamanchi A Vendrov and M S Runge ldquoOxidativestress and vascular diseaserdquo Arteriosclerosis Thrombosis andVascular Biology vol 25 no 1 pp 29ndash38 2005

[26] E Matsuura F Atzeni P Sarzi-Puttini M Turiel L R Lopezand M T Nurmohamed ldquoIs atherosclerosis an autoimmunediseaserdquo BMCMedicine vol 12 no 1 article 47 2014

[27] A Pirillo G D Norata and A L Catapano ldquoLOX-1 OxLDLand atherosclerosisrdquo Mediators of Inflammation vol 2013Article ID 152786 12 pages 2013

[28] C L Wang M Meng S B Liu L R Wang L H Hou and XH Cao ldquochemically sulfated polysaccharide from Grifola fron-dos induces HepG2 cell apoptosis by notch1-NF-120581B pathwayrdquoCarbohydrate Polymers vol 95 no 1 pp 282ndash287 2013

[29] WWongchana and T Palaga ldquoDirect regulation of interleukin-6 expression by Notch signaling in macrophagesrdquo Cellular andMolecular Immunology vol 9 no 2 pp 155ndash162 2012

[30] J C Choy D J Granville D W C Hunt and B M McManusldquoEndothelial cell apoptosis biochemical characteristics andpotential implications for atherosclerosisrdquo Journal of Molecularand Cellular Cardiology vol 33 no 9 pp 1673ndash1690 2001

[31] M Cuchel and D J Rader ldquoMacrophage reverse cholesteroltransport key to the regression of atherosclerosisrdquo Circulationvol 113 no 21 pp 2548ndash2555 2006

[32] A Yazdanyar C Yeang and X C Jiang ldquoRole of phospholipidtransfer protein in high-density lipoprotein-mediated reversecholesterol transportrdquo Current Atherosclerosis Reports vol 13no 3 pp 242ndash248 2011

[33] X Wang and D J Rader ldquoMolecular regulation of macrophagereverse cholesterol transportrdquo Current Opinion in Cardiologyvol 22 no 4 pp 368ndash372 2007

[34] S J Yang J M Choi L Kim et al ldquoNicotinamide improvesglucose metabolism and affects the hepatic NAD-sirtuin path-way in a rodent model of obesity and type 2 diabetesrdquo Journalof Nutritional Biochemistry vol 25 no 1 pp 66ndash72 2014

[35] L A Carlson L Oro and J Ostman ldquoEffect of a single dose ofnicotinic acid on plasma lipids in patients with hyperlipopro-teinemiardquo Acta Medica Scandinavica vol 183 no 5 pp 457ndash465 1968

[36] R H Stern J D Spence D J Freeman and A Parbtani ldquoTol-erance to nicotinic acid flushingrdquo Clinical Pharmacology andTherapeutics vol 50 no 1 pp 66ndash70 1991

[37] S M Grundy J I Cleeman C N Bairey Merz et al ldquoImpli-cations of recent clinical trials for the National CholesterolEducation Program Adult Treatment Panel III guidelinesrdquoCirculation vol 110 no 2 pp 227ndash239 2004

[38] M S Brown and J L Goldstein ldquoLowering plasma cholesterolby raising LDL receptorsrdquoNewEngland Journal ofMedicine vol305 no 9 pp 515ndash517 1981

[39] R Hampton D Dimster-Denk and J Rine ldquoThe biology ofHMG-CoA reductase the pros of contra-regulationrdquo Trends inBiochemical Sciences vol 21 no 4 pp 140ndash145 1996

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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 6: Research Article Niacin Inhibits Vascular Inflammation via ...downloads.hindawi.com/journals/mi/2014/263786.pdf · Research Article Niacin Inhibits Vascular Inflammation via Downregulating

6 Mediators of Inflammation

BlankoxLDL

0mM niacin 025mM niacin 05mM niacin 1mM niacin961 plusmn 063

702 plusmn 085

2551 plusmn 462

2132 plusmn 194

1844 plusmn 340

1463 plusmn 268

1371 plusmn 232

902 plusmn 084

1429 plusmn 282

750 plusmn 132

104

103

102

101

100

104103102101100

PI

Annexin-Y104103102101100

Annexin-Y104103102101100

Annexin-Y104103102101100

Annexin-Y104103102101100

Annexin-Y

(a)

002040608

112141618

2

oxLDL

lowast

lowastlowastlowastlowast

++++minus

Niacin (mM) 0 0 025 05 1

Relat

ive l

evel

of T

NF-120572

secr

etio

n

(b)

002040608

1121416

Relat

ive l

evel

of I

L-6

secr

etio

n

lowastlowast

oxLDL ++++minus

Niacin (mM) 0 0 025 05 1

(c)

Histone H3

NF-120581B p65

oxLDL ++++minus

Niacin (mM) 0 0 025 05 1

(d)

Notch1

120573-Actin

oxLDL ++++minus

Niacin (mM) 0 0 025 05 1

(e)

0

05

1

15

2

25

3

35

Relat

ive p

rote

in le

vel o

fN

F-120581

lowastlowast

lowastlowastlowastlowast

oxLDL ++++minus

Niacin (mM) 0 0 025 05 1

B p6

5in

nuc

lei o

f HU

VEC

s

(f)

0

05

1

15

2

Rela

tive p

rote

in le

vel o

fno

tch1

in H

UV

ECs

lowastlowastlowastlowast

lowastlowast

oxLDL ++++minus

Niacin (mM) 0 0 025 05 1

(g)

Figure 4 Niacin inhibited oxLDL-stimulated apoptosis and inflammation in HUVECs (a) shows the ratio of apoptotic HUVECs stainedwith annexin V-FITC and PI Representative data of flow cytometry analysis are presented (b) and (c) show the relative levels of TNF-120572 andIL-6 in the medium of HUVECs The concentrations of IL-6 and TNF-120572 were determined by ELISA kit (d) and (e) show the representativeimages of NF-120581B p65 and notch1 protein expression in HUVECs by western blot (f) and (g) show the IOD ratios of NF-120581B p65 and notch1expression respectively Data are presented as mean plusmn SD

119875 lt 001 versus blank group lowast119875 lt 005 lowastlowast119875 lt 001 versus oxLDL-treatedgroup without niacin

Mediators of Inflammation 7

0

05

1

15

2

25

0

02

04

06

08

1

12

14

Relat

ive l

evel

of I

L-6

secr

etio

n

0

02

04

06

08

1

12

14

Relat

ive p

rote

in le

vel o

f not

ch1

0

02

04

06

08

1

12

14

(a) (b)

(c)

(e)

(d)

(f)

oxLDL

lowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

++++minus

Niacin (mM) 0 0 025 05 1oxLDL ++++minus

Niacin (mM) 0 0 025 05 1

Relat

ive l

evel

of T

NF-120572

secr

etio

n

Histone H3

Notch1

Relat

ive p

rote

in le

vel o

fN

F-120581

Bp65

in n

ucle

i

NF-120581B p65

120573-Actin

oxLDL ++++minus

Niacin (mM) 0 0 025 05 1

oxLDL ++++minus

Niacin (mM) 0 0 025 05 1

oxLDL ++++minus

Niacin (mM) 0 0 025 05 1oxLDL ++++minus

Niacin (mM) 0 0 025 05 1

Figure 5 Niacin decreased the secretion of TNF-120572 and IL-6 and inhibited NF-120581B and notch1 expression in oxLDL-stimulated THP-1macrophages (a) and (b) show the relative levels of TNF-120572 and IL-6 secretion in the medium of THP-1 macrophages The concentrations ofIL-6 and TNF-120572 were determined by ELISA kit (c) and (d) show the representative images of NF-120581B p65 and notch1 protein expression inTHP-1 macrophages by western blot (e) and (f) show the IOD ratios of NF-120581B p65 and notch1 expression respectively Data are presented asmean plusmn SD

119875 lt 001 versus blank group lowast119875 lt 005 lowastlowast119875 lt 001 versus oxLDL-treated group without niacin

with that of HFD group niacin and simvastatin significantlydecreased the percentages of stained area to the total cross-sectional vessel wall by 56 and 67 respectively (Figure 6)The effect of simvastatin was superior to that of niacin

342 Niacin Increased HDL-C and ApoA I Levels andDecreased TG and Non-HDL-C Levels in Plasma of GuineaPigs Fed High Fat Diet As shown in Figure 7 after highfat diet for 8 weeks the levels of plasma TC TG HDL-Cand non-HDL-C were significantly increased in HFD groupcompared with CD group (119875 lt 001) which indicated asuccessful hyperlipidemic model in guinea pigs Comparedwith HFD group niacin decreased the levels of TG and

non-HDL-C by 27 and 12 respectively and increasedHDL level by 21 Niacin had no statistical influence onTC level in plasma Compared with HFD group simvastatindecreased the levels of TG non-HDL-C and TC by 1853 and 51 respectively Simvastatin had no significantinfluence on HDL-C level

The level of apoA I in plasma was also detected by SDS-PAGE in this study Comparedwith that ofHFDgroup niacinsignificantly promoted the level of apoA I by 42 whereassimvastatin had no significant influence on apoA I (Figure 8)

343 Niacin Significantly Upregulated the mRNA Amountof CYP7A1 in Liver Cholesterol metabolism in liver is a

8 Mediators of Inflammation

CD HFD

HFD-N HFD-S

(a)

0

05

1

15

2

CD HFD HFD-N HFD-S

Oil

red

O st

aine

d ar

ea (

)

lowastlowast

lowastlowast

(b)

Figure 6 Niacin and simvastatin significantly lessened lipid depo-sition in the arterial wall of guinea pigs fed high fat diet Lipiddeposition in the aorta wall was analyzed by oil red O staining aftertreatment for 8 weeksThe quantification of stained lipids was deter-mined by calculating the percentage of the positive area to the totalcross-sectional vessel wall area by Image-Pro Plus software Data arepresented as mean plusmn SD (119899 = 8)

119875 lt 001 versus CD grouplowastlowast

119875 lt 001 versus HFD group

complicated homeostasis involving multiple steps includingcholesterol ingression synthesis and conversion SR-B1 andLDL-R in liver play a critical role in cholesterol ingressionSR-B1 is the HDL receptor on the hepatocyte surface LDL-R can bind to LDL and VLDL and internalize them intohepatocytes [17] In the study we determined the effect ofniacin on the expressions of SR-B1 and LDL-R mRNA inliver As shown in Figures 9(a) and 9(b) after treatment withhigh fat diet for 8 weeks the LDL-R mRNA level was down-regulated (119875 lt 001) and the SR-B1 mRNA level wasnot significantly changed in HFD group Compared withHFD group niacin had no significant effect on SR-B1 and

LDL-R mRNA levels but simvastatin upregulated LDL-RmRNA level by 71

Cholesterol in liver can be converted into bile acidthrough cytochrome P450-meidiated oxidation The rate-limiting enzyme for the dominant pathway of bile acidsynthesis is cytochrome P450 7A1 (CYP7A1) As shown inFigure 9(c) compared with HFD group niacin significantlyupregulated the CYP7A1 mRNA level by 59 whereassimvastatin had no significant influence on its level HMGCRis the rate-limiting enzyme in the process of cholesterolsynthesis Compared with that of CD group the mRNA levelof HMGCR was significantly decreased in HFD group (119875 lt001) Compared with HFD group simvastatin upregulatedthe HMGCR mRNA levels by 46 whereas niacin had nosignificant influence on its level (Figure 9(d))

4 Discussion

For the first time to our knowledge this report demonstratesniacin inhibited vascular inflammation in guinea pig fedhigh fat diet and suppressed oxLDL-stimulated inflammatoryresponse even injury of endothelial cells and macrophagesin vitro The result indicates a new mechanism for niacinrsquosprotective action on cardiovascular disease in addition to itsestablished effects on lipid metabolism

The augmentation of inflammatory response has beenclearly documented in pathogenesis of vascular impairmentThe chronic inflammatory pathogenesis in the arterial wall isas follows Harmful substances in blood such as hypercholes-terolemia can induce endothelial dysfunction This causesthe production of ROS and the secretion of cellular adhesionmolecules (CAMs) cytokines and chemokines which facili-tate adherence and endothelial transmigration of leukocytes(monocytes and T helper lymphocytes) Monocytes in thearterial wall will be activated by proinflammatory cytokinesand differentiated into macrophages Activated macrophagesincrease the expression of CAMs and cytokines which resultsin recruitment of more leukocytes into the arterial wall acti-vates the complement pathways of immune system and theacute phase response stimulates proliferation and migrationof smooth muscle cells (SMCs) and promotes fibrous tissuedeposition [18] In progress the signaling molecule NF-120581Bis a proinflammatory major switch that can upregulate theexpression of lots of cytokines [19] Activated NF-120581B canresult in the enhanced efflux of TNF-120572 and IL-6 in serum[20] Early events in AS are usually driven by NF-120581B and thedisruption of NF-120581B signaling pathway has been shown toslow down the vascular impairment [21]

In the present study we demonstrate that niacin attenu-ated vascular inflammation induced by high fat diet in vivoThe involved evidences are as follows (1) Niacin lowered thenumber of macrophages (CD68 positive cells) in the arterialwall and significantly downregulated the inflammatory fac-tors (IL-6 and TNF-120572) levels in plasma of guinea pigs fed highfat diet (2) Both immunohistochemistry and western blotanalysis indicated niacin suppressed the expression of activeNF-120581B p65 in nuclei of the arterial wall The activated NF-120581Bis reported to form a heterodimer which usually consists oftwo proteins p65 and p50 subunitsThe p65 subunit has been

Mediators of Inflammation 9

0

50

100

150

200

250

CD HFD HFD-N HFD-S

TG (m

gdL

)

lowastlowastlowastlowast

(a)

0

500

1000

1500

2000

CD HFD HFD-N HFD-S

TC (m

gdL

)

lowastlowast

(b)

0

20

40

60

80

100

120

CD HFD HFD-N HFD-S

HD

L-C

(mg

dL)

lowast

(c)

0

500

1000

1500

2000

CD HFD HFD-N HFD-S

Non

-HD

L-C

(mg

dL)

lowastlowast

lowast

(d)

Figure 7 Effect of niacin and simvastatin on plasma lipid of guinea pigs fed high fat diet The levels of TG (a) TC (b) HDL-C (c) andnon-HDL-C (d) in plasma of guinea pigs were determined by enzyme method after 8 weeksrsquo treatment Data are presented as mean plusmn SD(119899 = 8)

119875 lt 001 versus CD group lowast119875 lt 005 lowastlowast119875 lt 001 versus HFD group

apoAI20

30

40

50

6080

160110

Mar

ker

CD HFD

HFD

-N

HFD

-S

Hum

an

(a)

0

05

1

15

2

CD HFD HFD-N HFD-S

Fold

indu

ctio

n of

apoA

I in

HD

L

lowastlowast

(b)

Figure 8 Niacin upregulated apoA I level in plasma of guinea pigs fed high fat diet HDL (density = 109ndash124 gmL) was separatedfrom plasma of guinea pigs by sequential ultracentrifugation The SDS-PAGE was performed on 15 SDS polyacrylamide gel and theapolipoproteins were stained with coomassie brilliant blue Densitometric quantitation of SDS-PAGE image was analyzed by Image-Pro Plussoftware (a) shows the representative SDS-PAGE image ofHDL (b) shows the relative level of apoA I in plasma by densitometric quantitationData are presented as mean plusmn SD of at least three independent experiments

119875 lt 005 versus CD group lowastlowast119875 lt 001 versus HFD group

10 Mediators of Inflammation

0

02

04

06

08

1

12

14

16

CD HFD HFD-N HFD-S

Relat

ive m

RNA

leve

l of S

R-B1

(a)

0

02

04

06

08

1

12

CD HFD HFD-N HFD-S

Relat

ive m

RNA

leve

l of L

DL-

R

lowastlowast

(b)

0

05

1

15

2

CD HFD HFD-N HFD-S

Relat

ive m

RNA

leve

l of C

YP7A

1 lowastlowast

(c)

0

02

04

06

08

1

12

14

CD HFD HFD-N HFD-S

Relat

ive m

RNA

leve

l of H

MG

CR

lowastlowast

(d)

Figure 9 Effect of niacin and simvastatin on the mRNA abundance of SR-B1 LDL-R CYP7A1 and HMGCR in liver of guinea pigs fedhigh fat dietThe mRNA levels which were analyzed by quantitative real-time PCR were calculated after being adjusted for 120573-actin usingthe 2minusΔΔCt method Data are presented as mean plusmn SD of at least three independent experiments

119875 lt 005 119875 lt 001 versus CD group

lowastlowast

119875 lt 001 versus HFD group

demonstrated to exert critical activity on the transcriptionof many inflammatory genes including adhesion moleculescytokines and chemokines [22] (3) CRP an early acute phasereactant is closely relevant to inflammation Baseline level ofCRP is a strong independent predictor of the risk of futuremyocardial infarction peripheral vascular disease strokeand vascular death amonghealthy individualswithout knownvascular disease [23] Kuvin et al have shown that niacindecreased CRP level by 15 in patients with stable coronaryartery disease [5] In patients with metabolic syndromeafter treatment with extended-release niacin (1 gday) for 52weeks their endothelial function was improved by 22 andthere was a decrease in CRP level by 20 [24] Our resultsalso showed niacin slightly lowered CRP level but hadno statistical difference (Table 1) (4) Oxidative stress wassuppressed by niacin Oxidative stress is closely related to theinflammation in the arterial wall Increased ROS productioncan initiate a cascade of signal transduction which results inendothelial dysfunction changes in vascular tone vascularremodeling and vascular inflammatory responses [25]

