Research Article Modulating the p66shc Signaling Pathway...

Research ArticleModulating the p66shc Signaling Pathway withProtocatechuic Acid Protects the Intestine fromIschemia-Reperfusion Injury and Alleviates SecondaryLiver Damage

Lingfei Ma1 Guangzhi Wang1 Zhao Chen1 Zhenlu Li1 Jihong Yao2 Haidong Zhao1

Shu Wang1 Zhenhai Ma1 Hong Chang1 and Xiaofeng Tian1

1 Department of General Surgery The Second Hospital of Dalian Medical University Dalian 116023 China2Department of Pharmacology Dalian Medical University Dalian 116044 China

Correspondence should be addressed to Xiaofeng Tian elsevier txfhotmailcom

Received 10 November 2013 Accepted 16 February 2014 Published 16 March 2014

Academic Editors E de Bree and M de Luca

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

Intestinal ischemia-reperfusion (IR) injury is a serious clinical pathophysiological process that may result in acute local intestineand remote liver injury Protocatechuic acid (PCA) which has beenwidely studied as a polyphenolic compound induces expressionof antioxidative genes that combat oxidative stress and cell apoptosis In this study we investigated the effect of PCA pretreatmentfor protecting intestinal IR-induced local intestine and remote liver injury in mice Intestinal IR was established by superiormesenteric artery occlusion for 45min followed by reperfusion for 90min After the reperfusion period PCA pretreatmentmarkedly alleviated intestine and liver injury induced by intestinal IR as indicated by histological alterations decreases inserological damage parameters and nuclear factor-kappa B and phospho-foxo3a protein expression levels and increases inglutathione glutathione peroxidase manganese superoxide dismutase protein expression and Bcl-xL protein expression in theintestine and liverThese parameters were accompanied by PCA-induced adaptor protein p66shc suppressionThese results suggestthat PCA has a significant protective effect in the intestine and liver following injury induced by intestinal IRThe protective effectof PCAmay be attributed to the suppression of p66shc and the regulation of p66shc-related antioxidative and antiapoptotic factors

1 Introduction

Intestinal ischemia-reperfusion (IR) injury is a complexsurgical and pathophysiological process which is secondaryto many clinical conditions such as shock small boweltransplantation disseminated intravascular coagulation andsepsis [1ndash4] Intestinal IR injury is an extremely criticalcondition since restoring the blood supply after mesentericischemia results in desquamation of the intestinal epitheliumbacteria translocation and systemic inflammatory response[5 6] Patients of such processes often suffer from fatal con-ditions including systemic inflammatory response syndrome(SIRS) and multiple organ dysfunction syndrome (MODS)[7] MODS is characterized as progressive physiologic

dysfunction of two or more organ systems The organsthat most commonly show potential dysfunction includethe local intestine [8] liver [9] lungs [10] and kidneys[8] Of these distant organs the liver is the first distantorgan involved after intestinal IR presumably because thevasculature of the liver is coupled in series with intestinalcirculation [9 11ndash13] Mechanism studies revealed that themagnitude of intestinal IR-induced liver injury dependson inflammatory infiltration reactive oxidant species (ROS)accumulation and the response of apoptosis [9 12 14] Aseries of studies has suggested that systemic inflammatoryresponse oxidative stress and cell apoptosis play importantroles during the development of intestinal IR injury andsubsequent liver damage Probable mechanisms involved in

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 387640 11 pageshttpdxdoiorg1011552014387640

2 The Scientific World Journal

this process include glutathione (GSH) depletion nuclearfactor-kappa B (NF-120581B) pathway activation and suppres-sion of Nf-E2-related factor 2 (Nrf2) nuclear translocation[14ndash16] However detailed mechanisms of intestinal IRremain obscure Recent studies showed that genetic abla-tion of the adaptor protein p66shc (p66shc) in the mousereduced ROS production and consequently prolonged thelife span by 30 [17] while deletion of p66shc protectedmice from IR-induced brain injury through blunted produc-tion of free radicals [18] However it is unknown whetherp66shc is involved in intestinal IR injury and subsequentliver dysfunction Therefore we examined the redox-relatedenzyme p66shc and hypothesized that targeted suppressionof p66shc would protect against damage during intestinalIR

p66shc is one of three isoforms derived from alternativesplicing of the Shc locus It is ubiquitously expressed inmammals and functions as a redox enzyme implicated inmitochondrial ROS generation [19] The other two isoformsof this family p46shc and p52shc have been described asadaptor proteins that can alter mitogen-activated proteinkinase and Fos activation upon stimulation with epidermalgrowth factor and platelet-derived growth factor [20] p66shcfunctionally differentiates from these isoforms Due to itsunique NH

2-terminal region it has a serine 36 residue that

can be phosphorylated allowing p66shc to be transferredfrom the cytosol to the mitochondria p66shc translocationcan lead to blunting of Ca2+ responses and opening ofthe mitochondrial permeability transition pore and thusinducing intracellular ROS overgeneration and cell apoptosisEvidence is increasingly accumulating to show that p66shcmay play a critical role in ROS production and cell apoptosis[17 21] p66shc-null mice unlike wild-typemice were shownto be protected against atherogenesis high glucose-inducedrenal injury and aging [17 22 23] Additionally knock-down of p66shc expression or phospho-p66shc suppressionameliorated cardiac ischemia-reperfusion induced oxidativestress injury in vivo or improved mouse kidney proximaltubule cells surviving during H

2O2-induced oxidative stress

injury in vitro [24 25]Protocatechuic acid (PCA) which has been widely stud-

ied as a polyphenolic compound is derived from greentea fruits and various Chinese herbal medicines such asSalvia miltiorrhiza [26] Recent studies have shown that PCAinduced high expression of antioxidative genes and playeda significant role in fighting against oxidative stress injuryand cell apoptosis [26 27] PCA pretreatment can preventcells from oxidative stress injury by relieving GSH depletionand enhancing the activity of glutathione peroxidase (GSH-PX) and glutathione reductase which show marked antiox-idative activity [27] Additionally studies showed that PCApretreatment significantly attenuated PC12 cell apoptosis bylimiting caspase-3 activity and inducing Bcl-2 expression[28] Notably a recent study indicated that PCA pretreatmentcan downregulate p66shc expression and protect againstcell apoptosis induced by lipid peroxidation in Caco-2 cells[27] However there was no evidence that PCA pretreatment

protects against acute intestinal stress injury and liver dys-function induced by intestinal IR

Therefore in this study we used an intestinal IR modelinmice to examine the hypotheses that (1) p66shc-dependentoxidative stress and apoptosis mechanisms are involved inlocal organ damage and remote liver injury induced byintestinal IR (2) p66shc suppression by PCA pretreatmentmay protect against intestinal injury and liver dysfunctioninduced by intestinal IR (3) the protective effect of PCApretreatment may be related to p66shc-related foxo3a andBcl-xL signaling pathways

2 Materials and Methods

21 Experimental Design The study was performed in adultmale ICR mice (18ndash22 g) (Animal Centre of Dalian MedicalUniversity Dalian China) The mice were housed at a tem-perature of 22plusmn 2∘C kept on a 1212-h photoperiod and wereall provided with food and water ad libitum Procedures wereconducted in accordance with the institutional guidelines forthe care and use of laboratory animals and the study wasapproved by the Institutional Animal Care Committee ofDalian Medical University (Dalian China)

22 Intestinal IR Model and Experimental Procedures Themouse intestinal IR model was established according topreviously standardized methods [29] Briefly after generalanesthesia a midline laparotomy was performed and thesuperior mesenteric artery (SMA) was isolated gently at itsorigin and occluded with an atraumatic microvascular clampfor 45min and then followed by reperfusion for 90min Theocclusion was confirmed by complete pulse cessation and theintestines became pale then the reperfusion was confirmedby the return of pulsatile flow to the mesenteric artery andits branches Sham animals underwent the same protocolbut the SMA was not occluded Fifty mice were dividedrandomly into five groups (119899 = 10) (i) a sham group whichunderwent sham surgery (ii) a IR group which underwentintestinal IR model as previously mentioned (iii) a sham +PCA high dose (H) group which underwent sham surgeryand pretreated with PCA (JampK Chemical Germany) at adose of 80mgkg intraperitoneally (ip) for 3 consecutivedays (iv) a IR + PCA low dose (L) group which underwentintestinal IR model as previously mentioned PCA waspretreated at a dose of 40mgkg ip for 3 consecutive days(v) a IR + PCA high dose (H) group which underwentintestinal IR model as previously mentioned and receivedPCA pretreatment at a dose of 80mgkg ip for 3 consecutivedays The dose and route of administration were determinedaccording to a previous study [30] PCA was dissolved insterile normal saline before administration and the mice inthe sham and IR groups were treated with an equal volumeof sterile normal saline for parallel Blood and tissue foranalysis were obtained by placing the mice under anesthesiaand reopening the abdominal midline incision After beingsacrificed by abdominal aortic blood drawing intestine andliver tissue samples were procured from a standard location

