Evaluation of the Effect of Andrographolide on … areas of alpha-SMA stain cell was microscopically...

Research ArticleEvaluation of the Effect of Andrographolide on AtheroscleroticRabbits Induced by Porphyromonas gingivalis

Rami Al Batran1 Fouad Al-Bayaty1 Mazen M Jamil Al-Obaidi1

Saba F Hussain2 and Tengku Z Mulok3

1 Center of Periodontology Studies Faculty of Dentistry Universiti Teknologi Mara (UiTM)40450 Shah Alam Selangor Darul Ehsan Malaysia

2 Center of Paediatric Dentistry and Orthodontics Studies Faculty of Dentistry Universiti Teknologi MARA (UiTM)40450 Shah Alam Selangor Darul Ehsan Malaysia

3 Center of Biomolecular Science Faculty of Applied Science Universiti Teknologi MARA (UiTM)40450 Shah Alam Selangor Darul Ehsan Malaysia

Correspondence should be addressed to Fouad Al-Bayaty drfouadhmyahoocom

Received 8 April 2014 Revised 2 July 2014 Accepted 4 July 2014 Published 18 August 2014

Academic Editor Kazuhiko Kotani

Copyright copy 2014 Rami Al Batran et alThis 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

Epidemiologic evidence has demonstrated significant associations between atherosclerosis and Porphyromonas gingivalis (Pg) Wehad investigated the effect of andrographolide (AND) on atherosclerosis induced by Pg in rabbits For experimental purpose weseparated thirty male white New Zealand rabbits into 5 groups Group 1 received standard food pellets Groups 2ndash5 were orallychallenged with Pg Group 3 received atorvastatin (AV 5mgkg) and Groups 4-5 received 10 and 20mgkg of AND respectivelyover 12 weeks Groups treated with AND showed significant decrease in TC TG and LDL levels (119875 lt 005) and significant increasein HDL level in the serum of rabbits Furthermore the treated groups (G3ndashG5) exhibited reductions in interleukins (IL-1120573 andIL-6) and C-reactive protein (CRP) as compared to atherogenicgroup (G2) The histological results showed that the thickening ofatherosclerotic plaques were less significant in treated groups (G3ndashG5) compared with atherogenicgroup (G2) Also alpha-smoothmuscle actin (120572-SMA) staining decreased within the plaques of atherogenicgroup (G2) while it was increased in treated groups(G3ndashG5) Lastly groups treated with AV andAND (G3ndashG5) showed significant reduction of CD36 expression (119875 lt 005) comparedto atherogenicgroup (G2) These results substantially proved that AND contain antiatherogenic activity

1 Introduction

Periodontal disease and heart disease are inflammatory con-ditions Generally it is considered that periodontal diseaseand heart disease are connected with each other however ithas not been clear whether having periodontal disease leadsto heart disease or vice versa According to the journal of theAmerican Medical Association [1] patients who have peri-odontal disease and at least one risk factor for heart diseaseshould have a medical evaluation for heart problems On theother hand patients who have heart disease should regularlycheck for signs of periodontal disease Pg is a nonmotile androd-shaped anaerobic gram-negative bacterium that can be

found in the mouth of an individual This bacterium is theprincipal cause of periodontal disease [2] Besides causinginfections to humans the way that it operates is very uniquesince it is a gram-negative bacterium it can attach to thesubgingival area of periodontal pocket and cause an inflam-mation of the periodontal tissue [3] Recent epidemiologicalstudies have demonstrated an association among periodontaldisease cardiovascular disease and atherosclerosis [4] Anassociation between atherosclerosis and Pg a major peri-odontopathogen has been shown [5] However the questionof whether this relationship is causal or coincidental stillexists Many individuals with evidence of atherosclerosishave demonstrated seropositivity to this pathogen Recently

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 724718 11 pageshttpdxdoiorg1011552014724718

2 BioMed Research International

evidence from diverse sources has suggested that Pg canactivate host innate immune responses associated withatherosclerosis [6] Various studies have confirmed thatthe inflammatory response to Pg could exacerbate vascularinflammation via secreted cytokines andor chemokineswhich ultimately develop atherosclerosis Meanwhile thecytokine and chemokines interact in the progression ofatherosclerosis Moreover the reaction of endothelial cellsin response to Pg and its various virulence factors arediverse and the expression of chemokine differs throughdifferent signal transduction pathways accordingly Resultsfrom these studies have reinforced the connection betweenPg and atherosclerosis and the role of Pg in the initiation andprogression of atherosclerosis has been presented [7]

A number of studies have reported the use of a largevariety of medicinal plants for treating several ailments [8ndash12] Andrographis paniculata (Burmf) Nees (Acanthaceae)is a long-established therapeutic plant commonly found inSouthEastAsia andoriginated from India to Indo-ChinaTheplant is generally known as ldquoking of bitterrdquoThemain bioactivecompound is AND as the core dynamic code and othercodes such as 14-deoxy-11 12-didehydroandrographolideand 14-deoxyandrographolide Recent studies on AND haveshown several pharmacological effects such as hypotensive[13] antihyperglycemic [14] accelerates wound healing [15]gastroprotective [16] antioxidant [11] and anticancer [17]Thepresent study has been aimed at assessing the antiatherogenicactivity of AND in atherosclerotic rabbits

2 Materials and Methods

21 Chemicals For the purpose of this study AND waspurchased from Sigma Aldrich (USA) and Pg strain 33277from ATCC (USA) whereas AV was acquired from theUniversity Malaya Medical Centre (UMMC) Pharmacy andused as the reference drug at a dose of 5mgkg body weight

22 Culturing Bacteria Under anaerobic condition PgATCC strain 33277 was cultured on anaerobic blood agarplates (Becton Dickinson Co) in an aerobic chamber (CoyLaboratory Products Inc) with 85N

2 5H

2 and 10CO

2

for 3 to 5 days and then inoculated into Schaedler broth(Difco Laboratories) containing hemin and menadione for24 hours according to the previous protocol [7] with somemodifications

23 Animal Ethics Thirty healthy male white New Zealandrabbits (3ndash35 kg) were acquired from the Animal HouseUniversity Technology Mara and the experiment wasapproved by the Committee of Faculty of Dentistry Uni-versity Technology Mara Ethic number [28052013 600-FF(PT 52)] The experimental rabbits were placed in separatecages and maintained on a twelve-hour daynight cycle atan ambient temperature with ad libitum access to food andwater

24 Maximum Tolerated Dose (MTD) Sixteen rabbits wereused in this experiment and were divided into two groups

comprising eight male and eight female rabbits All rabbitshad free access to water and given normal diet All animalswere clinically examined on daily basis during the experi-ments AND was diluted with water and orally administeredto different groups at doses of 50 100 and 500mgkg bodyweight daily for four weeks During the course of experimentwe had observed and recorded any possible changes onphysical appearance body weight and mortality

25 Animal Grouping Rabbits were divided into the follow-ing 5 groups (119873 = 6 per group)

Group 1 (normal control) Rabbits were continuously fed withthe standard pellets for 12 weeks

Group 2 (atherogenic control) Rabbits were continuouslychallenged orally with Pg ATCC 33277 (02mL of 15 times 1011990912bacterial cellsmL in 2CMCwith PBS) five times a week for12 weeks

Group 3 (atherogenic + AV) Rabbits were continuouslychallenged orally with Pg ATCC 33277 (02mL of 15 times 1011990912bacterial cellsmL in 2 CMC with PBS) five times a weekand continuously fed daily with AV (5mgkg) for 12 weeks

Group 4 (atherogenic + AND LD) Rabbits were continuouslychallenged orally with Pg ATCC 33277 (02mL of 15 times 1011990912bacterial cellsmL in 2CMCwithPBS) five times aweek andcontinuously fed daily with AND low dose (10mgkg) for 12weeks