To further confirm the direct anti-inflammatory prop-erty of niacin its effect on oxLDL-induced inflammatoryresponse of endothelial cells and macrophages was studiedoxLDL is pivotal in the development of AS and representsa crucial proinflammatory stimulus [26] Upon enteringinto the intima of arteries oxLDL activates endothelial

cells and upregulates adhesion molecule expression andinflammatory factors secretion all of which contribute tothe recruitment of circulating leukocytes Monocytes andormacrophages infiltrating the arterial wall take up oxLDL andform ldquofoam cellsrdquo which in turn promote further secretionof inflammatory mediators [27] Our data indicated niacinremarkably downregulated the secretion of TNF-120572 and IL-6stimulated by oxLDL in HUVECs and THP-1 macrophagesNotch1 signal pathway is an evolutionarily highly conservedmechanism for communication which can improve NF-120581Bactivity and have a positive correlation with the developmentof inflammation [28] A previous study demonstrates thatnotch knockout in macrophage markedly downregulates theexpression of proinflammatory factors Instead when thenotch signal pathway is activatedNF-120581B expression increasesand inflammatory factors secretion is also promoted [29]Our findings indicated niacin decreased the expressions ofNF-120581B and notch1 in oxLDL-induced inflammation Vas-cular endothelial apoptosis plays an important role in theinitiation of AS It may compromise vessel wall permeabilityto cytokines growth factors lipids and immune cells andincrease coagulation [30] In this study we indicated niacinattenuated oxLDL-induced apoptosis of HUVECs

Taken together these observations lend initial but com-pelling evidence that niacin has protective effects on car-diovascular disease through its vascular anti-inflammatory

Mediators of Inflammation 11

properties in addition to its established effects on lipidmetabolism

Reverse cholesterol transport (RCT) is a potent defensemechanism against AS It involves transporting cholesterolfromperipheral tissues and cells to liver transforming choles-terol into bile acid and finally eliminating it from the body[31] HDL and apoA play a key role in RCT They can drivecholesterol efflux from macrophage foam cells that residein vessel intima [32 33] Similar to previous studies ourfindings also indicated that niacin increased the levels ofHDL and apoA I in plasma significantly In the process ofRCT CYP7A1 is an important regulatory factor which canconvert cholesterol to bile acid in liver and be further excretedinto intestine for elimination from body with feces Here weshowed that the mRNA abundance of CYP7A1 in liver wassignificantly upregulated by niacin treatment These findingssuggest that niacin may modulate plasma lipid and exertantiatherosclerotic effects by promoting RCT progress

In clinical practice the oral dose of niacin for lipid-lowering purpose is usually 1ndash3 g per day equivalently 14ndash43mgkg per day According to the formula of Meeh-Rubnerequation we calculate the dose for guinea pig to be about80ndash250mgkg per day In addition a recent report showedthat up to 400mgkg per day of niacin treatment for 7 daysand 100mgkg per day of niacin treatment for 4 weeks aresafe and do not cause toxicity in rodent models [34] In thisstudy the applied dose of niacin for guinea pig is 100mgkgdwhich has pharmacologic effects and does not have varioustoxicities based on the changes in food intake body weightand behaviors In human plasma level of niacin was foundto be about 03mM after oral ingestion of 1 g niacin [3536] Since the reported plasma concentrations are one timemeasurement it is unclear whether these levels reflect troughor peak values Furthermore there is no reported clinicaldata available regarding plasma concentration of niacin afteringestion of 3 g niacin Extrapolation of the available datausing oral dose of 1 g niacin would indicate that the 3 g niacinoral administration may lead to the plasma concentrations ofniacin in the range of 08ndash1mM In our in-vitro studies weused 025 to 1mM niacin

Statins are the first-line therapy in the treatment ofcholesterol-induced atherosclerotic cardiovascular diseases[37] They suppress the conversion of HMG-CoA to meval-onate by competitively blocking HMGCR therefore theendogenous cholesterol synthesis in the liver is remarkablyreduced Consequently it results in the increase of LDL-Rexpression on hepatic cell surfaces and the decrease of LDL-Cin plasma [38]Meanwhile inhibition of cholesterol synthesiscauses an overexpression of HMGCR due to a feedback-regulatory mechanism [39] Similar to previous studies ourfindings also revealed that simvastatin significantly decreasedthe level of non-HDL-C in plasma followed by upregulatingLDL-R expression in liver and the HMGCR mRNA level inliver was increased in a compensatory way

5 Conclusion

In summary our data presented herein support the novelconcept that niacin has vascular anti-inflammatory and

potentially vascular-protective property which is indepen-dent of its effect on lipid regulation The anti-inflammatoryproperty of niacin is realized by downregulating nucleartranscription factor-120581B signaling pathway Meanwhile ourfinding also suggests that niacin modulates plasma lipid bystimulating the expression of factors involved in the processof RCT and promoting RCT

Conflict of Interests

The authors declare that they have no conflict of interest

Authorsrsquo Contribution

Yanhong Si and Ying Zhang have equally contributed to thisstudy

Acknowledgments

This research was supported by the financial supportsfrom the Taishan scholar foundation of Shandong Province(200867) and the Natural Science Foundations of China(81170785 and 81371234)

References

[1] R Altschul A Hoffer and J D Stephen ldquoInfluence of nicotinicacid on serum cholesterol in manrdquoArchives of Biochemistry andBiophysics vol 54 no 2 pp 558ndash559 1955

[2] C D Meyers V S Kamanna and M L Kashyap ldquoNiacin ther-apy in atherosclerosisrdquo Current Opinion in Lipidology vol 15no 6 pp 659ndash665 2004

[3] L A Carlson ldquoNicotinic acid the broad-spectrum lipid drug A50th anniversary reviewrdquo Journal of Internal Medicine vol 258no 2 pp 94ndash114 2005

[4] S H Ganji S Qin L Zhang V S Kamanna andM L KashyapldquoNiacin inhibits vascular oxidative stress redox-sensitive genesand monocyte adhesion to human aortic endothelial cellsrdquoAtherosclerosis vol 202 no 1 pp 68ndash75 2009

[5] J T Kuvin D M Dave K A Sliney et al ldquoEffects of extended-release niacin on lipoprotein particle size distribution andinflammatorymarkers in patients with coronary artery diseaserdquoAmerican Journal of Cardiology vol 98 no 6 pp 743ndash745 2006

[6] R Yang P Guo X Song F Liu and N Gao ldquoHyperlipidemicguinea pig model mechanisms of triglyceride metabolismdisorder and comparison to ratrdquo Biological and PharmaceuticalBulletin vol 34 no 7 pp 1046ndash1051 2011

[7] X Li Y Liu H Zhang L Ren Q Li and N Li ldquoAnimal modelsfor the atherosclerosis research a reviewrdquo Protein and Cell vol2 no 3 pp 189ndash201 2011

[8] J O Leite U Vaishnav M Puglisi H Fraser J Trias and M LFernandez ldquoA-002 (Varespladib) a phospholipase A2 inhibitorreduces atherosclerosis in guinea pigsrdquo BMC CardiovascularDisorders vol 9 article 7 2009

[9] J Auwerx ldquoThe human leukemia cell line THP-1 a multi-facetted model for the study of monocyte-macrophage differ-entiationrdquo Experientia vol 47 no 1 pp 22ndash31 1991

[10] M J Chapman G L Mills and J H Ledford ldquoThe distributionand partial characterization of the serum apolipoproteins in the

12 Mediators of Inflammation

guinea pigrdquo Biochemical Journal vol 149 no 2 pp 423ndash4361975

[11] X C Jiang C Bruce J Mar et al ldquoTargetedmutation of plasmaphospholipid transfer protein gene markedly reduces high-density lipoprotein levelsrdquo Journal of Clinical Investigation vol103 no 6 pp 907ndash914 1999

[12] T Collins and M I Cybulsky ldquoNF-120581B pivotal mediator orinnocent bystander in atherogenesisrdquo Journal of Clinical Inves-tigation vol 107 no 3 pp 255ndash264 2001

[13] Y Tsubosaka T Murata K Yamada D Uemura M Horiand H Ozaki ldquoHalichlorine reduces monocyte adhesion toendothelium through the suppression of nuclear factor-120581Bactivationrdquo Journal of Pharmacological Sciences vol 113 no 3pp 208ndash213 2010

[14] H Tsutsui ldquoMitochondrial oxidative stress and heart failurerdquoInternal Medicine vol 45 no 13 pp 809ndash813 2006

[15] M Seddon Y H Looi and A M Shah ldquoOxidative stressand redox signalling in cardiac hypertrophy and heart failurerdquoHeart vol 93 no 8 pp 903ndash907 2007

[16] WWongchana and T Palaga ldquoDirect regulation of interleukin-6 expression by Notch signaling in macrophagesrdquo Cellular andMolecular Immunology vol 9 no 2 pp 155ndash162 2012

[17] J Chen H Zhao X Ma et al ldquoThe effects of jiang-zhi-ningand itsmain components on cholesterol metabolismrdquo Evidence-Based Complementary and Alternative Medicine vol 2012Article ID 928234 10 pages 2012

[18] G Francisco C Hernandez and R Simo ldquoSerum markers ofvascular inflammation in dyslipemiardquo Clinica Chimica Actavol 369 no 2 pp 1ndash16 2006

[19] J Andersson P Libby and G K Hansson ldquoAdaptive immunityand atherosclerosisrdquoClinical Immunology vol 134 no 1 pp 33ndash46 2010

[20] W J Oh U Jung H S Eom H J Shin and H R Park ldquoInhibi-tion of lipopolysaccharide-induced proinflammatory responsesby Buddleja officinalis extract in BV-2 microglial cells vianegative regulation of NF-kB and ERK12 signalingrdquoMoleculesvol 18 no 8 pp 9195ndash9206 2013

[21] T M Wang C J Chen T S Lee et al ldquoDocosahexaenoic acidattenuates VCAM-1 expression and NF-120581B activation in TNF-120572-treated human aortic endothelial cellsrdquo Journal of NutritionalBiochemistry vol 22 no 2 pp 187ndash194 2011

[22] M S Islam H Yoshida N Matsuki et al ldquoAntioxidant freeradical-scavenging and NF-120581B-inhibitory activities of phy-tosteryl ferulates structure-activity studiesrdquo Journal of Pharma-cological Sciences vol 111 no 4 pp 328ndash337 2009

[23] P M Ridker M J Stampfer and N Rifai ldquoNovel risk factorsfor systemic atherosclerosis a comparison ofC-reactive proteinfibrinogen homocysteine lipoprotein(a) and standard choles-terol screening as predictors of peripheral arterial diseaserdquoJournal of the AmericanMedical Association vol 285 no 19 pp2481ndash2485 2001

[24] M Thoenes A Oguchi S Nagamia et al ldquoThe effects ofextended-release niacin on carotid intimal media thicknessendothelial function and inflammatory markers in patientswith the metabolic syndromerdquo International Journal of ClinicalPractice vol 61 no 11 pp 1942ndash1948 2007

[25] N R Madamanchi A Vendrov and M S Runge ldquoOxidativestress and vascular diseaserdquo Arteriosclerosis Thrombosis andVascular Biology vol 25 no 1 pp 29ndash38 2005

[26] E Matsuura F Atzeni P Sarzi-Puttini M Turiel L R Lopezand M T Nurmohamed ldquoIs atherosclerosis an autoimmunediseaserdquo BMCMedicine vol 12 no 1 article 47 2014

[27] A Pirillo G D Norata and A L Catapano ldquoLOX-1 OxLDLand atherosclerosisrdquo Mediators of Inflammation vol 2013Article ID 152786 12 pages 2013

[28] C L Wang M Meng S B Liu L R Wang L H Hou and XH Cao ldquochemically sulfated polysaccharide from Grifola fron-dos induces HepG2 cell apoptosis by notch1-NF-120581B pathwayrdquoCarbohydrate Polymers vol 95 no 1 pp 282ndash287 2013

[29] WWongchana and T Palaga ldquoDirect regulation of interleukin-6 expression by Notch signaling in macrophagesrdquo Cellular andMolecular Immunology vol 9 no 2 pp 155ndash162 2012

[30] J C Choy D J Granville D W C Hunt and B M McManusldquoEndothelial cell apoptosis biochemical characteristics andpotential implications for atherosclerosisrdquo Journal of Molecularand Cellular Cardiology vol 33 no 9 pp 1673ndash1690 2001

[31] M Cuchel and D J Rader ldquoMacrophage reverse cholesteroltransport key to the regression of atherosclerosisrdquo Circulationvol 113 no 21 pp 2548ndash2555 2006

[32] A Yazdanyar C Yeang and X C Jiang ldquoRole of phospholipidtransfer protein in high-density lipoprotein-mediated reversecholesterol transportrdquo Current Atherosclerosis Reports vol 13no 3 pp 242ndash248 2011

[33] X Wang and D J Rader ldquoMolecular regulation of macrophagereverse cholesterol transportrdquo Current Opinion in Cardiologyvol 22 no 4 pp 368ndash372 2007

[34] S J Yang J M Choi L Kim et al ldquoNicotinamide improvesglucose metabolism and affects the hepatic NAD-sirtuin path-way in a rodent model of obesity and type 2 diabetesrdquo Journalof Nutritional Biochemistry vol 25 no 1 pp 66ndash72 2014

[35] L A Carlson L Oro and J Ostman ldquoEffect of a single dose ofnicotinic acid on plasma lipids in patients with hyperlipopro-teinemiardquo Acta Medica Scandinavica vol 183 no 5 pp 457ndash465 1968

[36] R H Stern J D Spence D J Freeman and A Parbtani ldquoTol-erance to nicotinic acid flushingrdquo Clinical Pharmacology andTherapeutics vol 50 no 1 pp 66ndash70 1991

[37] S M Grundy J I Cleeman C N Bairey Merz et al ldquoImpli-cations of recent clinical trials for the National CholesterolEducation Program Adult Treatment Panel III guidelinesrdquoCirculation vol 110 no 2 pp 227ndash239 2004

[38] M S Brown and J L Goldstein ldquoLowering plasma cholesterolby raising LDL receptorsrdquoNewEngland Journal ofMedicine vol305 no 9 pp 515ndash517 1981

[39] R Hampton D Dimster-Denk and J Rine ldquoThe biology ofHMG-CoA reductase the pros of contra-regulationrdquo Trends inBiochemical Sciences vol 21 no 4 pp 140ndash145 1996

Submit your manuscripts athttpwwwhindawicom

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Disease Markers

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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 7: Research Article Niacin Inhibits Vascular Inflammation via ...downloads.hindawi.com/journals/mi/2014/263786.pdf · Research Article Niacin Inhibits Vascular Inflammation via Downregulating

Mediators of Inflammation 7

0

05

1

15

2

25

0

02

04

06

08

1

12

14

Relat

ive l

evel

of I

L-6

secr

etio

n

0

02

04

06

08

1

12

14

Relat

ive p

rote

in le

vel o

f not

ch1

0

02

04

06

08

1

12

14

(a) (b)

(c)

(e)

(d)

(f)

oxLDL

lowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

++++minus

Niacin (mM) 0 0 025 05 1oxLDL ++++minus

Niacin (mM) 0 0 025 05 1

Relat

ive l

evel

of T

NF-120572

secr

etio

n

Histone H3

Notch1

Relat

ive p

rote

in le

vel o

fN

F-120581

Bp65

in n

ucle

i

NF-120581B p65

120573-Actin

oxLDL ++++minus

Niacin (mM) 0 0 025 05 1

oxLDL ++++minus

Niacin (mM) 0 0 025 05 1

oxLDL ++++minus

Niacin (mM) 0 0 025 05 1oxLDL ++++minus

Niacin (mM) 0 0 025 05 1

Figure 5 Niacin decreased the secretion of TNF-120572 and IL-6 and inhibited NF-120581B and notch1 expression in oxLDL-stimulated THP-1macrophages (a) and (b) show the relative levels of TNF-120572 and IL-6 secretion in the medium of THP-1 macrophages The concentrations ofIL-6 and TNF-120572 were determined by ELISA kit (c) and (d) show the representative images of NF-120581B p65 and notch1 protein expression inTHP-1 macrophages by western blot (e) and (f) show the IOD ratios of NF-120581B p65 and notch1 expression respectively Data are presented asmean plusmn SD

119875 lt 001 versus blank group lowast119875 lt 005 lowastlowast119875 lt 001 versus oxLDL-treated group without niacin

with that of HFD group niacin and simvastatin significantlydecreased the percentages of stained area to the total cross-sectional vessel wall by 56 and 67 respectively (Figure 6)The effect of simvastatin was superior to that of niacin

