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Research Article3541015840-Tri-O-acetylresveratrol AttenuatesLipopolysaccharide-Induced Acute Respiratory DistressSyndrome via MAPKSIRT1 Pathway

Lijie Ma1 Yilin Zhao1 Ruixuan Wang1 Tingting Chen2 Wangping Li1

Yandong Nan1 Xueying Liu3 and Faguang Jin1

1Department of Respiration Tangdu Hospital Fourth Military Medical University Xirsquoan 710038 China2School of Accounting Xijing University Xirsquoan 710032 China3Department of Medicinal Chemistry School of Pharmacy Fourth Military Medical University Xirsquoan 710032 China

Correspondence should be addressed to Xueying Liu weiyingxuan427163com and Faguang Jin jinfagfmmueducn

Received 5 July 2015 Revised 12 October 2015 Accepted 22 October 2015

Academic Editor Kang-Yun Lee

Copyright copy 2015 Lijie Ma et alThis is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The aim of the present research was to investigate the protecting effects of 3541015840-tri-O-acetylresveratrol (AC-Rsv) on LPS-inducedacute respiratory distress syndrome (ARDS) Lung injuries have been evaluated by histological examination wet-to-dry weightratios and cell count and protein content in bronchoalveolar lavage fluid Inflammation was assessed by MPO activities andcytokine secretion in lungs and cells The results showed that AC-Rsv significantly reduced the mortality of mice stimulated withLPS Pretreatment of AC-Rsv attenuated LPS-induced histological changes alleviated pulmonary edema reduced blood vascularleakage and inhibited theMPOactivities in lungsWhatwasmore AC-Rsv andRsv treatment reduced the secretion of TNF-120572 IL-6and IL-1120573 in lungs andNR8383 cells respectively Further exploration revealed thatAC-Rsv andRsv treatment relieved LPS-inducedinhibition on SIRT1 expression and restrained the activation effects of LPS on MAPKs and NF-120581B activation both in vitro and invivo More importantly in vivo results have also demonstrated that the protecting effects of Rsv on LPS-induced inflammationwould be neutralized when SIRT1 was in-hibited by EX527 Taken together these results indicated that AC-Rsv protected lungtissue against LPS-induced ARDS by attenuating inflammation via p38 MAPKSIRT1 pathway

1 Introduction

Acute respiratory distress syndrome (ARDS) is a commonand devastating complication which is mainly caused bydirect lung injuries such as inhalation of toxic substancesand indirect systemic diseases including sepsis and severetrauma ARDS leads to high morbidity high mortality andexorbitant health care costs [1] Despite adoption of lung-protective ventilation and overall intensive care unit (ICU)the hospital mortality of ARDS patients is still higher than40 [2] Sepsis is believed to be one of the most importantcauses of occurrence and development of ARDS Numerousresearches have declared that inflammation may directly andindirectly affect lung endothelia and the pulmonary hemody-namics by activating inflammatory cells such as neutrophils

monocytes and lymphocytes and proinflammatory medi-ators including cytokines proteases and cyclooxygenases[3 4] Although there is a variety of anti-inflammatoryinterventions and treatments against the inflammatoryprocess in lungs the mortality of ARDS remains higher thanacceptable [5]

Endotoxin also known as lipopolysaccharide (LPS) isone of the main ingredients formatting cell wall of Gram-negative bacteria which has also been recognized as the mostimportant pathogen for incurable inflammation Evidenceshave revealed that LPS could activate neutrophils airwayepithelial cells and alveolar macrophages followed by exces-sive generation of chemokines such as NO COX-2 TNF-120572IL-1120573 and IL-6 Several studies have addressed that activationof nuclear factor- (NF-) 120581B and mitogen-activated protein

Hindawi Publishing CorporationMediators of InflammationVolume 2015 Article ID 143074 12 pageshttpdxdoiorg1011552015143074

2 Mediators of Inflammation

kinase (MAPK) pathways was responsible for the excessivegeneration of proinflammatory cytokines [6 7] What wasmore plenty of evidence indicated that TNF-120572 and IL-1120573may degrade IkBs (IkB120572 in particular) and translocate NF-120581B from the cytoplasm into the nucleus in minutes whichwould further amplify the inflammatory process [8] Besidesovergeneration of inflammatory mediators played a key rolein damaging the alveolar-capillary barrier and permeabilityTherefore it may be of great significance to find new waysto interfere with the inflammatory process in treatment ofARDS

Resveratrol (Rsv) has been known for hundreds of yearsas powdered root of Polygonum cuspidatum and nowadayswidely known to exist in grapes nuts and red wine [9] Alsoresveratrol is one of the most intensively researched naturalcompounds since it exhibited protecting effects on multiplediseases such as cardiovascular disorders [10 11] cancers[12 13] and inflammation [14 15] There are also plentyof evidences indicating that resveratrol exhibited its phar-macological properties by interfering with the expressionand activity of silent information regulator type-1 (SIRT1)which has been demonstrated to play a key role in transcrip-tional and posttranscriptional regulation of gene expressionthrough the deacetylation of histone and nonhistone proteins[16] Although resveratrol preserved multiple bioactivities ithas never been adopted as a clinical drug for its poor pharma-cokinetic and bioavailability properties [17] while 3541015840-tri-O-acetylresveratrol (AC-Rsv) a prodrug of resveratrol firstlyreported in 2002 [18] has overcome some of the shortagesand results in the accumulation of Rsv in lung [19] Moreimportantly researches from our laboratory and other teamshave revealed that AC-Rsv attenuated seawater inhalationinduced lung injury in rat and reduced 120574-irradiation relateddeath in mice [20 21]

In the present research we have shown that AC-Rsvexerted protective roles in LPS exposure induced ARDS inmice by modulating SIRT1 expression Furthermore ourresults have demonstrated that the protective effects of AC-Rsv on lung tissue might be through attenuating the pulmo-nary edema and lung inflammation by inhibiting the p38mitogen-activated protein kinase (MAPK) pathway

2 Materials and Methods

21 Animals and Agents Adultmale Kunmingmice (18ndash22 g)were obtained from the Animal Center of the FourthMilitaryMedical University (FMMU Xirsquoan China) The mice werecaptured in air-filtered temperature-controlled units withequaled light-dark cycles and had free access to food andwater All experimental processes and treatments to animalshave been approved by the Institutional Animal Care andUseCommittee of the FMMU according to the Declaration of theNational Institutes of Health Guide for Care and Use of Lab-oratory Animals (Publication Number 85-23 revised 1985)

LPS (Escherichia coli lipopolysaccharide 055B5) andEX527 were obtained from the Sigma Chemical Company(St Louis MO USA) Resveratrol (3541015840-trihydroxystilbene)

O

O

O

O

O

O

Figure 1 The chemical structure of 3541015840-tri-O-acetylresveratrol(AC-Rsv)

was purchased from Xirsquoan Grass Plant Technology Cor-poration (Xirsquoan China) with purity above 98 3541015840-Tri-O-acetylresveratrol (AC-Rsv structure showed in Figure 1)was synthesized by the Pharmacy Department of MedicinalChemistry of FMMU with HPLC purity gt 99 Enzyme-linked immunosorbent assay (ELISA) kits for TNF-120572 IL-6and IL-1120573 have been purchased from the RampD Corporation(RampD Systems Inc) Myeloperoxidase (MPO) activity ana-lyzing kit has been purchased from Jiancheng BioengineeringInstitute (Nanjing China) Antibodies including anti-p-NF-120581B anti-NF-120581B anti-p-p38 MAPK anti-p38 MAPK anti-SIRT1 anti-p-ERK anti-ERK and anti-120573-actin have beenpurchased from the Santa Cruz Biotechnology Inc (SantaCruz CA USA)

22 Modeling and Grouping In order to explore the pro-tecting effects of AC-Rsv on the mortality mice received anintraperitoneal injection of 20mgkg LPS (dissolved in 09saline and filtered through a 022mm membrane) with orwithout pretreatment of different doses of AC-Rsv (25 50or 100mgkg body weight) for 7 days The mortality of micefrom different groups (119899 = 20) was recorded every 12 h for84 h after LPS exposure

In the research on the protecting effects of AC-Rsv onLPS-induced ARDS mice were randomly divided into 4groups control LPS (5mgkg) only LPS (5mgkg) + AC-Rsv (50mgkg) AC-Rsv (50mgkg) Mice in the LPS + AC-Rsv group were pretreated with 50mgkg AC-Rsv for 7 daysand LPS was injected 90min after the last administrationof AC-Rsv since our previous results have indicated thatconcentration of Rsv in blood reached the peak 90minafter oral administration of AC-Rsv Mice from all groupswere sacrificed 12 h after LPS injection In addition theconcentration of LPS (20mgkg and 5mgkg) was selectedaccording to previous researches [22]

