Protective Effect And Mechanism Of Bergenin On Hepatic Ischemia-reperfusion Injury

Objective:The study aims to explore the pharmacological mechanism of Bergenin that affects PPAR-? in inhibiting autophagy and apoptosis by eliminating ROS based on the successful establishment of liver ischemia-reperfusion model.The results provide a new theoretical basis for clinical medication.Methods:1.A total of 24 Balb/C male mice were randomly divided into the normal saline control group(control group),sham operation group(sham group),sham operation+saline control group(sham+saline group)and simple drug group(sham+bergenin group).The sham operation group was given abdominal opening and closing treatment.Three days before the operation,normal saline or drugs(40 mg/kg)were given,and serum and liver tissues of all mice are collected to analyze the levels of liver enzymes,pathological changes,inflammatory factors,apoptotic and autophagy related markers by biochemical analysis,HE staining,ELISA and Western blot.2.A total of 120 Balb/C male mice were randomly divided into sham operation group(sham group),model group(IR group)and drug treatment group(IR+bergenin group)after being weighing.Three days before the establishment of the model,different concentrations of drugs(10 mg/kg,20 mg/kg,40 mg/kg)were administrated by gavage.Serum and liver tissues were collected at 2 h,8 h and 24 h after abdominal closure(24 rats in each group,8 rats at each time point).Serum ALT and AST were separated and measured to evaluate the level of pathological damage.The total protein and RNA were extracted from the most severely damaged 8 hours.The expression levels of related inflammatory factors(TNF-?,IL-1?,IL-6),pathway related molecules(PPAR-?,RXR-?,NF-?B,p38 MAPK),apoptosis and autophagy related proteins(Bcl-2,Bax,cleaved caspase-9,Beclin-1 and P62)were detected by ROS staining,ELISA,PCR,immunohistochemistry and Western blot to show the action mechanism of Bergenin3.The normal hepatocytes(LO2)were cultured.The cells were divided into normal group,model group,drug group,inhibitor group according to bergenin(3 mM)and GW9662(10 ?M)treatments,and all cells were treated by hypoxia and reoxygenation.The CCK-8 assay,PCR and Western blot were used to detect the cell survival ratio and expression level of pathway related molecules(PPAR-y,RXR-?)that aims to further verify the mechanism of BergeninResults:1.There was no significant differences in the levels of ALT and AST between the drug group and the control group(P>0.05);there was no significant pathological necrosis in the liver tissue of each group;there was no significant differences in the levels of serum inflammatory factors(TNF-?,IL-1?,IL-6)and related proteins(Bcl-2,Bax,Beclin-1 and PPAR-y)between each group(P>0.05)2.The levels of ALT and AST in the model group were significantly higher than those in the control group(P<0.05),but after pretreatment with different concentrations of Bergenin,the levels of liver enzymes decreased to a certain extent at 2 h,8 h and 24 h,especially at 8 h;consistent with the above results,HE staining showed that the model group had sheet edema and necrosis,while the drug treatment group(40mg/kg)had significantly reduced necrosis area.The difference between groups was significant(P<0.05)3.The time point 8h showed the most serious injury were selected for further study ROS staining showed that the fluorescence of the representative marker molecules in the model group was bright red,but after the treatment with bergenin,the fluorescence brightness decreased in a dose-dependent manner.The difference between the groups was statistically significant(P<0.05).The detection of inflammatory factors by ELISA and PCR showed that TNF-?,IL-6 and IL-1? appeared significantly in the IR group and the difference was statistically significant(P<0.05).TNF-? and IL-6 were further verified by immunohistochemistry,and the results were consistent with the above results4.The levels of Bcl-2,Bax,Cleaved Caspase-9,Beclin-1,LC-3 and p62 were detected by PCR and Western blot.The levels of Bax and Cleaved Caspase-9 in the model group was significantly lower than that in the control group,but decreased after drug treatment.On the contrary,Bcl-2 decreased in the model group and increased in the drug treatment group,the difference was statistically significant(P<0.05);similarly,positive Beclin-1 and LC-3 correlated with autophagy were consistent with Bax,while negative P62 correlated with Bcl-2;TUNEL staining and electron microscopy further demonstrated that the proportion of apoptotic cells and autophagic bodies in the model group was significantly higher than that in the control group,while the proportion of apoptotic cells and autophagic bodies in the drug treatment group decreased with the increase of concentration,the difference was statistically significant(P<0.05)5.The gene and protein levels of PPAR-y,RXR-?,NF-?B and p38 MAPK were detected by PCR and Western blot.The results showed that PPAR-y and RXR-? in the model group were significantly lower than those in the control group,while bergenin increased their expression level,the difference was statistically significant(P<0.05);at the same time,the phosphorylation level of NF-?B,p38 MAPK and other pathway proteins was significantly increased in the model group,but decreased in the drug treatment group Immunohistochemistry technology vertied above results through the detection of PPAR-y and p-P38 and the differences were all statistically significant(P<0.05)6.Normal hepatocytes were treated with Bergenin(3 mM)and PPAR-y inhibitor GW9662(10 ?M),respectively.After anoxia and reoxygenation,CCK-8 assay showed that the cell survival rate of the model group was significantly reduced,while that of the drug treatment group was increased.After GW9662 was added,the cell survival rate was significantly decreased,the difference was statistically significant(P<0.05).At the same time,we further analyzed the correlation by PCR and Western blot.The results showed that PPAR-y level decreased.GW9662 increased cell death while drug treatment significantly weakened the injury effect.The difference between the above results was statistically significant(P<0.05)Conclusion:1.Bergenin can effectively reduce the liver enzyme levels and improve the liver pathological changes induced by liver ischemia-reperfusion injury2.Bergenin can reduce the release of inflammatory factors(TNF-?,IL-1?,IL-6)by eliminating ROS and regulating p38 MAPK/PPAR-y pathway,and effectively inhibit liver injury induced by hepatocyte apoptosis and autophagy.