342 Niacin Increased HDL-C and ApoA I Levels andDecreased TG and Non-HDL-C Levels in Plasma of GuineaPigs Fed High Fat Diet As shown in Figure 7 after highfat diet for 8 weeks the levels of plasma TC TG HDL-Cand non-HDL-C were significantly increased in HFD groupcompared with CD group (119875 lt 001) which indicated asuccessful hyperlipidemic model in guinea pigs Comparedwith HFD group niacin decreased the levels of TG and

non-HDL-C by 27 and 12 respectively and increasedHDL level by 21 Niacin had no statistical influence onTC level in plasma Compared with HFD group simvastatindecreased the levels of TG non-HDL-C and TC by 1853 and 51 respectively Simvastatin had no significantinfluence on HDL-C level

The level of apoA I in plasma was also detected by SDS-PAGE in this study Comparedwith that ofHFDgroup niacinsignificantly promoted the level of apoA I by 42 whereassimvastatin had no significant influence on apoA I (Figure 8)

343 Niacin Significantly Upregulated the mRNA Amountof CYP7A1 in Liver Cholesterol metabolism in liver is a

8 Mediators of Inflammation

CD HFD

HFD-N HFD-S

(a)

0

05

1

15

2

CD HFD HFD-N HFD-S

Oil

red

O st

aine

d ar

ea (

)

lowastlowast

lowastlowast

(b)

Figure 6 Niacin and simvastatin significantly lessened lipid depo-sition in the arterial wall of guinea pigs fed high fat diet Lipiddeposition in the aorta wall was analyzed by oil red O staining aftertreatment for 8 weeksThe quantification of stained lipids was deter-mined by calculating the percentage of the positive area to the totalcross-sectional vessel wall area by Image-Pro Plus software Data arepresented as mean plusmn SD (119899 = 8)

119875 lt 001 versus CD grouplowastlowast

119875 lt 001 versus HFD group

complicated homeostasis involving multiple steps includingcholesterol ingression synthesis and conversion SR-B1 andLDL-R in liver play a critical role in cholesterol ingressionSR-B1 is the HDL receptor on the hepatocyte surface LDL-R can bind to LDL and VLDL and internalize them intohepatocytes [17] In the study we determined the effect ofniacin on the expressions of SR-B1 and LDL-R mRNA inliver As shown in Figures 9(a) and 9(b) after treatment withhigh fat diet for 8 weeks the LDL-R mRNA level was down-regulated (119875 lt 001) and the SR-B1 mRNA level wasnot significantly changed in HFD group Compared withHFD group niacin had no significant effect on SR-B1 and

LDL-R mRNA levels but simvastatin upregulated LDL-RmRNA level by 71

Cholesterol in liver can be converted into bile acidthrough cytochrome P450-meidiated oxidation The rate-limiting enzyme for the dominant pathway of bile acidsynthesis is cytochrome P450 7A1 (CYP7A1) As shown inFigure 9(c) compared with HFD group niacin significantlyupregulated the CYP7A1 mRNA level by 59 whereassimvastatin had no significant influence on its level HMGCRis the rate-limiting enzyme in the process of cholesterolsynthesis Compared with that of CD group the mRNA levelof HMGCR was significantly decreased in HFD group (119875 lt001) Compared with HFD group simvastatin upregulatedthe HMGCR mRNA levels by 46 whereas niacin had nosignificant influence on its level (Figure 9(d))

4 Discussion

For the first time to our knowledge this report demonstratesniacin inhibited vascular inflammation in guinea pig fedhigh fat diet and suppressed oxLDL-stimulated inflammatoryresponse even injury of endothelial cells and macrophagesin vitro The result indicates a new mechanism for niacinrsquosprotective action on cardiovascular disease in addition to itsestablished effects on lipid metabolism

The augmentation of inflammatory response has beenclearly documented in pathogenesis of vascular impairmentThe chronic inflammatory pathogenesis in the arterial wall isas follows Harmful substances in blood such as hypercholes-terolemia can induce endothelial dysfunction This causesthe production of ROS and the secretion of cellular adhesionmolecules (CAMs) cytokines and chemokines which facili-tate adherence and endothelial transmigration of leukocytes(monocytes and T helper lymphocytes) Monocytes in thearterial wall will be activated by proinflammatory cytokinesand differentiated into macrophages Activated macrophagesincrease the expression of CAMs and cytokines which resultsin recruitment of more leukocytes into the arterial wall acti-vates the complement pathways of immune system and theacute phase response stimulates proliferation and migrationof smooth muscle cells (SMCs) and promotes fibrous tissuedeposition [18] In progress the signaling molecule NF-120581Bis a proinflammatory major switch that can upregulate theexpression of lots of cytokines [19] Activated NF-120581B canresult in the enhanced efflux of TNF-120572 and IL-6 in serum[20] Early events in AS are usually driven by NF-120581B and thedisruption of NF-120581B signaling pathway has been shown toslow down the vascular impairment [21]

In the present study we demonstrate that niacin attenu-ated vascular inflammation induced by high fat diet in vivoThe involved evidences are as follows (1) Niacin lowered thenumber of macrophages (CD68 positive cells) in the arterialwall and significantly downregulated the inflammatory fac-tors (IL-6 and TNF-120572) levels in plasma of guinea pigs fed highfat diet (2) Both immunohistochemistry and western blotanalysis indicated niacin suppressed the expression of activeNF-120581B p65 in nuclei of the arterial wall The activated NF-120581Bis reported to form a heterodimer which usually consists oftwo proteins p65 and p50 subunitsThe p65 subunit has been

Mediators of Inflammation 9

0

50

100

150

200

250

CD HFD HFD-N HFD-S

TG (m

gdL

)

lowastlowastlowastlowast

(a)

0

500

1000

1500

2000

CD HFD HFD-N HFD-S

TC (m

gdL

)

lowastlowast

(b)

0

20

40

60

80

100

120

CD HFD HFD-N HFD-S

HD

L-C

(mg

dL)

lowast

(c)

0

500

1000

1500

2000

CD HFD HFD-N HFD-S

Non

-HD

L-C

(mg

dL)

lowastlowast

lowast

(d)

Figure 7 Effect of niacin and simvastatin on plasma lipid of guinea pigs fed high fat diet The levels of TG (a) TC (b) HDL-C (c) andnon-HDL-C (d) in plasma of guinea pigs were determined by enzyme method after 8 weeksrsquo treatment Data are presented as mean plusmn SD(119899 = 8)

119875 lt 001 versus CD group lowast119875 lt 005 lowastlowast119875 lt 001 versus HFD group

apoAI20

30

40

50

6080

160110

Mar

ker

CD HFD

HFD

-N

HFD

-S

Hum

an

(a)

0

05

1

15

2

CD HFD HFD-N HFD-S

Fold

indu

ctio

n of

apoA

I in

HD

L

lowastlowast

(b)

Figure 8 Niacin upregulated apoA I level in plasma of guinea pigs fed high fat diet HDL (density = 109ndash124 gmL) was separatedfrom plasma of guinea pigs by sequential ultracentrifugation The SDS-PAGE was performed on 15 SDS polyacrylamide gel and theapolipoproteins were stained with coomassie brilliant blue Densitometric quantitation of SDS-PAGE image was analyzed by Image-Pro Plussoftware (a) shows the representative SDS-PAGE image ofHDL (b) shows the relative level of apoA I in plasma by densitometric quantitationData are presented as mean plusmn SD of at least three independent experiments

119875 lt 005 versus CD group lowastlowast119875 lt 001 versus HFD group

10 Mediators of Inflammation

0

02

04

06

08

1

12

14

16

CD HFD HFD-N HFD-S

Relat

ive m

RNA

leve

l of S

R-B1

(a)

0

02

04

06

08

1

12

CD HFD HFD-N HFD-S

Relat

ive m

RNA

leve

l of L

DL-

R

lowastlowast

(b)

0

05

1

15

2

CD HFD HFD-N HFD-S

Relat

ive m

RNA

leve

l of C

YP7A

1 lowastlowast

(c)

0

02

04

06

08

1

12

14

CD HFD HFD-N HFD-S

Relat

ive m

RNA

leve

l of H

MG

CR

lowastlowast

(d)

Figure 9 Effect of niacin and simvastatin on the mRNA abundance of SR-B1 LDL-R CYP7A1 and HMGCR in liver of guinea pigs fedhigh fat dietThe mRNA levels which were analyzed by quantitative real-time PCR were calculated after being adjusted for 120573-actin usingthe 2minusΔΔCt method Data are presented as mean plusmn SD of at least three independent experiments

119875 lt 005 119875 lt 001 versus CD group

lowastlowast

119875 lt 001 versus HFD group

demonstrated to exert critical activity on the transcriptionof many inflammatory genes including adhesion moleculescytokines and chemokines [22] (3) CRP an early acute phasereactant is closely relevant to inflammation Baseline level ofCRP is a strong independent predictor of the risk of futuremyocardial infarction peripheral vascular disease strokeand vascular death amonghealthy individualswithout knownvascular disease [23] Kuvin et al have shown that niacindecreased CRP level by 15 in patients with stable coronaryartery disease [5] In patients with metabolic syndromeafter treatment with extended-release niacin (1 gday) for 52weeks their endothelial function was improved by 22 andthere was a decrease in CRP level by 20 [24] Our resultsalso showed niacin slightly lowered CRP level but hadno statistical difference (Table 1) (4) Oxidative stress wassuppressed by niacin Oxidative stress is closely related to theinflammation in the arterial wall Increased ROS productioncan initiate a cascade of signal transduction which results inendothelial dysfunction changes in vascular tone vascularremodeling and vascular inflammatory responses [25]

To further confirm the direct anti-inflammatory prop-erty of niacin its effect on oxLDL-induced inflammatoryresponse of endothelial cells and macrophages was studiedoxLDL is pivotal in the development of AS and representsa crucial proinflammatory stimulus [26] Upon enteringinto the intima of arteries oxLDL activates endothelial

cells and upregulates adhesion molecule expression andinflammatory factors secretion all of which contribute tothe recruitment of circulating leukocytes Monocytes andormacrophages infiltrating the arterial wall take up oxLDL andform ldquofoam cellsrdquo which in turn promote further secretionof inflammatory mediators [27] Our data indicated niacinremarkably downregulated the secretion of TNF-120572 and IL-6stimulated by oxLDL in HUVECs and THP-1 macrophagesNotch1 signal pathway is an evolutionarily highly conservedmechanism for communication which can improve NF-120581Bactivity and have a positive correlation with the developmentof inflammation [28] A previous study demonstrates thatnotch knockout in macrophage markedly downregulates theexpression of proinflammatory factors Instead when thenotch signal pathway is activatedNF-120581B expression increasesand inflammatory factors secretion is also promoted [29]Our findings indicated niacin decreased the expressions ofNF-120581B and notch1 in oxLDL-induced inflammation Vas-cular endothelial apoptosis plays an important role in theinitiation of AS It may compromise vessel wall permeabilityto cytokines growth factors lipids and immune cells andincrease coagulation [30] In this study we indicated niacinattenuated oxLDL-induced apoptosis of HUVECs

Taken together these observations lend initial but com-pelling evidence that niacin has protective effects on car-diovascular disease through its vascular anti-inflammatory

Mediators of Inflammation 11

properties in addition to its established effects on lipidmetabolism

Reverse cholesterol transport (RCT) is a potent defensemechanism against AS It involves transporting cholesterolfromperipheral tissues and cells to liver transforming choles-terol into bile acid and finally eliminating it from the body[31] HDL and apoA play a key role in RCT They can drivecholesterol efflux from macrophage foam cells that residein vessel intima [32 33] Similar to previous studies ourfindings also indicated that niacin increased the levels ofHDL and apoA I in plasma significantly In the process ofRCT CYP7A1 is an important regulatory factor which canconvert cholesterol to bile acid in liver and be further excretedinto intestine for elimination from body with feces Here weshowed that the mRNA abundance of CYP7A1 in liver wassignificantly upregulated by niacin treatment These findingssuggest that niacin may modulate plasma lipid and exertantiatherosclerotic effects by promoting RCT progress

In clinical practice the oral dose of niacin for lipid-lowering purpose is usually 1ndash3 g per day equivalently 14ndash43mgkg per day According to the formula of Meeh-Rubnerequation we calculate the dose for guinea pig to be about80ndash250mgkg per day In addition a recent report showedthat up to 400mgkg per day of niacin treatment for 7 daysand 100mgkg per day of niacin treatment for 4 weeks aresafe and do not cause toxicity in rodent models [34] In thisstudy the applied dose of niacin for guinea pig is 100mgkgdwhich has pharmacologic effects and does not have varioustoxicities based on the changes in food intake body weightand behaviors In human plasma level of niacin was foundto be about 03mM after oral ingestion of 1 g niacin [3536] Since the reported plasma concentrations are one timemeasurement it is unclear whether these levels reflect troughor peak values Furthermore there is no reported clinicaldata available regarding plasma concentration of niacin afteringestion of 3 g niacin Extrapolation of the available datausing oral dose of 1 g niacin would indicate that the 3 g niacinoral administration may lead to the plasma concentrations ofniacin in the range of 08ndash1mM In our in-vitro studies weused 025 to 1mM niacin

Statins are the first-line therapy in the treatment ofcholesterol-induced atherosclerotic cardiovascular diseases[37] They suppress the conversion of HMG-CoA to meval-onate by competitively blocking HMGCR therefore theendogenous cholesterol synthesis in the liver is remarkablyreduced Consequently it results in the increase of LDL-Rexpression on hepatic cell surfaces and the decrease of LDL-Cin plasma [38]Meanwhile inhibition of cholesterol synthesiscauses an overexpression of HMGCR due to a feedback-regulatory mechanism [39] Similar to previous studies ourfindings also revealed that simvastatin significantly decreasedthe level of non-HDL-C in plasma followed by upregulatingLDL-R expression in liver and the HMGCR mRNA level inliver was increased in a compensatory way

5 Conclusion

In summary our data presented herein support the novelconcept that niacin has vascular anti-inflammatory and

potentially vascular-protective property which is indepen-dent of its effect on lipid regulation The anti-inflammatoryproperty of niacin is realized by downregulating nucleartranscription factor-120581B signaling pathway Meanwhile ourfinding also suggests that niacin modulates plasma lipid bystimulating the expression of factors involved in the processof RCT and promoting RCT

Conflict of Interests

The authors declare that they have no conflict of interest

Authorsrsquo Contribution

Yanhong Si and Ying Zhang have equally contributed to thisstudy

Acknowledgments

This research was supported by the financial supportsfrom the Taishan scholar foundation of Shandong Province(200867) and the Natural Science Foundations of China(81170785 and 81371234)

References

[1] R Altschul A Hoffer and J D Stephen ldquoInfluence of nicotinicacid on serum cholesterol in manrdquoArchives of Biochemistry andBiophysics vol 54 no 2 pp 558ndash559 1955

[2] C D Meyers V S Kamanna and M L Kashyap ldquoNiacin ther-apy in atherosclerosisrdquo Current Opinion in Lipidology vol 15no 6 pp 659ndash665 2004

[3] L A Carlson ldquoNicotinic acid the broad-spectrum lipid drug A50th anniversary reviewrdquo Journal of Internal Medicine vol 258no 2 pp 94ndash114 2005

[4] S H Ganji S Qin L Zhang V S Kamanna andM L KashyapldquoNiacin inhibits vascular oxidative stress redox-sensitive genesand monocyte adhesion to human aortic endothelial cellsrdquoAtherosclerosis vol 202 no 1 pp 68ndash75 2009

[5] J T Kuvin D M Dave K A Sliney et al ldquoEffects of extended-release niacin on lipoprotein particle size distribution andinflammatorymarkers in patients with coronary artery diseaserdquoAmerican Journal of Cardiology vol 98 no 6 pp 743ndash745 2006

[6] R Yang P Guo X Song F Liu and N Gao ldquoHyperlipidemicguinea pig model mechanisms of triglyceride metabolismdisorder and comparison to ratrdquo Biological and PharmaceuticalBulletin vol 34 no 7 pp 1046ndash1051 2011

[7] X Li Y Liu H Zhang L Ren Q Li and N Li ldquoAnimal modelsfor the atherosclerosis research a reviewrdquo Protein and Cell vol2 no 3 pp 189ndash201 2011

[8] J O Leite U Vaishnav M Puglisi H Fraser J Trias and M LFernandez ldquoA-002 (Varespladib) a phospholipase A2 inhibitorreduces atherosclerosis in guinea pigsrdquo BMC CardiovascularDisorders vol 9 article 7 2009

[9] J Auwerx ldquoThe human leukemia cell line THP-1 a multi-facetted model for the study of monocyte-macrophage differ-entiationrdquo Experientia vol 47 no 1 pp 22ndash31 1991

[10] M J Chapman G L Mills and J H Ledford ldquoThe distributionand partial characterization of the serum apolipoproteins in the

12 Mediators of Inflammation

guinea pigrdquo Biochemical Journal vol 149 no 2 pp 423ndash4361975

[11] X C Jiang C Bruce J Mar et al ldquoTargetedmutation of plasmaphospholipid transfer protein gene markedly reduces high-density lipoprotein levelsrdquo Journal of Clinical Investigation vol103 no 6 pp 907ndash914 1999