The Scientific World Journal 3

in the terminal ileum and left lobe of liver Blood and tissuesamples were collected for future analysis

23 Intestine and Liver Morphologic Evaluation For his-topathological analysis the intestine and liver specimenswere instantly fixed in 10 buffered formalin phosphateand embedded in paraffin sectioned transversely (4 120583m)and then hematoxylin eosin (HampE) stained The mucosalinjury score was graded on a six-tiered scale modified fromChiursquos method [31] Liver pathologic scores were evaluatedaccording to Eckhoff rsquos reports [32]

24 Measurement of Serum Alanine Aminotransferase (ALT)Aspartate Aminotransferase (AST) Tumor Necrosis Factor(TNF)-120572 and Interleukin (IL)-6 Levels Collected blood wascentrifuged (1000 g 10min 4∘C) and the serumwas obtainedALT andAST levels in the serumweremeasured according tothemanufacturerrsquos instructions suppliedwith the commercialassay kits (Jiancheng Bioengineering BiotechnologyNanjingChina) TNF-120572 and IL-6 in the serumwere determined usingcommercially available enzyme-linked immunosorbent assaykits according to the manufacturerrsquos instructions (BOSTERBio-Engineering Limited Company Wuhan China)

25The Intestinal andHepatic Antioxidant SystemAssay Thelevel of GSH and the activities of GSH-PX were analysedusing commercial assay kits according to the manufacturerrsquosinstructions (Jiancheng Bioengineering Institute NanjingChina) and were expressed as mg GSHg protein and Ugprotein respectively

26 Semiquantitative Polymerase Chain Reaction (RT-PCR)for mRNA Analysis The levels of mRNA expression ofp66shc in the intestine and liver were determined by RT-PCR assays Total RNA was extracted from mouse intestineand liver using TRIZOL reagent (Invitrogen Carlsbad CAUSA) according to the manufacturerrsquos instructions Reversetranscription into cDNA was performed using a TAKARARNA PCR Kit (AMV) Ver 30 (TAKARA Dalian China)for PCR analysis The following primers were used p66shcsense 51015840-ACTACCCTGTGTTCCTTCTTTC-31015840 antisense51015840-TCGGTGGATTCCTGAGATACTGT-31015840 120573-actin sense51015840-AGAGGGAAATCGTGC-31015840 antisense 51015840-CAATAG-TGATGACCTGGCCGT-31015840 All primers were synthesizedby TAKARA Five microliters of each RT-PCR were elec-trophoresed in a 15 agarose gel and stained with ethidiumbromide A BioSpectrum-410 multispectral imaging systemwith Chemi HR camera 410 (UVP Upland CA USA) wasused to analyze the intensity of the DNA bandsThe intensityof each band was quantified by densitometry using a geldocumentation and analysis system and normalized to valuesfor 120573-actin

27 Protein Extraction and Western Blotting Tissues werehomogenized in mammalian tissue protein extractionreagent (KeyGEN Nanjing China) supplemented withprotease inhibitor and phosphatase inhibitor For westernimmunoblotting 25 to 60 120583g of protein was separated by

10ndash15 Bis-Tris protein gel (Bio-Rad Hercules CA USA)transferred to polyvinylidene difluoride membranes(Millipore Bedford MA USA) and blocked with5 nonfat milk or 1 bovine serum albumin in tris-buffered saline tween-20 for 2 h at room temperature Themembranes were then incubated overnight with antibodiesagainst p66shc phospho-p66shc foxo3a manganesesuperoxide dismutase (MnSOD) (Abcam Ltd CambridgeUK) phospho-foxo3a (Beyotime Institute of BiotechnologyHangzhou China) NF-120581B (Santa Cruz BiotechnologyCA USA) Bcl-xL (Proteintech group Wuhan China)cleaved-caspase-3 (Bioworld Minneapolis MN USA)Histone H31 (Santa Cruz Biotechnology) or 120573-actin(Beyotime Institute of Biotechnology) After incubation withappropriate horseradish peroxidase-conjugated secondaryantibodies membranes were developed with ECL plus sub-strate (Beyotime Institute of Biotechnology) Images weredocumented with a BioSpectrum-410 multispectral imagingsystem with a Chemi HR camera 410 (UVP Upland CAUSA) and quantitated with the Gel-Pro Analyzer Version 40(Media Cybernetics Rockville MD USA)

28 Statistical Analysis Data were expressed as mean plusmnstandard deviation (SD) The statistical analysis was carriedout using the SPSS160 statistical software package (SPSS IncChicago IL USA) Statistical comparisons were analyzedby Kruskal-Wallis test for nonnormal distributions followedby Wilcoxon Rank Sum test with Bonferroni adjustmentsfor multiple comparisonsor a one-way analysis of variance(ANOVA) followed by Student-Newman-Keuls (SNK) testfor normal distributions P value lt 005 was consideredstatistically significant

3 Results

31 PCA Pretreatment Decreased Histopathologic Damagesof Intestine and Liver after Intestinal IR There were noobvious morphologic changes in the intestine or liver tis-sues in the sham and sham + PCA groups whereas mor-phological changes from the IR group showed signifi-cant histopathologic abnormalities compared with the shamgroup including haemorrhage severe villi irregularities andedema in the intestine (Figure 1) Meanwhile liver tissuewasmarkedly damaged and showed cytoplasmic vacuolationextensive nuclear pyknosis and disintegration of hepaticcords (Figure 2) In the PCA pretreatment groups the sever-ity of histopathologic changes in the intestine and liver tissueswas weaker than that in the IR group (Figures 1 and 2)Theseresults indicate that PCA confers protection against intestineand liver injury induced by intestinal IR

32 PCA Pretreatment Protected against Inflammation Dam-age and Liver Injury Induced by Intestinal IR To determinewhether intestinal IR and PCA administration affect sys-temic inflammation we measured TNF-120572 and IL-6 concen-trations in serum Compared with the sham group TNF-120572 and IL-6 concentrations were significantly elevated in theserum of the IR group In contrast our results showed


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Figure 1 PCApretreatment improved intestinal IR-induced intestine histopathologic injury Tissueswere harvested at the end of reperfusionandwere stainedwith hematoxylin eosin Slides were examined under lightmicroscopy at 200xmagnification bars = 50120583mHistologic injuryscores in groups were quantified Results are presented as the mean plusmn SD 119899 = 10 lowastlowast119875 lt 001 versus sham group 119875 lt 001 versus IR group

that PCA pretreatment could prevent the increase in TNF-120572 and IL-6 production (Figure 3(a)) Serum ALT and ASTlevels were measured to assess liver function As shown inFigure 3(b) after a 45-minute intestinal ischemia followedby a 90-minute reperfusion ALT and AST levels in theserum were significantly increased compared with the shamgroup after PCA pretreatment a significant reduction inthese parameters was observed in both groups compared tothe IR group In addition the nuclear expression of NF-120581B p65 subunit is critical for a wide range of processes suchas inflammation [33] We found that intestinal IR-inducedintestine and liver NF-120581B p65 subunit expression in thenuclear were inhibited by PCA pretreatment (Figures 3(c)-3(d))

33 PCA Pretreatment Improved the Antioxidative Capacity ofthe Intestine and Liver after Intestinal IR The GSH systemis generally considered to be a sensitive indicator of freeradical scavenging ability In this study intestinal IR causeda dramatic decrease in GSH and GSH-PX activities in the

intestine and liver however PCA pretreatment improvedGSH and GSH-PX activities in a dose-dependent manner(Table 1)

34 Expressions of p66shc in the Intestine and Liver WereRestrained by PCA Pretreatment after Intestinal IR Todetermine whether p66shc plays a role in intestinal IRinjury and subsequent acute liver injury as well as exploringthe p66shc-regulation effect of PCA administration RT-PCR and western blot analysis were carried out Expressionlevels of p66shc mRNA were upregulated due to intestinalIR in the intestine and liver whereas PCA pretreatmentresulted in a significant dose-dependent decrease in p66shcmRNA levels compared to IR treatment There were nodifferences in the sham + PCA groups compared withthe sham group (Figure 4) Similarly changes in the levelof p66shc phosphorylation in the intestine and liver weresignificantly increased after intestinal IR PCA pretreatmentcould attenuate p66shc phosphorylation after intestinal IR(Figure 5) These data show that intestine and liver p66shc