23 Histological Evaluation Lung tissues of the same lobefrom different groups were fixed with 4 paraformaldehydefor 24 h and then embedded in paraffin Lung samples werecut into 5 120583m thick sections after being deparaffinized anddehydrated After that slices of lung samples were stainedwith hematoxylin and eosin Finally all slices were examinedand captured under microscope (Leica Germany)

Mediators of Inflammation 3

24 Lung Wet-to-Dry Weight Ratios Lung samples wereobtained 12 h after injection of LPS and weighed as soon aspossible after that all samples were dried to constant weightin an oven at 70∘C for 72 h and weighed again At last thewet-to-dry weight ratios were calculated by dividing the wetweight of each sample by the dry one

25 Bronchoalveolar Lavage Fluid (BALF) Analysis At theend of experiments lungs were removed intactly from miceand lavaged 5 times with 1mL ice-cold PBS (phosphatebuffered saline) The recovery ratio of lavaging fluid wasmore than 90Then the BALF was centrifuged at 1000 rpmfor 5min at 4∘C The supernatant was collected and proteinconcentration was determined by BCA method the resultswere calculated according to a standard curve and datawere expressed as 120583g protein per mL (120583gmL) On the otherhand the cell mass was resuspended in red blood cell-lysisbuffer and total cell amount was determined by using ahemocytometer

26 Analysis of MPO Activity MPO activity in lung tissueswas evaluated to represent the accumulation of neutrophilsin lungs challenged by LPS or not Briefly lung sampleswere homogenized in cold PBS (lung tissue to PBS 1 10)and homogenate supernatant was collected by centrifugingat 1000 rpm for 5min at 4∘C MPO activity was evaluatedaccording to the manufacturerrsquos instructions and values weremeasured with a spectrophotometer at 460 nm

27 Cell Culture and Treatment The alveolar macrophagecell line NR8383 was purchased from the ATCC (USA) andmaintained in Hamrsquos F12 medium containing 10 fetal calfserum at 37∘C in a humidified atmosphere containing 5CO2and 95 air

In order to evaluate the protecting effects of Rsv on LPSstimulated NR8383 cells cells were divided into four groupscontrol LPS (1 120583gmL) only LPS (1 120583gmL) + resveratrol(40 120583gmL) resveratrol (40 120583gmL) Cells from differentgroups were collected for further investigation after beingtreated for 12 h The concentration of LPS in this experimentwas selected according to previous researches [22]

In the research of exploring the relationship betweenSIRT1 expression and the protecting effects of Rsv on LPSstimulated NR8383 cells cells were divided into four groupsand treated with normal Hamrsquos F12 medium (control) LPS(1 120583gmL) LPS (1 120583gmL) + EX527 (1 120583M) + resveratrol(40 120583gmL) and LPS (1 120583gmL) + resveratrol (40 120583gmL) for12 h Cells were then collected for further examination By theway EX527 was first dissolved in dimethyl sulfoxide (DMSO)to 1mM and then diluted to the working concentration(1 120583M) with Hamrsquos F12 medium

28 Measurement of Cytokines Although it is usually con-cerning for the distortion of results ELISA was chosen toanalyze the content of TNF-120572 IL-6 and IL-1120573 in lungs andcells in order to provide supplementary data for the current

research Briefly lung tissues from each group were homoge-nized in cold PBS (lung tissue to PBS 1 5) by using a Tissue-Tearor and cells treated as described abovewere homogenizedby repeated frozen and dissolvedmethodHomogenates fromtissue and cells were centrifuged at 12000 rpm for 5min at4∘C Contents of TNF-120572 IL-6 and IL-1120573 in supernatantsfrom tissue and cell samples were measured according to themanufacturerrsquos instructions

29 Western Blot At the end of the experiment cell and tis-sue samples were collected and total proteins were extractedaccording to themanufacturerrsquos instructions (Beyotime Insti-tute of Biotechnology Jiangsu China) Protein concentra-tions were determined by BCA method with an assay kit(Beyotime) After boiling equal amounts of proteins fromeach group were separated on SDS-PAGE gel and trans-ferred to polyvinylidene fluoride membranes by wet transfermethodThemembranewas blockedwith 5nonfat drymilkin Tris-buffered saline with Tween 20 followed by incubationwith monoclonal antibodies overnight at 4∘C against p-NF-120581B (1 200) p-p38 MAPK (1 300) SIRT1 (1 200) p-ERK(1 200) and 120573-actin (1 5000) Following the incubationof antibodies were repeated washing and incubation ofsecondary antibody for 2 h at room temperature Finallyimmune complexes were visualized via the enhanced chemi-luminescence (ECL) system (Amersham Pharmacia BiotechArlington Heights IL USA)

210 Statistical Analysis Statistical analysis was performedwith SPSS 170 for Windows Numeric variables wereexpressed as means plusmn SD Differences between groupswere performed by one-way analysis of variance (ANOVA)followed by Dunnettrsquos test after the distribution of data wasconfirmed to be a normal distribution The Kaplan-Meiermethod and the log-rank test were used to analyze survivaldata Statistical significance was accepted as 119875 lt 005

3 Results

31 The Effect of AC-Rsv on LPS-Induced Mortality in MiceIn order to assess the protecting effects of AC-Rsv onendotoxemia we firstly evaluated the effects of AC-Rsv onLPS-induced mortality in mice As shown in Figure 2 theaccumulative mortalities of mice in middle-dose (50mgkg)andhigh-dose (100mgkg) groupswere 55and 65 respec-tively which were significantly lower than that of LPS group(80 mice dead) (119875 lt 005) Those data indicated that AC-Rsv protected mice from LPS-induced death and 50mgkgof AC-Rsv exhibited the best protecting effects which wasadopted to carry out further researches

32 Effects of AC-Rsv on LPS-Induced Lung MorphologicalChanges Histopathological results showed that lung samplesfrom control (Figure 3(a)) and AC-Rsv (Figure 3(d)) groupsexhibited a normal structure with clear pulmonary alveoliwhile LPS exposure led to infiltration of inflammatory cellsdamage of alveoli thickened alveolar wall and formationof hyaline membranes (Figure 3(b)) However LPS-induced


Time (hours)

Surv

ival

()

20 40 60 8000

20

40

60

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100

LPS25mgkg

50mgkg100mgkg

Figure 2 Effects of AC-Rsv on LPS-inducedmortality inmiceMicefrom each experimental group were injected with 20mgkg of LPSwith or without pretreatment of AC-Rsv (25 50 and 100mgkg)for 7 days Alive mice in each group were counted every 12 hThe accumulative mortalities of mice in middle-dose (50mgkg)and high-dose (100mgkg) groups were 55 and 65 respectivelywhich were significantly lower than that of LPS group (80 micedead) (119875 lt 005) More importantly 50mgkg of AC-Rsv exhibitedthe best protecting effect 119875 lt 005 119899 = 20

changes in lung structure have been dramatically attenuatedby AC-Rsv pretreatment (Figure 3(c))

33 Effects of AC-Rsv on Lung Edema Induced by LPS Thewet-to-dry weight ratios of lung samples from differentgroups have been calculated in order to assess the pulmonaryedema in mice challenged by LPS with or without pretreat-ment ofAC-Rsv As shown in Figure 3(e) thewet-to-dry ratiowas significantly increased in lungs from LPS group com-paredwith that of control (119899 = 6119875 lt 005) However admin-istration of AC-Rsv dramatically reduced water content inlungs 12 h after LPS injection The result also indicated thatAC-Rsv treatment alone did not affect water content in lungs

34 Effects of AC-Rsv on LPS-Induced Lung Vascular LeakageCell and protein content in BALF from all four groups wereevaluated in order to assess the vascular leakage in lungsstimulated with LPS as well as determine the protectingeffects of AC-Rsv in this respect As shown in Figures 3(f)and 3(g) LPS injection increased the cell and protein contentin BALF compared with those of control (119875 lt 005) whileAC-Rsv restricted cell and protein leakage from pulmonaryvessels into the alveoli when stimulated with LPS (119875 lt 005)Also there was no significant difference between control andAC-Rsv group

35 Effects of AC-Rsv on Inflammation Induced by LPS inLungs In order to evaluate the effects of AC-Rsv on LPS-induced inflammation in lungs MPO activity (Figure 4) andcontents of TNF-120572 IL-6 and IL-1120573 (Figures 5(a)ndash5(c)) havebeen measured The results revealed that LPS injection sig-nificantly upregulated the activity of MPO and increased the

contents of TNF-120572 IL-6 and IL-1120573 in lungs compared withthose of control (119875 lt 005) However pretreatment of AC-Rsv dramatically decreased MPO activity and inhibited theconcentration of TNF-120572 IL-6 and IL-1120573 in lungs (119875 lt 005)Also the results indicated that AC-Rsv treatment alone didnot affect theMPO activity and content of cytokines in lungs