[12] T Collins and M I Cybulsky ldquoNF-120581B pivotal mediator orinnocent bystander in atherogenesisrdquo Journal of Clinical Inves-tigation vol 107 no 3 pp 255ndash264 2001

[13] Y Tsubosaka T Murata K Yamada D Uemura M Horiand H Ozaki ldquoHalichlorine reduces monocyte adhesion toendothelium through the suppression of nuclear factor-120581Bactivationrdquo Journal of Pharmacological Sciences vol 113 no 3pp 208ndash213 2010

[14] H Tsutsui ldquoMitochondrial oxidative stress and heart failurerdquoInternal Medicine vol 45 no 13 pp 809ndash813 2006

[15] M Seddon Y H Looi and A M Shah ldquoOxidative stressand redox signalling in cardiac hypertrophy and heart failurerdquoHeart vol 93 no 8 pp 903ndash907 2007

[16] WWongchana and T Palaga ldquoDirect regulation of interleukin-6 expression by Notch signaling in macrophagesrdquo Cellular andMolecular Immunology vol 9 no 2 pp 155ndash162 2012

[17] J Chen H Zhao X Ma et al ldquoThe effects of jiang-zhi-ningand itsmain components on cholesterol metabolismrdquo Evidence-Based Complementary and Alternative Medicine vol 2012Article ID 928234 10 pages 2012

[18] G Francisco C Hernandez and R Simo ldquoSerum markers ofvascular inflammation in dyslipemiardquo Clinica Chimica Actavol 369 no 2 pp 1ndash16 2006

[19] J Andersson P Libby and G K Hansson ldquoAdaptive immunityand atherosclerosisrdquoClinical Immunology vol 134 no 1 pp 33ndash46 2010

[20] W J Oh U Jung H S Eom H J Shin and H R Park ldquoInhibi-tion of lipopolysaccharide-induced proinflammatory responsesby Buddleja officinalis extract in BV-2 microglial cells vianegative regulation of NF-kB and ERK12 signalingrdquoMoleculesvol 18 no 8 pp 9195ndash9206 2013

[21] T M Wang C J Chen T S Lee et al ldquoDocosahexaenoic acidattenuates VCAM-1 expression and NF-120581B activation in TNF-120572-treated human aortic endothelial cellsrdquo Journal of NutritionalBiochemistry vol 22 no 2 pp 187ndash194 2011

[22] M S Islam H Yoshida N Matsuki et al ldquoAntioxidant freeradical-scavenging and NF-120581B-inhibitory activities of phy-tosteryl ferulates structure-activity studiesrdquo Journal of Pharma-cological Sciences vol 111 no 4 pp 328ndash337 2009

[23] P M Ridker M J Stampfer and N Rifai ldquoNovel risk factorsfor systemic atherosclerosis a comparison ofC-reactive proteinfibrinogen homocysteine lipoprotein(a) and standard choles-terol screening as predictors of peripheral arterial diseaserdquoJournal of the AmericanMedical Association vol 285 no 19 pp2481ndash2485 2001

[24] M Thoenes A Oguchi S Nagamia et al ldquoThe effects ofextended-release niacin on carotid intimal media thicknessendothelial function and inflammatory markers in patientswith the metabolic syndromerdquo International Journal of ClinicalPractice vol 61 no 11 pp 1942ndash1948 2007

[25] N R Madamanchi A Vendrov and M S Runge ldquoOxidativestress and vascular diseaserdquo Arteriosclerosis Thrombosis andVascular Biology vol 25 no 1 pp 29ndash38 2005

[26] E Matsuura F Atzeni P Sarzi-Puttini M Turiel L R Lopezand M T Nurmohamed ldquoIs atherosclerosis an autoimmunediseaserdquo BMCMedicine vol 12 no 1 article 47 2014

[27] A Pirillo G D Norata and A L Catapano ldquoLOX-1 OxLDLand atherosclerosisrdquo Mediators of Inflammation vol 2013Article ID 152786 12 pages 2013

[28] C L Wang M Meng S B Liu L R Wang L H Hou and XH Cao ldquochemically sulfated polysaccharide from Grifola fron-dos induces HepG2 cell apoptosis by notch1-NF-120581B pathwayrdquoCarbohydrate Polymers vol 95 no 1 pp 282ndash287 2013

[29] WWongchana and T Palaga ldquoDirect regulation of interleukin-6 expression by Notch signaling in macrophagesrdquo Cellular andMolecular Immunology vol 9 no 2 pp 155ndash162 2012

[30] J C Choy D J Granville D W C Hunt and B M McManusldquoEndothelial cell apoptosis biochemical characteristics andpotential implications for atherosclerosisrdquo Journal of Molecularand Cellular Cardiology vol 33 no 9 pp 1673ndash1690 2001

[31] M Cuchel and D J Rader ldquoMacrophage reverse cholesteroltransport key to the regression of atherosclerosisrdquo Circulationvol 113 no 21 pp 2548ndash2555 2006

[32] A Yazdanyar C Yeang and X C Jiang ldquoRole of phospholipidtransfer protein in high-density lipoprotein-mediated reversecholesterol transportrdquo Current Atherosclerosis Reports vol 13no 3 pp 242ndash248 2011

[33] X Wang and D J Rader ldquoMolecular regulation of macrophagereverse cholesterol transportrdquo Current Opinion in Cardiologyvol 22 no 4 pp 368ndash372 2007

[34] S J Yang J M Choi L Kim et al ldquoNicotinamide improvesglucose metabolism and affects the hepatic NAD-sirtuin path-way in a rodent model of obesity and type 2 diabetesrdquo Journalof Nutritional Biochemistry vol 25 no 1 pp 66ndash72 2014

[35] L A Carlson L Oro and J Ostman ldquoEffect of a single dose ofnicotinic acid on plasma lipids in patients with hyperlipopro-teinemiardquo Acta Medica Scandinavica vol 183 no 5 pp 457ndash465 1968

[36] R H Stern J D Spence D J Freeman and A Parbtani ldquoTol-erance to nicotinic acid flushingrdquo Clinical Pharmacology andTherapeutics vol 50 no 1 pp 66ndash70 1991

[37] S M Grundy J I Cleeman C N Bairey Merz et al ldquoImpli-cations of recent clinical trials for the National CholesterolEducation Program Adult Treatment Panel III guidelinesrdquoCirculation vol 110 no 2 pp 227ndash239 2004

[38] M S Brown and J L Goldstein ldquoLowering plasma cholesterolby raising LDL receptorsrdquoNewEngland Journal ofMedicine vol305 no 9 pp 515ndash517 1981

[39] R Hampton D Dimster-Denk and J Rine ldquoThe biology ofHMG-CoA reductase the pros of contra-regulationrdquo Trends inBiochemical Sciences vol 21 no 4 pp 140ndash145 1996

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 8: Research Article Niacin Inhibits Vascular Inflammation via ...downloads.hindawi.com/journals/mi/2014/263786.pdf · Research Article Niacin Inhibits Vascular Inflammation via Downregulating

8 Mediators of Inflammation

CD HFD

HFD-N HFD-S

(a)

0

05

1

15

2

CD HFD HFD-N HFD-S

Oil

red

O st

aine

d ar

ea (

)

lowastlowast

lowastlowast

(b)

Figure 6 Niacin and simvastatin significantly lessened lipid depo-sition in the arterial wall of guinea pigs fed high fat diet Lipiddeposition in the aorta wall was analyzed by oil red O staining aftertreatment for 8 weeksThe quantification of stained lipids was deter-mined by calculating the percentage of the positive area to the totalcross-sectional vessel wall area by Image-Pro Plus software Data arepresented as mean plusmn SD (119899 = 8)

119875 lt 001 versus CD grouplowastlowast

119875 lt 001 versus HFD group

complicated homeostasis involving multiple steps includingcholesterol ingression synthesis and conversion SR-B1 andLDL-R in liver play a critical role in cholesterol ingressionSR-B1 is the HDL receptor on the hepatocyte surface LDL-R can bind to LDL and VLDL and internalize them intohepatocytes [17] In the study we determined the effect ofniacin on the expressions of SR-B1 and LDL-R mRNA inliver As shown in Figures 9(a) and 9(b) after treatment withhigh fat diet for 8 weeks the LDL-R mRNA level was down-regulated (119875 lt 001) and the SR-B1 mRNA level wasnot significantly changed in HFD group Compared withHFD group niacin had no significant effect on SR-B1 and

LDL-R mRNA levels but simvastatin upregulated LDL-RmRNA level by 71

Cholesterol in liver can be converted into bile acidthrough cytochrome P450-meidiated oxidation The rate-limiting enzyme for the dominant pathway of bile acidsynthesis is cytochrome P450 7A1 (CYP7A1) As shown inFigure 9(c) compared with HFD group niacin significantlyupregulated the CYP7A1 mRNA level by 59 whereassimvastatin had no significant influence on its level HMGCRis the rate-limiting enzyme in the process of cholesterolsynthesis Compared with that of CD group the mRNA levelof HMGCR was significantly decreased in HFD group (119875 lt001) Compared with HFD group simvastatin upregulatedthe HMGCR mRNA levels by 46 whereas niacin had nosignificant influence on its level (Figure 9(d))

4 Discussion

For the first time to our knowledge this report demonstratesniacin inhibited vascular inflammation in guinea pig fedhigh fat diet and suppressed oxLDL-stimulated inflammatoryresponse even injury of endothelial cells and macrophagesin vitro The result indicates a new mechanism for niacinrsquosprotective action on cardiovascular disease in addition to itsestablished effects on lipid metabolism

The augmentation of inflammatory response has beenclearly documented in pathogenesis of vascular impairmentThe chronic inflammatory pathogenesis in the arterial wall isas follows Harmful substances in blood such as hypercholes-terolemia can induce endothelial dysfunction This causesthe production of ROS and the secretion of cellular adhesionmolecules (CAMs) cytokines and chemokines which facili-tate adherence and endothelial transmigration of leukocytes(monocytes and T helper lymphocytes) Monocytes in thearterial wall will be activated by proinflammatory cytokinesand differentiated into macrophages Activated macrophagesincrease the expression of CAMs and cytokines which resultsin recruitment of more leukocytes into the arterial wall acti-vates the complement pathways of immune system and theacute phase response stimulates proliferation and migrationof smooth muscle cells (SMCs) and promotes fibrous tissuedeposition [18] In progress the signaling molecule NF-120581Bis a proinflammatory major switch that can upregulate theexpression of lots of cytokines [19] Activated NF-120581B canresult in the enhanced efflux of TNF-120572 and IL-6 in serum[20] Early events in AS are usually driven by NF-120581B and thedisruption of NF-120581B signaling pathway has been shown toslow down the vascular impairment [21]

In the present study we demonstrate that niacin attenu-ated vascular inflammation induced by high fat diet in vivoThe involved evidences are as follows (1) Niacin lowered thenumber of macrophages (CD68 positive cells) in the arterialwall and significantly downregulated the inflammatory fac-tors (IL-6 and TNF-120572) levels in plasma of guinea pigs fed highfat diet (2) Both immunohistochemistry and western blotanalysis indicated niacin suppressed the expression of activeNF-120581B p65 in nuclei of the arterial wall The activated NF-120581Bis reported to form a heterodimer which usually consists oftwo proteins p65 and p50 subunitsThe p65 subunit has been

Mediators of Inflammation 9

0

50

100

150

200

250

CD HFD HFD-N HFD-S

TG (m

gdL

)

lowastlowastlowastlowast

(a)

0

500

1000

1500

2000

CD HFD HFD-N HFD-S

TC (m

gdL

)

lowastlowast

(b)

0

20

40

60

80

100

120

CD HFD HFD-N HFD-S

HD

L-C

(mg

dL)

lowast

(c)

0

500

1000

1500

2000

CD HFD HFD-N HFD-S

Non

-HD

L-C

(mg

dL)

lowastlowast

lowast

(d)

Figure 7 Effect of niacin and simvastatin on plasma lipid of guinea pigs fed high fat diet The levels of TG (a) TC (b) HDL-C (c) andnon-HDL-C (d) in plasma of guinea pigs were determined by enzyme method after 8 weeksrsquo treatment Data are presented as mean plusmn SD(119899 = 8)

119875 lt 001 versus CD group lowast119875 lt 005 lowastlowast119875 lt 001 versus HFD group

apoAI20

30

40

50

6080

160110

Mar

ker

CD HFD

HFD

-N

HFD

-S

Hum

an

(a)

0

05

1

15

2

CD HFD HFD-N HFD-S

Fold

indu

ctio

n of

apoA

I in

HD

L

lowastlowast

(b)

Figure 8 Niacin upregulated apoA I level in plasma of guinea pigs fed high fat diet HDL (density = 109ndash124 gmL) was separatedfrom plasma of guinea pigs by sequential ultracentrifugation The SDS-PAGE was performed on 15 SDS polyacrylamide gel and theapolipoproteins were stained with coomassie brilliant blue Densitometric quantitation of SDS-PAGE image was analyzed by Image-Pro Plussoftware (a) shows the representative SDS-PAGE image ofHDL (b) shows the relative level of apoA I in plasma by densitometric quantitationData are presented as mean plusmn SD of at least three independent experiments

119875 lt 005 versus CD group lowastlowast119875 lt 001 versus HFD group

10 Mediators of Inflammation

0

02

04

06

08

1

12

14

16

CD HFD HFD-N HFD-S

Relat

ive m

RNA

leve

l of S

R-B1

(a)

0

02

04

06

08

1

12

CD HFD HFD-N HFD-S

Relat

ive m

RNA

leve

l of L

DL-

R

lowastlowast

(b)

0

05

1

15

2

CD HFD HFD-N HFD-S

Relat

ive m

RNA

leve

l of C

YP7A

1 lowastlowast

(c)

0

02

04

06

08

1

12

14

CD HFD HFD-N HFD-S

Relat

ive m

RNA

leve

l of H

MG

CR

lowastlowast

(d)

Figure 9 Effect of niacin and simvastatin on the mRNA abundance of SR-B1 LDL-R CYP7A1 and HMGCR in liver of guinea pigs fedhigh fat dietThe mRNA levels which were analyzed by quantitative real-time PCR were calculated after being adjusted for 120573-actin usingthe 2minusΔΔCt method Data are presented as mean plusmn SD of at least three independent experiments

119875 lt 005 119875 lt 001 versus CD group

lowastlowast

119875 lt 001 versus HFD group

demonstrated to exert critical activity on the transcriptionof many inflammatory genes including adhesion moleculescytokines and chemokines [22] (3) CRP an early acute phasereactant is closely relevant to inflammation Baseline level ofCRP is a strong independent predictor of the risk of futuremyocardial infarction peripheral vascular disease strokeand vascular death amonghealthy individualswithout knownvascular disease [23] Kuvin et al have shown that niacindecreased CRP level by 15 in patients with stable coronaryartery disease [5] In patients with metabolic syndromeafter treatment with extended-release niacin (1 gday) for 52weeks their endothelial function was improved by 22 andthere was a decrease in CRP level by 20 [24] Our resultsalso showed niacin slightly lowered CRP level but hadno statistical difference (Table 1) (4) Oxidative stress wassuppressed by niacin Oxidative stress is closely related to theinflammation in the arterial wall Increased ROS productioncan initiate a cascade of signal transduction which results inendothelial dysfunction changes in vascular tone vascularremodeling and vascular inflammatory responses [25]

To further confirm the direct anti-inflammatory prop-erty of niacin its effect on oxLDL-induced inflammatoryresponse of endothelial cells and macrophages was studiedoxLDL is pivotal in the development of AS and representsa crucial proinflammatory stimulus [26] Upon enteringinto the intima of arteries oxLDL activates endothelial

cells and upregulates adhesion molecule expression andinflammatory factors secretion all of which contribute tothe recruitment of circulating leukocytes Monocytes andormacrophages infiltrating the arterial wall take up oxLDL andform ldquofoam cellsrdquo which in turn promote further secretionof inflammatory mediators [27] Our data indicated niacinremarkably downregulated the secretion of TNF-120572 and IL-6stimulated by oxLDL in HUVECs and THP-1 macrophagesNotch1 signal pathway is an evolutionarily highly conservedmechanism for communication which can improve NF-120581Bactivity and have a positive correlation with the developmentof inflammation [28] A previous study demonstrates thatnotch knockout in macrophage markedly downregulates theexpression of proinflammatory factors Instead when thenotch signal pathway is activatedNF-120581B expression increasesand inflammatory factors secretion is also promoted [29]Our findings indicated niacin decreased the expressions ofNF-120581B and notch1 in oxLDL-induced inflammation Vas-cular endothelial apoptosis plays an important role in theinitiation of AS It may compromise vessel wall permeabilityto cytokines growth factors lipids and immune cells andincrease coagulation [30] In this study we indicated niacinattenuated oxLDL-induced apoptosis of HUVECs

Taken together these observations lend initial but com-pelling evidence that niacin has protective effects on car-diovascular disease through its vascular anti-inflammatory