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Figure 2 PCA pretreatment improved intestinal IR-induced liver histopathologic injury Tissues were harvested at the end of reperfusionandwere stainedwith hematoxylin eosin Slides were examined under lightmicroscopy at 200xmagnification bars = 50120583mHistologic injuryscores in groups were quantified Results are presented as the mean plusmn SD 119899 = 10 lowastlowast119875 lt 001 versus sham group 119875 lt 005 versus IR group119875 lt 001 versus IR group

mRNA expression and phosphorylation levels were elevatedafter intestinal IR while PCA pretreatment showed efficientalleviation of p66shc mRNA and phosphorylated proteinlevels particularly in the 80mgkg PCA pretreatment group

35 Effects of PCAPretreatment onOxidative-Stress Regulatorsof p66shc Signaling Pathway Foxo3a is a mammalian fork-head protein that tightly regulates the transcription of antiox-idant genes such as MnSOD and catalase Since previousreports indicated that foxo3a phosphorylation is associatedwith p66shc activation in response to oxidative stress [19] wefurther examined whether phosphorylation of foxo3a in theintestine and liver is involved in p66shc activation in responseto intestinal IR As shown in Figure 6 expression levels ofphospho-foxo3a were upregulated due to intestinal IR inthe intestine and liver whereas PCA pretreatment resultedin a significant dose-dependent decrease in phospho-foxo3alevels compared to IR treatment However there were nodifferences in the sham + PCA groups compared with the

sham group In contrast to phospho-foxo3a expression ofMnSOD in the intestine and liver after intestinal IR followingPCA pretreatment resulted in a significant dose-dependentincrease in MnSOD levels compared to IR exposure

36 Effects of PCA on Apoptotic Factors in the p66shc Sig-naling Pathway Several studies have shown that p66shc is acritical apoptotic component in a variety of pathophysiolog-ical processes For example high glucose levels inducedgreater caspase-3 activation in empty vector cells than inp66shc knockdown cells [34] In the present study intesti-nal IR resulted in a significant increase in the intestineand liver cleaved-caspase-3 expression as compared to thesham-operated group while PCA pretreatment preventedthe intestinal IR-induced increase of cleaved-caspase-3expression Moreover expression of the antiapoptotic factorBcl-xL in the intestine and liver was restrained becauseof intestinal IR However PCA pretreatment markedly


IL-6TNF-120572

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Figure 3 PCA pretreatment ameliorated intestinal IR-induced systemic inflammation hepatic dysfunction and NF120581B activation ((a) and(b)) Alterations of IL-6 TNF-120572 ALT and AST in the serum 119899 = 10 (c) Western blot analysis of nuclear NF120581B p65 in the intestine 119899 = 3(d) Western blot analysis of nuclear NF120581B p65 in the liver 119899 = 3 Results are presented as the mean plusmn SD lowastlowast119875 lt 001 versus sham group119875 lt 005 versus IR group 119875 lt 001 versus IR group 1 indicates Sham 2 IR 3 Sham + PCA (H) 4 IR + PCA (L) 5 IR + PCA (H)


IntestineLiver

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Figure 4 PCA pretreatment suppressed the mRNA expression of p66shc in the intestine and liver after intestinal IR Results are presentedas the mean plusmn SD 119899 = 3 lowastlowast119875 lt 001 versus sham group 119875 lt 001 versus IR group

Table 1 PCA pretreatment ameliorated intestinal IR induced GSH system dysfunction in the intestine and liver

Group Intestine GSH(mgg protein)

Intestine GSH-PX(Ug protein)

Liver GSH(mgg protein)

Liver GSH-PX(Ug protein)

Sham 371 plusmn 048 8773 plusmn 1051 245 plusmn 017 42431 plusmn 2206

IR 225 plusmn 055lowastlowast

4354 plusmn 861lowastlowast



Sham + PCA (H) 381 plusmn 056 9438 plusmn 1945 229 plusmn 023 41856 plusmn 1572

IR + PCA (L) 275 plusmn 053 6985 plusmn 1058

176 plusmn 008 35701 plusmn 851

IR + PCA (H) 323 plusmn 016

7240 plusmn 1103

207 plusmn 017

37942 plusmn 439

Results are presented as the mean plusmn SD 119899 = 10 lowastlowast119875 lt 001 versus sham 119875 lt 001 versus IR


P-p66shc

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Figure 5 PCA pretreatment suppressed the phosphorylation of p66shc in the intestine and liver after intestinal IR Results are presented asthe mean plusmn SD 119899 = 3 lowastlowast119875 lt 001 versus sham group 119875 lt 001 versus IR group

enhanced expression of Bcl-xL compared with the IR group(Figure 6)

4 Discussion

Intestinal IR which is thought to be a triggering event in thedevelopment of local and distant organ dysfunction remainsa critical problem [35] Multiple organ failure particularlyremote liver injury is a recurrent complication of intestinalIR injury and contributes to its highmorbidity andmortality[9 11ndash13] In this study we determined the effect of PCAon intestinal IR injury and secondary severe liver injurymediated by p66shc suppression Since PCA is commonlyused as an antioxidative and stress-protective agent duringmultiple pathophysiological processes [26ndash28] we examined

the protective effect of this agent during these processesThis study demonstrated that PCA pretreatment significantlyattenuated intestinal IR induced local intestine and remoteliver morphological damage oxidative stress response sys-temic inflammation and hepatic dysfunction all of whichresulted in improvement of the morbid state Our resultsalso demonstrated that PCA pretreatment had significantrestraining effects on p66shc mRNA expression and proteinphosphorylation after intestinal IR in the intestine and liveraccompanied by p66shc-related oxidative stress regulatorsand apoptotic protein alteration

A number of changes may occur in the intestinal mucosaand hepatocytes after acute intestinal IR leading to intesti-nal barrier dysfunction bacterial translocation systemicinflammation and accompanying hypohepatia [8 9 11


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Figure 6 PCA pretreatment regulated the oxidative-stress regulators and apoptotic factors of p66shc pathway Western blot analysis ofphospho-foxo3a (p-foxo3a) foxo3a MnSOD cleaved-caspase-3 (C-caspase-3) and Bcl-xL in the intestine and liver Results are presented asthe mean plusmn SD 119899 = 3 lowastlowast119875 lt 001 versus sham group 119875 lt 005 versus IR group 119875 lt 001 versus IR group 1 indicates Sham 2 IR 3Sham + PCA (H) 4 IR + PCA (L) 5 IR + PCA (H)

12] In the present study our results indicated that severeintestinal mucosal lesions and hepatic cell damage occurredafter intestinal IR whereas PCA pretreatment significantlydecreased these negative changes This suggests that PCAcan improve morphological alterations in the intestinalmucosa and hepatocytes in a murine model of intestinalIR

Several reports have indicated that impaired intestinalreperfusion after acute ischemia resulted in bacteria translo-cation into blood and other organs by damaging the intestinalbarrier The consequences of this process include inflamma-tory cytokine release oxidative stress induction and SIRS orMODS initiation [1 4] Most importantly IL-6 and TNF-120572are inflammatory cytokines and are commonly known to beelevated in the serum after intestinal IR [15] Once secondaryliver injury is increased ALT and AST are the clearest indica-tors of this process [14] In this study intestinal IR resulted inthe release of systemic inflammatory cytokines (IL-6 TNF-120572)as well as elevation of liver enzymes (ALT AST) Secondly ithas been demonstrated that activation of NF-120581B is a criticalevent for a wide range of processes such as inflammation [33]We found that NF-120581B p65 was activated in the intestine andliver after intestinal IR Moreover GSH and GSH-PX aregenerally used as critical indicators of antioxidative capacity[36] Giovannini et al [27] indicated that an impairedGSH antioxidant system promoted Caco-2 cells towards anapoptotic-prone phenotype by activating p66Shc expressionSimilarly our data showed that intestinal IR resulted in

reduction in the antioxidative capacity in the intestine andliver manifested as GSH depletion and a reduction in GSH-PX activity These alterations parallel the enhanced p66shcmRNA expression and phospho-p66shc upregulation In thisstudy intestinal and liver p66shc mRNA expression andprotein phosphorylation levels were suppressed by PCA andthe indicators described above were significantly improvedIn summary suppressing expression of p66shc by PCApretreatment can improve the inflammatory response liverdysfunction and organ oxidative stress injury in the intestineand liver after intestinal IR