36 Effects of AC-Rsv on the Expression of MAPKSIRT1 Path-way in Lung In order to further illustrate the mechanismsof how AC-Rsv exhibited the protecting effects against theendotoxemia induced by LPS the expression ofMAPKSIRT1pathway was evaluated by western blotThe results (Figure 6)showed that administration of LPS significantly decreasedthe SIRT1 expression and increased the expression of p-p38MAPK p-ERK and p-NF-120581B (119875 lt 005) However pre-treatment ofAC-Rsv dramatically decreased p-p38MAPK p-ERK and p-NF-120581B expression and enhanced the expressionof SIRT1 in lungs stimulated with LPS (119875 lt 005)

37 Effects of Resveratrol on Generation of InflammatoryMediators in NR8383 Cells Stimulated by LPS Based on thefindings that AC-Rsv attenuated LPS-induced lung injury wefurther evaluated the effects of its intermediate metaboliteresveratrol on NR8383 cells challenge by LPS because allAC-Rsv would become Rsv in the body [19] The results(Figures 5(d)ndash5(f)) showed that LPS stimulation increasedthe generation of TNF-120572 IL-6 and IL-1120573 in NR8383 cellscompared with that of control (119875 lt 005) while resveratrolsignificantly reversed this trend and decreased the formationof TNF-120572 IL-6 and IL-1120573 (119875 lt 005) inNR8383 cells exposedto LPS What was more there were no significant differencesin the content of chemokine between control and resveratrolgroups

38 Effects of Resveratrol on the Expression of MAPKSIRT1Pathway in NR8383 Cells We further evaluated whetherresveratrol could modulate the expression of MAPKSIRT1pathway in NR8383 cells like AC-Rsv did in LPS chal-lenged lungs The expressions of SIRT1 p-p38 MAPK p-ERK and p-NF-120581B have been evaluated in cells stimulatedby LPS together with treatment of Rsv or not and theresults (Figure 7) showed that LPS stimulation inhibited theexpression of SIRT1 and upregulated p-p38 MAPK p-ERKand p-NF-120581B expression in NR8383 cells (119875 lt 005) whileresveratrol treatment reversed this trend just like AC-Rsv didin LPS stimulated lungs

39 SIRT1 Inhibitor Neutralized the Protecting Effects ofResveratrol on NR8383 Cells Stimulated by LPS NR8383 cellswere exposed to normal culture LPS LPS + EX527 + Rsvand LPS + Rsv for 12 h and then the expression of SIRT1 andgeneration of cytokines were evaluated by western blot andELISAmethod respectivelyThe results (Figure 8(a)) showedthat SIRT1 expression was dramatically suppressed by LPScomparedwith that of control while Rsv treatment effectivelyreversed the decrease of SIRT1 expression Astonishinglycotreatment of EX527 and Rsv counterbalanced the elevatingeffects of Rsv on the expression of SIRT1


(a) (b) (c) (d)

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Figure 3 Protecting effects of AC-Rsv on the LPS exposure induced lung injuries (andashd) Morphological changes were evaluated 12 h afterLPS exposure by HampE staining LPS stimulation group (b) showed increasing lung edema alveolar hemorrhage neutrophil infiltration anddestroyed epithelialendothelial cell structures compared with those of control (a) while significant improvement was observed in samplesfrom the LPS + AC-Rsv group (c) AC-Rsv treatment alone barely affected the structure of lungs (d) (e) Wet-to-dry ratios of lung samplesdata are expressed as mean plusmn SD 119899 = 8 LPS injection significantly increased the WD ratios of lung samples compared with that of controllowast

119875 lt 005 versus control while pretreatment of AC-Rsv dramatically decreased theWD ratios of lung samples stimulated by LPS lowastlowast119875 lt 005versus lowast119875 ((f) and (g)) Protein concentration (a) and cell count (b) in BALF Data are expressed as mean plusmn SD 119899 = 8 LPS exposuresignificantly increased cell and protein content in BALF compared with those of control lowast119875 lt 005 versus control and pretreatment ofAC-Rsv decreased the cell and protein content in BALF from lung stimulated by LPS lowastlowast119875 lt 005 versus lowast119875


00

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activ

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g p

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Figure 4 Effects of AC-Rsv on MPO activity in lungs stimulatedby LPS Data are expressed as mean plusmn SD 119899 = 8 LPS injectionsignificantly increased theMPO activity in lung compared with thatof control lowast119875 lt 005 versus control while pretreatment of AC-Rsvdramatically inhibited the elevating effects of LPS on MPO activitylowastlowast

119875 lt 005 versus lowast119875

On the other hand the results (Figures 8(b)ndash8(d)) fromthe evaluation on the generation of cytokines revealed thatLPS stimulation increased the generation of TNF-120572 IL-1120573and IL-6 and Rsv treatment reversed this trend Similar tothe effects on SIRT1 expression coincubation of EX527 andRsv did not inhibit the generation of those cytokines

4 Discussion

ARDS is a serious manifestation of systemic inflammationinduced by varied factors which may further result in multi-ple organs dysfunction syndromes (MODS) with a very highmortality [2] Previous researches have revealed that over-activated inflammatory cells and secretion of inflammatorymediators were the primary pathogenesis for ARDS [23 24]There were also researches on the mechanism and treatmentof ARDS in vitro and in vivo and substantial progressionhas been made in understanding of this disease Howeveravailable treatments were still limited in clinic and it wasthus meaningful to explore new pharmacological treatmentfor ARDS

As a natural polyphenol resveratrol (Rsv) has beenconfirmed to possess a number of pharmacological functions[10ndash15] Rsv has never been adopted as a clinical drug forits short half-life low bioavailability and poor targetingHowever those shortages have been overcome to some extentby AC-Rsv and administration of AC-Rsv increased theconcentration of Rsv in lungs 20-fold compared with thatof administration of Rsv [19] Therefore we investigated theprotecting effects of AC-Rsv on LPS-induced ARDS and theresults revealed that AC-Rsv could reduce the mortality rateof mice challenged by LPS and attenuate lung injury byrestricting leakage of fluid from blood vessels into pulmonaryalveoli inhibiting the concentration of cytokines and allevi-ating the abnormal expression of MAPKSIRT1 expressioninduced by LPS

As a hallmark of ARDS [25] pulmonary edema wascaused by dysfunction of alveolar-capillary barriers andincreased filtration of protein-rich fluid into the alveolarspaces [26] In the present research cell content and proteinconcentration in BALF and lung edema have been measuredin order to quantify the magnitude of pulmonary edema andsmall vascular permeability The results revealed that admin-istration of LPS increased water content in lung tissues andenhanced infiltration of cells and leakage of protein-rich fluidfrom blood vessels into alveoli while pretreatment of AC-Rsv significantly reduced the lung WD ratio decreased theprotein concentration in BALF and inhibited the exudationof cells from blood vessels into alveoli induced by LPS

It has been confirmed that inflammation was anothercharacter for ARDS in addition neutrophils and macropha-ges were the main executive cells in LPS-induced pulmonaryinflammation [27] Myeloperoxidase (MPO) was a uniqueconstituent of neutrophil cytoplasmic granules which wasindispensable for the killing of phagocytosed pathogensTherefore MPO activity was markedly relevant to neu-trophils accumulation and served as the marker of inflam-mation [28] In the current research it has been found thatMPO activity was dramatically increased in lungs 12 h afterLPS exposure while pretreatment of AC-Rsv significantlydecreased the MPO activity In addition histopathologicalstudy has also revealed that AC-Rsv pretreatment markedlyreduced the neutrophil infiltration from blood vessels intopulmonary alveoli

LPS stimulation as well as activated neutrophils couldfurther induce the generation and secretion of numerousinflammatory mediators and chemotactic cytokines amongwhich TNF-120572 IL-1120573 and IL-6 were characterized mediatorsparticipating in the occurrence and development of ARDS[29] Those chemokines were also related to the influx accu-mulation and activation of highly destructive cells involvedin inflammation more importantly the positive feedbackbetween TNF-120572 and IL-1120573 and NF-120581B would facilitate moreNF-120581B expression phosphorylation and translocation intonucleus which could further elicit the inflammatory cascadeleading to severe damage to lung tissues [30 31] In thepresent research it has been found that TNF-120572 IL-1120573 and IL-6 increased remarkably in lungs and NR8383 cells after LPSexposure while treatment of AC-Rsv and Rsv significantlydecreased the generation and secretion of TNF-120572 IL-1120573 andIL-6 in lung tissues and NR8383 cells respectively

