Ischemic Preconditioning-induced SOCS-1 Activation And Degradation Of TRAF6 To Protect Rat Intestinal Ischemia Reperfusion Injury

bjective: To investigate the expression of TLR4(Toll like receptor 4)-TRAF6(tumor necrosis factor receptor associated-factor 6)signaling pathway in intestinal ischemia and reperfusion injury in rats,and the expression changes and significance of TRAF6 and SOCS-1(suppressor of cytokine signaling-1)after ischemic preconditioning in small intestine and distant organs in rats.We detected the expression levels of apoptosis-related genes in vital organs after intestinal ischemia and reperfusion injury,and observed its relationship with intestinal injury.Methods: A total of 48 Sprague-Dawley(SD)male rats(250-300g)were randomly divided into sham group,ischemia group,ischemia-reperfusion group and ischemic preconditioning group(n = 8).Sham operation group(Sham),rats received only the superior mesenteric artery(superior mesenteric artery,SMA)anatomy and then the abdomen was closed for 2h;further divided into ischemia 1h(I1),3h(I3),6h(I6)group,using noninvasive vascular clamp to clamp SMA and induce intestinal ischemia model;ischemia-reperfusion group(IR),clamping SMA for 1h and 1h reperfusion;ischemic preconditioning group(IP),by clipping SMA 10 minutes and 10 minutes reperfusion,then clipping SMA 1h and 1h reperfusion.Animal inferior vena blood was collected immediately,then all animals were sacrificed immediately after surgery,and at the same time collecting organizations.Intestinal histologic injury was observed by H & E staining,TUNEL(TdT-mediated dUTP Nick-End Labeling)staining to detect apoptotic cells,TRAF6 and SOCS-1 expression was detected by immunohistochemistry in the intestine tissue,meanwhile,the plasma myeloperoxidase(MPO)levels were been detected.Via Real-time quantitative PCR,Western blot to detect TLR4,TRAF6,RIP1(receptor interacting protein1),cleaved-Caspase-3(cysteinyl aspartate specific proteinase-3),and SOCS-1 expression in the intestine and distant organs tissues.Correlation analysis between TRAF6 mRNA and RIP1 mRNA,SOCS-1mRNA levels was made.Results:(1)Inferior vena plasma MPO levels increased in comparison with the Sham group at all time points(p<0.05),which reached the peak in ischemia-reperfusion group,after ischemic preconditioning,the MPO vitalilty were significantly decreased compared with the IR group(p <0.05).(2)H & E staining showed that intestinal villus damaged most serious after ischemia-reperfusion,after ischemic preconditioning,there is a little intestine villi damage.TUNEL staining detected that apoptosis rate increased after intestinal ischemia and ischemia-reperfusion injury,after ischemic preconditioning,the apoptosis rate reduced.(3)In ischemia and ischemia-reperfusion group,the expression of TLR4,TRAF6,RIP1,cleaved-Caspase 3 increased in intestine tissue which were detected by Real-time quantitative PCR and Western blot,the expression of SOCS-1 decreased,the difference between ischemia 3h,6h,IR group and Sham group was statistically significant(p <0.05).After ischemia preconditioning,the expression of intestine TLR4,TRAF6,RIP1,cleaved-Caspase-3 was significantly reduced compared with the IR group(p <0.05).the expression of SOCS-1 was increased(p <0.05),and the correlation analysis showed that TRAF6 mRNA increased with the rise of RIP1 mRNA which was positively correlated(r = 0.84433406,p <0.05),and TRAF6 mRNA was negatively correlated with the expression of SOCS-1mRNA(r =-0.76229915,p <0.05).(4)Immunohistochemistry showed that the expression of TRAF6 in intestinal ischemia and ischemia-reperfusion group enhanced,and the expression of SOCS-1 decreased,however,in the ischemic preconditioning group,the expression of TRAF6 decreased,the expression of SOCS-1 increased,and the expression of positive position were mainly in small intestinal mucosa cell cytoplasm.(5)In each group,the expression of TLR4,TRAF6,RIP1,SOCS-1,cleaved-Caspase-3 in lung,liver and kidney tissue were similar with those in intestinal expression.Conclusion:(1)TLR4-TRAF6 signaling pathway and SOCS-1 involved in apoptosis in intestinal ischemia and reperfusion injury;(2)Intestine ischemia preconditioning may activate SOCS-1 signal,which inhibited the TLR4-TRAF6 signaling pathway,and play a important role in anti-apoptotic effects.