Group 5 (atherogenic + ANDHD) Rabbits were continuouslychallenged orally with Pg ATCC 33277 (02mL of 15 times 1011990912bacterial cellsmL in 2 CMC with PBS) five times a weekand continuously fed daily with AND high dose (20mgkg)for 12 weeks

26 Biochemical Measurement The experimental rabbitswere kept under fasting condition for at least 12 hoursbefore blood sampling to allow the relevant estimationof lipid profile levels Blood was taken from the ear veinof nonanaesthetized rabbit into an open plain tube andcentrifuged at 3500 g for 15min to obtain serum The levelsof total cholesterol (TC) triglycerides (TG) low-densitylipoprotein-cholesterol (LDL) high-density lipoprotein-cholesterol (HDL) and C-reactive protein (CRP) wereanalyzed from the serum of rabbits at the Universityof Malaya Medical Centre to evaluate the changes inbiomarkers using an automatic biochemistry analyzer(Dimension AR DuPont USA)

27 Proinflammatory Cytokines Measurement IL-1120573 and IL-6 concentrations were measured in serum using ELISA(enzyme-linked immunosorbent assay) (Abcam USA) withsensitivity of 1 pgmL and without cross reactivity againstother cytokines according to the manufacturerrsquos recommen-dations

BioMed Research International 3

Table 1 Significance was defined as lowast119875 lt 005 compared to atherogenicgroup (G2) 119875 lt 005 indicates significant difference versus normalcontrol (G1)

Animal group TC (mmolL) LDL (mmolL) HDL (mmolL) TG (mmolL)Group 1 144 plusmn 001 058 plusmn 002 057 plusmn 001 053 plusmn 001

Group 2 2621 plusmn 029

2469 plusmn 014

044 plusmn 001

131 plusmn 002

Group 3 1521 plusmn 012lowast

1365 plusmn 016lowast











TC total cholesterol LDL low-density lipoprotein HDL High-density lipoprotein TG triglyceridesGroup 1 normal control Group 2 atherogenic control Group 3 atherogenic + AV Group 4 atherogenic + AND LD Group 5 atherogenic + AND HDStatistical analysis of the data was carried out using one-way analysis of variance (ANOVA) and Tukeyrsquos post hoc test for average comparison on SPSS 180Mean values plusmn SEM (119873 = 6) were used Significance difference was defined as lowast119875 lt 005 compared to atherogenic group (G2) 119875 lt 005 compared to normalcontrol (G1)

28 Histopathological Study of Aorta Aorta was harvestedfrom the rabbits and placed in formaldehyde 10 24 h lateraorta was embedded in paraffin cut at 4 120583m stained withhematoxylin and eosin (HampE) and then scanned to assesspathological changes

29 Immunohistochemistry Study Shortly 4-120583m sections ofaortic tissue blocks were placed on poly-L-lysine coated slidesfor the purpose of performing immunohistochemistry Theslide sections were immersed in target retrieval solution(DAKO Lot 10069393) heated in microwave oven at 98∘Cfor 20 minutes (maximum power 700W) and then cooled atroom temperature anti-alpha smooth muscle actin antibody(abcam ab7817) 150 dilution was used as primary anti-body as previously described [18] Briefly the sections wereincubated with each primary antibody as mentioned abovefor 1 hour after rinsing thrice in Dako wash buffer (TBS)they were incubated with biotinylated secondary antibodyby following kit (LSAB system2-HRP) (Lot 10069908) for 1hour at room temperature After TBS rinses the sectionswereincubated with streptavidin-horseradish peroxidase conju-gate for additional 30 minutes at room temperature (DAKO)followed by a course of incubation in diaminobenzidine(DAB) DAKO (lot 10067468) Control immunohistochem-istry reactions were performed to evaluate the specificityof the labels omitting the primary antibody Staining withhematoxylin was performed and used as a reference of thecytoarchitecture of the tissue

210 Morphometric Analysis Images were captured usinga Nikon microscope (Y-THS Japan) by two experiencedobservers in an independent and blinded fashion with theatherogenic control and treatment groups The mean per-centage of thickening of the intimal layer foam cells anddensity areas of alpha-SMA stain cell was microscopicallyassessed Several sections were microscopically analyzedunder high power fields (HPF) for each experimental groupusing an optical image analyzer (ImagePro Plus 45 MediaCybernetics Silver Spring MD)

211 Western Blotting Homogenized samples from the aortawere separated on 4ndash20 sodium dodecyl sulphate (SDS-PAGE) gels and the proteins were transferred to polyvinyli-dene fluoride (PVDF) membrane Briefly the membranes

were blocked with 5 nonfat milk followed by primary anti-bodies recognizing anti-CD36 antibody (ab78054) 11000dilution and incubated at 4∘C overnight The membraneswere washed and incubated with HRP-conjugated goat anti-rabbit IgGThe densities were normalized to the total amountof protein loaded in eachwell as determined by densitometryanalysis of PVDF membranes and stained with AmershamECL PrimeWestern Blotting Detection Reagent (GE Health-care) The proteins were visualized by chemiluminescence(UVP Bio Spectrum USA) and the densities of specificbands were quantitated by densitometry using (Image J 137software NIH USA) [19] Housekeeping protein 120573-actin(1 1000) was used as loading control

212 Statistical Analysis All values were reported as mean plusmnSEM Data were analyzed by one-way ANOVA and Tukeyrsquospost hoc test for multiple comparison using SPSS 18 (SPSSInc Chicago Illinois USA) Significancewas defined as lowast119875 lt005 compared to atherogenic group (G2)

3 Results

31 Maximum Tolerated Dose (MTD) of AND The resultspresented in supplementary data (see Tables S1 and S2 inSupplementary Material available online at httpdxdoiorg1011552014724718) show maximum tolerated dose of ANDin male and female rabbits Toxicology was assessed on themortality rateThe dosages of AND at 50 100 and 500mgkgbody weight did not produce any mortality to the rabbitsduring four weeks

32 Effect of AND on Lipid Profile Table 1 shows the levelof lipid parameters in serum among different experimen-tal groups Treated groups (G3ndashG5) showed a significantreduction of TG level (119875 lt 005) as against atherogenicgroup (G2) at a value of 131 plusmn 001mmolL Meanwhileatherogenic group (G2) had higher level of TG comparedto normal control group (G1) AV group (G3) had the mostsignificant reduction of TG level followed by AND HD andAND LD Atherogenic group (G2) had higher level of TCcompared to normal control group (G1)The total cholesterollevel (TC) value for AV group (G3) was 1521 plusmn 012mmolLwhich was the most significant reduction of TC level (119875 lt005) followed by AND HD group (G5) at value of 1620 plusmn


018mmolL compared to atherogenic group (G2) at valueof 2621 plusmn 029mmolL Furthermore AND LD (G4) hadsignificantly reduced the TC level compared to atherogenicgroup (G2) however it was less effective than AV and ANDHD (G3 and G5)

The LDL level was significantly lower (119875 lt 005) inthe AV and AND HD groups (G3 G5) at values of 1365 plusmn016mmolL and 1437 plusmn 022mmolL compared with theatherogenic group (G2) Atherogenic group (G2) had higherlevel of LDL compared to normal control group (G1) TheLDL level for AND LD (G4) was significantly decreasedcompared to atherogenic group (G2) The HDL level in theatherogenic group (G2) was significantly decreased (119875 lt005) with a value of 044plusmn001mmolL compared to normalcontrol group (G1) Meanwhile the AND HD group (G5)had significantly increased HDL level at value of 064 plusmn001mmolL compared to atherogenic group (G2) The AVgroup (G3) had a HDL level of 065 plusmn 001mmolL that wassignificantly higher than the atherogenic group (G2) Basedon the lipid profile results AV group (G3) had showed thebest results followed by AND HD (G5) and AND LD (G4)compared to atherogenic group (G2)