In addition to regulating the expression of inflammatorycytokines AC-Rev should have affected the activation ofenzymes and mediators involved in pathogenesis of inflam-mation Mitogen-activated protein kinase (MAPK) was aconserved cellular energy status sensor which could be acti-vated by LPS [32 33] What was more activation of MAPKsmay further lead to secretion of cytokines such as TNF-120572and IL-1120573 and expression of inflammatory mediators such asinducible nitric oxide synthase (iNOS) and cyclooxygenase-2(COX-2) by enhancing transcription of NF-120581B [34] Togetherwith MAPKs SIRT1 has also been regarded as a key sen-sor and metabolic modulator for inflammation especiallyARDS Besides the corporation between SIRT1 and MAPKin mediating the molecule response to stimuli without or


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pgm

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Figure 5 Effects of AC-Rsv on content of cytokines in LPS stimulated lungs and NR8383 cells have been measured by ELISA (andashc) TNF-120572IL-6 and IL-1120573 levels in lungs (dndashf) TNF-120572 IL-6 and IL-1120573 levels in NR8383 cells Data are expressed as mean plusmn SD 119899 = 8 LPS stimulationincreased the content of TNF-120572 IL-6 and IL-1120573 in lungs and NR8383 cells compared with that of control lowast119875 lt 005 versus control whileAC-Rsv and Rsv treatment significantly inhibited the formation of those cytokines in lung and NR8383 cells respectively lowastlowast119875 lt 005 versuslowast

119875

within pretreatment was closely related to NF-120581B [35 36]Based on those clues we explored the expression of SIRT1 andMAPK pathway in LPS-induced ARDS the results showedthat LPS exposure inhibited SIRT1 expression and enhancedthe phosphorylation of p38 MAPK and ERK followed byincreased p-NF-120581B expression in vitro and in vivo Howeverpretreatment of AC-Rsv relieved LPS-induced inhibition on

SIRT1 expression and restrained the effects of LPS on p38MAPK ERK and NF-120581B in lungs and NR8383 cells

To further confirm the protective role of AC-Rsv on LPS-induced ARDS via interfering with the expression of SIRT1in vitro experiment has been carried out by cotreatmentof EX527 and Rsv for LPS stimulated NR8383 cells EX527is a new type of highly selective inhibitor for SIRT1 which


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Figure 6 Effects of AC-Rsv on the expression ofMAPKSIRT1 axis in lungs were evaluated bywestern blot Relative expression levels of p-p38MAPK p-ERK and p-NF-120581Bwere normalized to the expression levels of their total forms SIRT1 expressionwas expressed by comparingwiththat of 120573-actin Administration of LPS significantly decreased the SIRT1 expression and increased the expression of p-p38 MAPK p-ERKand p-NF-120581B in lungs compared with that of control lowast119875 lt 005 versus control while AC-Rsv treatment dramatically decreased expressionof p-p38 MAPK p-ERK and p-NF-120581B and enhanced the SIRT1 expression in lungs when challenged by LPS lowastlowast119875 lt 005 versus lowast119875


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lowast

RsvLPS + Rsv(e)

Figure 7 Effects of Rsv on the expression of MAPKSIRT1 pathway in NR8383 cells stimulated by LPS Relative expression levels of p-p38MAPK p-ERK and p-NF-120581B were normalized to the expression levels of their total forms SIRT1 expression was expressed by comparingwith that of 120573-actin Stimulation of LPS significantly decreased the SIRT1 expression and increased the expression of p-p38 MAPK p-ERKand p-NF-120581B in NR8383 cells compared with that of control lowast119875 lt 005 versus control while Rsv treatment dramatically decreased p-p38MAPK p-ERK and p-NF-120581B expression and enhanced the expression of SIRT1 in NR8383 cells when challenged by LPS lowastlowast119875 lt 005 versuslowast

119875


00

02

03

01

SIRT1

120573-actin

SIRT

1120573

-act

in

Control LPSLPS + RsvLPS + EX527+ Rsv

lowastlowastlowast

lowastlowast

lowast

(a)

0

100

200

300


lowastlowastlowast

lowastlowast

lowast

TNF-120572

(pg

mL)

(b)

IL-1120573

(pg

mL)

0

50

100

150

200


lowastlowast

lowastlowast

lowastlowast

(c)

IL-6

(pg

mL)

0

50

100

150


lowastlowast

lowastlowastlowastlowast

(d)

Figure 8 Neutralizing effects of EX527 on the protecting effects of Rsv on LPS stimulatedNR8383 cells (a) Expression of SIRT1 was evaluatedby western blot and normalized to the expression level of 120573-actin LPS stimulation inhibited the expression of SIRT1 compared with that ofcontrol lowast119875 lt 005 versus control while Rsv treatment significantly reversed the inhibiting effects lowastlowast119875 lt 005 versus lowast119875 on the contraryEX527 and Rsv cotreatment counterbalanced the enhancing effects of Rsv on the expression of SIRT1 lowastlowastlowast119875 lt 005 versus lowastlowast119875 (bndashd) Contentsof TNF-120572 IL-6 and IL-1120573 were evaluated by ELISA LPS stimulation increased the generation of TNF-120572 IL-6 and IL-1120573 in NR8383 cellscompared with that of control lowast119875 lt 005 versus control while Rsv treatment significantly inhibited the formation of those cytokines inNR8383 cells when stimulated by LPS lowastlowast119875 lt 005 versus lowast119875 astonishingly EX527 and Rsv cotreatment neutralized the inhibiting effects ofRsv on the generation of TNF-120572 IL-6 and IL-1120573 lowastlowastlowast119875 lt 005 versus lowastlowast119875

could effectively inhibit the acetylation enzyme activity ofSIRT1 (IC

5038 nmolL) [37] As expected LPS stimulation

decreased SIRT1 expression and increased the generation ofTNF-120572 IL-1120573 and IL-6 while Rsv treatment could reversethis trend to some extent However cotreatment of EX527and Rsv counterbalanced the attenuating effects of Rsv ondecreased expression of SIRT1 and accelerated generationof TNF-120572 IL-1120573 and IL-6 resulting from LPS stimulationTaken together those results indicated that the activation ofSIRT1 by Rsv attenuated the inflammation in NR8383 cellsstimulated by LPS while the inhibition of SIRT1 by EX527could neutralize the protecting effects of Rsv on LPS-inducedinflammation and lung injury

In summary results from the present research indicatedthat AC-Rsv pretreatment therapeutically attenuated LPS-inducedARDS by suppressing lung edema inhibiting leakage

of protein-rich fluid from blood vessels into pulmonaryalveoli and depressing inflammatory process in mice Themechanisms underlying the therapeutic effects of AC-Rsvwere probably via the MAPKSIRT1 pathways Although fur-ther researches were needed the present research indicatedthat AC-Rsv may be a potential therapeutic agent for ARDS

Conflict of Interests

The authors have no conflict of interests to declare

Authorsrsquo Contribution

Lijie Ma and Yilin Zhao contributed to the paper equally


References

[1] Y Koh ldquoUpdate in acute respiratory distress syndromerdquo Journalof Intensive Care vol 2 article 2 2014

[2] J Villar J Blanco J M Anon et al ldquoThe ALIEN study inci-dence and outcome of acute respiratory distress syndrome inthe era of lung protective ventilationrdquo Intensive Care Medicinevol 37 no 12 pp 1932ndash1941 2011

[3] FMafra de Lima K TNaves AHMachado R Albertini A BVillaverde and F Aimbire ldquoLung inflammation and endothelialcell damage are decreased after treatment with phototherapy(PhT) in amodel of acute lung injury induced byEscherichia colilipopolysaccharide in the ratrdquoCell Biology International vol 33no 12 pp 1212ndash1221 2009

[4] W Jing M Chunhua and W Shumin ldquoEffects of acteoside onlipopolysaccharide-induced inflammation in acute lung injuryvia regulation of NF-kappaB pathway in vivo and in vitrordquoToxicology and Applied Pharmacology vol 285 no 2 pp 128ndash135 2015

[5] D G Geboers F M de Beer A M Boer et al ldquoPlasma suPARas a prognostic biological marker for ICU mortality in ARDSpatientsrdquo Intensive Care Medicine vol 41 no 7 pp 1281ndash12902015

[6] S Khan R J Choi O Shehzad et al ldquoMolecular mechanismof capillarisin-mediated inhibition of MyD88TIRAP inflam-matory signaling in in vitro and in vivo experimental modelsrdquoJournal of Ethnopharmacology vol 145 no 2 pp 626ndash637 2013

[7] N Bhaskaran S Shukla J K Srivastava and S GuptaldquoChamomile an anti-inflammatory agent inhibits induciblenitric oxide synthase expression by blocking RelAp65 activityrdquoInternational Journal of Molecular Medicine vol 26 no 6 pp935ndash940 2010

[8] M S Hayden and S Ghosh ldquoNF- kappaB in immunobiologyrdquoCell Research vol 21 no 2 pp 223ndash244 2011