Mediators of Inflammation 11

properties in addition to its established effects on lipidmetabolism

Reverse cholesterol transport (RCT) is a potent defensemechanism against AS It involves transporting cholesterolfromperipheral tissues and cells to liver transforming choles-terol into bile acid and finally eliminating it from the body[31] HDL and apoA play a key role in RCT They can drivecholesterol efflux from macrophage foam cells that residein vessel intima [32 33] Similar to previous studies ourfindings also indicated that niacin increased the levels ofHDL and apoA I in plasma significantly In the process ofRCT CYP7A1 is an important regulatory factor which canconvert cholesterol to bile acid in liver and be further excretedinto intestine for elimination from body with feces Here weshowed that the mRNA abundance of CYP7A1 in liver wassignificantly upregulated by niacin treatment These findingssuggest that niacin may modulate plasma lipid and exertantiatherosclerotic effects by promoting RCT progress

In clinical practice the oral dose of niacin for lipid-lowering purpose is usually 1ndash3 g per day equivalently 14ndash43mgkg per day According to the formula of Meeh-Rubnerequation we calculate the dose for guinea pig to be about80ndash250mgkg per day In addition a recent report showedthat up to 400mgkg per day of niacin treatment for 7 daysand 100mgkg per day of niacin treatment for 4 weeks aresafe and do not cause toxicity in rodent models [34] In thisstudy the applied dose of niacin for guinea pig is 100mgkgdwhich has pharmacologic effects and does not have varioustoxicities based on the changes in food intake body weightand behaviors In human plasma level of niacin was foundto be about 03mM after oral ingestion of 1 g niacin [3536] Since the reported plasma concentrations are one timemeasurement it is unclear whether these levels reflect troughor peak values Furthermore there is no reported clinicaldata available regarding plasma concentration of niacin afteringestion of 3 g niacin Extrapolation of the available datausing oral dose of 1 g niacin would indicate that the 3 g niacinoral administration may lead to the plasma concentrations ofniacin in the range of 08ndash1mM In our in-vitro studies weused 025 to 1mM niacin

Statins are the first-line therapy in the treatment ofcholesterol-induced atherosclerotic cardiovascular diseases[37] They suppress the conversion of HMG-CoA to meval-onate by competitively blocking HMGCR therefore theendogenous cholesterol synthesis in the liver is remarkablyreduced Consequently it results in the increase of LDL-Rexpression on hepatic cell surfaces and the decrease of LDL-Cin plasma [38]Meanwhile inhibition of cholesterol synthesiscauses an overexpression of HMGCR due to a feedback-regulatory mechanism [39] Similar to previous studies ourfindings also revealed that simvastatin significantly decreasedthe level of non-HDL-C in plasma followed by upregulatingLDL-R expression in liver and the HMGCR mRNA level inliver was increased in a compensatory way

5 Conclusion

In summary our data presented herein support the novelconcept that niacin has vascular anti-inflammatory and

potentially vascular-protective property which is indepen-dent of its effect on lipid regulation The anti-inflammatoryproperty of niacin is realized by downregulating nucleartranscription factor-120581B signaling pathway Meanwhile ourfinding also suggests that niacin modulates plasma lipid bystimulating the expression of factors involved in the processof RCT and promoting RCT

Conflict of Interests

The authors declare that they have no conflict of interest

Authorsrsquo Contribution

Yanhong Si and Ying Zhang have equally contributed to thisstudy

Acknowledgments

This research was supported by the financial supportsfrom the Taishan scholar foundation of Shandong Province(200867) and the Natural Science Foundations of China(81170785 and 81371234)

References

[1] R Altschul A Hoffer and J D Stephen ldquoInfluence of nicotinicacid on serum cholesterol in manrdquoArchives of Biochemistry andBiophysics vol 54 no 2 pp 558ndash559 1955

[2] C D Meyers V S Kamanna and M L Kashyap ldquoNiacin ther-apy in atherosclerosisrdquo Current Opinion in Lipidology vol 15no 6 pp 659ndash665 2004

[3] L A Carlson ldquoNicotinic acid the broad-spectrum lipid drug A50th anniversary reviewrdquo Journal of Internal Medicine vol 258no 2 pp 94ndash114 2005

[4] S H Ganji S Qin L Zhang V S Kamanna andM L KashyapldquoNiacin inhibits vascular oxidative stress redox-sensitive genesand monocyte adhesion to human aortic endothelial cellsrdquoAtherosclerosis vol 202 no 1 pp 68ndash75 2009

[5] J T Kuvin D M Dave K A Sliney et al ldquoEffects of extended-release niacin on lipoprotein particle size distribution andinflammatorymarkers in patients with coronary artery diseaserdquoAmerican Journal of Cardiology vol 98 no 6 pp 743ndash745 2006

[6] R Yang P Guo X Song F Liu and N Gao ldquoHyperlipidemicguinea pig model mechanisms of triglyceride metabolismdisorder and comparison to ratrdquo Biological and PharmaceuticalBulletin vol 34 no 7 pp 1046ndash1051 2011

[7] X Li Y Liu H Zhang L Ren Q Li and N Li ldquoAnimal modelsfor the atherosclerosis research a reviewrdquo Protein and Cell vol2 no 3 pp 189ndash201 2011

[8] J O Leite U Vaishnav M Puglisi H Fraser J Trias and M LFernandez ldquoA-002 (Varespladib) a phospholipase A2 inhibitorreduces atherosclerosis in guinea pigsrdquo BMC CardiovascularDisorders vol 9 article 7 2009

[9] J Auwerx ldquoThe human leukemia cell line THP-1 a multi-facetted model for the study of monocyte-macrophage differ-entiationrdquo Experientia vol 47 no 1 pp 22ndash31 1991

[10] M J Chapman G L Mills and J H Ledford ldquoThe distributionand partial characterization of the serum apolipoproteins in the

12 Mediators of Inflammation

guinea pigrdquo Biochemical Journal vol 149 no 2 pp 423ndash4361975

[11] X C Jiang C Bruce J Mar et al ldquoTargetedmutation of plasmaphospholipid transfer protein gene markedly reduces high-density lipoprotein levelsrdquo Journal of Clinical Investigation vol103 no 6 pp 907ndash914 1999

[12] T Collins and M I Cybulsky ldquoNF-120581B pivotal mediator orinnocent bystander in atherogenesisrdquo Journal of Clinical Inves-tigation vol 107 no 3 pp 255ndash264 2001

[13] Y Tsubosaka T Murata K Yamada D Uemura M Horiand H Ozaki ldquoHalichlorine reduces monocyte adhesion toendothelium through the suppression of nuclear factor-120581Bactivationrdquo Journal of Pharmacological Sciences vol 113 no 3pp 208ndash213 2010

[14] H Tsutsui ldquoMitochondrial oxidative stress and heart failurerdquoInternal Medicine vol 45 no 13 pp 809ndash813 2006

[15] M Seddon Y H Looi and A M Shah ldquoOxidative stressand redox signalling in cardiac hypertrophy and heart failurerdquoHeart vol 93 no 8 pp 903ndash907 2007

[16] WWongchana and T Palaga ldquoDirect regulation of interleukin-6 expression by Notch signaling in macrophagesrdquo Cellular andMolecular Immunology vol 9 no 2 pp 155ndash162 2012

[17] J Chen H Zhao X Ma et al ldquoThe effects of jiang-zhi-ningand itsmain components on cholesterol metabolismrdquo Evidence-Based Complementary and Alternative Medicine vol 2012Article ID 928234 10 pages 2012

[18] G Francisco C Hernandez and R Simo ldquoSerum markers ofvascular inflammation in dyslipemiardquo Clinica Chimica Actavol 369 no 2 pp 1ndash16 2006

[19] J Andersson P Libby and G K Hansson ldquoAdaptive immunityand atherosclerosisrdquoClinical Immunology vol 134 no 1 pp 33ndash46 2010

[20] W J Oh U Jung H S Eom H J Shin and H R Park ldquoInhibi-tion of lipopolysaccharide-induced proinflammatory responsesby Buddleja officinalis extract in BV-2 microglial cells vianegative regulation of NF-kB and ERK12 signalingrdquoMoleculesvol 18 no 8 pp 9195ndash9206 2013

[21] T M Wang C J Chen T S Lee et al ldquoDocosahexaenoic acidattenuates VCAM-1 expression and NF-120581B activation in TNF-120572-treated human aortic endothelial cellsrdquo Journal of NutritionalBiochemistry vol 22 no 2 pp 187ndash194 2011

[22] M S Islam H Yoshida N Matsuki et al ldquoAntioxidant freeradical-scavenging and NF-120581B-inhibitory activities of phy-tosteryl ferulates structure-activity studiesrdquo Journal of Pharma-cological Sciences vol 111 no 4 pp 328ndash337 2009

[23] P M Ridker M J Stampfer and N Rifai ldquoNovel risk factorsfor systemic atherosclerosis a comparison ofC-reactive proteinfibrinogen homocysteine lipoprotein(a) and standard choles-terol screening as predictors of peripheral arterial diseaserdquoJournal of the AmericanMedical Association vol 285 no 19 pp2481ndash2485 2001

[24] M Thoenes A Oguchi S Nagamia et al ldquoThe effects ofextended-release niacin on carotid intimal media thicknessendothelial function and inflammatory markers in patientswith the metabolic syndromerdquo International Journal of ClinicalPractice vol 61 no 11 pp 1942ndash1948 2007

[25] N R Madamanchi A Vendrov and M S Runge ldquoOxidativestress and vascular diseaserdquo Arteriosclerosis Thrombosis andVascular Biology vol 25 no 1 pp 29ndash38 2005

[26] E Matsuura F Atzeni P Sarzi-Puttini M Turiel L R Lopezand M T Nurmohamed ldquoIs atherosclerosis an autoimmunediseaserdquo BMCMedicine vol 12 no 1 article 47 2014

[27] A Pirillo G D Norata and A L Catapano ldquoLOX-1 OxLDLand atherosclerosisrdquo Mediators of Inflammation vol 2013Article ID 152786 12 pages 2013

[28] C L Wang M Meng S B Liu L R Wang L H Hou and XH Cao ldquochemically sulfated polysaccharide from Grifola fron-dos induces HepG2 cell apoptosis by notch1-NF-120581B pathwayrdquoCarbohydrate Polymers vol 95 no 1 pp 282ndash287 2013

[29] WWongchana and T Palaga ldquoDirect regulation of interleukin-6 expression by Notch signaling in macrophagesrdquo Cellular andMolecular Immunology vol 9 no 2 pp 155ndash162 2012

[30] J C Choy D J Granville D W C Hunt and B M McManusldquoEndothelial cell apoptosis biochemical characteristics andpotential implications for atherosclerosisrdquo Journal of Molecularand Cellular Cardiology vol 33 no 9 pp 1673ndash1690 2001

[31] M Cuchel and D J Rader ldquoMacrophage reverse cholesteroltransport key to the regression of atherosclerosisrdquo Circulationvol 113 no 21 pp 2548ndash2555 2006

[32] A Yazdanyar C Yeang and X C Jiang ldquoRole of phospholipidtransfer protein in high-density lipoprotein-mediated reversecholesterol transportrdquo Current Atherosclerosis Reports vol 13no 3 pp 242ndash248 2011

[33] X Wang and D J Rader ldquoMolecular regulation of macrophagereverse cholesterol transportrdquo Current Opinion in Cardiologyvol 22 no 4 pp 368ndash372 2007

[34] S J Yang J M Choi L Kim et al ldquoNicotinamide improvesglucose metabolism and affects the hepatic NAD-sirtuin path-way in a rodent model of obesity and type 2 diabetesrdquo Journalof Nutritional Biochemistry vol 25 no 1 pp 66ndash72 2014

[35] L A Carlson L Oro and J Ostman ldquoEffect of a single dose ofnicotinic acid on plasma lipids in patients with hyperlipopro-teinemiardquo Acta Medica Scandinavica vol 183 no 5 pp 457ndash465 1968

[36] R H Stern J D Spence D J Freeman and A Parbtani ldquoTol-erance to nicotinic acid flushingrdquo Clinical Pharmacology andTherapeutics vol 50 no 1 pp 66ndash70 1991

[37] S M Grundy J I Cleeman C N Bairey Merz et al ldquoImpli-cations of recent clinical trials for the National CholesterolEducation Program Adult Treatment Panel III guidelinesrdquoCirculation vol 110 no 2 pp 227ndash239 2004

[38] M S Brown and J L Goldstein ldquoLowering plasma cholesterolby raising LDL receptorsrdquoNewEngland Journal ofMedicine vol305 no 9 pp 515ndash517 1981

[39] R Hampton D Dimster-Denk and J Rine ldquoThe biology ofHMG-CoA reductase the pros of contra-regulationrdquo Trends inBiochemical Sciences vol 21 no 4 pp 140ndash145 1996

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 9: Research Article Niacin Inhibits Vascular Inflammation via ...downloads.hindawi.com/journals/mi/2014/263786.pdf · Research Article Niacin Inhibits Vascular Inflammation via Downregulating

Mediators of Inflammation 9

0

50

100

150

200

250

CD HFD HFD-N HFD-S

TG (m

gdL

)

lowastlowastlowastlowast

(a)

0

500

1000

1500

2000

CD HFD HFD-N HFD-S

TC (m

gdL

)

lowastlowast

(b)

0

20

40

60

80

100

120

CD HFD HFD-N HFD-S

HD

L-C

(mg

dL)

lowast

(c)

0

500

1000

1500

2000

CD HFD HFD-N HFD-S

Non

-HD

L-C

(mg

dL)

lowastlowast

lowast

(d)

Figure 7 Effect of niacin and simvastatin on plasma lipid of guinea pigs fed high fat diet The levels of TG (a) TC (b) HDL-C (c) andnon-HDL-C (d) in plasma of guinea pigs were determined by enzyme method after 8 weeksrsquo treatment Data are presented as mean plusmn SD(119899 = 8)

119875 lt 001 versus CD group lowast119875 lt 005 lowastlowast119875 lt 001 versus HFD group

apoAI20

30

40

50

6080

160110

Mar

ker

CD HFD

HFD

-N

HFD

-S

Hum

an

(a)

0

05

1

15

2

CD HFD HFD-N HFD-S

Fold

indu

ctio

n of

apoA

I in

HD

L

lowastlowast

(b)

Figure 8 Niacin upregulated apoA I level in plasma of guinea pigs fed high fat diet HDL (density = 109ndash124 gmL) was separatedfrom plasma of guinea pigs by sequential ultracentrifugation The SDS-PAGE was performed on 15 SDS polyacrylamide gel and theapolipoproteins were stained with coomassie brilliant blue Densitometric quantitation of SDS-PAGE image was analyzed by Image-Pro Plussoftware (a) shows the representative SDS-PAGE image ofHDL (b) shows the relative level of apoA I in plasma by densitometric quantitationData are presented as mean plusmn SD of at least three independent experiments

119875 lt 005 versus CD group lowastlowast119875 lt 001 versus HFD group

10 Mediators of Inflammation

0

02

04

06

08

1

12

14

16

CD HFD HFD-N HFD-S

Relat

ive m

RNA

leve

l of S

R-B1

(a)

0

02

04

06

08

1

12

CD HFD HFD-N HFD-S

Relat

ive m

RNA

leve

l of L

DL-

R

lowastlowast

(b)

0

05

1

15

2

CD HFD HFD-N HFD-S

Relat

ive m

RNA

leve

l of C

YP7A

1 lowastlowast

(c)

0

02

04

06

08

1

12

14

CD HFD HFD-N HFD-S

Relat

ive m

RNA

leve

l of H

MG

CR

lowastlowast

(d)

Figure 9 Effect of niacin and simvastatin on the mRNA abundance of SR-B1 LDL-R CYP7A1 and HMGCR in liver of guinea pigs fedhigh fat dietThe mRNA levels which were analyzed by quantitative real-time PCR were calculated after being adjusted for 120573-actin usingthe 2minusΔΔCt method Data are presented as mean plusmn SD of at least three independent experiments

119875 lt 005 119875 lt 001 versus CD group

lowastlowast

119875 lt 001 versus HFD group

demonstrated to exert critical activity on the transcriptionof many inflammatory genes including adhesion moleculescytokines and chemokines [22] (3) CRP an early acute phasereactant is closely relevant to inflammation Baseline level ofCRP is a strong independent predictor of the risk of futuremyocardial infarction peripheral vascular disease strokeand vascular death amonghealthy individualswithout knownvascular disease [23] Kuvin et al have shown that niacindecreased CRP level by 15 in patients with stable coronaryartery disease [5] In patients with metabolic syndromeafter treatment with extended-release niacin (1 gday) for 52weeks their endothelial function was improved by 22 andthere was a decrease in CRP level by 20 [24] Our resultsalso showed niacin slightly lowered CRP level but hadno statistical difference (Table 1) (4) Oxidative stress wassuppressed by niacin Oxidative stress is closely related to theinflammation in the arterial wall Increased ROS productioncan initiate a cascade of signal transduction which results inendothelial dysfunction changes in vascular tone vascularremodeling and vascular inflammatory responses [25]