p66shc functions to regulate the level of intracellularROS cell apoptosis and lifespan in mammals [19 2137] Its transduction properties in downstream signalinghave recently attracted significant interest in stress-relatedresearch [17 19] Reports demonstrated that animals with amutant p66shc gene induced stress resistance and prolongedlifespan [37] Foxo3a belongs to theO subclass of the forkheadfamily and is thought to be a regulator of longevity andoxidative stress [38] This gene encodes a variety of impor-tant antioxidant agents such as MnSOD and catalase [19]When oxidative stress occurs ROS signaling activates p66shcthrough phosphorylation of a serine 36 residue Phospho-p66shc can catalyze foxo3a phosphorylation andcytoplasmtranslocation resulting in suppression ofMnSOD expression[19] Nemoto and Finkel demonstrated that dephospho-rylation of foxo3a resulted in both an increase in ROSscavenging ability and oxidative stress resistance whereas


phosphorylation of foxo3a led to oxidative stress injury pro-motion However such injury induced by hydrogen peroxideis relieved in p66shcminusminus cells [19] Additionally AGE-inducedphosphorylation of p66Shc selectively phosphorylated foxo3aand downregulated MnSOD in Human Embryonic Kidney293 cells [39] In the present study p66shc was significantlyphosphorylated in the intestine and liver after acute intestinalIR suggesting that p66shc phosphorylation is involvedin the local intestine and remote liver injury induced byintestinal IR However PCApretreatmentmarkedly reducedphosphorylation of p66shc in the liver and intestine Addi-tionally our results revealed that intestinal IR can lead toan increase in intestinal and hepatic foxo3a phosphorylationand downregulation of the transcription product MnSODwhereas PCA pretreatment reversed these effects Our resultsindicate that phosphorylation of foxo3a may be mediatedin part by intestinal IR-induced p66shc phosphorylation inthe intestine and liver Because phosphorylation of p66shcwas suppressed PCA suppressed foxo3a phosphorylation andenhanced MnSOD expression

Increasing data support a viewpoint that varying degreesof multiple organ apoptosis occur during the pathogenesisof intestinal IR [9 10 40] Horie et al [14] found that ratsexposed to intestinal IR caused an increase in apoptotichepatocytes Previous reports demonstrated that cleaved-caspase-3 and Bcl-xL were sensitive indicators of p66shc-induced apoptosis [34 41] Therefore we detected cleaved-caspase-3 and Bcl-xL to reflect the proapoptosis and anti-apoptosis levels of the intestine and liver in the present studyThese results showed that intestinal IR-induced intestineand liver p66shc phosphorylation were accompanied bycleaved-caspase-3 upregulation and Bcl-xL downregulationwhereas PCA pretreatment significantly reversed this trendConsistent with previous reports [34 41] we inferred thatintestinal IR can result in cell apoptosis in the intestineand liver and such damages were characterized by p66shcphosphorylation-induced caspase-3 activation In contrastPCA pretreatment may protect intestinal epithelial cells andhepatocyte from apoptosis induced by intestinal IR throughupregulation of the antiapoptotic gene Bcl-xL followingp66shc suppression

In summary we demonstrated that PCA had signifi-cant protective effects against local intestine and remoteliver injury induced by intestinal IR in mice and that itsprotective effects appeared to involve inhibition of p66shcFurthermore such protective effects may be mediated in partby foxo3a activation MnSOD upregulation and enhancedBcl-xL expression involved in the p66shc signaling pathwayAccordingly PCAmay be a potential candidate for improvingintestinal IR injury and secondary liver damage Addition-ally manipulation of p66shc phosphorylation may offer anattractive therapeutic means for alleviating multiple organinjury in intestinal IR patients

Conflict of Interests

All authors state that they have no conflict of interests

Authorsrsquo Contribution

Lingfei Ma and Guangzhi Wang are co-first authors for equalcontribution to the present study Lingfei Ma and GuangzhiWang wrote the paper Lingfei Ma Guangzhi Wang andJihong Yao designed the experiments Zhenlu Li Zhao ChenHaidong Zhao Shu Wang Zhenhai Ma and Hong Changperformed the experiments and analyzed the data XiaofengTian and Jihong Yao edited the paper

Acknowledgment

This work was supported by the Chinese National NaturalScience Foundation (Grant no 81171850)

References

[1] N Kalia N J Brown R FMWood KHopkinson B Fairburnand A G Pockley ldquoEffects of intestinal ischemia-reperfusioninjury on rat peripheral blood neutrophil activationrdquo DigestiveDiseases and Sciences vol 48 no 9 pp 1677ndash1684 2003

[2] A Pierro and S Eaton ldquoIntestinal ischemia reperfusion injuryand multisystem organ failurerdquo Seminars in Pediatric Surgeryvol 13 no 1 pp 11ndash17 2004

[3] H T Hassoun B C Kone D W Mercer F G Moody N WWeisbrodt and F AMoore ldquoPost-injurymultiple organ failurethe role of the gutrdquo Shock vol 15 no 1 pp 1ndash10 2001

[4] J B Antonsson and R G Fiddian-Green ldquoThe role of the gutin shock and multiple system organ failurerdquo European Journalof Surgery vol 157 no 1 pp 3ndash12 1991

[5] Y-Z Lu C-C Wu Y-C Huang et al ldquoNeutrophil primingby hypoxic preconditioning protects against epithelial bar-rier damage and enteric bacterial translocation in intestinalischemiareperfusionrdquo Laboratory Investigation vol 92 no 5pp 783ndash796 2012

[6] M E Johansson and G C Hansson ldquoThe goblet cell a keyplayer in ischaemia-reperfusion injuryrdquo Gut vol 62 no 2 pp188ndash189 2013

[7] B D Kubiak S P Albert L A Gatto et al ldquoPeritoneal negativepressure therapy prevents multiple organ injury in a chronicporcine sepsis and ischemiareperfusion modelrdquo Shock vol 34no 5 pp 525ndash534 2010

[8] K Kilic V Hanci S Selek et al ldquoThe effects of dexmedetomi-dine on mesenteric arterial occlusion-associated gut ischemiaand reperfusion-induced gut and kidney injury in rabbitsrdquoJournal of Surgical Research vol 178 no 1 pp 223ndash232 2012

[9] O Collange A L Charles J Bouitbir et al ldquoMethylene blueprotects liver oxidative capacity after gut ischaemia-reperfusionin the ratrdquo European Journal of Vascular amp EndovascularSurgery vol 45 no 2 pp 168ndash175 2013

[10] S Matsuo W L Yang M Aziz A Jacob and P Wang ldquoCyclicarginine-glycine-aspartate attenuates acute lung injury in miceafter intestinal ischemiareperfusionrdquo Critical Care vol 17 no1 article R19 2013

[11] S Towfigh T Heisler D A Rigberg et al ldquoIntestinal ischemiaand the gut-liver axis an in vitro modelrdquo Journal of SurgicalResearch vol 88 no 2 pp 160ndash164 2000

[12] Y Horie and H Ishii ldquoLiver dysfunction elicited by gutischemia-reperfusionrdquo Pathophysiology vol 8 no 1 pp 11ndash202001


[13] I Leister E M Mbachu S Post et al ldquoVasoactive intestinalpolypeptide and gastrin-releasing peptide attenuate hepaticmicrovasculatory disturbances following intestinal ischemiaand reperfusionrdquo Digestion vol 66 no 3 pp 186ndash192 2002

[14] Y Horie Y Yamagishi S Kato M Kajihara H Kimura and HIshii ldquoLow-dose ethanol attenuates gut ischemiareperfusion-induced liver injury in rats via nitric oxide productionrdquo Journalof Gastroenterology and Hepatology vol 18 no 2 pp 211ndash2172003

[15] H Jing G Shen G Wang et al ldquoMG132 alleviates liver injuryinduced by intestinal ischemiareperfusion in rats involvementof the AhR and NF120581B pathwaysrdquo Journal of Surgical Researchvol 176 no 1 pp 63ndash73 2012

[16] H-D Zhao F Zhang G Shen et al ldquoSulforaphane protectsliver injury induced by intestinal ischemia reperfusion throughNrf2-ARE pathwayrdquo World Journal of Gastroenterology vol 16no 24 pp 3002ndash3010 2010

[17] C Napoli I Martin-Padura F de Nigris et al ldquoDeletion ofthe p66119878ℎ119888 longevity gene reduces systemic and tissue oxidativestress vascular cell apoptosis and early atherogenesis in micefed a high-fat dietrdquo Proceedings of the National Academy ofSciences of the United States of America vol 100 no 4 pp 2112ndash2116 2003

