| Object: Hepatic ischemia-reperfusion injury(IRI)affects the effect of liver transplantation.A variety of mechanisms are involved in this process.Among them,Kupffer cells(KCs)play an important role in hepatic ischemia-reperfusion injury by regulating liver immune rejection,but the specific mechanism remains to be further explored.Suberanilide hydroxamic acid(SAHA)is a broad-spectrum histone deacetylase inhibitor.Studies have shown that SAHA can effectively inhibit the inflammation of macrophages and induce autophagy in a variety of cells through the AKT / mTOR pathway.But its role in liver transplantation-induced hepatic ischemia-reperfusion injury remains unknown.In this study,the rats with liver transplantation were treated with SAHA,the role of SAHA in this model was explored,and we hope to lay a foundation for the drug treatment of IRI.Method: Male Sprague Dawley(SD)rats were used to construct a model of liver transplantation-induced hepatic ischemia-reperfusion injury.The experimental animals were randomly divided into 6 groups: 1.Sham group;2.ischemia-reperfusion group(IR group);3.IR + DMSO group;4.IR + SAHA group;5.IR + SAHA + CL group;6.IR + SAHA + CQ group.Peripheral blood and liver tissues of each group were obtained at 24 h after reperfusion.Total liver and KCs proteins were extracted.Inflammatory factors,apoptosis-related proteins,and AKT / mTOR pathway protein levels were measured by western blotting assay.Liver tissue pathology was measured by HE.Apoptosis of hepatocytes was measured by TUNEL assay.KCs of normal SD rats were extracted and randomly divided into 6 groups: Normal group,LPS group,LPS + Scramble-siRNA group(LPS + Scramble),LPS + DMSO group,LPS + SAHA group,LPS + SAHA + ATG5-siRNA group,The proportion of M1 type-polarized cells in each group was detected by flow cytometry,immunofluorescence was used to detect the expression of autophagy marker LC3 B in each group,transmission electron microscopy was used for the detection of KCs autophagosomes,the autophagy-related factors,inflammatory factors,AKT / mTOR pathway and P65 activation levels were detected by western blotting or RT-qPCR.Results: Compared with the IR + DMSO group(743.60±90.64 U/L),the transaminase levels in the SAHA treatment group(423.60±95.81 U/L)was effectively reduced(n=5,P<0.05),and the apoptosis of hepatocytes was significantly reduced(IR+DMSO: 129.70±13.21,IR+SAHA: 42.67±9.71,n=3,P<0.05),and IL-1?(IR+DMSO: 0.61±0.05,IR+SAHA: 0.22±0.02,n=3,P<0.05),TNF-?(IR+DMSO: 0.52±0.03,IR+SAHA: 0.24±0.04,n=3,P<0.05),P65(IR+DMSO: 0.59±0.06,IR+SAHA: 0.28±0.06,n=3,P<0.05)were significantly inhibited in the in vivo test.What's more,SAHA promoted the expression of LC3B(IR+DMSO: 0.19±0.05,IR+SAHA: 0.68±0.07,n=3,P<0.05),ATG5(IR+DMSO: 0.16±0.04,IR+SAHA: 0.61±0.06,n=3,P<0.05),and inhibited the level of P62(IR+DMSO: 0.60±0.04,IR+SAHA: 0.18±0.06,n=3,P<0.05).SAHA relieved the inflammation and edema of ischemia-reperfused hepatocytes,it also effectively promotes the formation of autophagosomes in KCs(IR+DMSO: 2.10±0.21,IR+SAHA: 5.21±0.57,n=3,P<0.05).After being treated by CL,the protective effect of SAHA on liver was significantly weakened(IR+SAHA: 3.21±0.45,IR+SAHA+CL: 6.19±0.35,n=5,P<0.05).What's more,SAHA increased the proportion of CD68 + CD86 + KCs(LPS+Scramble: 21.25±1.71%,LPS+SAHA: 9.92±0.87%,n=3,P<0.05).Conclusion: SAHA may reduce the degree of P65 activation through the AKT / mTOR autophagy pathway and thus inhibit KCs activation,thereby reducing the degree of ischemia-reperfusion injury after liver transplantation in rats. |
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