To further confirm the direct anti-inflammatory prop-erty of niacin its effect on oxLDL-induced inflammatoryresponse of endothelial cells and macrophages was studiedoxLDL is pivotal in the development of AS and representsa crucial proinflammatory stimulus [26] Upon enteringinto the intima of arteries oxLDL activates endothelial

cells and upregulates adhesion molecule expression andinflammatory factors secretion all of which contribute tothe recruitment of circulating leukocytes Monocytes andormacrophages infiltrating the arterial wall take up oxLDL andform ldquofoam cellsrdquo which in turn promote further secretionof inflammatory mediators [27] Our data indicated niacinremarkably downregulated the secretion of TNF-120572 and IL-6stimulated by oxLDL in HUVECs and THP-1 macrophagesNotch1 signal pathway is an evolutionarily highly conservedmechanism for communication which can improve NF-120581Bactivity and have a positive correlation with the developmentof inflammation [28] A previous study demonstrates thatnotch knockout in macrophage markedly downregulates theexpression of proinflammatory factors Instead when thenotch signal pathway is activatedNF-120581B expression increasesand inflammatory factors secretion is also promoted [29]Our findings indicated niacin decreased the expressions ofNF-120581B and notch1 in oxLDL-induced inflammation Vas-cular endothelial apoptosis plays an important role in theinitiation of AS It may compromise vessel wall permeabilityto cytokines growth factors lipids and immune cells andincrease coagulation [30] In this study we indicated niacinattenuated oxLDL-induced apoptosis of HUVECs

Taken together these observations lend initial but com-pelling evidence that niacin has protective effects on car-diovascular disease through its vascular anti-inflammatory

Mediators of Inflammation 11

properties in addition to its established effects on lipidmetabolism

Reverse cholesterol transport (RCT) is a potent defensemechanism against AS It involves transporting cholesterolfromperipheral tissues and cells to liver transforming choles-terol into bile acid and finally eliminating it from the body[31] HDL and apoA play a key role in RCT They can drivecholesterol efflux from macrophage foam cells that residein vessel intima [32 33] Similar to previous studies ourfindings also indicated that niacin increased the levels ofHDL and apoA I in plasma significantly In the process ofRCT CYP7A1 is an important regulatory factor which canconvert cholesterol to bile acid in liver and be further excretedinto intestine for elimination from body with feces Here weshowed that the mRNA abundance of CYP7A1 in liver wassignificantly upregulated by niacin treatment These findingssuggest that niacin may modulate plasma lipid and exertantiatherosclerotic effects by promoting RCT progress

In clinical practice the oral dose of niacin for lipid-lowering purpose is usually 1ndash3 g per day equivalently 14ndash43mgkg per day According to the formula of Meeh-Rubnerequation we calculate the dose for guinea pig to be about80ndash250mgkg per day In addition a recent report showedthat up to 400mgkg per day of niacin treatment for 7 daysand 100mgkg per day of niacin treatment for 4 weeks aresafe and do not cause toxicity in rodent models [34] In thisstudy the applied dose of niacin for guinea pig is 100mgkgdwhich has pharmacologic effects and does not have varioustoxicities based on the changes in food intake body weightand behaviors In human plasma level of niacin was foundto be about 03mM after oral ingestion of 1 g niacin [3536] Since the reported plasma concentrations are one timemeasurement it is unclear whether these levels reflect troughor peak values Furthermore there is no reported clinicaldata available regarding plasma concentration of niacin afteringestion of 3 g niacin Extrapolation of the available datausing oral dose of 1 g niacin would indicate that the 3 g niacinoral administration may lead to the plasma concentrations ofniacin in the range of 08ndash1mM In our in-vitro studies weused 025 to 1mM niacin

Statins are the first-line therapy in the treatment ofcholesterol-induced atherosclerotic cardiovascular diseases[37] They suppress the conversion of HMG-CoA to meval-onate by competitively blocking HMGCR therefore theendogenous cholesterol synthesis in the liver is remarkablyreduced Consequently it results in the increase of LDL-Rexpression on hepatic cell surfaces and the decrease of LDL-Cin plasma [38]Meanwhile inhibition of cholesterol synthesiscauses an overexpression of HMGCR due to a feedback-regulatory mechanism [39] Similar to previous studies ourfindings also revealed that simvastatin significantly decreasedthe level of non-HDL-C in plasma followed by upregulatingLDL-R expression in liver and the HMGCR mRNA level inliver was increased in a compensatory way

5 Conclusion

In summary our data presented herein support the novelconcept that niacin has vascular anti-inflammatory and

potentially vascular-protective property which is indepen-dent of its effect on lipid regulation The anti-inflammatoryproperty of niacin is realized by downregulating nucleartranscription factor-120581B signaling pathway Meanwhile ourfinding also suggests that niacin modulates plasma lipid bystimulating the expression of factors involved in the processof RCT and promoting RCT

Conflict of Interests

The authors declare that they have no conflict of interest

Authorsrsquo Contribution

Yanhong Si and Ying Zhang have equally contributed to thisstudy

Acknowledgments

This research was supported by the financial supportsfrom the Taishan scholar foundation of Shandong Province(200867) and the Natural Science Foundations of China(81170785 and 81371234)

References

[1] R Altschul A Hoffer and J D Stephen ldquoInfluence of nicotinicacid on serum cholesterol in manrdquoArchives of Biochemistry andBiophysics vol 54 no 2 pp 558ndash559 1955

[2] C D Meyers V S Kamanna and M L Kashyap ldquoNiacin ther-apy in atherosclerosisrdquo Current Opinion in Lipidology vol 15no 6 pp 659ndash665 2004

[3] L A Carlson ldquoNicotinic acid the broad-spectrum lipid drug A50th anniversary reviewrdquo Journal of Internal Medicine vol 258no 2 pp 94ndash114 2005

[4] S H Ganji S Qin L Zhang V S Kamanna andM L KashyapldquoNiacin inhibits vascular oxidative stress redox-sensitive genesand monocyte adhesion to human aortic endothelial cellsrdquoAtherosclerosis vol 202 no 1 pp 68ndash75 2009

[5] J T Kuvin D M Dave K A Sliney et al ldquoEffects of extended-release niacin on lipoprotein particle size distribution andinflammatorymarkers in patients with coronary artery diseaserdquoAmerican Journal of Cardiology vol 98 no 6 pp 743ndash745 2006

[6] R Yang P Guo X Song F Liu and N Gao ldquoHyperlipidemicguinea pig model mechanisms of triglyceride metabolismdisorder and comparison to ratrdquo Biological and PharmaceuticalBulletin vol 34 no 7 pp 1046ndash1051 2011

[7] X Li Y Liu H Zhang L Ren Q Li and N Li ldquoAnimal modelsfor the atherosclerosis research a reviewrdquo Protein and Cell vol2 no 3 pp 189ndash201 2011

[8] J O Leite U Vaishnav M Puglisi H Fraser J Trias and M LFernandez ldquoA-002 (Varespladib) a phospholipase A2 inhibitorreduces atherosclerosis in guinea pigsrdquo BMC CardiovascularDisorders vol 9 article 7 2009

[9] J Auwerx ldquoThe human leukemia cell line THP-1 a multi-facetted model for the study of monocyte-macrophage differ-entiationrdquo Experientia vol 47 no 1 pp 22ndash31 1991

[10] M J Chapman G L Mills and J H Ledford ldquoThe distributionand partial characterization of the serum apolipoproteins in the

12 Mediators of Inflammation

guinea pigrdquo Biochemical Journal vol 149 no 2 pp 423ndash4361975

[11] X C Jiang C Bruce J Mar et al ldquoTargetedmutation of plasmaphospholipid transfer protein gene markedly reduces high-density lipoprotein levelsrdquo Journal of Clinical Investigation vol103 no 6 pp 907ndash914 1999

[12] T Collins and M I Cybulsky ldquoNF-120581B pivotal mediator orinnocent bystander in atherogenesisrdquo Journal of Clinical Inves-tigation vol 107 no 3 pp 255ndash264 2001

[13] Y Tsubosaka T Murata K Yamada D Uemura M Horiand H Ozaki ldquoHalichlorine reduces monocyte adhesion toendothelium through the suppression of nuclear factor-120581Bactivationrdquo Journal of Pharmacological Sciences vol 113 no 3pp 208ndash213 2010

[14] H Tsutsui ldquoMitochondrial oxidative stress and heart failurerdquoInternal Medicine vol 45 no 13 pp 809ndash813 2006

[15] M Seddon Y H Looi and A M Shah ldquoOxidative stressand redox signalling in cardiac hypertrophy and heart failurerdquoHeart vol 93 no 8 pp 903ndash907 2007

[16] WWongchana and T Palaga ldquoDirect regulation of interleukin-6 expression by Notch signaling in macrophagesrdquo Cellular andMolecular Immunology vol 9 no 2 pp 155ndash162 2012

[17] J Chen H Zhao X Ma et al ldquoThe effects of jiang-zhi-ningand itsmain components on cholesterol metabolismrdquo Evidence-Based Complementary and Alternative Medicine vol 2012Article ID 928234 10 pages 2012

[18] G Francisco C Hernandez and R Simo ldquoSerum markers ofvascular inflammation in dyslipemiardquo Clinica Chimica Actavol 369 no 2 pp 1ndash16 2006

[19] J Andersson P Libby and G K Hansson ldquoAdaptive immunityand atherosclerosisrdquoClinical Immunology vol 134 no 1 pp 33ndash46 2010

[20] W J Oh U Jung H S Eom H J Shin and H R Park ldquoInhibi-tion of lipopolysaccharide-induced proinflammatory responsesby Buddleja officinalis extract in BV-2 microglial cells vianegative regulation of NF-kB and ERK12 signalingrdquoMoleculesvol 18 no 8 pp 9195ndash9206 2013

[21] T M Wang C J Chen T S Lee et al ldquoDocosahexaenoic acidattenuates VCAM-1 expression and NF-120581B activation in TNF-120572-treated human aortic endothelial cellsrdquo Journal of NutritionalBiochemistry vol 22 no 2 pp 187ndash194 2011

[22] M S Islam H Yoshida N Matsuki et al ldquoAntioxidant freeradical-scavenging and NF-120581B-inhibitory activities of phy-tosteryl ferulates structure-activity studiesrdquo Journal of Pharma-cological Sciences vol 111 no 4 pp 328ndash337 2009

[23] P M Ridker M J Stampfer and N Rifai ldquoNovel risk factorsfor systemic atherosclerosis a comparison ofC-reactive proteinfibrinogen homocysteine lipoprotein(a) and standard choles-terol screening as predictors of peripheral arterial diseaserdquoJournal of the AmericanMedical Association vol 285 no 19 pp2481ndash2485 2001

[24] M Thoenes A Oguchi S Nagamia et al ldquoThe effects ofextended-release niacin on carotid intimal media thicknessendothelial function and inflammatory markers in patientswith the metabolic syndromerdquo International Journal of ClinicalPractice vol 61 no 11 pp 1942ndash1948 2007

[25] N R Madamanchi A Vendrov and M S Runge ldquoOxidativestress and vascular diseaserdquo Arteriosclerosis Thrombosis andVascular Biology vol 25 no 1 pp 29ndash38 2005

[26] E Matsuura F Atzeni P Sarzi-Puttini M Turiel L R Lopezand M T Nurmohamed ldquoIs atherosclerosis an autoimmunediseaserdquo BMCMedicine vol 12 no 1 article 47 2014

[27] A Pirillo G D Norata and A L Catapano ldquoLOX-1 OxLDLand atherosclerosisrdquo Mediators of Inflammation vol 2013Article ID 152786 12 pages 2013

[28] C L Wang M Meng S B Liu L R Wang L H Hou and XH Cao ldquochemically sulfated polysaccharide from Grifola fron-dos induces HepG2 cell apoptosis by notch1-NF-120581B pathwayrdquoCarbohydrate Polymers vol 95 no 1 pp 282ndash287 2013

[29] WWongchana and T Palaga ldquoDirect regulation of interleukin-6 expression by Notch signaling in macrophagesrdquo Cellular andMolecular Immunology vol 9 no 2 pp 155ndash162 2012

[30] J C Choy D J Granville D W C Hunt and B M McManusldquoEndothelial cell apoptosis biochemical characteristics andpotential implications for atherosclerosisrdquo Journal of Molecularand Cellular Cardiology vol 33 no 9 pp 1673ndash1690 2001

[31] M Cuchel and D J Rader ldquoMacrophage reverse cholesteroltransport key to the regression of atherosclerosisrdquo Circulationvol 113 no 21 pp 2548ndash2555 2006

[32] A Yazdanyar C Yeang and X C Jiang ldquoRole of phospholipidtransfer protein in high-density lipoprotein-mediated reversecholesterol transportrdquo Current Atherosclerosis Reports vol 13no 3 pp 242ndash248 2011

[33] X Wang and D J Rader ldquoMolecular regulation of macrophagereverse cholesterol transportrdquo Current Opinion in Cardiologyvol 22 no 4 pp 368ndash372 2007

[34] S J Yang J M Choi L Kim et al ldquoNicotinamide improvesglucose metabolism and affects the hepatic NAD-sirtuin path-way in a rodent model of obesity and type 2 diabetesrdquo Journalof Nutritional Biochemistry vol 25 no 1 pp 66ndash72 2014

[35] L A Carlson L Oro and J Ostman ldquoEffect of a single dose ofnicotinic acid on plasma lipids in patients with hyperlipopro-teinemiardquo Acta Medica Scandinavica vol 183 no 5 pp 457ndash465 1968

[36] R H Stern J D Spence D J Freeman and A Parbtani ldquoTol-erance to nicotinic acid flushingrdquo Clinical Pharmacology andTherapeutics vol 50 no 1 pp 66ndash70 1991

[37] S M Grundy J I Cleeman C N Bairey Merz et al ldquoImpli-cations of recent clinical trials for the National CholesterolEducation Program Adult Treatment Panel III guidelinesrdquoCirculation vol 110 no 2 pp 227ndash239 2004

[38] M S Brown and J L Goldstein ldquoLowering plasma cholesterolby raising LDL receptorsrdquoNewEngland Journal ofMedicine vol305 no 9 pp 515ndash517 1981

[39] R Hampton D Dimster-Denk and J Rine ldquoThe biology ofHMG-CoA reductase the pros of contra-regulationrdquo Trends inBiochemical Sciences vol 21 no 4 pp 140ndash145 1996

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 10: Research Article Niacin Inhibits Vascular Inflammation via ...downloads.hindawi.com/journals/mi/2014/263786.pdf · Research Article Niacin Inhibits Vascular Inflammation via Downregulating

10 Mediators of Inflammation

0

02

04

06

08

1

12

14

16

CD HFD HFD-N HFD-S

Relat

ive m

RNA

leve

l of S

R-B1

(a)

0

02

04

06

08

1

12

CD HFD HFD-N HFD-S

Relat

ive m

RNA

leve

l of L

DL-

R

lowastlowast

(b)

0

05

1

15

2

CD HFD HFD-N HFD-S

Relat

ive m

RNA

leve

l of C

YP7A

1 lowastlowast

(c)

0

02

04

06

08

1

12

14

CD HFD HFD-N HFD-S

Relat

ive m

RNA

leve

l of H

MG

CR

lowastlowast

(d)

Figure 9 Effect of niacin and simvastatin on the mRNA abundance of SR-B1 LDL-R CYP7A1 and HMGCR in liver of guinea pigs fedhigh fat dietThe mRNA levels which were analyzed by quantitative real-time PCR were calculated after being adjusted for 120573-actin usingthe 2minusΔΔCt method Data are presented as mean plusmn SD of at least three independent experiments

119875 lt 005 119875 lt 001 versus CD group

lowastlowast

119875 lt 001 versus HFD group

demonstrated to exert critical activity on the transcriptionof many inflammatory genes including adhesion moleculescytokines and chemokines [22] (3) CRP an early acute phasereactant is closely relevant to inflammation Baseline level ofCRP is a strong independent predictor of the risk of futuremyocardial infarction peripheral vascular disease strokeand vascular death amonghealthy individualswithout knownvascular disease [23] Kuvin et al have shown that niacindecreased CRP level by 15 in patients with stable coronaryartery disease [5] In patients with metabolic syndromeafter treatment with extended-release niacin (1 gday) for 52weeks their endothelial function was improved by 22 andthere was a decrease in CRP level by 20 [24] Our resultsalso showed niacin slightly lowered CRP level but hadno statistical difference (Table 1) (4) Oxidative stress wassuppressed by niacin Oxidative stress is closely related to theinflammation in the arterial wall Increased ROS productioncan initiate a cascade of signal transduction which results inendothelial dysfunction changes in vascular tone vascularremodeling and vascular inflammatory responses [25]