[18] R D Spescha Y Shi S Wegener et al ldquoDeletion of theageing gene p66119878ℎ119888 reduces early stroke size following ischaemiareperfusion brain injuryrdquo European Heart Journal vol 34 no2 pp 96ndash103 2013

[19] SNemoto andT Finkel ldquoRedox regulation of forkhead proteinsthrough a p66shc-dependent signaling pathwayrdquo Science vol295 no 5564 pp 2450ndash2452 2002

[20] H-C Ku H-S Liu P-F Hung et al ldquoGreen tea (minus)-epigallocatechin gallate inhibits IGF-I and IGF-IIstimulation of3T3-L1 preadipocyte mitogenesis via the 67-kDa laminin recep-tor but not AMP-activated protein kinase pathwayrdquo MolecularNutrition amp Food Research vol 56 no 4 pp 580ndash592 2012

[21] S Pacini M Pellegrini E Migliaccio et al ldquop66SHC promotesapoptosis and antagonizes mitogenic signaling in T cellsrdquoMolecular and Cellular Biology vol 24 no 4 pp 1747ndash17572004

[22] Y Shi F Cosentino G G Camici et al ldquoOxidized low-density lipoprotein activates p66119878ℎ119888 via lectin-like oxidizedlow-density lipoprotein receptor-1 protein kinase C-120573 andc-jun N-terminal kinase kinase in human endothelial cellsrdquoArteriosclerosis Thrombosis and Vascular Biology vol 31 no 9pp 2090ndash2097 2011

[23] L Sun L Xiao J Nie et al ldquop66Shc mediates high-glucoseand angiotensin II-induced oxidative stress renal tubular injuryvia mitochondrial-dependent apoptotic pathwayrdquo AmericanJournal of PhysiologymdashRenal Physiology vol 299 no 5 ppF1014ndashF1025 2010

[24] I Arany A Faisal Y Nagamine and R L Safirstein ldquop66119904ℎ119888inhibits pro-survival epidermal growth factor receptorERKsignaling during severe oxidative stress inmouse renal proximaltubule cellsrdquoThe Journal of Biological Chemistry vol 283 no 10pp 6110ndash6117 2008

[25] A Carpi R Menabo N Kaludercic P Pelicci F di Lisa andMGiorgio ldquoThe cardioprotective effects elicited by p66119878ℎ119888 ablationdemonstrate the crucial role of mitochondrial ROS formationin ischemiareperfusion injuryrdquoBiochimica et BiophysicaActamdashBioenergetics vol 1787 no 7 pp 774ndash780 2009

[26] R Masella C Santangelo M DrsquoArchivio V G Li C Giovan-nini and F Galvano ldquoProtocatechuic acid and human disease

prevention biological activities and molecular mechanismsrdquoCurrentMedicinal Chemistry vol 19 no 18 pp 2901ndash2917 2012

[27] C Giovannini B Scazzocchio P Matarrese et al ldquoApoptosisinduced by oxidized lipids is associated with up-regulation ofp66Shc in intestinal Caco-2 cells protective effects of phenoliccompoundsrdquo Journal of Nutritional Biochemistry vol 19 no 2pp 118ndash128 2008

[28] Y-M Liu B Jiang Y-M Bao and L-J An ldquoProtocatechuic acidinhibits apoptosis by mitochondrial dysfunction in rotenone-induced PC12 cellsrdquo Toxicology in Vitro vol 22 no 2 pp 430ndash437 2008

[29] J S Lane K E Todd M P N Lewis et al ldquoInterleukin-10reduces the systemic inflammatory response in a murine modelof intestinal ischemiareperfusionrdquo Surgery vol 122 no 2 pp288ndash294 1997

[30] V Krajka-Kuzniak H Szaefer and W Baer-Dubowska ldquoHep-atic and extrahepatic expression of glutathione S-transferaseisozymes in mice and its modulation by naturally occurringphenolic acidsrdquo Environmental Toxicology and Pharmacologyvol 25 no 1 pp 27ndash32 2008

[31] C J Chiu A H McArdle R Brown H J Scott and F NGurd ldquoIntestinal mucosal lesion in low-flow states I A mor-phological hemodynamic and metabolic reappraisalrdquo Archivesof Surgery vol 101 no 4 pp 478ndash483 1970

[32] D E Eckhoff G Bilbao L Frenette J A Thompson and JL Contreras ldquo17-Beta-estradiol protects the liver against warmischemiareperfusion injury and is associated with increasedserum nitric oxide and decreased tumor necrosis factor-alphardquoSurgery vol 132 no 2 pp 302ndash309 2002

[33] S-I Kim Y-B Kim K M Koh et al ldquoActivation of NF-kappaB pathway in oral buccal mucosa during small intestinalischemia-reperfusion injuryrdquo Journal of Surgical Research vol179 no 1 pp 99ndash105 2013

[34] S Menini L Amadio G Oddi et al ldquoDeletion of p66119878ℎ119888longevity gene protects against experimental diabetic glomeru-lopathy by preventing diabetes-induced oxidative stressrdquo Dia-betes vol 55 no 6 pp 1642ndash1650 2006

[35] H Yasuhara ldquoAcute mesenteric ischemia the challenge ofgastroenterologyrdquo Surgery Today vol 35 no 3 pp 185ndash1952005

[36] R Franco and J A Cidlowski ldquoGlutathione efflux and celldeathrdquo Antioxidants amp Redox Signaling vol 17 no 12 pp 1694ndash1713 2012

[37] E Migliaccio M Giogio S Mele et al ldquoThe p66119904ℎ119888 adaptorprotein controls oxidative stress response and life span inmammalsrdquo Nature vol 402 no 6759 pp 309ndash313 1999

[38] H Huang and D J Tindall ldquoFOXO factors a matter of life anddeathrdquo Future Oncology vol 2 no 1 pp 83ndash89 2006

[39] W Cai J C He L Zhu X Chen G E Striker and HVlassara ldquoAGE-receptor-1 counteracts cellular oxidant stressinduced by AGEs via negative regulation of p66119904ℎ119888-dependentFKHRL1 phosphorylationrdquo American Journal of PhysiologymdashCell Physiology vol 294 no 1 pp C145ndashC152 2008

[40] S-H Wen Y-H Ling Y Li et al ldquoIschemic postconditioningduring reperfusion attenuates oxidative stress and intestinalmucosal apoptosis induced by intestinal ischemiareperfusionvia aldose reductaserdquo Surgery vol 153 no 4 pp 555ndash564 2013

[41] S Haga K Terui M Fukai et al ldquoPreventing hypox-iareoxygenation damage to hepatocytes by p66119904ℎ119888 ablation up-regulation of anti-oxidant and anti-apoptotic proteinsrdquo Journalof Hepatology vol 48 no 3 pp 422ndash432 2008

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


this process include glutathione (GSH) depletion nuclearfactor-kappa B (NF-120581B) pathway activation and suppres-sion of Nf-E2-related factor 2 (Nrf2) nuclear translocation[14ndash16] However detailed mechanisms of intestinal IRremain obscure Recent studies showed that genetic abla-tion of the adaptor protein p66shc (p66shc) in the mousereduced ROS production and consequently prolonged thelife span by 30 [17] while deletion of p66shc protectedmice from IR-induced brain injury through blunted produc-tion of free radicals [18] However it is unknown whetherp66shc is involved in intestinal IR injury and subsequentliver dysfunction Therefore we examined the redox-relatedenzyme p66shc and hypothesized that targeted suppressionof p66shc would protect against damage during intestinalIR

p66shc is one of three isoforms derived from alternativesplicing of the Shc locus It is ubiquitously expressed inmammals and functions as a redox enzyme implicated inmitochondrial ROS generation [19] The other two isoformsof this family p46shc and p52shc have been described asadaptor proteins that can alter mitogen-activated proteinkinase and Fos activation upon stimulation with epidermalgrowth factor and platelet-derived growth factor [20] p66shcfunctionally differentiates from these isoforms Due to itsunique NH

2-terminal region it has a serine 36 residue that

can be phosphorylated allowing p66shc to be transferredfrom the cytosol to the mitochondria p66shc translocationcan lead to blunting of Ca2+ responses and opening ofthe mitochondrial permeability transition pore and thusinducing intracellular ROS overgeneration and cell apoptosisEvidence is increasingly accumulating to show that p66shcmay play a critical role in ROS production and cell apoptosis[17 21] p66shc-null mice unlike wild-typemice were shownto be protected against atherogenesis high glucose-inducedrenal injury and aging [17 22 23] Additionally knock-down of p66shc expression or phospho-p66shc suppressionameliorated cardiac ischemia-reperfusion induced oxidativestress injury in vivo or improved mouse kidney proximaltubule cells surviving during H

2O2-induced oxidative stress

injury in vitro [24 25]Protocatechuic acid (PCA) which has been widely stud-

ied as a polyphenolic compound is derived from greentea fruits and various Chinese herbal medicines such asSalvia miltiorrhiza [26] Recent studies have shown that PCAinduced high expression of antioxidative genes and playeda significant role in fighting against oxidative stress injuryand cell apoptosis [26 27] PCA pretreatment can preventcells from oxidative stress injury by relieving GSH depletionand enhancing the activity of glutathione peroxidase (GSH-PX) and glutathione reductase which show marked antiox-idative activity [27] Additionally studies showed that PCApretreatment significantly attenuated PC12 cell apoptosis bylimiting caspase-3 activity and inducing Bcl-2 expression[28] Notably a recent study indicated that PCA pretreatmentcan downregulate p66shc expression and protect againstcell apoptosis induced by lipid peroxidation in Caco-2 cells[27] However there was no evidence that PCA pretreatment

protects against acute intestinal stress injury and liver dys-function induced by intestinal IR

Therefore in this study we used an intestinal IR modelinmice to examine the hypotheses that (1) p66shc-dependentoxidative stress and apoptosis mechanisms are involved inlocal organ damage and remote liver injury induced byintestinal IR (2) p66shc suppression by PCA pretreatmentmay protect against intestinal injury and liver dysfunctioninduced by intestinal IR (3) the protective effect of PCApretreatment may be related to p66shc-related foxo3a andBcl-xL signaling pathways

2 Materials and Methods

21 Experimental Design The study was performed in adultmale ICR mice (18ndash22 g) (Animal Centre of Dalian MedicalUniversity Dalian China) The mice were housed at a tem-perature of 22plusmn 2∘C kept on a 1212-h photoperiod and wereall provided with food and water ad libitum Procedures wereconducted in accordance with the institutional guidelines forthe care and use of laboratory animals and the study wasapproved by the Institutional Animal Care Committee ofDalian Medical University (Dalian China)

22 Intestinal IR Model and Experimental Procedures Themouse intestinal IR model was established according topreviously standardized methods [29] Briefly after generalanesthesia a midline laparotomy was performed and thesuperior mesenteric artery (SMA) was isolated gently at itsorigin and occluded with an atraumatic microvascular clampfor 45min and then followed by reperfusion for 90min Theocclusion was confirmed by complete pulse cessation and theintestines became pale then the reperfusion was confirmedby the return of pulsatile flow to the mesenteric artery andits branches Sham animals underwent the same protocolbut the SMA was not occluded Fifty mice were dividedrandomly into five groups (119899 = 10) (i) a sham group whichunderwent sham surgery (ii) a IR group which underwentintestinal IR model as previously mentioned (iii) a sham +PCA high dose (H) group which underwent sham surgeryand pretreated with PCA (JampK Chemical Germany) at adose of 80mgkg intraperitoneally (ip) for 3 consecutivedays (iv) a IR + PCA low dose (L) group which underwentintestinal IR model as previously mentioned PCA waspretreated at a dose of 40mgkg ip for 3 consecutive days(v) a IR + PCA high dose (H) group which underwentintestinal IR model as previously mentioned and receivedPCA pretreatment at a dose of 80mgkg ip for 3 consecutivedays The dose and route of administration were determinedaccording to a previous study [30] PCA was dissolved insterile normal saline before administration and the mice inthe sham and IR groups were treated with an equal volumeof sterile normal saline for parallel Blood and tissue foranalysis were obtained by placing the mice under anesthesiaand reopening the abdominal midline incision After beingsacrificed by abdominal aortic blood drawing intestine andliver tissue samples were procured from a standard location










3 Results




0

1

2

3

4

5

Inte

stine

Chi

ursquos sc

ore

Sham IR

Sham

+PC

A (H

)

IR+

PCA

(L)

IR+

PCA

(H)

lowastlowast











0

1

2

3

4

Sham IR

Sham

+PC

A (H

)

IR+

PCA

(L)

IR+

PCA

(H)

lowastlowast

Live

r inj

ury

scor

e







IL-6TNF-120572

1 2 3 4 5 1 2 3 4 5

Seru

m cy

toki

ne le

vels

(pg

mL)

0

100

200

300

400

lowastlowast

lowastlowast

(a)

1 2 3 4 5 1 2 3 4 5

Seru

m A

LT an

d A

ST le

vels

(UL

)

ALTAST

0

50

100

150

200

lowastlowast

lowastlowast

(b)


NF120581B p65

Histone

NF120581

B p65

hist

one (

of sh

am)

0

200

400

600

800

Sham IR

Sham

+PC

A (H

)

IR+

PCA

(L)

IR+

PCA

(H)

lowastlowast

(c)


NF120581B p65

Histone

0

200

400

600

NF120581

B p65

hist

one (

of sh

am)

Sham IR

Sham

+PC

A (H

)

IR+

PCA

(L)

IR+

PCA

(H)

lowastlowast

(d)



IntestineLiver

P66

shc120573

-act

in (

of sh

am)

P66shc

P66shc

120573-Actin

120573-Actin

0

50

100

150

200

250

0

100

200

300lowastlowast

lowastlowast

Sham

IR Sham

+PC

A (H

)

IR+

PCA

(L)

IR+

PCA

(H)

Sham IR

Sham

+PC

A (H

)

IR+

PCA

(L)

IR+

PCA

(H)

Sham IR

Sham

+PC

A (H

)

IR+

PCA

(L)

IR+

PCA

(H)

Intestine

Liver

P66

shc120573

-act

in (

of sh

am)














176 plusmn 008 35701 plusmn 851


7240 plusmn 1103

207 plusmn 017

37942 plusmn 439



P-p66shc

P-p66shc

Total-p66shc

Total-p66shc

0

100

200

300

0

100

200

300

400

Sham IR

Sham

+PC

A (H

)

IR+

PCA

(L)

IR+

PCA

(H)

Sham IR

Sham

+PC

A (H

)

IR+

PCA

(L)

IR+

PCA

(H)

P-p66

shc

tota

l-p66

shc (

of sh

am)

P-p66

shc

tota

l-p66

shc (

of sh

am)


Intestine

Liver

Sham

IR Sham

+PC

A (H

)

IR+

PCA

(L)

IR+

PCA

(H)

Intestine Liver



4 Discussion





Inte

stine

Live

r

1 42 3 5

1 42 3 5

P-foxo3aFoxo3a

MnSODC-caspase-3

Bcl-xL120573-Actin

P-foxo3aFoxo3a

MnSODC-caspase-3

Bcl-xL120573-Actin

0

200

400

600

800

1000

0

50

100

150

0

200

400

600

800

1000

0

50

100

150

P-fo

xo3

afo

xo3

a(

of sh

am)

MnS

OD

120573-a

ctin

(of

sham

)

C-ca

spas

e-3

120573-a

ctin

(of

sham

)

Bcl-x

L120573

-act

in(

of sh

am)

1 42 3 5

1 42 3 51 42 3 5

IntestineLiver

IntestineLiver

Sham

IR Sham

+PC

A (H

)

IR+

PCA

(L)

IR+

PCA

(H)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast














Acknowledgment


References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























3 Results




0

1

2

3

4

5

Inte

stine

Chi

ursquos sc

ore

Sham IR

Sham

+PC

A (H

)

IR+

PCA

(L)

IR+

PCA

(H)

lowastlowast











0

1

2

3

4

Sham IR

Sham

+PC

A (H

)

IR+

PCA

(L)

IR+

PCA

(H)

lowastlowast

Live

r inj

ury

scor

e







IL-6TNF-120572

1 2 3 4 5 1 2 3 4 5

Seru

m cy

toki

ne le

vels

(pg

mL)

0

100

200

300

400

lowastlowast

lowastlowast

(a)

1 2 3 4 5 1 2 3 4 5

Seru

m A

LT an

d A

ST le

vels

(UL

)

ALTAST

0

50

100

150

200

lowastlowast

lowastlowast

(b)


NF120581B p65

Histone

NF120581

B p65

hist

one (

of sh

am)

0

200

400

600

800

Sham IR

Sham

+PC

A (H

)

IR+

PCA

(L)

IR+

PCA

(H)

lowastlowast

(c)


NF120581B p65

Histone

0

200

400

600

NF120581

B p65

hist

one (

of sh

am)

