| Objective: To investigate the protective effect of Pinocembrin(PIN)on hepatic ischemia reperfusion injury(HIRI)and its possible molecular mechanism.Methods: The experiment was divided into two parts: in vivo experiment and in vitro experiment.The first part is the in vivo experiments:(1)Establishment of a mouse liver ischemia-reperfusion model: forty C57 mice(8 weeks)were selected to establish a liver ischemia-reperfusion model.The portal vein branches draining the left lateral lobe and median lobe were ligated to induce a warm 70% liver ischemia.They were randomly divided into five groups: sham operation control(Sham),ischemia reperfusion group(I/R),low concentration PIN preconditioning group(I/R + PIN40),high concentration PIN preconditioning group(I/R + PIN80).Reperfusion was resumed for 6hours after 1 hour of ischemia,and then blood and liver tissue samples were collected.(2)The contents of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)in serum of each group were detected by kit.(3)sample of the liver tissue was collected,processed and H&E stained to be examined under an optical microscope for possible tissue damage.(4)The contents of superoxide dismutase(SOD),glutathione(GSH)and malondialdehyde(MDA)in the liver tissue of each group were detected by kit.(5)Tunel staining was used to detect apoptosis in liver tissue.(6)Immunohistochemistry was used to detect the expression of TLR4 in liver tissues,and western blot was used to detect the expression of apoptosis-related proteins(bcl-2 and Bax)and HMGB1/TLR4 inflammatory pathway proteins in liver tissues.The second part was in vitro experiments:(1)cck-8 method was used to detect the effect of different concentrations of the hormone on the proliferation of AML12 mouse cells in vitro.(2)The H2O2 stimulated mouse cell model was established to simulate the cell response during hepatic ischemia/reperfusion.They were divided into blank control group(Control),Pinocembrin group(PIN40 group),hydrogen peroxide group(H2O2800)and Pinocembrin + hydrogen peroxide group(PIN40+H2O2800).(3)DCFH-DA staining were used to detect reactive oxygen species content in liver cell models of each group.(4)Hoechst staining was used to detect the apoptosis of hepatocytes in each group.(5)Western blot was used to detect apoptosis-related proteins(Bcl-2 and Bax)and HMGB1/TLR4 inflammatory pathway protein expression.Results: The results of in vivo experiments showed that:(1)The animal model of mouse HIRI was successfully established.(2)Compared with I/R group,the AST and ALT contents in serum of the PIN preconditioning group were significantly decreased.(3)The pathological changes of liver tissue structure in the PIN preconditioning group were significantly lighter than those in the I / R group.(4)The content of GSH and SOD in liver tissue in the PIN preconditioning group was significantly higher than that in I / R group,while the content of MDA was significantly lower than that in I / R group.(5)Compared with the I /R group,the expression of anti-apoptotic protein Bcl-2 in liver tissue was significantly increased,and the expression of pro-apoptotic protein Bax was significantly decreased in the PIN preconditioning group.(6)Compared with the I/R group,the expression level of HMGB1/TLR4 inflammatory pathway protein in the liver tissue of the PIN preconditioning group was significantly decreased.The results of in vitro experiments showed:(1)In different time periods of 12,24 and 48 hours,the PIN solution in the concentration range of 0 to 40 mol/L had no significant effect on the proliferation of mouse liver cells.(2)The content of ROS in the PIN40 + H2O2800 group was significantly decreased after the preconditioning with PIN compared with the hydrogen peroxide group(H2O2800).(3)Compared with the H2O2800 group,the apoptotic cells of the liver cells in the PIN40+H2O2800 group was significantly decreased.(4)Compared with H2O2800 group,the expression of anti-apoptotic protein Bcl-2 in liver cells was significantly increased,and the expression of pro-apoptotic protein Bax was significantly decreased in the PIN40+H2O2800 group.(5)Compared with the H2O2800 group,the expression level of HMGB1/TLR4 inflammatory pathway protein in the liver cells of the PIN40 + H2O2800 group was significantly decreased.Conclusion: The preconditioning with PIN can improve the liver injury caused by HIRI.The possible mechanism is related to PIN reducing oxidative stress,inhibiting apoptosis and inhibiting the activation of HMGB1 / TLR4 inflammation pathway. |
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