Role Of Pyroptosis In Hepatic Ischemia-reperfusion Injury In Mice

Objective: To explore whether pyroptosis is involved in the development of mouse hepatic ischemia-reperfusion injury by establishing the model of HIRI in mice and detecting the expression of pyroptosis and inflammatory relevant indicators.Methods:(1)Establishment of partial(70%)HIRI model in mice: 24 male C57BL/6 mice(8 to 10 weeks)weighing 18 to 22 g were randomly divided into Sham operation control group(Sham group,n=6),6h reperfusion group(HIR 6 h group,n=6),12 h reperfusion group(HIR 12 h group,n=6),and 24 h reperfusion group(HIR24 h group,n=6).The “ligation method” was used to ligate the common trunk of the middle lobe and the left lobe of the liver,the blood flow was restored after blocking the blood flow of the two lobes of the liver for 90 minutes,the mice were executed to collect blood and liver tissue samples at the above-mentioned time points.(2)The expression levels of ALT,AST and LDH were detected by automatic biochemical instrument after blood samples were extracted,and then the mice with the most severe liver function injury were selected to continue the follow-up study.(3)HE staining was used to observe the pathological changes of liver tissue in the sham-operated control group and the selected experimental group.(4)Real-time quantitative PCR(RT-PCR)was used to detect the expression of cell death and inflammation-related mRNA in the liver tissues of the two groups.(5)Western blot method was used to detect the expression of focal death-related proteins in liver tissues of two groups.(6)The expression and localization of focal death key protein GSDMD in liver tissues were detected by immunohistochemistry.Results:(1)Model establishment: 70% mouse liver ischemia-reperfusion injury model was successfully established.(2)Liver function results: With the prolongation of reperfusion time,the levels of ALT,AST and LDH in mice increased first and then decreased.It reached its peak at HIR 12 h.The differences between the experimental groups in the Sham group were statistically significant.The HIR 12 h group was selected as the subject of subsequent study.(3)HE staining results: microscopically,the hepatic lobules in the Sham group showed complete structure and normal hepatocyte morphology.In the HIR 12 h group,large necrotic areas were seen,hepatic sinus congestion and dilatation were obvious,and there were a lot of inflammatory cell infiltration.(4)Pyroptosis and inflammation-related mRNA expression: NLRP3,ASC,associated with cell coke in HIR 12 h group,The expression levels of caspase-11,IL-1? mRNA and inflammation-related TNF-?,MCP-1 and CXCL-1/2/10 mRNA were significantly higher than those in Sham group,and the difference wasstatistically significant(P<0.05).(5)Expression of pyroptosis-related proteins: related proteins involved in the classical pyroptosis pathway in the HIR 12 h group,NLRP3,AIM2,ASC,caspase-1,and caspase involved in the non-classical pyroptosis pathway,compared with the Sham group The expression levels of GSD and IL-1? in-11 and cell cocaine were significantly increased,and the difference was statistically significant(P<0.05).(6)GADMD immunohistochemistry results: a large number of Kupffer cells with nuclear staining could be seen in the HIR 12 h group.Conclusions:(1)Establishment of partial(70%)HIRI model in mice is feasible by “ligation method”.(2)Pyroptosis is involved in the development of mouse HIRI.(3)Some cytokines and chemokines participated in the inflammatory response of mouse HIRI process.