Isoflurane Preconditioning Protects Hepatic Ischemia-Reperfusion Injury Via Suppressing Kupffer Cells Noncanonical Pyroptosis Pathway

Objective:To investigate the protective effect of isoflurane preconditioning on hepatic ischemia-reperfusion injury by inhibiting the noncanonical pyroptosis pathway of Kupffer cells.Methods:In vivo experiment:HIRI mouse model was established.30 healthy C57BL/6 mice were randomly divided into sham-operation group(sham group),isoflurane group(iso group)and Hepatic ischemia-reperfusion injury group(HIRI group),with 10 mice in each group.Mice in the iso group were inhaled with 1.4%isoflurane for 30 minutes and then underwent hepatic ischemia-reperfusion surgery at an interval of 30 minutes.The protective effect of isoflurane preconditioning on the hepatic ischemia-reperfusion injury was detected pathological score of HE and serumALT/AST.Serum IL-1? and IL-18 were detected by ELISA to evaluate the inhibitory effect of isoflurane preconditioning on pyroptosis.Western Blot was used to detect the protein expression of caspase-11 in liver tissues,and to evaluate the inhibitory effect of isoflurane preconditioning on noncanonical pyroptosis pathway.The expression of caspase-11 in Kupffer cells was detected by double-labeled immunofluorescence assay to evaluate the inhibitory effect of isoflurane preconditioning on noncanonical pyroptosis pathway in Kupffer cells.In vitro experiments:Kupffer cells were extracted from the liver of 30 healthy C57BL/6 mice,and the cells were divided into control group(control group),emisoflurane group(emiso group)and endotoxin group(LPS group).Cells of the control group were routinely cultured in medium;the emiso group were incubated in supernatant with 0.28mmol/L emisoflurane for 30 minutes,followed by lug/ml LPS in supernatant;The LPS group cells were incubated in supernatant containing lug/ml LPS.Cells in the 3 groups were cultured for 24 hours.The levels of IL-1? and IL-18 in cell culture supernatant were detected by ELISA.Western Blot and immunofluorescence assay were used to detect the expression of caspase-11 in Kupffer cells from the protein level.RT-PCR was used to detect the expression of caspase-11 in Kupffer cells in each group from the mRNA level,and to verify the inhibitory effect of isoflurane preconditioning on noncanonical pyroptosis pathway in Kupffer cells.Results:In vivo experiments,The Suzuki's score of HE staining section showed that the score in the HIRI group was significantly higher than that in the sham group(P=0.001),while the score in the iso group was significantly lower than that in the HIRI group(P=0.033).The result of serum ALT/AST showed that the serum level of HIRI group was significantly higher than that of sham group(ALT:P<0.001;AST:P<0.001),while that of iso group was significantly lower than that of HIRI group(ALT:P<0.001;AST:P=0.002).The results of ELISA suggested that the expression of IL-1?and IL-18 in serum in the HIRI group was significantly higher than that in the sham group(IL-1?:P<0.001;IL-18:P=0.005),while the expression of IL-1? and IL-18 in the iso group was significantly lower than that in the HIRI group(IL-1?:P=0.006;IL-18:P=0.039).The results of Western Blot indicated that the expression of caspase-11 in the liver tissues of mice in the HIRI group was significantly higher than that in the control group(P<0.001),while the expression of caspase-11 in the liver tissues of iso group was significantly lower than that in the HIRI group(P<0.001).Immunofluorescence results suggested that the expression of caspase-11 in the labeled Kupffer cells of HIRI group was significantly higher than that of sham group(P<0.001),while the iso group was lower than that of HIRI group(P<0.001).In vitro experiments,ELISA results suggested that IL-1? and IL-18 in Kupffer cells supernatant in LPS group were significantly higher than that in control group(IL-1?:P<0.001;IL-18:P<0.001),while 1L-1? and IL-18 in emiso group were significantly lower than those in LPS group(IL-1?:P<0.001;IL-18:P<0.001).Western Blot and immunofluorescence results showed that the expression of caspase-11 in Kupffer cells in LPS group was significantly higher than that in control group(P<0.001),and the expression of caspase-11 in Kupffer cells in emiso group was significantly lower than that in LPS group(P<0.001).RT-PCR results showed that compared with control group,the caspase-11 mRNA level of Kupffer cells in LPS group was significantly increased(P<0.001),while the caspase-11 mRNA level in emiso group was significantly lower than that in LPS group(P<0.001).Conclusion:Isoflurane preconditioning has a protective effect on hepatic ischemia-reperfusion injury,which is related to the inhibition of the noncanonical pyroptosis pathway of Kupffer cells,the resident macrophages of the liver.