33 Effect of AND on C-Reactive Protein C-reactive pro-tein (CRP) level in serum was measured to determine theinflammation status of the experimental groups as shownin Figure 1 Generally the atherogenic group (G2) hadsignificantly higher levels of CRP (119875 lt 005) at value392 plusmn 009 (120583gmL) compared with the normal group (G1)Outstandingly the AV and AND HD groups (G3 and G5)had significantly lower levels of CRP (119875 lt 005) at values306 plusmn 003 (120583gmL) and 314 plusmn 001 (120583gmL) respectivelycompared with the atherogenic group (G2) The CRP levelfor AND LD (G4) was significantly decreased compared toatherogenic group (G2) but was at higher value than the AVand AND HD groups (G3 and G5)

34 Effect of AND on Proinflammatory Cytokines The effectof AND on interleukin 1 (IL-1120573) activity in the differentexperimental groups is shown in Figure 2 Generally rabbitsin the atherogenic group (G2) had significantly higher (119875 lt005) levels of IL-1120573 at a value of 8433 plusmn 067 (pgmL)compared with the normal control group (G1) Notably theexperimental rabbits treatedwith ANDLD andHD (G4 G5)had significantly lower levels of IL-1120573 at values of 6245plusmn143(pgmL) and 5595 plusmn 116 (pgmL) respectively compared tothe atherogenic group (G2) Moreover there were significantdifferences in the tested IL-1120573 level between AND HD (G5)and AV (G3) at a value of 5207 plusmn 071 (pgmL)

The effect of AND on interleukin 1 (IL-6) activity amongthe different experimental groups is shown in Figure 3Generally the IL-6 results were similar to the IL-1120573 resultsThe IL-6 level in the atherogenic group (G2)was the highest ata value of 44264 plusmn 075 (pgmL) among the groups Howeverthe AV group (G3) had the lowest IL-6 levels at a value of31535plusmn 078 (pgmL) compared with the atherogenic group(G2) The AND HD group (G5) had significantly (119875 lt 005)decreased IL-6 levels compared to the atherogenic group

000050100150200250300350400450

G1 G2 G3 G4 G5Animal groups

CRP(120583

gm

L)

lowast lowast lowast

Figure 1 The effect of AND on C-reactive protein (CRP) inatherosclerotic rabbits Group 1 normal control Group 2 athero-genic control Group 3 atherogenic + AV Group 4 atherogenic +ANDLDGroup 5 atherogenic +ANDHD Statistical analysis of thedata was carried out using one-way analysis of variance (ANOVA)andTukeyrsquos post hoc test for average comparison on SPSS 180Meanvaluesplusmn SEM (119873 = 6) were used Significance differencewas definedas lowast119875 lt 005 compared to atherogenic group (G2)

119875 lt 005

compared to normal control (G1)

000100020003000400050006000700080009000

10000


IL-1120573

(pg

mL)

lowast

lowastlowast

Figure 2 The effect of AND on interleukin 1 (IL-1120573) in serum inatherosclerotic rabbits Group 1 normal control Group 2 athero-genic control Group 3 atherogenic + AV Group 4 atherogenic +ANDLDGroup 5 atherogenic +ANDHD Statistical analysis of thedata was carried out using one-way analysis of variance (ANOVA)andTukeyrsquos post hoc test for average comparison on SPSS 180Meanvaluesplusmn SEM (119873 = 6) were used Significance differencewas definedas lowast119875 lt 005 compared to atherogenic group (G2)

119875 lt 005


(G2) Furthermore AND LD (G4) had shown significantlydecreased IL-6 levels (119875 lt 005) compared to the atherogenicgroup (G2) but was at higher value than AV and AND HD(G3 and G5)

35 Histopathological Findings Histopathological examina-tion of aorta sections from experimental groups is shownin Figure 4 Histological examination of the hematoxylinand eosin-stained section of aorta of normal group (G1)(rabbits fed with a normal diet) showed normal histologicalexamination of aorta with no aortic lesion or presence offoam cells while the aorta examination of the atherogenic


0005000

100001500020000250003000035000400004500050000


lowast

lowastlowast

IL-6

(pg

mL)


119875 lt 005


group (G2) showed thickening of the intimal layer andaccumulation of lipids leading to format foam cells signalingthe early phase in the development of atherosclerosis Theaorta of the groups treated with AV and AND (G3 G4and G5) showed reduced accumulation of atheroscleroticlesion due to decreased number of foam cells and cholesteroldeposits in the aorta The morphometric analysis of aortasection results had showed that the thickening of the intimallayer and foam cells was significantly increased (119875 lt 005)in atherogenic group (G2) compared to normal controlgroup (G1) Furthermore treated groups (G3ndashG5) showedsignificantly decreased intimal layer and foam cells (119875 lt005) compared with atherogenic group (G2)

36 Immunohistochemistry Assessment Immunohistochem-ical evaluation of alpha-smooth muscle actin (120572-SMA) pro-tein expression in aorta section and histomorphometricanalysis from experimental groups are shown in Figure 5Atherogenic group (G2) showed lesser 120572-SMA stainingcompared to normal control group (G1) which had morestain intake of 120572-SMA On other hand AV group (G3)and AND HD group (G5) showed strong 120572-SMA proteinexpression compared to atherogenic group (G2) while ANDLD (G4) showed less 120572-SMA protein expression comparedto AV group (G3) and AND HD group (G5) Atherogenicgroup (G2) had demonstrated significant reduction (119875 lt005) of 120572-SMA protein expression in comparison to normalcontrol group (G1) However treated groups (G3ndashG5) hadshowed significantly elevated protein expression (119875 lt 005)compared to atherogenic group (G2)

37 Western Blot Analysis The effect of AND on CD36expression in aortic tissue homogenate in different experi-mental groups is shown in Figure 6Throughout the westernblot analysis we undertook a comparable analysis of CD36

protein in the tissue homogenates of each group where the120573-actin protein was considered as the loading control Theresult showed that CD36 was significantly inhibited in thoserabbits which received the AV and AND (G3 G4 and G5)compared to the atherogenic group (G2) On the other handthe expression of CD36 protein was the lowest in AV group(G3) followed by the AND HD group (G5) and then ANDLD group (G4) These results had confirmed the role ofAND in inhibiting macrophage and lipid accumulation inatherosclerotic rabbits

4 Discussion

MTD was administrated at various doses (50 100 and500mgkgday of AND) for four weeks in male and femalerabbits However AND did not produce any mortality tothe rabbits From this experiment it can be concluded thatthe rabbits did not have mortality even at maximum dosageof 500mgkg body weight The experimental induction ofatherosclerosis using rabbits is widely accepted in studiesof atherosclerosis The rabbitrsquos abdominal aorta is a perfectmodel for atherosclerosis which can imitate human lesions ifcorrect food anddietary conditions are given to the rabbit [2021] Using experimental animals such as rabbits is crucialas it allows for consideration of factors such as diet lengthof experiment pathogenesis of disease and the impact ofdosage and the duration of feeding which later yields betterand more reliable results Humans have a specific range ofblood lipid profiles which indicates whether patients aresuffering from various blood and lipid disorders Unlikerabbits rabbits do not have specific reference range of bloodlipid profiles level for atherosclerotic condition [22] There-fore lipid profiles in atherosclerotic rabbits were undertakenbased on a comparison of significant andnonsignificant levelsof lipid profile among the five different groups (G1ndashG5)

This present study had showed that challenging with Pgfive times a week was sufficient to induce atherosclerosisin rabbits Furthermore Pg had significantly increased thetotal cholesterol (TC) level in experimental groups Physio-logically a high TC level is caused by excessive loading ofcholesterol in the liver These cholesterols are supposed tobe metabolized in the liver however the excess cholesterolis then recycled in the circulating blood As the rabbitswere in a hypercholesterolemic state it was noted that ANDcould significantly reduce the TC level (119875 lt 005) whencompared to the atherogenic group (G2) this had showedthat AND has a positive effect in lowering the TC level ina hypercholesterolemic state Lowering the total cholesterolserum is the primary objective of any treatment in CVDin attenuating hypercholesterolemic conditions [23] further-more 10 reduction on the concentration of total cholesterolserum can reduce the mortality rate to 15 [24] totalcholesterol could be reduced in several ways which includesintake of a sufficient dose somehowmanaged to increase LDLreceptor proteins in the liver tissue [25ndash27] At the same timethere was an increase of LDL receptor mRNA as the functionof receptor is to eliminate LDL in the blood which furthertranslates into a lower total cholesterol level The higher LDL


05

1015202530354045

G1 G2 G3 G4 G5

lowast

lowastlowast

G3 G4 G5

TI TITI

TM TMTM

G1 G2

TI

TI

TM TM

Thickening of the intimal layer ()Foam cells ()

Figure 4 The effect of AND on histological sections of aorta in atherosclerotic rabbits (HampE) TI tunica intima TM tunica mediaarrowheads indicated the foam cells Group 1 normal control Group 2 atherogenic control Group 3 atherogenic + AV Group 4 atherogenic+ AND LD Group 5 atherogenic + AND HD Image analysis was accomplished using an optical image analyzer (ImagePro Plus 45 MediaCybernetics Silver Spring MD) The data are expressed as percentage means plusmn SEM (119899 = 6) and were analysed using one-way analysis ofvariance (ANOVA) followed by Tukeyrsquos post hoc test for average comparisons on SPSS 180 Significance difference was defined as lowast119875 lt 005compared to atherogenic group (G2) 119875 lt 005 compared to normal control (G1)

level can be attributed to the reduction of LDL receptorsby cholesterol in the liver [28] It is postulated that Pg caninduce an elevation of TC and LDL levels and reduce HDLlevels as well [6] The reduction in the LDL can be attributedto AND which has an antihyperlipidemic property [10]Furthermore the antioxidant activity of AND might protectLDL against coppermediatedmodification by decreasing thebinding of Cu2+ to LDL The decreased binding of Cu2+ toLDLoccurred when antioxidants reactedwith specific aminoacid residue on apolipoprotein B which normally bondswith Cu2 [29] Competition between antioxidants and Cu2+over apolipoprotein B leads to a decreased affinity betweenapolipoprotein B and Cu2+ ions Moreover AND can be

taken as a supplement to prevent elevation of LDL as thislipoprotein can easily cause health complications (eg CVD)The level of HDL in the body indicates a risk factor for thedevelopment of atherosclerosis The lower the HDL levelthe higher the risk of having atherosclerosis PhysiologicallyHDL transports cholesterol from the peripheral tissue tothe liver for catabolism to be converted into bile salt [30]This physiological pathway involving HDL as cholesterolcarrier is important in reducing the cholesterol level in theblood In this study the rabbits treated with AND and AVhad shown increased HDL level in their blood as againstthe atherogenic group (G2) This is consistent with clinicalstudies conducted on simvastatin which concluded that the


000

500

1000

1500

2000

2500

3000

3500

4000

G1 G2 G3 G4 G5

lowastlowast

lowast

120572-S

MC

area

()

G1 G2

G3 G4 G5

Figure 5 The effect of AND on 120572-smooth muscle actin of aorta in atherosclerotic rabbits Arrows indicated were the positive staining of120572-SMA Group 1 normal control Group 2 atherogenic control Group 3 atherogenic + AV Group 4 atherogenic + AND LD Group 5atherogenic + AND HD Image analysis was accomplished using an optical image analyzer (ImagePro Plus 45 Media Cybernetics SilverSpring MD) The data are expressed as percentage means plusmn SEM (119899 = 6) and were analysed using one-way analysis of variance (ANOVA)followed by Tukeyrsquos post hoc test for average comparisons on SPSS 180 Significance difference was defined as lowast119875 lt 005 compared toatherogenic group (G2) 119875 lt 005 compared to normal control (G1)

latter was able to moderately increase the HDL level [31]Furthermore Hernandez-Presa et al [32] have discoveredthat there was a reduction of plasma TG levels in rabbitwhich had been induced with hypercholesterolemia aftertreatment with simvastatin this finding is consistent withour results which had revealed that AND might reduce thehypercholesterolemia status due to the antioxidant propertiesof AND

Vascular inflammation is mediated by numerous celltypes that communicate with each other through a seriesof cytokine-receptor-mediated interactions allowing bidirec-tional crosstalk between resident vascular cells and inflam-matory cells [33] For effective communication to takeplace these disparate cell types evolved a common set ofsoluble cytokine ligands and specific membrane receptors

which allow them to transmit their effects into the cellMost cytokines initiate a complex and varied repertoire ofresponses in their target cells and can initiate advance andpotentially resolve atherogenic inflammationThemicroenvi-ronment of the atherosclerotic plaque is a dynamic collectionof resident vascular and infiltrating inflammatory cells andtheir cytokine products This complex milieu consists ofmultiple cytokines with redundant pleiotropic and oppos-ing effects and the balance of pro- and anti-inflammatorycytokines often determines plaque severity and stabilityCells present in the lesion must interrogate and respond tothis multitude of factors in an appropriate fashion whichtogether often result in the development and progressionof atherosclerosis [34 35] The inflammatory process in theatherosclerotic artery may lead to increased blood levels of


0

01

02

03

04

05

06

07

08

09

G1 G2 G3 G4 G5

lowast

lowast

lowast

CD36

CD36

120573-Actin

G1 G2 G3 G4 G5

53kDa

43kDa

Figure 6 The effect of AND on CD36 expression in aortic tissue homogenate in atherosclerotic rabbits Group 1 normal control Group 2atherogenic control Group 3 atherogenic + AV Group 4 atherogenic + AND LD Group 5 atherogenic + ANDHDThe data were analyzedby density color and displayed as mean plusmn SEM Significance difference was defined as lowast119875 lt 005 compared to atherogenic group (G2)119875 lt 005 compared to normal control (G1)

inflammatory cytokines and other acute-phase reactants [36]In our study we had noticed an elevation in the plasmaCRP of those rabbits orally challenged with Pg A similarfinding was observed by other studies [37] CRP has thoughtto be synthesized solely in the liver after stimulation bycytokines such as IL-6 and TNF-120572 [38] Previous studieshave found that high dietary cholesterol intake can increasethe production of atherogenic inflammatory cytokines suchas IL-6 and TNF120572 [39] In addition hypercholesterolemiamay induce CRP secretions in adipocytes by reducingthe expression of peroxisome proliferator-activated receptor(PPAR-120574) [40] Therefore administration of AND over 12weeks could reduce the levels of CRP compared with theatherogenic group (G2) and that could be due to the anti-inflammatory activity of AND This is consistent with Al-Aubaidy et al [41] who have concluded that administrationof Aliskiren that has antioxidant and anti-inflammatoryeffects could reduce the TNF120572 and CRP in hyperlipidemicrabbit

The IL-1 family is composed of four proteins that sharesequence homology IL-1120572 IL-1120573 IL1 receptor antagonistand IL-18 IL-1120573 immune reactivity is found in monocytemacrophage EC and VSMC in human and experimentalatherosclerotic plaque According to several epidemiologicaland experimental studies natural or synthetic productshaving anti-inflammatory action are proven to have a strongpreventive effect on the development of atherosclerosis [42]To investigate the potential anti-inflammatory activity ofAND as an antiatherogenic agent we had evaluated therole of AND on the inflammatory progression of early

atherosclerosis in rabbits After 12 weeks of AND treatmentalong with oral challenge with Pg the levels of IL-1120573 inserum were dropped due to the treatment of AND Thisindicates that AND can prevent atherosclerosis by alleviatingvascular inflammation This is consistent with Kong et al[43] who have demonstrated that Kaempferol has an anti-inflammatory effect on early atherosclerosis in rabbits feda high cholesterol diets Furthermore AV had significantlyreduced the level of IL-1120573 this is due to the fact thatAV belongs to the statin category and statins can act asanti-inflammatory agents as well as having lipid-loweringeffects [44] Emerging data suggest that the proinflammatorycytokine IL-6 secreted by adipocytes plays an importantrole in atherosclerosis In the present study we have showedthat treatment with AV and AND had significantly reducedIL-6 This result has demonstrated that AND has an anti-inflammatory effect in atherosclerosis which may reduce IL-6 secretion from adipocytes in atherosclerotic rabbits Thisfinding is in line with that of Zhao and Zhang [45] whohave demonstrated that AV has decreased IL-6 secretion inhypercholesterolemic rabbits Recent studies have demon-strated that atherosclerosis may affect the expressions of awide variety of adhesion molecules which increases the pro-duction of atherogenic inflammatory cytokines [46] Thesestudies indicate that Pg may induce a systemic inflammationresponse We have observed that changes in the circulatingconcentration of IL-6 were nearly identical to the changesin plasma cholesterol concentration throughout the studyproviding further evidence that atherosclerosis might inducesystemic inflammatory responses directly


Macrophages SMCs and T cells have been considered asthe key cells involved in atherosclerosis lesion developmentMacrophages evolve into foam cells after lipid uptake whichfinally leads to early fatty streak formation T cells themain cells in cellular immunity reach intima at an earlystage of atherosclerosis formation Activated T cells producecytokines taking part in regulating lesion formation [47]Meanwhile as atherosclerosis progresses vascular SMCs arein proximity to and physically interact with inflammatorycell types for example monocytes and macrophages whichplay a very important role in further exacerbating the disease[48] The normal cell structure and morphology of normalhealthy aorta can be seen in the normal group (G1) Theresults showed that foam cells aortic lesion or accumulationof cholesterol deposits was not present in the aortas of normalgroup (G1) because the rabbits in the normal group weregiven only a normal diet In the atherogenic group (G2)the histology results showed that there are thick plaquesand lipids accumulation in the tunica intima regions ofthe aorta The structure of smooth muscle cells in themedia was changed The intima was irregularly thickenedand multilaminated The internal elastic lamina was alsothickened Fatty streaks were developed at the intimal layerand progressed into plaque Overall the plaque in the picturewas considered as well as developed atheromatous plaquewith a fibrous cap encircling the top of the plaque Thefibrous cap was composed of proliferating smooth musclecellsmacrophages lymphocytes foamcells and extracellularmatrix Atherogenic group was characterized by an increasedpopulation of macrophage-derived foam cells formed due toincreased uptake of oxidized LDL by smooth muscle cellsand macrophages The formation of foam cells signals theearly phase in the development of atherosclerosis [49] Theintimal elastic layer in atherogenic group was not clearlyseen between intima and media layer as the smooth musclewas infiltrated with macrophages and inflammatory cells Itwas observed that thickness was prominent in the foam cellsin the atherogenic group (G2) compared with the groupstreated with AV and AND This result is consistent with astudy conducted on rabbits fed with cholesterol and treatedwith Nifedipine byHenry and Bentley [50]The aortic lesionsthat were present in the groups treated with AV and ANDwere visibly thinner than those in atherogenic group (G2)Histopathological examination of treated groups revealed alesser presence of foam cells Less cholesterol deposits wereseen in the aorta of the AV and AND groups with very littledeposits observed in the intima tunica and medica tunicaregion and no cholesterol deposit was seen in the adventitiatunica region this could be due to the anti-inflammatoryproperty of AV and AND Same phenomenon has been seenin study conducted by Bobek andGalbavy using oystermush-room in rabbits [51] Decreased depositions of cholesterol intreated group were represented by lesser lipid laden foamcells which were originally derived from circulating totalcholesterol The accumulations of atherosclerotic plaques arerelated to the amount of serum total cholesterol [52]This wasclearly seen when cholesterol found within the arterial wallwas proportional to the serum cholesterol level in the lipidprofile test Furthermore immunohistochemical andwestern

blot tests have confirmed stronger positive expressions ofSMCs and reduction of macrophage in aortas of groupstreated with AV and AND This is consistent with a studyconducted by Rosenfeld and Ross on smoothmuscle cell pro-liferation in atherosclerotic lesions in hypercholesterolemicfat-fed rabbits [53]

5 Conclusion

In summary this study had revealed that AND has the abilityto improve or inhibit ailments such as hypercholesterolemiaand atherosclerosis induced in rabbits orally challenged withPg Furthermore AND has showed the ability to significantlyinhibit the formation and progression of atherosclerosisinduced by Pg which is one of the pathogenic microorgan-isms in chronic periodontitis This potential effect could beattributed to the anti-inflammatory effect of AND which wasinvolved in the reduction of proinflammatory cytokines

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This study was financially supported by The Ministry ofHigher Education (MOHE) [Grant No 600-RMIFRGS53(302014)] and Universiti Teknologi Mara (UiTM) ShahAlam Selangor Malaysia

References

[1] P P Hujoel M Drangsholt C Spiekerman and T A DeRouenldquoPeriodontal disease and coronary heart disease riskrdquo Journal ofthe AmericanMedical Association vol 284 no 11 pp 1406ndash14102000

[2] S C Holt L Kesavalu S Walker and C A Genco ldquoVirulencefactors of Porphyromonas gingivalisrdquo Periodontology 2000 vol20 no 1 pp 168ndash238 1999

[3] L Li E Messas E L Batista Jr R A Levine and S AmarldquoPorphyromonas gingivalis infection accelerates the progressionof atherosclerosis in a heterozygous apolipoprotein E-deficientmurine modelrdquo Circulation vol 105 no 7 pp 861ndash867 2002

[4] M Leinonen and P Saikku ldquoEvidence for infectious agents incardiovascular disease and atherosclerosisrdquo The Lancet Infec-tious Diseases vol 2 no 1 pp 11ndash17 2002

[5] J Beck R Garcia G Heiss P S Vokonas and S OffenbacherldquoPeriodontal disease and cardiovascular diseaserdquo Journal ofPeriodontology vol 67 no 10 pp 1123ndash1137 1996

[6] F C Gibson III C Hong H Chou et al ldquoInnate immunerecognition of invasive bacteria accelerates atherosclerosis inapolipoprotein E-deficient micerdquo Circulation vol 109 no 22pp 2801ndash2806 2004

[7] F C Gibson III H Yumoto Y Takahashi H-H Chouand C A Genco ldquoInnate immune signaling and Porphy-romonas gingivalis-accelerated atherosclerosisrdquo Journal of Den-tal Research vol 85 no 2 pp 106ndash121 2006


[8] R Al Batran F Al-BayatyMAmeenAbdulla et al ldquoGastropro-tective effects ofCorchorus olitorius leaf extract against ethanol-induced gastric mucosal hemorrhagic lesions in ratsrdquo Journal ofGastroenterology and Hepatology vol 28 no 8 pp 1321ndash13292013

[9] R Al Batran F Al-Bayaty MM Jamil Al-Obaidi et al ldquoIn vivoantioxidant and antiulcer activity of Parkia speciosa ethanolicleaf extract against ethanol-induced gastric ulcer in ratsrdquo PLoSONE vol 8 no 5 Article ID e64751 2013

[10] R Al Batran F Al-Bayaty M M J Al-Obaidi and M AAbdulla ldquoAcute toxicity and the effect of andrographolideon porphyromonas gingivalis-induced hyperlipidemia in ratsrdquoBioMed Research International vol 2013 Article ID 594012 7pages 2013

[11] R Al Batran F H Al-Bayaty and M M J Al-Obaidi ldquoIn-Vivoeffect of andrographolide on alveolar bone resorption inducedby Porphyromonas gingivalis and its relation with antioxidantenzymesrdquo BioMed Research International vol 2013 Article ID276329 6 pages 2013

[12] S M Dhiyaaldeen M A Alshawsh S M Salama et al ldquoPoten-tial activity of 3-(2-Chlorophenyl)-1-phenyl-propenonein accel-erating wound healing in ratsrdquo BioMed Research Internationalvol 2014 Article ID 792086 10 pages 2014

[13] C Y Zhang andB Tan ldquoHypotensive activity of aqueous extractof Andrographis paniculata in ratsrdquo Clinical and ExperimentalPharmacology and Physiology vol 23 no 8 pp 675ndash678 1996

[14] X Zhang and B K Tan ldquoAntihyperglycaemic and anti-oxidantproperties of Andrographis paniculata in normal and diabeticratsrdquo Clinical and Experimental Pharmacology and Physiologyvol 27 no 5-6 pp 358ndash363 2000

[15] F H Al-Bayaty M A Abdulla M I A Hassan and H MAli ldquoEffect of Andrographis paniculata leaf extract on woundhealing in ratsrdquoNatural Product Research vol 26 no 5 pp 423ndash429 2012

[16] S Q Wasman A A Mahmood L S Chua M A Alshawshand S Hamdan ldquoAntioxidant and gastroprotective activities ofAndrographis paniculata (Hempedu Bumi) in Sprague Dawleyratsrdquo Indian Journal of Experimental Biology vol 49 no 10 pp767ndash772 2011

[17] E Amroyan E Gabrielian A Panossian G Wikman andH Wagner ldquoInhibitory effect of andrographolide from Andro-graphis paniculata on PAF-induced platelet aggregationrdquo Phy-tomedicine vol 6 no 1 pp 27ndash31 1999

[18] E J Sohn C Kim Y S Kim et al ldquoEffects of magnolol(551015840-diallyl-221015840-dihydroxybiphenyl) on diabetic nephropathyin type 2 diabetic Goto-Kakizaki ratsrdquo Life Sciences vol 80 no5 pp 468ndash475 2007

[19] M Kumar S K Prasad S Krishnamurthy and S HemalathaldquoAntihyperglycemic activity of Houttuynia cordata Thunb instreptozotocin-induced diabetic ratsrdquo Advances in Pharmaco-logical Sciences vol 2014 Article ID 809438 12 pages 2014

[20] A Orlandi MMarcellini and L G Spagnoli ldquoAging influencesdevelopment and progression of early aortic atheroscleroticlesions in cholesterol-fed rabbitsrdquo Arteriosclerosis Thrombosisand Vascular Biology vol 20 no 4 pp 1123ndash1136 2000

[21] S G Cremers S J Wolffram and P D Weinberg ldquoAthero-protective effects of dietary l-arginine increase with age incholesterol-fed rabbitsrdquo British Journal of Nutrition vol 105 no10 pp 1439ndash1447 2011

[22] A Sarkar S C Lavania D N Pandey andM C Pant ldquoChangesin the blood lipid profile after administration of Ocimum

sanctum (Tulsi) leaves in the normal albino rabbitsrdquo IndianJournal of Physiology and Pharmacology vol 38 no 4 pp 311ndash312 1994

[23] M Seed F Hoppichler D Reaveley et al ldquoRelation of serumlipoprotein(a) concentration and apolipoprotein(a) phenotypeto coronary heart disease in patients with familial hypercholes-terolemiardquo The New England Journal of Medicine vol 322 no21 pp 1494ndash1499 1990

[24] A LGould J E RossouwNC Santanello J FHeyse andCDFurberg ldquoCholesterol reduction yields clinical benefit impactof statin trialsrdquo Circulation vol 97 no 10 pp 946ndash952 1998

[25] E Reihner M Rudling D Stahlberg et al ldquoInfluence ofpravastatin a specific inhibitor of HMG-CoA reductase onhepatic metabolism of cholesterolrdquoThe New England Journal ofMedicine vol 323 no 4 pp 224ndash228 1990

[26] NKume T Kita AMikami et al ldquoInduction ofmRNA for low-density lipoprotein receptors in heterozygous watanabe herita-ble hyperlipidemic rabbits treatedwithCS-514 (pravastatin) andcholestyraminerdquo Circulation vol 79 no 5 pp 1084ndash1090 1989

[27] A Miyazaki and T Koga ldquoLipid lowering effects of pravastatinin common marmosetsrdquo Arzneimittel-Forschung vol 48 no 2pp 154ndash160 1998

[28] V A Mustad T D Etherton A D Cooper et al ldquoReducingsaturated fat intake is associated with increased levels of LDLreceptors on mononuclear cells in healthy men and womenrdquoJournal of Lipid Research vol 38 no 3 pp 459ndash468 1997

[29] K L Retsky M W Freeman and B Frei ldquoAscorbic acidoxidation product(s) protect human low density lipoproteinagainst atherogenic modification Anti- rather than prooxidantactivity of vitamin C in the presence of transition metal ionsrdquoThe Journal of Biological Chemistry vol 268 no 2 pp 1304ndash1309 1993

[30] G Assmann H Schulte A Von Eckardstein and Y HuangldquoHigh-density lipoprotein cholesterol as a predictor of coro-nary heart disease risk The PROCAM experience and patho-physiological implications for reverse cholesterol transportrdquoAtherosclerosis vol 124 pp S11ndashS20 1996

[31] N Tahara H Kai M Ishibashi et al ldquoSimvastatin Attenu-ates Plaque Inflammation evaluation by FluorodeoxyglucosePositron Emission Tomographyrdquo Journal of the American Col-lege of Cardiology vol 48 no 9 pp 1825ndash1831 2006

[32] M A Hernandez-Presa M O Ortego J Tunon et al ldquoSim-vastatin reduces NF-120581B activity in peripheral mononuclear andin plaque cells of rabbit atheroma more markedly than lipidlowering dietrdquo Cardiovascular Research vol 57 no 1 pp 168ndash177 2003

[33] G K Hansson and P Libby ldquoThe immune response inatherosclerosis a double-edged swordrdquo Nature ReviewsImmunology vol 6 no 7 pp 508ndash519 2006

[34] M V Autieri ldquoPro- and anti-inflammatory cytokine networksin atherosclerosisrdquo ISRN Vascular Medicine vol 2012 ArticleID 987629 17 pages 2012

[35] K Takahashi M Takeya and N Sakashita ldquoMultifunctionalroles of macrophages in the development and progression ofatherosclerosis in humans and experimental animalsrdquo MedicalElectron Microscopy vol 35 no 4 pp 179ndash203 2002

[36] H Lu D L Rateri D L Feldman et al ldquoRenin inhibitionreduces hypercholesterolemia-induced atherosclerosis inmicerdquoJournal of Clinical Investigation vol 118 no 3 pp 984ndash9932008

[37] A Pejcic L J Kesic and J Milasin ldquoC-reactive protein as asystemic marker of inflammation in periodontitisrdquo European


Journal of Clinical Microbiology and Infectious Diseases vol 30no 3 pp 407ndash414 2011

[38] H J Moshage H M J Roelofs J F Van Pelt et al ldquoTheeffect of interleukin-1 interleukin-6 and its interrelationship onthe synthesis of serum amyloid A and C-reactive protein inprimary cultures of adult human hepatocytesrdquo Biochemical andBiophysical Research Communications vol 155 no 1 pp 112ndash1171988

[39] S M A Rahman A-M van Dam M Schultzberg and MCrisby ldquoHigh cholesterol diet results in increased expressionof interleukin-6 and caspase-1 in the brain of apolipoprotein Eknockout andwild typemicerdquo Journal ofNeuroimmunology vol169 no 1 pp 59ndash67 2005

[40] D Zhang D Che S Zhao and Y Sun ldquoEffects of atorvastatinon C-reactive protein secretions by adipocytes in hypercholes-terolemic rabbitsrdquo Journal of Cardiovascular Pharmacology vol50 no 3 pp 281ndash285 2007

[41] H A Al-Aubaidy H A Sahib B A Mohammad N R Hadiand S M Abas ldquoAntiatherosclerotic potential of aliskiren itsantioxidant and anti-inflammatory effects in rabbits a random-ized controlled trialrdquo Journal of Pharmaceutical Technology andDrug Research vol 2 no 1 p 11 2013

[42] R Altman H Ł Luciardi J Muntaner et al ldquoEfficacy assess-ment of meloxicam a preferential cyclooxygenase-2 inhibitorin acute coronary syndromes without ST-segment elevationthe nonsteroidal anti-inflammatory drugs in unstable anginatreatment-2 (NUT-2) pilot studyrdquo Circulation vol 106 no 2pp 191ndash195 2002

[43] L Kong C Luo X Li Y Zhou and H He ldquoThe anti-inflammatory effect of kaempferol on early atherosclerosis inhigh cholesterol fed rabbitsrdquo Lipids in Health and Disease vol12 no 1 article 115 2013

[44] JMusial A Undas P GajewskiM JankowskiW Sydor andASzczeklik ldquoAnti-inflammatory effects of simvastatin in subjectswith hypercholesterolemiardquo International Journal of Cardiologyvol 77 no 2-3 pp 247ndash253 2001

[45] S Zhao and D Zhang ldquoAtorvastatin reduces interleukin-6plasma concentration and adipocyte secretion of hypercholes-terolemic rabbitsrdquo Clinica Chimica Acta vol 336 no 1-2 pp103ndash108 2003

[46] S N Han L S Leka A H Lichtenstein L M AusmanE J Schaefer and S N Meydani ldquoEffect of hydrogenatedand saturated relative to polyunsaturated fat on immune andinflammatory responses of adults with moderate hypercholes-terolemiardquo Journal of Lipid Research vol 43 no 3 pp 445ndash4522002

[47] Z Mallat S Taleb H Ait-Oufella and A Tedgui ldquoThe role ofadaptive T cell immunity in atherosclerosisrdquo Journal of LipidResearch vol 50 pp S364ndashS369 2009

[48] H C Stary A B Chandler S Glagov et al ldquoA definition ofinitial fatty streak and intermediate lesions of atherosclerosis areport from the committee on vascular lesions of the council onarteriosclerosis American Heart Associationrdquo Arteriosclerosisand Thrombosis vol 14 no 5 pp 840ndash856 1994

[49] W B Kannel W P Castelli and T Gordon ldquoCholesterol in theprediction of atherosclerotic disease new perspectives based onthe Framingham studyrdquoAnnals of InternalMedicine vol 90 no1 pp 85ndash91 1979

[50] P D Henry and K I Bentley ldquoSuppression of atherogenesisin cholesterol-fed rabbit treated with nifedipinerdquo Journal ofClinical Investigation vol 68 no 5 pp 1366ndash1369 1981

[51] P Bobek and S Galbavy ldquoHypocholesterolemic and antiathero-genic effect of oystermushroom (Pleurotus ostreatus) in rabbitsrdquoFoodNahrung vol 43 no 5 pp 339ndash342 1999

[52] E B Smith ldquoTransport interactions and retention of plasmaproteins in the intima the barrier function of the internal elasticlaminardquo European Heart Journal vol 11 pp 72ndash81 1990

[53] M E Rosenfeld and R Ross ldquoMacrophage and smooth musclecell proliferation in atherosclerotic lesions of WHHL and com-parably hypercholesterolemic fat-fed rabbitsrdquo ArteriosclerosisThrombosis and Vascular Biology vol 10 no 5 pp 680ndash6871990

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


evidence from diverse sources has suggested that Pg canactivate host innate immune responses associated withatherosclerosis [6] Various studies have confirmed thatthe inflammatory response to Pg could exacerbate vascularinflammation via secreted cytokines andor chemokineswhich ultimately develop atherosclerosis Meanwhile thecytokine and chemokines interact in the progression ofatherosclerosis Moreover the reaction of endothelial cellsin response to Pg and its various virulence factors arediverse and the expression of chemokine differs throughdifferent signal transduction pathways accordingly Resultsfrom these studies have reinforced the connection betweenPg and atherosclerosis and the role of Pg in the initiation andprogression of atherosclerosis has been presented [7]

A number of studies have reported the use of a largevariety of medicinal plants for treating several ailments [8ndash12] Andrographis paniculata (Burmf) Nees (Acanthaceae)is a long-established therapeutic plant commonly found inSouthEastAsia andoriginated from India to Indo-ChinaTheplant is generally known as ldquoking of bitterrdquoThemain bioactivecompound is AND as the core dynamic code and othercodes such as 14-deoxy-11 12-didehydroandrographolideand 14-deoxyandrographolide Recent studies on AND haveshown several pharmacological effects such as hypotensive[13] antihyperglycemic [14] accelerates wound healing [15]gastroprotective [16] antioxidant [11] and anticancer [17]Thepresent study has been aimed at assessing the antiatherogenicactivity of AND in atherosclerotic rabbits

2 Materials and Methods

21 Chemicals For the purpose of this study AND waspurchased from Sigma Aldrich (USA) and Pg strain 33277from ATCC (USA) whereas AV was acquired from theUniversity Malaya Medical Centre (UMMC) Pharmacy andused as the reference drug at a dose of 5mgkg body weight

22 Culturing Bacteria Under anaerobic condition PgATCC strain 33277 was cultured on anaerobic blood agarplates (Becton Dickinson Co) in an aerobic chamber (CoyLaboratory Products Inc) with 85N

2 5H

2 and 10CO

2

for 3 to 5 days and then inoculated into Schaedler broth(Difco Laboratories) containing hemin and menadione for24 hours according to the previous protocol [7] with somemodifications

23 Animal Ethics Thirty healthy male white New Zealandrabbits (3ndash35 kg) were acquired from the Animal HouseUniversity Technology Mara and the experiment wasapproved by the Committee of Faculty of Dentistry Uni-versity Technology Mara Ethic number [28052013 600-FF(PT 52)] The experimental rabbits were placed in separatecages and maintained on a twelve-hour daynight cycle atan ambient temperature with ad libitum access to food andwater

24 Maximum Tolerated Dose (MTD) Sixteen rabbits wereused in this experiment and were divided into two groups

comprising eight male and eight female rabbits All rabbitshad free access to water and given normal diet All animalswere clinically examined on daily basis during the experi-ments AND was diluted with water and orally administeredto different groups at doses of 50 100 and 500mgkg bodyweight daily for four weeks During the course of experimentwe had observed and recorded any possible changes onphysical appearance body weight and mortality

25 Animal Grouping Rabbits were divided into the follow-ing 5 groups (119873 = 6 per group)

Group 1 (normal control) Rabbits were continuously fed withthe standard pellets for 12 weeks