[9] Y-C Chi S-P Lin and Y-C Hou ldquoA new herb-drug interac-tion of Polygonum cuspidatum a resveratrol-rich nutraceuticalwith carbamazepine in ratsrdquoToxicology andApplied Pharmacol-ogy vol 263 no 3 pp 315ndash322 2012

[10] R K Vella C Pullen F R Coulson andA S Fenning ldquoResver-atrol prevents cardiovascular complications in the SHRSTZ ratby reductions in oxidative stress and inflammationrdquo BioMedResearch International vol 2015 Article ID 918123 8 pages2015

[11] Q Xu and L-Y Si ldquoResveratrol role in cardiovascular andmetabolic health andpotentialmechanisms of actionrdquoNutritionResearch vol 32 no 9 pp 648ndash658 2012

[12] P Liu H Liang Q Xia et al ldquoResveratrol induces apoptosis ofpancreatic cancers cells by inhibitingmiR-21 regulation of BCL-2 expressionrdquo Clinical and Translational Oncology vol 15 no 9pp 741ndash746 2013

[13] Y-Q Xue J-M Di Y Luo K-J Cheng X Wei and Z ShildquoResveratrol oligomers for the prevention and treatment ofcancersrdquo Oxidative Medicine and Cellular Longevity vol 2014Article ID 765832 9 pages 2014

[14] M M Poulsen K Fjeldborg M J Ornstrup T N Kjaeligr MK Noslashhr and S B Pedersen ldquoResveratrol and inflammationchallenges in translating pre-clinical findings to improvedpatient outcomesrdquo Biochimica et Biophysica Acta vol 1852 no6 pp 1124ndash1136 2015

[15] Z Liu C Jiang J Zhang B Liu andQDu ldquoResveratrol inhibitsinflammation and ameliorates insulin resistant endothelial

dysfunction via regulation of AMP-activated protein kinase andsirtuin 1 activitiesrdquo Journal of Diabetes 2015

[16] T Finkel C-X Deng and R Mostoslavsky ldquoRecent progressin the biology and physiology of sirtuinsrdquo Nature vol 460 no7255 pp 587ndash591 2009

[17] TWalle F HsiehMH DeLegge J E Oatis Jr andU KWalleldquoHigh absorption but very low bioavailability of oral resveratrolin humansrdquoDrugMetabolism andDisposition vol 32 no 12 pp1377ndash1382 2004

[18] Y Dobrydneva R L Williams G Z Morris and P F Black-more ldquoDietary phytoestrogens and their synthetic structuralanalogues as calcium channel blockers in human plateletsrdquoJournal of Cardiovascular Pharmacology vol 40 no 3 pp 399ndash410 2002

[19] L Liang X Liu Q Wang S Cheng S Zhang and M ZhangldquoPharmacokinetics tissue distribution and excretion study ofresveratrol and its prodrug 3541015840-tri-O-acetylresveratrol inratsrdquo Phytomedicine vol 20 no 6 pp 558ndash563 2013

[20] L Ma Y Li Y Zhao et al ldquo3541015840-Tri-O-acetylresveratrol ame-liorates seawater exposure-induced lung injury by upregulatingconnexin 43 expression in lungrdquoMediators of Inflammation vol2013 Article ID 182132 8 pages 2013

[21] K Koide S Osman A L Garner et al ldquoThe use of 3541015840-tri-O-acetylresveratrol as a potential prodrug for resveratrol protectsmice from 120574-irradiation-induced deathrdquo ACS Medicinal Chem-istry Letters vol 2 no 4 pp 270ndash274 2011

[22] B Zhang Z-Y Liu Y-Y Li et al ldquoAntiinflammatory effects ofmatrine in LPS-induced acute lung injury in micerdquo EuropeanJournal of Pharmaceutical Sciences vol 44 no 5 pp 573ndash5792011

[23] D R Janz and L B Ware ldquoBiomarkers of ALIARDS patho-genesis discovery and relevance to clinical trialsrdquo Seminars inRespiratory and Critical Care Medicine vol 34 no 4 pp 537ndash548 2013

[24] L BWare ldquoAutopsy in ARDS insights into natural historyrdquoTheLancet Respiratory Medicine vol 1 no 5 pp 352ndash354 2013

[25] L W Soromou N Chen L Jiang et al ldquoAstragalin attenuateslipopolysaccharide-induced inflammatory responses by down-regulating NF-kappaB signaling pathwayrdquo Biochemical andBiophysical Research Communications vol 419 no 2 pp 256ndash261 2012

[26] S Herold N M Gabrielli and I Vadasz ldquoNovel concepts ofacute lung injury and alveolar-capillary barrier dysfunctionrdquoAmerican Journal of PhysiologymdashLung Cellular and MolecularPhysiology vol 305 no 10 pp L665ndashL681 2013

[27] T Zhu D-XWangW Zhang et al ldquoAndrographolide protectsagainst LPS-induced acute lung injury by inactivation of NF-120581Brdquo PLoS ONE vol 8 no 2 Article ID e56407 2013

[28] D El Kebir L Jozsef W Pan and J G Filep ldquoMyeloperoxidasedelays neutrophil apoptosis through CD11bCD18 integrins andprolongs inflammationrdquoCirculation Research vol 103 no 4 pp352ndash359 2008

[29] S K Cribbs and G S Martin ldquoStem cells in sepsis and acutelung injuryrdquo The American Journal of the Medical Sciences vol341 no 4 pp 325ndash332 2011

[30] I Muller S Beissert and D Kulms ldquoAnti-apoptotic NF-120581Band lsquogain of functionrsquo mutp53 in concert act pro-apoptotic inresponse to UVB+IL-1 via enhanced TNF productionrdquo Journalof Investigative Dermatology vol 135 no 3 pp 851ndash860 2015


[31] R Bianchi I Giambanco and R Donato ldquoS100BRAGE-dependent activation of microglia via NF-kappaB and AP-1 Co-regulation of COX-2 expression by S100B IL-1beta and TNF-alphardquo Neurobiology of Aging vol 31 no 4 pp 665ndash677 2010

[32] C X Jian M Z Li W Y Zheng et al ldquoTormentic acidinhibits LPS-induced inflammatory response in human gingivalfibroblasts via inhibition of TLR4-mediated NF-120581B and MAPKsignalling pathwayrdquo Archives of Oral Biology vol 60 no 9 pp1327ndash1332 2015

[33] J Yamana L Santos and E Morand ldquoEnhanced induction ofLPS-induced fibroblast MCP-1 by interferon-120574 involvement ofJNK and MAPK phosphatase-1rdquo Cellular Immunology vol 255no 1-2 pp 26ndash32 2009

[34] N W Lucchi and J M Moore ldquoLPS induces secretion ofchemokines by human syncytiotrophoblast cells in a MAPK-dependent mannerrdquo Journal of Reproductive Immunology vol73 no 1 pp 20ndash27 2007

[35] S Cetrullo S DrsquoAdamo B Tantini RM Borzi and F FlamignildquomTOR AMPK and Sirt1 key players in metabolic stressmanagementrdquo Critical Reviews in Eukaryotic Gene Expressionvol 25 no 1 pp 59ndash75 2015

[36] C Ehlting O Bohmer M J Hahnel et al ldquoOncostatin M reg-ulates SOCS3 mRNA stability via the MEKndashERK12-pathwayindependent of p38MAPKMK2rdquo Cellular Signalling vol 27 no3 pp 555ndash567 2015

[37] A D Napper J Hixon T McDonagh et al ldquoDiscovery ofindoles as potent and selective inhibitors of the deacetylaseSIRT1rdquo Journal ofMedicinal Chemistry vol 48 no 25 pp 8045ndash8054 2005


kinase (MAPK) pathways was responsible for the excessivegeneration of proinflammatory cytokines [6 7] What wasmore plenty of evidence indicated that TNF-120572 and IL-1120573may degrade IkBs (IkB120572 in particular) and translocate NF-120581B from the cytoplasm into the nucleus in minutes whichwould further amplify the inflammatory process [8] Besidesovergeneration of inflammatory mediators played a key rolein damaging the alveolar-capillary barrier and permeabilityTherefore it may be of great significance to find new waysto interfere with the inflammatory process in treatment ofARDS

Resveratrol (Rsv) has been known for hundreds of yearsas powdered root of Polygonum cuspidatum and nowadayswidely known to exist in grapes nuts and red wine [9] Alsoresveratrol is one of the most intensively researched naturalcompounds since it exhibited protecting effects on multiplediseases such as cardiovascular disorders [10 11] cancers[12 13] and inflammation [14 15] There are also plentyof evidences indicating that resveratrol exhibited its phar-macological properties by interfering with the expressionand activity of silent information regulator type-1 (SIRT1)which has been demonstrated to play a key role in transcrip-tional and posttranscriptional regulation of gene expressionthrough the deacetylation of histone and nonhistone proteins[16] Although resveratrol preserved multiple bioactivities ithas never been adopted as a clinical drug for its poor pharma-cokinetic and bioavailability properties [17] while 3541015840-tri-O-acetylresveratrol (AC-Rsv) a prodrug of resveratrol firstlyreported in 2002 [18] has overcome some of the shortagesand results in the accumulation of Rsv in lung [19] Moreimportantly researches from our laboratory and other teamshave revealed that AC-Rsv attenuated seawater inhalationinduced lung injury in rat and reduced 120574-irradiation relateddeath in mice [20 21]

In the present research we have shown that AC-Rsvexerted protective roles in LPS exposure induced ARDS inmice by modulating SIRT1 expression Furthermore ourresults have demonstrated that the protective effects of AC-Rsv on lung tissue might be through attenuating the pulmo-nary edema and lung inflammation by inhibiting the p38mitogen-activated protein kinase (MAPK) pathway

2 Materials and Methods

21 Animals and Agents Adultmale Kunmingmice (18ndash22 g)were obtained from the Animal Center of the FourthMilitaryMedical University (FMMU Xirsquoan China) The mice werecaptured in air-filtered temperature-controlled units withequaled light-dark cycles and had free access to food andwater All experimental processes and treatments to animalshave been approved by the Institutional Animal Care andUseCommittee of the FMMU according to the Declaration of theNational Institutes of Health Guide for Care and Use of Lab-oratory Animals (Publication Number 85-23 revised 1985)

LPS (Escherichia coli lipopolysaccharide 055B5) andEX527 were obtained from the Sigma Chemical Company(St Louis MO USA) Resveratrol (3541015840-trihydroxystilbene)

O

O

O

O

O

O

Figure 1 The chemical structure of 3541015840-tri-O-acetylresveratrol(AC-Rsv)

was purchased from Xirsquoan Grass Plant Technology Cor-poration (Xirsquoan China) with purity above 98 3541015840-Tri-O-acetylresveratrol (AC-Rsv structure showed in Figure 1)was synthesized by the Pharmacy Department of MedicinalChemistry of FMMU with HPLC purity gt 99 Enzyme-linked immunosorbent assay (ELISA) kits for TNF-120572 IL-6and IL-1120573 have been purchased from the RampD Corporation(RampD Systems Inc) Myeloperoxidase (MPO) activity ana-lyzing kit has been purchased from Jiancheng BioengineeringInstitute (Nanjing China) Antibodies including anti-p-NF-120581B anti-NF-120581B anti-p-p38 MAPK anti-p38 MAPK anti-SIRT1 anti-p-ERK anti-ERK and anti-120573-actin have beenpurchased from the Santa Cruz Biotechnology Inc (SantaCruz CA USA)

22 Modeling and Grouping In order to explore the pro-tecting effects of AC-Rsv on the mortality mice received anintraperitoneal injection of 20mgkg LPS (dissolved in 09saline and filtered through a 022mm membrane) with orwithout pretreatment of different doses of AC-Rsv (25 50or 100mgkg body weight) for 7 days The mortality of micefrom different groups (119899 = 20) was recorded every 12 h for84 h after LPS exposure

In the research on the protecting effects of AC-Rsv onLPS-induced ARDS mice were randomly divided into 4groups control LPS (5mgkg) only LPS (5mgkg) + AC-Rsv (50mgkg) AC-Rsv (50mgkg) Mice in the LPS + AC-Rsv group were pretreated with 50mgkg AC-Rsv for 7 daysand LPS was injected 90min after the last administrationof AC-Rsv since our previous results have indicated thatconcentration of Rsv in blood reached the peak 90minafter oral administration of AC-Rsv Mice from all groupswere sacrificed 12 h after LPS injection In addition theconcentration of LPS (20mgkg and 5mgkg) was selectedaccording to previous researches [22]

23 Histological Evaluation Lung tissues of the same lobefrom different groups were fixed with 4 paraformaldehydefor 24 h and then embedded in paraffin Lung samples werecut into 5 120583m thick sections after being deparaffinized anddehydrated After that slices of lung samples were stainedwith hematoxylin and eosin Finally all slices were examinedand captured under microscope (Leica Germany)












3 Results




Time (hours)

Surv

ival

()

20 40 60 8000

20

40

60

80

100

LPS25mgkg

50mgkg100mgkg












(a) (b) (c) (d)

Wet

-to-d

ry w

eigh

t rat

io

0

2

4

6


lowast

lowastlowast

(e)

00

02

04

06

08


Prot

ein

in B

ALF

(120583g120583

L)

lowastlowast

lowast

(f)

0

5

10

15

20

25

30

35


lowast

lowast

lowast

Cell

coun

t in

BALF

times10

4m

L

(g)




00

05

10

15

20

MPO

activ

ity (U

g p

rote

in)


lowast

lowastlowast




4 Discussion








0

100

200

300


TNF-120572

cont

ent (

pgm

L)

lowastlowast

lowast

(a)

0

50

100

150


lowastlowast

lowast

IL-1120573

cont

ent (

pgm

L)

(b)

IL-6

cont

ent (

pgm

L)

0

50

100

150


lowastlowast

lowast

(c)

TNF-120572

(pg

mL)

0

100

200

300

Control LPS Rsv

lowastlowast

lowast

LPS + Rsv(d)

IL-1120573

(pg

mL)

0

50

100

150

200

Control LPS

lowastlowast

lowast

RsvLPS + Rsv(e)

IL-6

(pg

mL)

0

30

60

90

120

150

Control LPS

lowastlowast

lowast

RsvLPS + Rsv(f)


119875





p-p38

t-p38

p-ERK

t-ERK

SIRT1

p-NF-120581B

t-NF-120581B

120573-actin

LPSAC-Rsv ++

++

minusminusminus

minus

(a)

p-p3

8p-

p38

00

02

04

06

08


lowastlowast

lowast

(b)

00

02

04

06

08

p-ER

Kt-E

RK


lowastlowast

lowast

(c)

00

02

04

06

08

p-N

F-120581

Bt-N

F-120581

B


lowastlowast

lowast

(d)

SIRT

1120573

-act

in

000

005

010

015


lowast

lowastlowast

(e)



p-p38

t-p38

p-ERK

t-ERK

SIRT1

p-NF-120581B

t-NF-120581B

120573-actin

LPSAC-Rsv ++

++ minus

minus

minus

minus

(a)

00

02

04

06

p-p3

8t-p

38

Control LPS

lowast

lowastlowast

RsvLPS + Rsv(b)

00

02

04

06

08

p-ER

Kt-E

RK

Control LPS

lowast

lowastlowast

RsvLPS + Rsv(c)

00

02

01

04

03

SIRT

1120573

-act

in

Control LPS

lowast

lowastlowast

RsvLPS + Rsv(d)

00

08

02

04

06

p-N

F-120581

Bt-N

F-120581

B

Control LPS

lowastlowast

lowast

RsvLPS + Rsv(e)


119875


00

02

03

01

SIRT1

120573-actin

SIRT

1120573

-act

in


lowastlowastlowast

lowastlowast

lowast

(a)

0

100

200

300


lowastlowastlowast

lowastlowast

lowast

TNF-120572

(pg

mL)

(b)

IL-1120573

(pg

mL)

0

50

100

150

200


lowastlowast

lowastlowast

lowastlowast

(c)

IL-6

(pg

mL)

0

50

100

150


lowastlowast


(d)












References



















































3 Results




Time (hours)

Surv

ival

()

20 40 60 8000

20

40

60

80

100

LPS25mgkg

50mgkg100mgkg












(a) (b) (c) (d)

Wet

-to-d

ry w

eigh

t rat

io

0

2

4

6


lowast

lowastlowast

(e)

00

02

04

06

08


Prot

ein

in B

ALF

(120583g120583

L)

lowastlowast

lowast

(f)

0

5

10

15

20

25

30

35


lowast

lowast

lowast

Cell

coun

t in

BALF

times10

4m

L

(g)




00

05

10

15

20

MPO

activ

ity (U

g p

rote

in)


lowast

lowastlowast




4 Discussion








0

100

200

300


TNF-120572

cont

ent (

pgm

L)

lowastlowast

lowast

(a)

0

50

100

150


lowastlowast

lowast

IL-1120573

cont

ent (

pgm

L)

(b)

IL-6

cont

ent (

pgm

L)

0

50

100

150


lowastlowast

lowast

(c)

TNF-120572

(pg

mL)

0

100

200

300

Control LPS Rsv

lowastlowast

lowast

LPS + Rsv(d)

IL-1120573

(pg

mL)

0

50

100

150

200

Control LPS

lowastlowast

lowast

RsvLPS + Rsv(e)

IL-6

(pg

mL)

0

30

60

90

120

150

Control LPS

lowastlowast

lowast

RsvLPS + Rsv(f)


119875





p-p38

t-p38

p-ERK

t-ERK

SIRT1

p-NF-120581B

t-NF-120581B

120573-actin

LPSAC-Rsv ++

++

minusminusminus

minus

(a)

p-p3

8p-

p38

00

02

04

06

08


lowastlowast

lowast

(b)

00

02

04

06

08

p-ER

Kt-E

RK


lowastlowast

lowast

(c)

00

02

04

06

08

p-N

F-120581

Bt-N

F-120581

B


lowastlowast

lowast

(d)

SIRT

1120573

-act

in

000

005

010

015


lowast

lowastlowast

(e)



p-p38

t-p38

p-ERK

t-ERK

SIRT1

p-NF-120581B

t-NF-120581B

120573-actin

LPSAC-Rsv ++

++ minus

minus

minus

minus

(a)

00

02

04

06

p-p3

8t-p

38

Control LPS

lowast

lowastlowast

RsvLPS + Rsv(b)

00

02

04

06

08

p-ER

Kt-E

RK

Control LPS

lowast

lowastlowast

RsvLPS + Rsv(c)

00

02

01

04

03

SIRT

1120573

-act

in

Control LPS

lowast

lowastlowast

RsvLPS + Rsv(d)

00

08

02

04

06

p-N

F-120581

Bt-N

F-120581

B

Control LPS

lowastlowast

lowast

RsvLPS + Rsv(e)


119875


00

02

03

01

SIRT1

120573-actin

SIRT

1120573

-act

in


lowastlowastlowast

lowastlowast

lowast

(a)

0

100

200

300


lowastlowastlowast

lowastlowast

lowast

TNF-120572

(pg

mL)

(b)

IL-1120573

(pg

mL)

0

50

100

150

200


lowastlowast

lowastlowast

lowastlowast

(c)

IL-6

(pg

mL)

0

50

100

150


lowastlowast


(d)












References









































Time (hours)

Surv

ival

()

20 40 60 8000

20

40

60

80

100

LPS25mgkg

50mgkg100mgkg












(a) (b) (c) (d)

Wet

-to-d

ry w

eigh

t rat

io

0

2

4

6


lowast

lowastlowast

(e)

00

02

04

06

08


Prot

ein

in B

ALF

(120583g120583

L)

lowastlowast

lowast

(f)

0

5

10

15

20

25

30

35


lowast

lowast

lowast

Cell

coun

t in

BALF

times10

4m

L

(g)




00

05

10

15

20

MPO

activ

ity (U

g p

rote

in)


lowast

lowastlowast




4 Discussion








0

100

200

300


TNF-120572

cont

ent (

pgm

L)

lowastlowast

lowast

(a)

0

50

100

150


lowastlowast

lowast

IL-1120573

cont

ent (

pgm

L)

(b)

IL-6

cont

ent (

pgm

L)

0

50

100

150


lowastlowast

lowast

(c)

TNF-120572

(pg

mL)

0

100

200

300

Control LPS Rsv

lowastlowast

lowast

LPS + Rsv(d)

IL-1120573

(pg

mL)

0

50

100

150

200

Control LPS

lowastlowast

lowast

RsvLPS + Rsv(e)

IL-6

(pg

mL)

0

30

60

90

120

150

Control LPS

lowastlowast

lowast

RsvLPS + Rsv(f)


119875





p-p38

t-p38

p-ERK

t-ERK

SIRT1

p-NF-120581B

t-NF-120581B

120573-actin

LPSAC-Rsv ++

++

minusminusminus

minus

(a)

p-p3

8p-

p38

00

02

04

06

08


lowastlowast

lowast

(b)

00

02

04

06

08

p-ER

Kt-E

RK


lowastlowast

lowast

(c)

00

02

04

06

08

p-N

F-120581

Bt-N

F-120581

B


lowastlowast

lowast

(d)

SIRT

1120573

-act

in

000

005

010

015


lowast

lowastlowast

(e)



p-p38

t-p38

p-ERK

t-ERK

SIRT1

p-NF-120581B

t-NF-120581B

120573-actin

LPSAC-Rsv ++

++ minus

minus

minus

minus

(a)

00

02

04

06

p-p3

8t-p

38

Control LPS

lowast

lowastlowast

RsvLPS + Rsv(b)

00

02

04

06

08

p-ER

Kt-E

RK

Control LPS

lowast

lowastlowast

RsvLPS + Rsv(c)

00

02

01

04

03

SIRT

1120573

-act

in

Control LPS

lowast

lowastlowast

RsvLPS + Rsv(d)

00

08

02

04

06

p-N

F-120581

Bt-N

F-120581

B

Control LPS

lowastlowast

lowast

RsvLPS + Rsv(e)


119875


00

02

03

01

SIRT1

120573-actin

SIRT

1120573

-act

in


lowastlowastlowast

lowastlowast

lowast

(a)

0

100

200

300


lowastlowastlowast

lowastlowast

lowast

TNF-120572

(pg

mL)

(b)

IL-1120573

(pg

mL)

0

50

100

150

200


lowastlowast

lowastlowast

lowastlowast

(c)

IL-6

(pg

mL)

0

50

100

150


lowastlowast


(d)












References









































(a) (b) (c) (d)

Wet

-to-d

ry w

eigh

t rat

io

0

2

4

6


lowast

lowastlowast

(e)

00

02

04

06

08


Prot

ein

in B

ALF

(120583g120583

L)

lowastlowast

lowast

(f)

0

5

10

15

20

25

30

35


lowast

lowast

lowast

Cell

coun

t in

BALF

times10

4m

L

(g)




00

05

10

15

20

MPO

activ

ity (U

g p

rote

in)


lowast

lowastlowast




4 Discussion








0

100

200

300


TNF-120572

cont

ent (

pgm

L)

lowastlowast

lowast

(a)

0

50

100

150


lowastlowast

lowast

IL-1120573

cont

ent (

pgm

L)

(b)

IL-6

cont

ent (

pgm

L)

0

50

100

150


lowastlowast

lowast

(c)

TNF-120572

(pg

mL)

0

100

200

300

Control LPS Rsv

lowastlowast

lowast

LPS + Rsv(d)

IL-1120573

(pg

mL)

0

50

100

150

200

Control LPS

lowastlowast

lowast

RsvLPS + Rsv(e)

IL-6

(pg

mL)

0

30

60

90

120

150

Control LPS

lowastlowast

lowast

RsvLPS + Rsv(f)


119875





p-p38

t-p38

p-ERK

t-ERK

SIRT1

p-NF-120581B

t-NF-120581B

120573-actin

LPSAC-Rsv ++

++

minusminusminus

minus

(a)

p-p3

8p-

p38

00

02

04

06

08


lowastlowast

lowast

(b)

00

02

04

06

08

p-ER

Kt-E

RK


lowastlowast

lowast

(c)

00

02

04

06

08

p-N

F-120581

Bt-N

F-120581

B


lowastlowast

lowast

(d)

SIRT

1120573

-act

in

000

005

010

015


lowast

lowastlowast

(e)



p-p38

t-p38

p-ERK

t-ERK

SIRT1

p-NF-120581B

t-NF-120581B

120573-actin

LPSAC-Rsv ++

++ minus

minus

minus

minus

(a)

00

02

04

06

p-p3

8t-p

38

Control LPS

lowast

lowastlowast

RsvLPS + Rsv(b)

00

02

04

06

08

p-ER

Kt-E

RK

Control LPS

lowast

lowastlowast

RsvLPS + Rsv(c)

00

02

01

04

03

SIRT

1120573

-act

in

Control LPS

lowast

lowastlowast

RsvLPS + Rsv(d)

00

08

02

04

06

p-N

F-120581

Bt-N

F-120581

B

Control LPS

lowastlowast

lowast

RsvLPS + Rsv(e)


119875


00

02

03

01

SIRT1

120573-actin

SIRT

1120573

-act

in


lowastlowastlowast

lowastlowast

lowast

(a)

0

100

200

300


lowastlowastlowast

lowastlowast

lowast

TNF-120572

(pg

mL)

(b)

IL-1120573

(pg

mL)

0

50

100

150

200


lowastlowast

lowastlowast

lowastlowast

(c)

IL-6

(pg

mL)

0

50

100

150


lowastlowast


(d)












References









































00

05

10

15

20

MPO

activ

ity (U

g p

rote

in)


lowast

lowastlowast




4 Discussion








0

100

200

300


TNF-120572

cont

ent (

pgm

L)

lowastlowast

lowast

(a)

0

50

100

150


lowastlowast

lowast

IL-1120573

cont

ent (

pgm

L)

(b)

IL-6

cont

ent (

pgm

L)

0

50

100

150


lowastlowast

lowast

(c)