To further confirm the direct anti-inflammatory prop-erty of niacin its effect on oxLDL-induced inflammatoryresponse of endothelial cells and macrophages was studiedoxLDL is pivotal in the development of AS and representsa crucial proinflammatory stimulus [26] Upon enteringinto the intima of arteries oxLDL activates endothelial

cells and upregulates adhesion molecule expression andinflammatory factors secretion all of which contribute tothe recruitment of circulating leukocytes Monocytes andormacrophages infiltrating the arterial wall take up oxLDL andform ldquofoam cellsrdquo which in turn promote further secretionof inflammatory mediators [27] Our data indicated niacinremarkably downregulated the secretion of TNF-120572 and IL-6stimulated by oxLDL in HUVECs and THP-1 macrophagesNotch1 signal pathway is an evolutionarily highly conservedmechanism for communication which can improve NF-120581Bactivity and have a positive correlation with the developmentof inflammation [28] A previous study demonstrates thatnotch knockout in macrophage markedly downregulates theexpression of proinflammatory factors Instead when thenotch signal pathway is activatedNF-120581B expression increasesand inflammatory factors secretion is also promoted [29]Our findings indicated niacin decreased the expressions ofNF-120581B and notch1 in oxLDL-induced inflammation Vas-cular endothelial apoptosis plays an important role in theinitiation of AS It may compromise vessel wall permeabilityto cytokines growth factors lipids and immune cells andincrease coagulation [30] In this study we indicated niacinattenuated oxLDL-induced apoptosis of HUVECs

Taken together these observations lend initial but com-pelling evidence that niacin has protective effects on car-diovascular disease through its vascular anti-inflammatory

Mediators of Inflammation 11

properties in addition to its established effects on lipidmetabolism

Reverse cholesterol transport (RCT) is a potent defensemechanism against AS It involves transporting cholesterolfromperipheral tissues and cells to liver transforming choles-terol into bile acid and finally eliminating it from the body[31] HDL and apoA play a key role in RCT They can drivecholesterol efflux from macrophage foam cells that residein vessel intima [32 33] Similar to previous studies ourfindings also indicated that niacin increased the levels ofHDL and apoA I in plasma significantly In the process ofRCT CYP7A1 is an important regulatory factor which canconvert cholesterol to bile acid in liver and be further excretedinto intestine for elimination from body with feces Here weshowed that the mRNA abundance of CYP7A1 in liver wassignificantly upregulated by niacin treatment These findingssuggest that niacin may modulate plasma lipid and exertantiatherosclerotic effects by promoting RCT progress

In clinical practice the oral dose of niacin for lipid-lowering purpose is usually 1ndash3 g per day equivalently 14ndash43mgkg per day According to the formula of Meeh-Rubnerequation we calculate the dose for guinea pig to be about80ndash250mgkg per day In addition a recent report showedthat up to 400mgkg per day of niacin treatment for 7 daysand 100mgkg per day of niacin treatment for 4 weeks aresafe and do not cause toxicity in rodent models [34] In thisstudy the applied dose of niacin for guinea pig is 100mgkgdwhich has pharmacologic effects and does not have varioustoxicities based on the changes in food intake body weightand behaviors In human plasma level of niacin was foundto be about 03mM after oral ingestion of 1 g niacin [3536] Since the reported plasma concentrations are one timemeasurement it is unclear whether these levels reflect troughor peak values Furthermore there is no reported clinicaldata available regarding plasma concentration of niacin afteringestion of 3 g niacin Extrapolation of the available datausing oral dose of 1 g niacin would indicate that the 3 g niacinoral administration may lead to the plasma concentrations ofniacin in the range of 08ndash1mM In our in-vitro studies weused 025 to 1mM niacin

Statins are the first-line therapy in the treatment ofcholesterol-induced atherosclerotic cardiovascular diseases[37] They suppress the conversion of HMG-CoA to meval-onate by competitively blocking HMGCR therefore theendogenous cholesterol synthesis in the liver is remarkablyreduced Consequently it results in the increase of LDL-Rexpression on hepatic cell surfaces and the decrease of LDL-Cin plasma [38]Meanwhile inhibition of cholesterol synthesiscauses an overexpression of HMGCR due to a feedback-regulatory mechanism [39] Similar to previous studies ourfindings also revealed that simvastatin significantly decreasedthe level of non-HDL-C in plasma followed by upregulatingLDL-R expression in liver and the HMGCR mRNA level inliver was increased in a compensatory way

5 Conclusion

In summary our data presented herein support the novelconcept that niacin has vascular anti-inflammatory and

potentially vascular-protective property which is indepen-dent of its effect on lipid regulation The anti-inflammatoryproperty of niacin is realized by downregulating nucleartranscription factor-120581B signaling pathway Meanwhile ourfinding also suggests that niacin modulates plasma lipid bystimulating the expression of factors involved in the processof RCT and promoting RCT

Conflict of Interests

The authors declare that they have no conflict of interest

Authorsrsquo Contribution

Yanhong Si and Ying Zhang have equally contributed to thisstudy

Acknowledgments

This research was supported by the financial supportsfrom the Taishan scholar foundation of Shandong Province(200867) and the Natural Science Foundations of China(81170785 and 81371234)

References

[1] R Altschul A Hoffer and J D Stephen ldquoInfluence of nicotinicacid on serum cholesterol in manrdquoArchives of Biochemistry andBiophysics vol 54 no 2 pp 558ndash559 1955

[2] C D Meyers V S Kamanna and M L Kashyap ldquoNiacin ther-apy in atherosclerosisrdquo Current Opinion in Lipidology vol 15no 6 pp 659ndash665 2004

[3] L A Carlson ldquoNicotinic acid the broad-spectrum lipid drug A50th anniversary reviewrdquo Journal of Internal Medicine vol 258no 2 pp 94ndash114 2005

[4] S H Ganji S Qin L Zhang V S Kamanna andM L KashyapldquoNiacin inhibits vascular oxidative stress redox-sensitive genesand monocyte adhesion to human aortic endothelial cellsrdquoAtherosclerosis vol 202 no 1 pp 68ndash75 2009

[5] J T Kuvin D M Dave K A Sliney et al ldquoEffects of extended-release niacin on lipoprotein particle size distribution andinflammatorymarkers in patients with coronary artery diseaserdquoAmerican Journal of Cardiology vol 98 no 6 pp 743ndash745 2006

[6] R Yang P Guo X Song F Liu and N Gao ldquoHyperlipidemicguinea pig model mechanisms of triglyceride metabolismdisorder and comparison to ratrdquo Biological and PharmaceuticalBulletin vol 34 no 7 pp 1046ndash1051 2011

[7] X Li Y Liu H Zhang L Ren Q Li and N Li ldquoAnimal modelsfor the atherosclerosis research a reviewrdquo Protein and Cell vol2 no 3 pp 189ndash201 2011

[8] J O Leite U Vaishnav M Puglisi H Fraser J Trias and M LFernandez ldquoA-002 (Varespladib) a phospholipase A2 inhibitorreduces atherosclerosis in guinea pigsrdquo BMC CardiovascularDisorders vol 9 article 7 2009

[9] J Auwerx ldquoThe human leukemia cell line THP-1 a multi-facetted model for the study of monocyte-macrophage differ-entiationrdquo Experientia vol 47 no 1 pp 22ndash31 1991

[10] M J Chapman G L Mills and J H Ledford ldquoThe distributionand partial characterization of the serum apolipoproteins in the

12 Mediators of Inflammation

guinea pigrdquo Biochemical Journal vol 149 no 2 pp 423ndash4361975

[11] X C Jiang C Bruce J Mar et al ldquoTargetedmutation of plasmaphospholipid transfer protein gene markedly reduces high-density lipoprotein levelsrdquo Journal of Clinical Investigation vol103 no 6 pp 907ndash914 1999

[12] T Collins and M I Cybulsky ldquoNF-120581B pivotal mediator orinnocent bystander in atherogenesisrdquo Journal of Clinical Inves-tigation vol 107 no 3 pp 255ndash264 2001

[13] Y Tsubosaka T Murata K Yamada D Uemura M Horiand H Ozaki ldquoHalichlorine reduces monocyte adhesion toendothelium through the suppression of nuclear factor-120581Bactivationrdquo Journal of Pharmacological Sciences vol 113 no 3pp 208ndash213 2010

[14] H Tsutsui ldquoMitochondrial oxidative stress and heart failurerdquoInternal Medicine vol 45 no 13 pp 809ndash813 2006

[15] M Seddon Y H Looi and A M Shah ldquoOxidative stressand redox signalling in cardiac hypertrophy and heart failurerdquoHeart vol 93 no 8 pp 903ndash907 2007

[16] WWongchana and T Palaga ldquoDirect regulation of interleukin-6 expression by Notch signaling in macrophagesrdquo Cellular andMolecular Immunology vol 9 no 2 pp 155ndash162 2012

[17] J Chen H Zhao X Ma et al ldquoThe effects of jiang-zhi-ningand itsmain components on cholesterol metabolismrdquo Evidence-Based Complementary and Alternative Medicine vol 2012Article ID 928234 10 pages 2012

[18] G Francisco C Hernandez and R Simo ldquoSerum markers ofvascular inflammation in dyslipemiardquo Clinica Chimica Actavol 369 no 2 pp 1ndash16 2006

[19] J Andersson P Libby and G K Hansson ldquoAdaptive immunityand atherosclerosisrdquoClinical Immunology vol 134 no 1 pp 33ndash46 2010

[20] W J Oh U Jung H S Eom H J Shin and H R Park ldquoInhibi-tion of lipopolysaccharide-induced proinflammatory responsesby Buddleja officinalis extract in BV-2 microglial cells vianegative regulation of NF-kB and ERK12 signalingrdquoMoleculesvol 18 no 8 pp 9195ndash9206 2013

[21] T M Wang C J Chen T S Lee et al ldquoDocosahexaenoic acidattenuates VCAM-1 expression and NF-120581B activation in TNF-120572-treated human aortic endothelial cellsrdquo Journal of NutritionalBiochemistry vol 22 no 2 pp 187ndash194 2011

[22] M S Islam H Yoshida N Matsuki et al ldquoAntioxidant freeradical-scavenging and NF-120581B-inhibitory activities of phy-tosteryl ferulates structure-activity studiesrdquo Journal of Pharma-cological Sciences vol 111 no 4 pp 328ndash337 2009

[23] P M Ridker M J Stampfer and N Rifai ldquoNovel risk factorsfor systemic atherosclerosis a comparison ofC-reactive proteinfibrinogen homocysteine lipoprotein(a) and standard choles-terol screening as predictors of peripheral arterial diseaserdquoJournal of the AmericanMedical Association vol 285 no 19 pp2481ndash2485 2001

[24] M Thoenes A Oguchi S Nagamia et al ldquoThe effects ofextended-release niacin on carotid intimal media thicknessendothelial function and inflammatory markers in patientswith the metabolic syndromerdquo International Journal of ClinicalPractice vol 61 no 11 pp 1942ndash1948 2007

[25] N R Madamanchi A Vendrov and M S Runge ldquoOxidativestress and vascular diseaserdquo Arteriosclerosis Thrombosis andVascular Biology vol 25 no 1 pp 29ndash38 2005

[26] E Matsuura F Atzeni P Sarzi-Puttini M Turiel L R Lopezand M T Nurmohamed ldquoIs atherosclerosis an autoimmunediseaserdquo BMCMedicine vol 12 no 1 article 47 2014

[27] A Pirillo G D Norata and A L Catapano ldquoLOX-1 OxLDLand atherosclerosisrdquo Mediators of Inflammation vol 2013Article ID 152786 12 pages 2013

[28] C L Wang M Meng S B Liu L R Wang L H Hou and XH Cao ldquochemically sulfated polysaccharide from Grifola fron-dos induces HepG2 cell apoptosis by notch1-NF-120581B pathwayrdquoCarbohydrate Polymers vol 95 no 1 pp 282ndash287 2013

[29] WWongchana and T Palaga ldquoDirect regulation of interleukin-6 expression by Notch signaling in macrophagesrdquo Cellular andMolecular Immunology vol 9 no 2 pp 155ndash162 2012

[30] J C Choy D J Granville D W C Hunt and B M McManusldquoEndothelial cell apoptosis biochemical characteristics andpotential implications for atherosclerosisrdquo Journal of Molecularand Cellular Cardiology vol 33 no 9 pp 1673ndash1690 2001

[31] M Cuchel and D J Rader ldquoMacrophage reverse cholesteroltransport key to the regression of atherosclerosisrdquo Circulationvol 113 no 21 pp 2548ndash2555 2006

[32] A Yazdanyar C Yeang and X C Jiang ldquoRole of phospholipidtransfer protein in high-density lipoprotein-mediated reversecholesterol transportrdquo Current Atherosclerosis Reports vol 13no 3 pp 242ndash248 2011

[33] X Wang and D J Rader ldquoMolecular regulation of macrophagereverse cholesterol transportrdquo Current Opinion in Cardiologyvol 22 no 4 pp 368ndash372 2007

[34] S J Yang J M Choi L Kim et al ldquoNicotinamide improvesglucose metabolism and affects the hepatic NAD-sirtuin path-way in a rodent model of obesity and type 2 diabetesrdquo Journalof Nutritional Biochemistry vol 25 no 1 pp 66ndash72 2014

[35] L A Carlson L Oro and J Ostman ldquoEffect of a single dose ofnicotinic acid on plasma lipids in patients with hyperlipopro-teinemiardquo Acta Medica Scandinavica vol 183 no 5 pp 457ndash465 1968

[36] R H Stern J D Spence D J Freeman and A Parbtani ldquoTol-erance to nicotinic acid flushingrdquo Clinical Pharmacology andTherapeutics vol 50 no 1 pp 66ndash70 1991

[37] S M Grundy J I Cleeman C N Bairey Merz et al ldquoImpli-cations of recent clinical trials for the National CholesterolEducation Program Adult Treatment Panel III guidelinesrdquoCirculation vol 110 no 2 pp 227ndash239 2004

[38] M S Brown and J L Goldstein ldquoLowering plasma cholesterolby raising LDL receptorsrdquoNewEngland Journal ofMedicine vol305 no 9 pp 515ndash517 1981

[39] R Hampton D Dimster-Denk and J Rine ldquoThe biology ofHMG-CoA reductase the pros of contra-regulationrdquo Trends inBiochemical Sciences vol 21 no 4 pp 140ndash145 1996

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 11: Research Article Niacin Inhibits Vascular Inflammation via ...downloads.hindawi.com/journals/mi/2014/263786.pdf · Research Article Niacin Inhibits Vascular Inflammation via Downregulating

Mediators of Inflammation 11

properties in addition to its established effects on lipidmetabolism

Reverse cholesterol transport (RCT) is a potent defensemechanism against AS It involves transporting cholesterolfromperipheral tissues and cells to liver transforming choles-terol into bile acid and finally eliminating it from the body[31] HDL and apoA play a key role in RCT They can drivecholesterol efflux from macrophage foam cells that residein vessel intima [32 33] Similar to previous studies ourfindings also indicated that niacin increased the levels ofHDL and apoA I in plasma significantly In the process ofRCT CYP7A1 is an important regulatory factor which canconvert cholesterol to bile acid in liver and be further excretedinto intestine for elimination from body with feces Here weshowed that the mRNA abundance of CYP7A1 in liver wassignificantly upregulated by niacin treatment These findingssuggest that niacin may modulate plasma lipid and exertantiatherosclerotic effects by promoting RCT progress

In clinical practice the oral dose of niacin for lipid-lowering purpose is usually 1ndash3 g per day equivalently 14ndash43mgkg per day According to the formula of Meeh-Rubnerequation we calculate the dose for guinea pig to be about80ndash250mgkg per day In addition a recent report showedthat up to 400mgkg per day of niacin treatment for 7 daysand 100mgkg per day of niacin treatment for 4 weeks aresafe and do not cause toxicity in rodent models [34] In thisstudy the applied dose of niacin for guinea pig is 100mgkgdwhich has pharmacologic effects and does not have varioustoxicities based on the changes in food intake body weightand behaviors In human plasma level of niacin was foundto be about 03mM after oral ingestion of 1 g niacin [3536] Since the reported plasma concentrations are one timemeasurement it is unclear whether these levels reflect troughor peak values Furthermore there is no reported clinicaldata available regarding plasma concentration of niacin afteringestion of 3 g niacin Extrapolation of the available datausing oral dose of 1 g niacin would indicate that the 3 g niacinoral administration may lead to the plasma concentrations ofniacin in the range of 08ndash1mM In our in-vitro studies weused 025 to 1mM niacin

Statins are the first-line therapy in the treatment ofcholesterol-induced atherosclerotic cardiovascular diseases[37] They suppress the conversion of HMG-CoA to meval-onate by competitively blocking HMGCR therefore theendogenous cholesterol synthesis in the liver is remarkablyreduced Consequently it results in the increase of LDL-Rexpression on hepatic cell surfaces and the decrease of LDL-Cin plasma [38]Meanwhile inhibition of cholesterol synthesiscauses an overexpression of HMGCR due to a feedback-regulatory mechanism [39] Similar to previous studies ourfindings also revealed that simvastatin significantly decreasedthe level of non-HDL-C in plasma followed by upregulatingLDL-R expression in liver and the HMGCR mRNA level inliver was increased in a compensatory way

5 Conclusion

In summary our data presented herein support the novelconcept that niacin has vascular anti-inflammatory and

potentially vascular-protective property which is indepen-dent of its effect on lipid regulation The anti-inflammatoryproperty of niacin is realized by downregulating nucleartranscription factor-120581B signaling pathway Meanwhile ourfinding also suggests that niacin modulates plasma lipid bystimulating the expression of factors involved in the processof RCT and promoting RCT

