Expression Of HSP70 And PARP-1 In Hepatic Ischemia-reperfusion Injury

Objective:By establishing rat heat treatment,HSP70 inhibition,PARP-1 inhibition,and hepatic ischemia-reperfusion model,To study the expression and correlation of HSP70 and PARP-1 during hepatic ischemia-reperfusion.,The protective mechanism of hepatic ischemia-reperfusion injury in rats was studied.?Method:Thirty male Sprague-Dawley rats,weighing between 350 g and 400 g,were randomly divided into five groups of 6 rats each;Group A(sham operation group): no treatment factor was applied,laparotomy was performed after successful anesthesia,and liver tissue and blood specimens were taken directly;Group N(heat treatment group): The rats were placed in a 40 °C water bath for heat treatment 1 hour before the operation.After anesthesia,the ischemia-reperfusion model was established according to the Pringle's method.The liver tissue and blood samples were taken after successful modeling.Group B(ischemic reperfusion group): After the anesthesia was successful,the ischemia-reperfusion model was established according to Pringle's method.After successful modeling,liver tissue and blood samples were taken.Group O(HSP70 inhibition group): quercetin solution(100mg/kg)was intraperitoneally injected 1 hour before operation,heat treatment was the same as N group,and the hepatic ischemia-reperfusion model was established according to Pringle's method after successful anesthesia.Liver tissue and blood specimens;Group P(PARP-1 inhibition group): AG14361(10 mg/kg)was intraperitoneally injected 5 days before surgery,and heat treatment was performed in the same group N one hour before the operation.After anesthesia was successful,the liver ischemia-reperfusion model was established according to Pringle's method.The serum biochemical parameters of rats were analyzed.The pathological changes of liver tissues were observed by HE staining.The expressions of HSP70 and PARP-1 were detected by Western blot.The expression positions of HSP70 and PARP-1 were observed by immunofluorescence.HSP70 and PARP-1 were detected by immunoprecipitation.Relationship.Result:1.Serum ALT and AKP detection: ALT and AKP were the lowest in group A,and ALT and AKP were the highest in group B.ALT and AKP values in group N,group O and group P were between group A and group B;group B was higher than group B.N group,A group,O group,P group,the difference was statistically significant(p<0.05),N group was lower than B group,O group,P group,the difference was statistically significant(p<0.05),O group and There was no significant difference in ALT and AKP between the P group(p>0.05).2.HE staining: Group A(sham operation group): normal liver tissue structure;hepatocytes no edema,degeneration,no cell necrosis;group B(ischemia-reperfusion group)hepatocyte structure disorder,edema,large degeneration and necrosis The liver tissue structure and hepatocytes of N group,O group and P group had different degrees of damage.The degree of injury was between A and B groups,and the damage degree of N group was lighter than that of O group and P group.The group and the P group had the same degree of damage.3.Western blot analysis: The expression of HSP70 and PARP-1 in each group was detected by extracting liver cell nuclear protein.The expression of HSP70 and PARP-1 protein was detected in each group.Statistical analysis showed:The expression of HSP70 protein was the highest in group N,and the expression of HSP70 protein was the lowest in group O.The expression of HSP70 protein in group A,group B and group P was between N group and O group;group N was better than group A and group B.The expressions of O group and P group were high,and the difference was statistically significant(p<0.05).The difference between group A and group O was not statistically significant(p>0.05).The difference between group B and group P was not statistical.Academic significance(p>0.05).The expression of PARP-1 protein was the highest in group N,and the expression of PARP-1 protein was the lowest in group P.The expression of PARP-1 protein in group A,group B and group O was between N group and P group;Compared with group A,group B,group O and group P,the expression was higher(p<0.05).There was no significant difference between group A and group P(p>0.05).Group B and O The difference was not statistically significant(p>0.05).It indicated that heat treatment,ischemia-reperfusion can induce the expression of HSP70 and PARP-1,quercetin inhibited the expression of HSP70,and AG14361 inhibited the expression of PARP-1.4.Immunofluorescence detection: The liver tissues of each group were observed under fluorescence microscope.It was observed that the nucleus showed blue after being stained by DAPI staining solution;the green fluorescent signal was FITC-labeled HSP70 protein,and the cytoplasm and nucleus were visible.The green fluorescent signal,HSP70 expression in cytoplasm and nucleus;red fluorescent signal is CY3 labeled PARP-1 protein,PARP-1 protein is mainly expressed in the nucleus,suggesting that HSP70 may enter the nucleus and PARP-1 binding.5.Co-immunoprecipitation assay: N-hepatocyte nucleoproteins with high expression of HSP70 and PARP-1 were extracted,and immunoprecipitated with HSP70 antibody.Western blot analysis was performed on the precipitate: the target band appeared at 70 KDa and 110 KDa,which was HSP70.And PARP-1 protein,confirmed the presence of HSP70 and PARP-1 conjugate,heat treatment and ischemia-reperfusion stress,HSP70 and PARP-1 binding play a protective role.Conclusion:Heat treatment,ischemia-reperfusion induced up-regulation of HSP70 and PARP-1 can reduce hepatic ischemia-reperfusion injury.This protective mechanism may be related to the colocalization of HSP70 and PARP-1 in the nucleus.