TNF-120572

(pg

mL)

0

100

200

300

Control LPS Rsv

lowastlowast

lowast

LPS + Rsv(d)

IL-1120573

(pg

mL)

0

50

100

150

200

Control LPS

lowastlowast

lowast

RsvLPS + Rsv(e)

IL-6

(pg

mL)

0

30

60

90

120

150

Control LPS

lowastlowast

lowast

RsvLPS + Rsv(f)


119875





p-p38

t-p38

p-ERK

t-ERK

SIRT1

p-NF-120581B

t-NF-120581B

120573-actin

LPSAC-Rsv ++

++

minusminusminus

minus

(a)

p-p3

8p-

p38

00

02

04

06

08


lowastlowast

lowast

(b)

00

02

04

06

08

p-ER

Kt-E

RK


lowastlowast

lowast

(c)

00

02

04

06

08

p-N

F-120581

Bt-N

F-120581

B


lowastlowast

lowast

(d)

SIRT

1120573

-act

in

000

005

010

015


lowast

lowastlowast

(e)



p-p38

t-p38

p-ERK

t-ERK

SIRT1

p-NF-120581B

t-NF-120581B

120573-actin

LPSAC-Rsv ++

++ minus

minus

minus

minus

(a)

00

02

04

06

p-p3

8t-p

38

Control LPS

lowast

lowastlowast

RsvLPS + Rsv(b)

00

02

04

06

08

p-ER

Kt-E

RK

Control LPS

lowast

lowastlowast

RsvLPS + Rsv(c)

00

02

01

04

03

SIRT

1120573

-act

in

Control LPS

lowast

lowastlowast

RsvLPS + Rsv(d)

00

08

02

04

06

p-N

F-120581

Bt-N

F-120581

B

Control LPS

lowastlowast

lowast

RsvLPS + Rsv(e)


119875


00

02

03

01

SIRT1

120573-actin

SIRT

1120573

-act

in


lowastlowastlowast

lowastlowast

lowast

(a)

0

100

200

300


lowastlowastlowast

lowastlowast

lowast

TNF-120572

(pg

mL)

(b)

IL-1120573

(pg

mL)

0

50

100

150

200


lowastlowast

lowastlowast

lowastlowast

(c)

IL-6

(pg

mL)

0

50

100

150


lowastlowast


(d)












References









































0

100

200

300


TNF-120572

cont

ent (

pgm

L)

lowastlowast

lowast

(a)

0

50

100

150


lowastlowast

lowast

IL-1120573

cont

ent (

pgm

L)

(b)

IL-6

cont

ent (

pgm

L)

0

50

100

150


lowastlowast

lowast

(c)

TNF-120572

(pg

mL)

0

100

200

300

Control LPS Rsv

lowastlowast

lowast

LPS + Rsv(d)

IL-1120573

(pg

mL)

0

50

100

150

200

Control LPS

lowastlowast

lowast

RsvLPS + Rsv(e)

IL-6

(pg

mL)

0

30

60

90

120

150

Control LPS

lowastlowast

lowast

RsvLPS + Rsv(f)


119875





p-p38

t-p38

p-ERK

t-ERK

SIRT1

p-NF-120581B

t-NF-120581B

120573-actin

LPSAC-Rsv ++

++

minusminusminus

minus

(a)

p-p3

8p-

p38

00

02

04

06

08


lowastlowast

lowast

(b)

00

02

04

06

08

p-ER

Kt-E

RK


lowastlowast

lowast

(c)

00

02

04

06

08

p-N

F-120581

Bt-N

F-120581

B


lowastlowast

lowast

(d)

SIRT

1120573

-act

in

000

005

010

015


lowast

lowastlowast

(e)



p-p38

t-p38

p-ERK

t-ERK

SIRT1

p-NF-120581B

t-NF-120581B

120573-actin

LPSAC-Rsv ++

++ minus

minus

minus

minus

(a)

00

02

04

06

p-p3

8t-p

38

Control LPS

lowast

lowastlowast

RsvLPS + Rsv(b)

00

02

04

06

08

p-ER

Kt-E

RK

Control LPS

lowast

lowastlowast

RsvLPS + Rsv(c)

00

02

01

04

03

SIRT

1120573

-act

in

Control LPS

lowast

lowastlowast

RsvLPS + Rsv(d)

00

08

02

04

06

p-N

F-120581

Bt-N

F-120581

B

Control LPS

lowastlowast

lowast

RsvLPS + Rsv(e)


119875


00

02

03

01

SIRT1

120573-actin

SIRT

1120573

-act

in


lowastlowastlowast

lowastlowast

lowast

(a)

0

100

200

300


lowastlowastlowast

lowastlowast

lowast

TNF-120572

(pg

mL)

(b)

IL-1120573

(pg

mL)

0

50

100

150

200


lowastlowast

lowastlowast

lowastlowast

(c)

IL-6

(pg

mL)

0

50

100

150


lowastlowast


(d)












References









































p-p38

t-p38

p-ERK

t-ERK

SIRT1

p-NF-120581B

t-NF-120581B

120573-actin

LPSAC-Rsv ++

++

minusminusminus

minus

(a)

p-p3

8p-

p38

00

02

04

06

08


lowastlowast

lowast

(b)

00

02

04

06

08

p-ER

Kt-E

RK


lowastlowast

lowast

(c)

00

02

04

06

08

p-N

F-120581

Bt-N

F-120581

B


lowastlowast

lowast

(d)

SIRT

1120573

-act

in

000

005

010

015


lowast

lowastlowast

(e)



p-p38

t-p38

p-ERK

t-ERK

SIRT1

p-NF-120581B

t-NF-120581B

120573-actin

LPSAC-Rsv ++

++ minus

minus

minus

minus

(a)

00

02

04

06

p-p3

8t-p

38

Control LPS

lowast

lowastlowast

RsvLPS + Rsv(b)

00

02

04

06

08

p-ER

Kt-E

RK

Control LPS

lowast

lowastlowast

RsvLPS + Rsv(c)

00

02

01

04

03

SIRT

1120573

-act

in

Control LPS

lowast

lowastlowast

RsvLPS + Rsv(d)

00

08

02

04

06

p-N

F-120581

Bt-N

F-120581

B

Control LPS

lowastlowast

lowast

RsvLPS + Rsv(e)


119875


00

02

03

01

SIRT1

120573-actin

SIRT

1120573

-act

in


lowastlowastlowast

lowastlowast

lowast

(a)

0

100

200

300


lowastlowastlowast

lowastlowast

lowast

TNF-120572

(pg

mL)

(b)

IL-1120573

(pg

mL)

0

50

100

150

200


lowastlowast

lowastlowast

lowastlowast

(c)

IL-6

(pg

mL)

0

50

100

150


lowastlowast


(d)












References









































p-p38

t-p38

p-ERK

t-ERK

SIRT1

p-NF-120581B

t-NF-120581B

120573-actin

LPSAC-Rsv ++

++ minus

minus

minus

minus

(a)

00

02

04

06

p-p3

8t-p

38

Control LPS

lowast

lowastlowast

RsvLPS + Rsv(b)

00

02

04

06

08

p-ER

Kt-E

RK

Control LPS

lowast

lowastlowast

RsvLPS + Rsv(c)

00

02

01

04

03

SIRT

1120573

-act

in

Control LPS

lowast

lowastlowast

RsvLPS + Rsv(d)

00

08

02

04

06

p-N

F-120581

Bt-N

F-120581

B

Control LPS

lowastlowast

lowast

RsvLPS + Rsv(e)


119875


00

02

03

01

SIRT1

120573-actin

SIRT

1120573

-act

in


lowastlowastlowast

lowastlowast

lowast

(a)

0

100

200

300


lowastlowastlowast

lowastlowast

lowast

TNF-120572

(pg

mL)

(b)

IL-1120573

(pg

mL)

0

50

100

150

200


lowastlowast

lowastlowast

lowastlowast

(c)

IL-6

(pg

mL)

0

50

100

150


lowastlowast


(d)












References









































00

02

03

01

SIRT1

120573-actin

SIRT

1120573

-act

in


lowastlowastlowast

lowastlowast

lowast

(a)

0

100

200

300


lowastlowastlowast

lowastlowast

lowast

TNF-120572

(pg

mL)

(b)

IL-1120573

(pg

mL)

0

50

100

150

200


lowastlowast

lowastlowast

lowastlowast

(c)

IL-6

(pg

mL)

0

50

100

150


lowastlowast


(d)












References









































References








































3,5,4′-Tri-O-acetylresveratrol Attenuates ... fileResearchArticle 3,5,4-Tri-O-acetylresveratrol...

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Transcript of 3,5,4′-Tri-O-acetylresveratrol Attenuates ... fileResearchArticle 3,5,4-Tri-O-acetylresveratrol...