Conflict of Interests

The authors declare that they have no conflict of interest

Authorsrsquo Contribution

Yanhong Si and Ying Zhang have equally contributed to thisstudy

Acknowledgments

This research was supported by the financial supportsfrom the Taishan scholar foundation of Shandong Province(200867) and the Natural Science Foundations of China(81170785 and 81371234)

References

[1] R Altschul A Hoffer and J D Stephen ldquoInfluence of nicotinicacid on serum cholesterol in manrdquoArchives of Biochemistry andBiophysics vol 54 no 2 pp 558ndash559 1955

[2] C D Meyers V S Kamanna and M L Kashyap ldquoNiacin ther-apy in atherosclerosisrdquo Current Opinion in Lipidology vol 15no 6 pp 659ndash665 2004

[3] L A Carlson ldquoNicotinic acid the broad-spectrum lipid drug A50th anniversary reviewrdquo Journal of Internal Medicine vol 258no 2 pp 94ndash114 2005

[4] S H Ganji S Qin L Zhang V S Kamanna andM L KashyapldquoNiacin inhibits vascular oxidative stress redox-sensitive genesand monocyte adhesion to human aortic endothelial cellsrdquoAtherosclerosis vol 202 no 1 pp 68ndash75 2009

[5] J T Kuvin D M Dave K A Sliney et al ldquoEffects of extended-release niacin on lipoprotein particle size distribution andinflammatorymarkers in patients with coronary artery diseaserdquoAmerican Journal of Cardiology vol 98 no 6 pp 743ndash745 2006

[6] R Yang P Guo X Song F Liu and N Gao ldquoHyperlipidemicguinea pig model mechanisms of triglyceride metabolismdisorder and comparison to ratrdquo Biological and PharmaceuticalBulletin vol 34 no 7 pp 1046ndash1051 2011

[7] X Li Y Liu H Zhang L Ren Q Li and N Li ldquoAnimal modelsfor the atherosclerosis research a reviewrdquo Protein and Cell vol2 no 3 pp 189ndash201 2011

[8] J O Leite U Vaishnav M Puglisi H Fraser J Trias and M LFernandez ldquoA-002 (Varespladib) a phospholipase A2 inhibitorreduces atherosclerosis in guinea pigsrdquo BMC CardiovascularDisorders vol 9 article 7 2009

[9] J Auwerx ldquoThe human leukemia cell line THP-1 a multi-facetted model for the study of monocyte-macrophage differ-entiationrdquo Experientia vol 47 no 1 pp 22ndash31 1991

[10] M J Chapman G L Mills and J H Ledford ldquoThe distributionand partial characterization of the serum apolipoproteins in the

12 Mediators of Inflammation

guinea pigrdquo Biochemical Journal vol 149 no 2 pp 423ndash4361975

[11] X C Jiang C Bruce J Mar et al ldquoTargetedmutation of plasmaphospholipid transfer protein gene markedly reduces high-density lipoprotein levelsrdquo Journal of Clinical Investigation vol103 no 6 pp 907ndash914 1999

[12] T Collins and M I Cybulsky ldquoNF-120581B pivotal mediator orinnocent bystander in atherogenesisrdquo Journal of Clinical Inves-tigation vol 107 no 3 pp 255ndash264 2001

[13] Y Tsubosaka T Murata K Yamada D Uemura M Horiand H Ozaki ldquoHalichlorine reduces monocyte adhesion toendothelium through the suppression of nuclear factor-120581Bactivationrdquo Journal of Pharmacological Sciences vol 113 no 3pp 208ndash213 2010

[14] H Tsutsui ldquoMitochondrial oxidative stress and heart failurerdquoInternal Medicine vol 45 no 13 pp 809ndash813 2006

[15] M Seddon Y H Looi and A M Shah ldquoOxidative stressand redox signalling in cardiac hypertrophy and heart failurerdquoHeart vol 93 no 8 pp 903ndash907 2007

[16] WWongchana and T Palaga ldquoDirect regulation of interleukin-6 expression by Notch signaling in macrophagesrdquo Cellular andMolecular Immunology vol 9 no 2 pp 155ndash162 2012

[17] J Chen H Zhao X Ma et al ldquoThe effects of jiang-zhi-ningand itsmain components on cholesterol metabolismrdquo Evidence-Based Complementary and Alternative Medicine vol 2012Article ID 928234 10 pages 2012

[18] G Francisco C Hernandez and R Simo ldquoSerum markers ofvascular inflammation in dyslipemiardquo Clinica Chimica Actavol 369 no 2 pp 1ndash16 2006

[19] J Andersson P Libby and G K Hansson ldquoAdaptive immunityand atherosclerosisrdquoClinical Immunology vol 134 no 1 pp 33ndash46 2010

[20] W J Oh U Jung H S Eom H J Shin and H R Park ldquoInhibi-tion of lipopolysaccharide-induced proinflammatory responsesby Buddleja officinalis extract in BV-2 microglial cells vianegative regulation of NF-kB and ERK12 signalingrdquoMoleculesvol 18 no 8 pp 9195ndash9206 2013

[21] T M Wang C J Chen T S Lee et al ldquoDocosahexaenoic acidattenuates VCAM-1 expression and NF-120581B activation in TNF-120572-treated human aortic endothelial cellsrdquo Journal of NutritionalBiochemistry vol 22 no 2 pp 187ndash194 2011

[22] M S Islam H Yoshida N Matsuki et al ldquoAntioxidant freeradical-scavenging and NF-120581B-inhibitory activities of phy-tosteryl ferulates structure-activity studiesrdquo Journal of Pharma-cological Sciences vol 111 no 4 pp 328ndash337 2009

[23] P M Ridker M J Stampfer and N Rifai ldquoNovel risk factorsfor systemic atherosclerosis a comparison ofC-reactive proteinfibrinogen homocysteine lipoprotein(a) and standard choles-terol screening as predictors of peripheral arterial diseaserdquoJournal of the AmericanMedical Association vol 285 no 19 pp2481ndash2485 2001

[24] M Thoenes A Oguchi S Nagamia et al ldquoThe effects ofextended-release niacin on carotid intimal media thicknessendothelial function and inflammatory markers in patientswith the metabolic syndromerdquo International Journal of ClinicalPractice vol 61 no 11 pp 1942ndash1948 2007

[25] N R Madamanchi A Vendrov and M S Runge ldquoOxidativestress and vascular diseaserdquo Arteriosclerosis Thrombosis andVascular Biology vol 25 no 1 pp 29ndash38 2005

[26] E Matsuura F Atzeni P Sarzi-Puttini M Turiel L R Lopezand M T Nurmohamed ldquoIs atherosclerosis an autoimmunediseaserdquo BMCMedicine vol 12 no 1 article 47 2014

[27] A Pirillo G D Norata and A L Catapano ldquoLOX-1 OxLDLand atherosclerosisrdquo Mediators of Inflammation vol 2013Article ID 152786 12 pages 2013

[28] C L Wang M Meng S B Liu L R Wang L H Hou and XH Cao ldquochemically sulfated polysaccharide from Grifola fron-dos induces HepG2 cell apoptosis by notch1-NF-120581B pathwayrdquoCarbohydrate Polymers vol 95 no 1 pp 282ndash287 2013

[29] WWongchana and T Palaga ldquoDirect regulation of interleukin-6 expression by Notch signaling in macrophagesrdquo Cellular andMolecular Immunology vol 9 no 2 pp 155ndash162 2012

[30] J C Choy D J Granville D W C Hunt and B M McManusldquoEndothelial cell apoptosis biochemical characteristics andpotential implications for atherosclerosisrdquo Journal of Molecularand Cellular Cardiology vol 33 no 9 pp 1673ndash1690 2001

[31] M Cuchel and D J Rader ldquoMacrophage reverse cholesteroltransport key to the regression of atherosclerosisrdquo Circulationvol 113 no 21 pp 2548ndash2555 2006

[32] A Yazdanyar C Yeang and X C Jiang ldquoRole of phospholipidtransfer protein in high-density lipoprotein-mediated reversecholesterol transportrdquo Current Atherosclerosis Reports vol 13no 3 pp 242ndash248 2011

[33] X Wang and D J Rader ldquoMolecular regulation of macrophagereverse cholesterol transportrdquo Current Opinion in Cardiologyvol 22 no 4 pp 368ndash372 2007

[34] S J Yang J M Choi L Kim et al ldquoNicotinamide improvesglucose metabolism and affects the hepatic NAD-sirtuin path-way in a rodent model of obesity and type 2 diabetesrdquo Journalof Nutritional Biochemistry vol 25 no 1 pp 66ndash72 2014

[35] L A Carlson L Oro and J Ostman ldquoEffect of a single dose ofnicotinic acid on plasma lipids in patients with hyperlipopro-teinemiardquo Acta Medica Scandinavica vol 183 no 5 pp 457ndash465 1968

[36] R H Stern J D Spence D J Freeman and A Parbtani ldquoTol-erance to nicotinic acid flushingrdquo Clinical Pharmacology andTherapeutics vol 50 no 1 pp 66ndash70 1991

[37] S M Grundy J I Cleeman C N Bairey Merz et al ldquoImpli-cations of recent clinical trials for the National CholesterolEducation Program Adult Treatment Panel III guidelinesrdquoCirculation vol 110 no 2 pp 227ndash239 2004

[38] M S Brown and J L Goldstein ldquoLowering plasma cholesterolby raising LDL receptorsrdquoNewEngland Journal ofMedicine vol305 no 9 pp 515ndash517 1981

[39] R Hampton D Dimster-Denk and J Rine ldquoThe biology ofHMG-CoA reductase the pros of contra-regulationrdquo Trends inBiochemical Sciences vol 21 no 4 pp 140ndash145 1996

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 12: Research Article Niacin Inhibits Vascular Inflammation via ...downloads.hindawi.com/journals/mi/2014/263786.pdf · Research Article Niacin Inhibits Vascular Inflammation via Downregulating

12 Mediators of Inflammation

guinea pigrdquo Biochemical Journal vol 149 no 2 pp 423ndash4361975

[11] X C Jiang C Bruce J Mar et al ldquoTargetedmutation of plasmaphospholipid transfer protein gene markedly reduces high-density lipoprotein levelsrdquo Journal of Clinical Investigation vol103 no 6 pp 907ndash914 1999

[12] T Collins and M I Cybulsky ldquoNF-120581B pivotal mediator orinnocent bystander in atherogenesisrdquo Journal of Clinical Inves-tigation vol 107 no 3 pp 255ndash264 2001

[13] Y Tsubosaka T Murata K Yamada D Uemura M Horiand H Ozaki ldquoHalichlorine reduces monocyte adhesion toendothelium through the suppression of nuclear factor-120581Bactivationrdquo Journal of Pharmacological Sciences vol 113 no 3pp 208ndash213 2010

[14] H Tsutsui ldquoMitochondrial oxidative stress and heart failurerdquoInternal Medicine vol 45 no 13 pp 809ndash813 2006

[15] M Seddon Y H Looi and A M Shah ldquoOxidative stressand redox signalling in cardiac hypertrophy and heart failurerdquoHeart vol 93 no 8 pp 903ndash907 2007

[16] WWongchana and T Palaga ldquoDirect regulation of interleukin-6 expression by Notch signaling in macrophagesrdquo Cellular andMolecular Immunology vol 9 no 2 pp 155ndash162 2012

[17] J Chen H Zhao X Ma et al ldquoThe effects of jiang-zhi-ningand itsmain components on cholesterol metabolismrdquo Evidence-Based Complementary and Alternative Medicine vol 2012Article ID 928234 10 pages 2012

[18] G Francisco C Hernandez and R Simo ldquoSerum markers ofvascular inflammation in dyslipemiardquo Clinica Chimica Actavol 369 no 2 pp 1ndash16 2006

[19] J Andersson P Libby and G K Hansson ldquoAdaptive immunityand atherosclerosisrdquoClinical Immunology vol 134 no 1 pp 33ndash46 2010

[20] W J Oh U Jung H S Eom H J Shin and H R Park ldquoInhibi-tion of lipopolysaccharide-induced proinflammatory responsesby Buddleja officinalis extract in BV-2 microglial cells vianegative regulation of NF-kB and ERK12 signalingrdquoMoleculesvol 18 no 8 pp 9195ndash9206 2013

[21] T M Wang C J Chen T S Lee et al ldquoDocosahexaenoic acidattenuates VCAM-1 expression and NF-120581B activation in TNF-120572-treated human aortic endothelial cellsrdquo Journal of NutritionalBiochemistry vol 22 no 2 pp 187ndash194 2011

[22] M S Islam H Yoshida N Matsuki et al ldquoAntioxidant freeradical-scavenging and NF-120581B-inhibitory activities of phy-tosteryl ferulates structure-activity studiesrdquo Journal of Pharma-cological Sciences vol 111 no 4 pp 328ndash337 2009

[23] P M Ridker M J Stampfer and N Rifai ldquoNovel risk factorsfor systemic atherosclerosis a comparison ofC-reactive proteinfibrinogen homocysteine lipoprotein(a) and standard choles-terol screening as predictors of peripheral arterial diseaserdquoJournal of the AmericanMedical Association vol 285 no 19 pp2481ndash2485 2001

[24] M Thoenes A Oguchi S Nagamia et al ldquoThe effects ofextended-release niacin on carotid intimal media thicknessendothelial function and inflammatory markers in patientswith the metabolic syndromerdquo International Journal of ClinicalPractice vol 61 no 11 pp 1942ndash1948 2007

[25] N R Madamanchi A Vendrov and M S Runge ldquoOxidativestress and vascular diseaserdquo Arteriosclerosis Thrombosis andVascular Biology vol 25 no 1 pp 29ndash38 2005

[26] E Matsuura F Atzeni P Sarzi-Puttini M Turiel L R Lopezand M T Nurmohamed ldquoIs atherosclerosis an autoimmunediseaserdquo BMCMedicine vol 12 no 1 article 47 2014

[27] A Pirillo G D Norata and A L Catapano ldquoLOX-1 OxLDLand atherosclerosisrdquo Mediators of Inflammation vol 2013Article ID 152786 12 pages 2013

[28] C L Wang M Meng S B Liu L R Wang L H Hou and XH Cao ldquochemically sulfated polysaccharide from Grifola fron-dos induces HepG2 cell apoptosis by notch1-NF-120581B pathwayrdquoCarbohydrate Polymers vol 95 no 1 pp 282ndash287 2013

[29] WWongchana and T Palaga ldquoDirect regulation of interleukin-6 expression by Notch signaling in macrophagesrdquo Cellular andMolecular Immunology vol 9 no 2 pp 155ndash162 2012

[30] J C Choy D J Granville D W C Hunt and B M McManusldquoEndothelial cell apoptosis biochemical characteristics andpotential implications for atherosclerosisrdquo Journal of Molecularand Cellular Cardiology vol 33 no 9 pp 1673ndash1690 2001

[31] M Cuchel and D J Rader ldquoMacrophage reverse cholesteroltransport key to the regression of atherosclerosisrdquo Circulationvol 113 no 21 pp 2548ndash2555 2006

[32] A Yazdanyar C Yeang and X C Jiang ldquoRole of phospholipidtransfer protein in high-density lipoprotein-mediated reversecholesterol transportrdquo Current Atherosclerosis Reports vol 13no 3 pp 242ndash248 2011

[33] X Wang and D J Rader ldquoMolecular regulation of macrophagereverse cholesterol transportrdquo Current Opinion in Cardiologyvol 22 no 4 pp 368ndash372 2007

[34] S J Yang J M Choi L Kim et al ldquoNicotinamide improvesglucose metabolism and affects the hepatic NAD-sirtuin path-way in a rodent model of obesity and type 2 diabetesrdquo Journalof Nutritional Biochemistry vol 25 no 1 pp 66ndash72 2014

[35] L A Carlson L Oro and J Ostman ldquoEffect of a single dose ofnicotinic acid on plasma lipids in patients with hyperlipopro-teinemiardquo Acta Medica Scandinavica vol 183 no 5 pp 457ndash465 1968

[36] R H Stern J D Spence D J Freeman and A Parbtani ldquoTol-erance to nicotinic acid flushingrdquo Clinical Pharmacology andTherapeutics vol 50 no 1 pp 66ndash70 1991

[37] S M Grundy J I Cleeman C N Bairey Merz et al ldquoImpli-cations of recent clinical trials for the National CholesterolEducation Program Adult Treatment Panel III guidelinesrdquoCirculation vol 110 no 2 pp 227ndash239 2004

[38] M S Brown and J L Goldstein ldquoLowering plasma cholesterolby raising LDL receptorsrdquoNewEngland Journal ofMedicine vol305 no 9 pp 515ndash517 1981

[39] R Hampton D Dimster-Denk and J Rine ldquoThe biology ofHMG-CoA reductase the pros of contra-regulationrdquo Trends inBiochemical Sciences vol 21 no 4 pp 140ndash145 1996

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 13: Research Article Niacin Inhibits Vascular Inflammation via ...downloads.hindawi.com/journals/mi/2014/263786.pdf · Research Article Niacin Inhibits Vascular Inflammation via Downregulating

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom