Ghrelin enhances autophagy to attenuate intestinal ischemia/reperfusion injury by activating Sirt1
Objective:To explore the role of Ghrelin and Sirt1 in intestinal ischemia/reperfusion(IIR)inju-ry.Methods:In vivo,24 SPF healthy male C57BL/6J mice,aged 6 to 8 weeks and weighing 20 to 25 g,were randomly divided into four groups,sham operation group(S group,n=6),intestinal isch-emia/reperfusion group(IIR group,n=6),intestinal ischemia/reperfusion+Ghrelin group(IIR+G group,n=6),and intestinal ischemia/reperfusion+Ghrelin+EX527 group(IIR+G+E group,n=6).The superior mesenteric artery(SMA)root was closed for 45 min and the blood flow was restored for 2 h to establish the IIR model.At the end of reperfusion,the mice were sacrificed to take the small intestine samples,HE staining was adopted to observe the histopathological changes,transmission electron microscopy(TEM)was adopted to observe the subcellular structure,the superoxide dis-mutase(SOD)activity,malondialdehyde(MDA)level,and ATP content of small intestines were as-sayed,Western Blot and qRT-PCR were adopted to detect the expression of GHSR-1α,Sirt1 and key molecules of autophagy,and immunofluorescence was adopted to detect the co-expression of Sirt1 and LC3B.Results:Compared with group S,group IIR showed increased Chiu's scores,obvi-ous mitochondrial swelling and significant vacuolization changes,severe destruction of cristae struc-ture,increased MDA production,decreased SOD activity and ATP content,increased autophago-somes in intestines,down-regulated Sqstm1 mRNA and p62 protein expression,up-regulated Becn1 mRNA and Beclin1 protein expression,Map1lc3b mRNA and LC3Ⅱ/LC3Ⅰ protein expression ratio were elevated,Ghsr-1α mRNA.And after IIR,GHSR-1α protein and Sirt1 mRNA,Sirt1 protein ex-pression were down-regulated(P<0.05).Compared with the above results of IIR group,the Chiu's scores of the IIR+G group reduced,the mitochondrial ultrastructure destruction was partially re-versed,energy metabolism was improved,ATP content increased significantly,MDA production de-creased,SOD activity increased,autophagosomes increased,Sqstm1 mRNA and p62 protein expres-sion were down-regulated,Becn1 mRNA and Beclin1 protein expression were up-regulated,Map1lc3b mRNA,LC3Ⅱ/LC3Ⅰ ratio increased,Ghsr-1α mRNA,GHSR-1α protein and Sirt1 mRNA,Sirt1 protein expression were up-regulated(P<0.05).Compared with the IIR+G group,the IIR+G+E group showed higher Chiu's scores,reduced mitochondria with increased structural disruption,increased MDA production,decreased SOD activity and ATP content,decreased au-tophagosomes,up-regulated expression of Sqstm1 mRNA and p62 protein expression,down-regulat-ed expression of Becn1 mRNA and Beclin1 protein expression.Map1lc3b mRNA and LC3Ⅱ/LC3Ⅰ protein expression ratio decreased,Sirt1 mRNA and Sirt1 protein expression was down-regulated(P<0.05),while Ghsr-1α mRNA and GHSR-1α protein expression was not different from that of IIR+G group.Conclusion:Ghrelin may attenuate IIR injury by enhancing autophagy through activat-ing Sirt1.
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