Protective Effect And Mechanisms Of Lycium Barbarum Polysaccharides On Chemical Hepatocyte Injury

Objectives:To explore the protective effect and mechanisms of lycium barbarum polysaccharides(LBPs)on chemical hepatocyte injury induced by ethanol and anti tuberculosis drugs,provide experimental and theoretical basis for the protection of hepatotoxicity from exogenous chemicals and the development and utilization of LBPs.Methods:1.The protective effect of LBPs on ethanol and antituberculosis drug-induced hepatocyte injury.Human normal hepatocytes(L-02 cells)were selected for this study Effect of LBPs on L-02 cells viability was investigated by MTT method,and the concentration of ethanol and antituberculosis drugs was screened to establish two hepatocyte injury models.Control group,ethanol injury group,LBPs protection group and silymarin positive control group were set up to study the protective effect of LBPs on ethanol-induced hepatocyte injury.Control group,isoniazid(INH)+rifampicin(RFP)injury group,LBPs protection group and silymarin positive control group were set up to study the protective effect of LBPs on antituberculosis drugs-induced hepatocyte injury.(1)Cell viability of L-02 cells in each group was determined by MTT method,and the molecular weight range LBPs with the highest cell viability was selected for subsequent study.(2)The activity of alanine aminotransferase(ALT),aspartate aminotransferase(AST),lactate dehydrogenase(LDH)in L-02 cells of each group was determined by spectrophotometry.2.Mechanisms of the protective effect of LBPs3 on ethanol and antituberculosis drugs-induced hepatocyte injury.(1)The level of reactive oxygen species(ROS)in L-02 cells of each group was determined by 2’,7’-dichlorodihydrofluorescein diacetate(DCFH-DA)method.(2)The activity of Catalase(CAT),superoxide dismutase(SOD),glutathione peroxidase(GSH-Px)and the content of glutathione(GSH),malondialdehyde(MDA)in L-02 cells of each group was determined by spectrophotometry.(3)The apoptosis rate of L-02 cells in each group was determined by Annexin V-FITC/PI double staining method.(4)The expression levels of related proteins in the lysates of L-02 cells in each group were determined by western blot method,including the expression levels of Nrf2,HO-1,NQO1,GCLC in the Nrf2 signaling pathway,and JNK,p-JNK in the JNK signaling pathways,and Bcl-2,Bax,cytochrome c,caspase-9,caspase-3 and other proteins in the mitochondrial apoptosis pathway.Results:1.The protective effect of LBPs3 on ethanol and antituberculosis drugs-induced hepatocyte injury.LBPs between 0 and 200μg/mL did not inhibit or promote the proliferation of L-02 cells.An ethanol-induced liver hepatocyte injury model was established by damaging L-02 cells with 5%ethanol for 4 h.(1)The cell activity of LBPs3 group with different concentrations was higher than that of other LBPs groups,12.5,25,and 100μg/mL LBPs3(low,medium and high LBPs3 groups)were selected to study their protective effect on ethanol-induced hepatocyte injury.(2)The activity of ALT,AST,and LDH in the cell culture medium of the medium and high LBPs3 groups was lower than that of the ethanol-injured group(P<0.05).An antituberculosis drugs-induced hepatocyte injury model was established by damaging L-02 cells with 100+200μg/mL INH+RFP for 24 h.(1)The cell activity of LBPs3 group with different concentrations was higher than that of other LBPs groups,12.5,25,and 50μg/mL LBPs3(low,medium and high LBPs3 groups)were selected to study their protective effect on antituberculosis drugs-induced hepatocyte injury.(2)The activity of ALT,AST,and LDH in the cell culture medium of the low,medium and high LBPs3 groups was lower than that of the INH+RFP injured group(P<0.05).2.Mechanisms of the protective effect of LBPs3 on ethanol and antituberculosis drugs-induced hepatocyte injury.Mechanisms of the protective effect of LBPs3 on ethanol-induced hepatocyte injury.Compared with the ethanol-injured group:(1)The intracellular ROS level decreased in the middle and high LBPs3 groups(P<0.05).(2)The activity of CAT,SOD and GSH-Px increased in the low,medium and high LBPs3 groups,the content of GSH increased in the medium and high LBPs3 groups,the content of MDA decreased in the medium and high LBPs3 groups(P<0.05).(3)The nuclear translocation level of Nrf2 protein and the expression of HO-1,NQO1,and GCLC proteins increased in the low,medium and high LBPs3 groups(P<0.05).(4)The apoptosis rate decreased in the low,medium and high LBPs3 groups(P<0.05).(5)The level of JNK protein phosphorylation decreased in the medium and high LBPs3 groups(P<0.05).The expression of caspase-3 protein decreased in the medium and high LBPs3 groups.The expression of Bax,cyt c and caspase-9 protein decreased in the low,medium and high LBPs3 groups.The expression of Bcl-2 protein increased in the medium and high LBPs3 groups.The ratio of Bcl-2/Bax increased in the low,medium and high LBPs3 groups(P<0.05).Mechanisms of the protective effect of LBPs3 on antituberculosis drugs-induced hepatocyte injury.Compared with the INH+RFP injured group:(1)The intracellular ROS level decreased in the low,middle and high LBPs3 groups(P<0.05).(2)The activity of CAT,SOD and the content of GSH increased in the medium and high LBPs3 groups,the activity of GSH-Px increased in the low,medium and high LBPs3 groups,the content of MDA decreased in the low,medium and high LBPs3 groups(P<0.05).(3)The nuclear translocation level of Nrf2 protein and the expression of HO-1 increased in the low,medium and high LBPs3 groups,the expression of NQO1 and GCLC increased in the high LBPs3 group(P<0.05).(4)The apoptosis rate decreased in the low,medium and high LBPs3 groups(P<0.05).(5)The level of JNK protein phosphorylation decreased in the low,medium and high LBPs3 groups(P<0.05).The expression of Bax protein decreased in the low,medium and high LBPs3 groups.The expression of caspase-3 protein decreased in the medium and high LBPs3 groups.The expression of Bcl-2 protein and the ratio of Bcl-2/Bax increased in the low,medium and high LBPs3 groups(P<0.05).Conclusions:(1)LBPs3 has protective effect on chemical hepatocyte injury induced by ethanol and antituberculosis drugs.(2)LBPs3 can exert its protective effect on ethanol and antituberculosis drugs-induced hepatocyte injury via inhibiting oxidative stress of hepatocytes,activating Nrf2 signaling pathway,inhibiting JNK pathway and mitochondrial-mediated apoptosis of hepatocytes.