Group 2 (atherogenic control) Rabbits were continuouslychallenged orally with Pg ATCC 33277 (02mL of 15 times 1011990912bacterial cellsmL in 2CMCwith PBS) five times a week for12 weeks

Group 3 (atherogenic + AV) Rabbits were continuouslychallenged orally with Pg ATCC 33277 (02mL of 15 times 1011990912bacterial cellsmL in 2 CMC with PBS) five times a weekand continuously fed daily with AV (5mgkg) for 12 weeks

Group 4 (atherogenic + AND LD) Rabbits were continuouslychallenged orally with Pg ATCC 33277 (02mL of 15 times 1011990912bacterial cellsmL in 2CMCwithPBS) five times aweek andcontinuously fed daily with AND low dose (10mgkg) for 12weeks

Group 5 (atherogenic + ANDHD) Rabbits were continuouslychallenged orally with Pg ATCC 33277 (02mL of 15 times 1011990912bacterial cellsmL in 2 CMC with PBS) five times a weekand continuously fed daily with AND high dose (20mgkg)for 12 weeks

26 Biochemical Measurement The experimental rabbitswere kept under fasting condition for at least 12 hoursbefore blood sampling to allow the relevant estimationof lipid profile levels Blood was taken from the ear veinof nonanaesthetized rabbit into an open plain tube andcentrifuged at 3500 g for 15min to obtain serum The levelsof total cholesterol (TC) triglycerides (TG) low-densitylipoprotein-cholesterol (LDL) high-density lipoprotein-cholesterol (HDL) and C-reactive protein (CRP) wereanalyzed from the serum of rabbits at the Universityof Malaya Medical Centre to evaluate the changes inbiomarkers using an automatic biochemistry analyzer(Dimension AR DuPont USA)

27 Proinflammatory Cytokines Measurement IL-1120573 and IL-6 concentrations were measured in serum using ELISA(enzyme-linked immunosorbent assay) (Abcam USA) withsensitivity of 1 pgmL and without cross reactivity againstother cytokines according to the manufacturerrsquos recommen-dations





2469 plusmn 014

044 plusmn 001

131 plusmn 002




















3 Results









000050100150200250300350400450


CRP(120583

gm

L)



119875 lt 005


000100020003000400050006000700080009000

10000


IL-1120573

(pg

mL)

lowast

lowastlowast


119875 lt 005





0005000

100001500020000250003000035000400004500050000


lowast

lowastlowast

IL-6

(pg

mL)


119875 lt 005






4 Discussion




05

1015202530354045

G1 G2 G3 G4 G5

lowast

lowastlowast

G3 G4 G5

TI TITI

TM TMTM

G1 G2

TI

TI

TM TM






000

500

1000

1500

2000

2500

3000

3500

4000

G1 G2 G3 G4 G5

lowastlowast

lowast

120572-S

MC

area

()

G1 G2

G3 G4 G5






0

01

02

03

04

05

06

07

08

09

G1 G2 G3 G4 G5

lowast

lowast

lowast

CD36

CD36

120573-Actin

G1 G2 G3 G4 G5

53kDa

43kDa








5 Conclusion




Acknowledgments


References































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















2469 plusmn 014

044 plusmn 001

131 plusmn 002




















3 Results









000050100150200250300350400450


CRP(120583

gm

L)



119875 lt 005


000100020003000400050006000700080009000

10000


IL-1120573

(pg

mL)

lowast

lowastlowast


119875 lt 005





0005000

100001500020000250003000035000400004500050000


lowast

lowastlowast

IL-6

(pg

mL)


119875 lt 005






4 Discussion




05

1015202530354045

G1 G2 G3 G4 G5

lowast

lowastlowast

G3 G4 G5

TI TITI

TM TMTM

G1 G2

TI

TI

TM TM






000

500

1000

1500

2000

2500

3000

3500

4000

G1 G2 G3 G4 G5

lowastlowast

lowast

120572-S

MC

area

()

G1 G2

G3 G4 G5






0

01

02

03

04

05

06

07

08

09

G1 G2 G3 G4 G5

lowast

lowast

lowast

CD36

CD36

120573-Actin

G1 G2 G3 G4 G5

53kDa

43kDa








5 Conclusion




Acknowledgments


References































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















000050100150200250300350400450


CRP(120583

gm

L)



119875 lt 005


000100020003000400050006000700080009000

10000


IL-1120573

(pg

mL)

lowast

lowastlowast


119875 lt 005





0005000

100001500020000250003000035000400004500050000


lowast

lowastlowast

IL-6

(pg

mL)


119875 lt 005






4 Discussion




05

1015202530354045

G1 G2 G3 G4 G5

lowast

lowastlowast

G3 G4 G5

TI TITI

TM TMTM

G1 G2

TI

TI

TM TM






000

500

1000

1500

2000

2500

3000

3500

4000

G1 G2 G3 G4 G5

lowastlowast

lowast

120572-S

MC

area

()

G1 G2

G3 G4 G5






0

01

02

03

04

05

06

07

08

09

G1 G2 G3 G4 G5

lowast

lowast

lowast

CD36

CD36

120573-Actin

G1 G2 G3 G4 G5

53kDa

43kDa








5 Conclusion




Acknowledgments


References































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0005000

100001500020000250003000035000400004500050000


lowast

lowastlowast

IL-6

(pg

mL)


119875 lt 005






4 Discussion




05

1015202530354045

G1 G2 G3 G4 G5

lowast

lowastlowast

G3 G4 G5

TI TITI

TM TMTM

G1 G2

TI

TI

TM TM






000

500

1000

1500

2000

2500

3000

3500

4000

G1 G2 G3 G4 G5

lowastlowast

lowast

120572-S

MC

area

()

G1 G2

G3 G4 G5






0

01

02

03

04

05

06

07

08

09

G1 G2 G3 G4 G5

lowast

lowast

lowast

CD36

CD36

120573-Actin

G1 G2 G3 G4 G5

53kDa

43kDa








5 Conclusion




Acknowledgments


References































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















05

1015202530354045

G1 G2 G3 G4 G5

lowast

lowastlowast

G3 G4 G5

TI TITI

TM TMTM

G1 G2

TI

TI

TM TM






000

500

1000

1500

2000

2500

3000

3500

4000

G1 G2 G3 G4 G5

lowastlowast

lowast

120572-S

MC

area

()

G1 G2

G3 G4 G5






0

01

02

03

04

05

06

07

08

09

G1 G2 G3 G4 G5

lowast

lowast

lowast

CD36

CD36

120573-Actin

G1 G2 G3 G4 G5

53kDa

43kDa








5 Conclusion




Acknowledgments


References































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















000

500

1000

1500

2000

2500

3000

3500

4000

G1 G2 G3 G4 G5

lowastlowast

lowast

120572-S

MC

area

()

G1 G2

G3 G4 G5






0

01

02

03

04

05

06

07

08

09

G1 G2 G3 G4 G5

lowast

lowast

lowast

CD36

CD36

120573-Actin

G1 G2 G3 G4 G5

53kDa

43kDa








5 Conclusion




Acknowledgments


References































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

01

02

03

04

05

06

07

08

09

G1 G2 G3 G4 G5

lowast

lowast

lowast

CD36

CD36

120573-Actin

G1 G2 G3 G4 G5

53kDa

43kDa








5 Conclusion




Acknowledgments


References































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















5 Conclusion




Acknowledgments


References































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of







































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Evaluation of the Effect of Andrographolide on … areas of alpha-SMA stain cell was microscopically...

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Transcript of Evaluation of the Effect of Andrographolide on … areas of alpha-SMA stain cell was microscopically...