Sham IR

Sham

+PC

A (H

)

IR+

PCA

(L)

IR+

PCA

(H)

lowastlowast

(d)



IntestineLiver

P66

shc120573

-act

in (

of sh

am)

P66shc

P66shc

120573-Actin

120573-Actin

0

50

100

150

200

250

0

100

200

300lowastlowast

lowastlowast

Sham

IR Sham

+PC

A (H

)

IR+

PCA

(L)

IR+

PCA

(H)

Sham IR

Sham

+PC

A (H

)

IR+

PCA

(L)

IR+

PCA

(H)

Sham IR

Sham

+PC

A (H

)

IR+

PCA

(L)

IR+

PCA

(H)

Intestine

Liver

P66

shc120573

-act

in (

of sh

am)














176 plusmn 008 35701 plusmn 851


7240 plusmn 1103

207 plusmn 017

37942 plusmn 439



P-p66shc

P-p66shc

Total-p66shc

Total-p66shc

0

100

200

300

0

100

200

300

400

Sham IR

Sham

+PC

A (H

)

IR+

PCA

(L)

IR+

PCA

(H)

Sham IR

Sham

+PC

A (H

)

IR+

PCA

(L)

IR+

PCA

(H)

P-p66

shc

tota

l-p66

shc (

of sh

am)

P-p66

shc

tota

l-p66

shc (

of sh

am)


Intestine

Liver

Sham

IR Sham

+PC

A (H

)

IR+

PCA

(L)

IR+

PCA

(H)

Intestine Liver



4 Discussion





Inte

stine

Live

r

1 42 3 5

1 42 3 5

P-foxo3aFoxo3a

MnSODC-caspase-3

Bcl-xL120573-Actin

P-foxo3aFoxo3a

MnSODC-caspase-3

Bcl-xL120573-Actin

0

200

400

600

800

1000

0

50

100

150

0

200

400

600

800

1000

0

50

100

150

P-fo

xo3

afo

xo3

a(

of sh

am)

MnS

OD

120573-a

ctin

(of

sham

)

C-ca

spas

e-3

120573-a

ctin

(of

sham

)

Bcl-x

L120573

-act

in(

of sh

am)

1 42 3 5

1 42 3 51 42 3 5

IntestineLiver

IntestineLiver

Sham

IR Sham

+PC

A (H

)

IR+

PCA

(L)

IR+

PCA

(H)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast














Acknowledgment


References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

1

2

3

4

5

Inte

stine

Chi

ursquos sc

ore

Sham IR

Sham

+PC

A (H

)

IR+

PCA

(L)

IR+

PCA

(H)

lowastlowast











0

1

2

3

4

Sham IR

Sham

+PC

A (H

)

IR+

PCA

(L)

IR+

PCA

(H)

lowastlowast

Live

r inj

ury

scor

e







IL-6TNF-120572

1 2 3 4 5 1 2 3 4 5

Seru

m cy

toki

ne le

vels

(pg

mL)

0

100

200

300

400

lowastlowast

lowastlowast

(a)

1 2 3 4 5 1 2 3 4 5

Seru

m A

LT an

d A

ST le

vels

(UL

)

ALTAST

0

50

100

150

200

lowastlowast

lowastlowast

(b)


NF120581B p65

Histone

NF120581

B p65

hist

one (

of sh

am)

0

200

400

600

800

Sham IR

Sham

+PC

A (H

)

IR+

PCA

(L)

IR+

PCA

(H)

lowastlowast

(c)


NF120581B p65

Histone

0

200

400

600

NF120581

B p65

hist

one (

of sh

am)

Sham IR

Sham

+PC

A (H

)

IR+

PCA

(L)

IR+

PCA

(H)

lowastlowast

(d)



IntestineLiver

P66

shc120573

-act

in (

of sh

am)

P66shc

P66shc

120573-Actin

120573-Actin

0

50

100

150

200

250

0

100

200

300lowastlowast

lowastlowast

Sham

IR Sham

+PC

A (H

)

IR+

PCA

(L)

IR+

PCA

(H)

Sham IR

Sham

+PC

A (H

)

IR+

PCA

(L)

IR+

PCA

(H)

Sham IR

Sham

+PC

A (H

)

IR+

PCA

(L)

IR+

PCA

(H)

Intestine

Liver

P66

shc120573

-act

in (

of sh

am)














176 plusmn 008 35701 plusmn 851


7240 plusmn 1103

207 plusmn 017

37942 plusmn 439



P-p66shc

P-p66shc

Total-p66shc

Total-p66shc

0

100

200

300

0

100

200

300

400

Sham IR

Sham

+PC

A (H

)

IR+

PCA

(L)

IR+

PCA

(H)

Sham IR

Sham

+PC

A (H

)

IR+

PCA

(L)

IR+

PCA

(H)

P-p66

shc

tota

l-p66

shc (

of sh

am)

P-p66

shc

tota

l-p66

shc (

of sh

am)


Intestine

Liver

Sham

IR Sham

+PC

A (H

)

IR+

PCA

(L)

IR+

PCA

(H)

Intestine Liver



4 Discussion





Inte

stine

Live

r

1 42 3 5

1 42 3 5

P-foxo3aFoxo3a

MnSODC-caspase-3

Bcl-xL120573-Actin

P-foxo3aFoxo3a

MnSODC-caspase-3

Bcl-xL120573-Actin

0

200

400

600

800

1000

0

50

100

150

0

200

400

600

800

1000

0

50

100

150

P-fo

xo3

afo

xo3

a(

of sh

am)

MnS

OD

120573-a

ctin

(of

sham

)

C-ca

spas

e-3

120573-a

ctin

(of

sham

)

Bcl-x

L120573

-act

in(

of sh

am)

1 42 3 5

1 42 3 51 42 3 5

IntestineLiver

IntestineLiver

Sham

IR Sham

+PC

A (H

)

IR+

PCA

(L)

IR+

PCA

(H)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast














Acknowledgment


References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















0

1

2

3

4

Sham IR

Sham

+PC

A (H

)

IR+

PCA

(L)

IR+

PCA

(H)

lowastlowast

Live

r inj

ury

scor

e







IL-6TNF-120572

1 2 3 4 5 1 2 3 4 5

Seru

m cy

toki

ne le

vels

(pg

mL)

0

100

200

300

400

lowastlowast

lowastlowast

(a)

1 2 3 4 5 1 2 3 4 5

Seru

m A

LT an

d A

ST le

vels

(UL

)

ALTAST

0

50

100

150

200

lowastlowast

lowastlowast

(b)


NF120581B p65

Histone

NF120581

B p65

hist

one (

of sh

am)

0

200

400

600

800

Sham IR

Sham

+PC

A (H

)

IR+

PCA

(L)

IR+

PCA

(H)

lowastlowast

(c)


NF120581B p65

Histone

0

200

400

600

NF120581

B p65

hist

one (

of sh

am)

Sham IR

Sham

+PC

A (H

)

IR+

PCA

(L)

IR+

PCA

(H)

lowastlowast

(d)



IntestineLiver

P66

shc120573

-act

in (

of sh

am)

P66shc

P66shc

120573-Actin

120573-Actin

0

50

100

150

200

250

0

100

200

300lowastlowast

lowastlowast

Sham

IR Sham

+PC

A (H

)

IR+

PCA

(L)

IR+

PCA

(H)

Sham IR

Sham

+PC

A (H

)

IR+

PCA

(L)

IR+

PCA

(H)

Sham IR

Sham

+PC

A (H

)

IR+

PCA

(L)

IR+

PCA

(H)

Intestine

Liver

P66

shc120573

-act

in (

of sh

am)














176 plusmn 008 35701 plusmn 851


7240 plusmn 1103

207 plusmn 017

37942 plusmn 439



P-p66shc

P-p66shc

Total-p66shc

Total-p66shc

0

100

200

300

0

100

200

300

400

Sham IR

Sham

+PC

A (H

)

IR+

PCA

(L)

IR+

PCA

(H)

Sham IR

Sham

+PC

A (H

)

IR+

PCA

(L)

IR+

PCA

(H)

P-p66

shc

tota

l-p66

shc (

of sh

am)

P-p66

shc

tota

l-p66

shc (

of sh

am)


Intestine

Liver

Sham

IR Sham

+PC

A (H

)

IR+

PCA

(L)

IR+

PCA

(H)

Intestine Liver



4 Discussion





Inte

stine

Live

r

1 42 3 5

1 42 3 5

P-foxo3aFoxo3a

MnSODC-caspase-3

Bcl-xL120573-Actin

P-foxo3aFoxo3a

MnSODC-caspase-3

Bcl-xL120573-Actin

0

200

400

600

800

1000

0

50

100

150

0

200

400

600

800

1000

0

50

100

150

P-fo

xo3

afo

xo3

a(

of sh

am)

MnS

OD

120573-a

ctin

(of

sham

)

C-ca

spas

e-3

120573-a

ctin

(of

sham

)

Bcl-x

L120573

-act

in(

of sh

am)

1 42 3 5

1 42 3 51 42 3 5

IntestineLiver

IntestineLiver

Sham

IR Sham

+PC

A (H

)

IR+

PCA

(L)

IR+

PCA

(H)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast














Acknowledgment


References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















IL-6TNF-120572

1 2 3 4 5 1 2 3 4 5

Seru

m cy

toki

ne le

vels

(pg

mL)

0

100

200

300

400

lowastlowast

lowastlowast

(a)

1 2 3 4 5 1 2 3 4 5

Seru

m A

LT an

d A

ST le

vels

(UL

)

ALTAST

0

50

100

150

200

lowastlowast

lowastlowast

(b)


NF120581B p65

Histone

NF120581

B p65

hist

one (

of sh

am)

0

200

400

600

800

Sham IR

Sham

+PC

A (H

)

IR+

PCA

(L)

IR+

PCA

(H)

lowastlowast

(c)


NF120581B p65

Histone

0

200

400

600

NF120581

B p65

hist

one (

of sh

am)

Sham IR

Sham

+PC

A (H

)

IR+

PCA

(L)

IR+

PCA

(H)

lowastlowast

(d)



IntestineLiver

P66

shc120573

-act

in (

of sh

am)

P66shc

P66shc

120573-Actin

120573-Actin

0

50

100

150

200

250

0

100

200

300lowastlowast

lowastlowast

Sham

IR Sham

+PC

A (H

)

IR+

PCA

(L)

IR+

PCA

(H)

Sham IR

Sham

+PC

A (H

)

IR+

PCA

(L)

IR+

PCA

(H)

Sham IR

Sham

+PC

A (H

)

IR+

PCA

(L)

IR+

PCA

(H)

Intestine

Liver

P66

shc120573

-act

in (

of sh

am)














176 plusmn 008 35701 plusmn 851


7240 plusmn 1103

207 plusmn 017

37942 plusmn 439



P-p66shc

P-p66shc

Total-p66shc

Total-p66shc

0

100

200

300

0

100

200

300

400

Sham IR

Sham

+PC

A (H

)

IR+

PCA

(L)

IR+

PCA

(H)

Sham IR

Sham

+PC

A (H

)

IR+

PCA

(L)

IR+

PCA

(H)

P-p66

shc

tota

l-p66

shc (

of sh

am)

P-p66

shc

tota

l-p66

shc (

of sh

am)


Intestine

Liver

Sham

IR Sham

+PC

A (H

)

IR+

PCA

(L)

IR+

PCA

(H)

Intestine Liver



4 Discussion





Inte

stine

Live

r

1 42 3 5

1 42 3 5

P-foxo3aFoxo3a

MnSODC-caspase-3

Bcl-xL120573-Actin

P-foxo3aFoxo3a

MnSODC-caspase-3

Bcl-xL120573-Actin

0

200

400

600

800

1000

0

50

100

150

0

200

400

600

800

1000

0

50

100

150

P-fo

xo3

afo

xo3

a(

of sh

am)

MnS

OD

120573-a

ctin

(of

sham

)

C-ca

spas

e-3

120573-a

ctin

(of

sham

)

Bcl-x

L120573

-act

in(

of sh

am)

1 42 3 5

1 42 3 51 42 3 5

IntestineLiver

IntestineLiver

Sham

IR Sham

+PC

A (H

)

IR+

PCA

(L)

IR+

PCA

(H)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast














Acknowledgment


References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















IntestineLiver

P66

shc120573

-act

in (

of sh

am)

P66shc

P66shc

120573-Actin

120573-Actin

0

50

100

150

200

250

0

100

200

300lowastlowast

lowastlowast

Sham

IR Sham

+PC

A (H

)

IR+

PCA

(L)

IR+

PCA

(H)

Sham IR

Sham

+PC

A (H

)

IR+

PCA

(L)

IR+

PCA

(H)

Sham IR

Sham

+PC

A (H

)

IR+

PCA

(L)

IR+

PCA

(H)

Intestine

Liver

P66

shc120573

-act

in (

of sh

am)














176 plusmn 008 35701 plusmn 851


7240 plusmn 1103

207 plusmn 017

37942 plusmn 439



P-p66shc

P-p66shc

Total-p66shc

Total-p66shc

0

100

200

300

0

100

200

300

400

Sham IR

Sham

+PC

A (H

)

IR+

PCA

(L)

IR+

PCA

(H)

Sham IR

Sham

+PC

A (H

)

IR+

PCA

(L)

IR+

PCA

(H)

P-p66

shc

tota

l-p66

shc (

of sh

am)

P-p66

shc

tota

l-p66

shc (

of sh

am)


Intestine

Liver

Sham

IR Sham

+PC

A (H

)

IR+

PCA

(L)

IR+

PCA

(H)

Intestine Liver



4 Discussion





Inte

stine

Live

r

1 42 3 5

1 42 3 5

P-foxo3aFoxo3a

MnSODC-caspase-3

Bcl-xL120573-Actin

P-foxo3aFoxo3a

MnSODC-caspase-3

Bcl-xL120573-Actin

0

200

400

600

800

1000

0

50

100

150

0

200

400

600

800

1000

0

50

100

150

P-fo

xo3

afo

xo3

a(

of sh

am)

MnS

OD

120573-a

ctin

(of

sham

)

C-ca

spas

e-3

120573-a

ctin

(of

sham

)

Bcl-x

L120573

-act

in(

of sh

am)

1 42 3 5

1 42 3 51 42 3 5

IntestineLiver

IntestineLiver

Sham

IR Sham

+PC

A (H

)

IR+

PCA

(L)

IR+

PCA

(H)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast














Acknowledgment


References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















P-p66shc

P-p66shc

Total-p66shc

Total-p66shc

0

100

200

300

0

100

200

300

400

Sham IR

Sham

+PC

A (H

)

IR+

PCA

(L)

IR+

PCA

(H)

Sham IR

Sham

+PC

A (H

)

IR+

PCA

(L)

IR+

PCA

(H)

P-p66

shc

tota

l-p66

shc (

of sh

am)

P-p66

shc

tota

l-p66

shc (

of sh

am)


Intestine

Liver

Sham

IR Sham

+PC

A (H

)

IR+

PCA

(L)

IR+

PCA

(H)

Intestine Liver



4 Discussion





Inte

stine

Live

r

1 42 3 5

1 42 3 5

P-foxo3aFoxo3a

MnSODC-caspase-3

Bcl-xL120573-Actin

P-foxo3aFoxo3a

MnSODC-caspase-3

Bcl-xL120573-Actin

0

200

400

600

800

1000

0

50

100

150

0

200

400

600

800

1000

0

50

100

150

P-fo

xo3

afo

xo3

a(

of sh

am)

MnS

OD

120573-a

ctin

(of

sham

)

C-ca

spas

e-3

120573-a

ctin

(of

sham

)

Bcl-x

L120573

-act

in(

of sh

am)

1 42 3 5

1 42 3 51 42 3 5

IntestineLiver

IntestineLiver

Sham

IR Sham

+PC

A (H

)

IR+

PCA

(L)

IR+

PCA

(H)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast














Acknowledgment


References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Inte

stine

Live

r

1 42 3 5

1 42 3 5

P-foxo3aFoxo3a

MnSODC-caspase-3

Bcl-xL120573-Actin

P-foxo3aFoxo3a

MnSODC-caspase-3

Bcl-xL120573-Actin

0

200

400

600

800

1000

0

50

100

150

0

200

400

600

800

1000

0

50

100

150

P-fo

xo3

afo

xo3

a(

of sh

am)

MnS

OD

120573-a

ctin

(of

sham

)

C-ca

spas

e-3

120573-a

ctin

(of

sham

)

Bcl-x

L120573

-act

in(

of sh

am)

1 42 3 5

1 42 3 51 42 3 5

IntestineLiver

IntestineLiver

Sham

IR Sham

+PC

A (H

)

IR+

PCA

(L)

IR+

PCA

(H)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast














Acknowledgment


References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























Acknowledgment


References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article Modulating the p66shc Signaling Pathway...

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Transcript of Research Article Modulating the p66shc Signaling Pathway...