Pharmacodynamic Evaluation And Mechanism Study Of Compound Monoammonium Glycyrrhizinate And Cysteine Hydrochloride In Improving Liver Fibrosis

Objective : To study the effect of compound monoammonium glycyrrhizinate and cysteine hydrochloride on improving liver fibrosis and its mechanismMethods:This experiment is divided into two parts: in vitro experiment and in vivo experiment.1.In vivo experimental,method of grouping animals: healthy male rats were randomly divided into 6 groups: blank control group(untreated group),carbon tetrachloride control group(model group),compound monoammonium glycyrrhizate and cysteine hydrochloride(15mg/kg,30mg/kg,60mg/kg)treatment group,bicyclol(positive drug group,200 mg/kg)treatment group.Except for the blank control group,the other groups were all injected with 50% CCl4 repeatedly within 8 weeks to establish a rat liver fibrosis model(1ml/kg,dissolved in olive oil,injected intraperitoneally,twice a week).During the establishment of the model,rats in the treatment group were treated by intraperitoneal injection of compound monoammonium glycyrrhizinate and cysteine hydrochloride.During the whole experiment,the blank control group was given normal water,and the other groups were given 6% ethanol aqueous solution.All rats were sacrificed at the end of the 8th week,and blood and living samples of rats were taken and stored at-80℃.Detect the content of ALT and AST in serum through the kit.The pathological changes in the liver of rats in each group were detected by Masson staining and Sirius red staining.Elisa kit detects changes in related indicators of oxidative stress such as MPO,SOD,MDA,GSH-PX,etc.Detect the changes of Nrf2,Nqo-1,HO-1 in the Nrf2 pathway by Western-Blot.2.In vitro experiment,compound monoammonium glycyrrhizinate,cysteine hydrochloride and DMEM complete medium were used to prepare medicated medium,and the medicated medium was used to culture rat hepatic stellate cells(HSC-T6 cells).Take different concentrations of drug-containing media to culture HSC-T6 cells,and MTT method to detect cell viability.After TGF-β1 activates HSC-T6 cells,select 0.1μg/ml,1μg/ml,10μg/ml drug-containing medium to culture the activated HSC-T6 cells.Subsequently,the cell survival rate was detected by MTT method,and the expression of α-SMA protein was detected by qPCR.Results : 1.In vivo experiment: The results of ALT and AST data showed that compared with the normal group,the ALT and AST levels of the model group were increased;Compared with the model group,the medium-dose group and high-dose group(30mg/kg,60mg/kg)of MG-CH significantly reduced the levels of ALT and AST in serum.The results of Masson staining and Sirius red staining showed that: compared with the normal group,the model group had steatosis,inflammatory cell infiltration and liver fibrosis formation;compared with the model group,both the MG-CH treatment group and the bicyclol group could improve the condition.The results of the Elisa kit showed that compared with the normal group,the expression of MPO and MDA protein in the liver of the model group increased,while the expression of SOD and GSH-PX protein decreased.Compared with the model group,the expressions of MPO and MDA in the MG-CH medium-dose group and MG-CH high-dose group(30mg/kg,60mg/kg)were significantly reduced,and the expression of SOD and GSH-PX proteins increased.The western blot results showed that compared with the blank group,the Nrf2 protein content in the nucleus,the nucleocytoplasmic ratio of Nrf2 protein,and the mRNA levels of HO-1 and NQO1 in the model group had no significant changes compared with the blank group.Compared with the model group,the MG-CH middle-dose group and MG-CH high-dose group(30mg/kg,60mg/kg)increased the content of Nrf2 protein in the nucleus and the ratio of Nrf2 protein to nucleoplasm.And their mRNA expression of HO-1 and NQO1 also increased significantly 2.In vitro experiment: MTT results showed that the concentrations of drug containing medium(50μg/ m L,10μg/ m L,1μg/ m L,0.1μg/ m L)did not affect the non-activated HSC-T6 cells.TGF-β1 can activate HSC-T6 cells.After culturing activated HSC-T6 cells with medicated medium at concentrations of 0.1 μg/ml,1 μg/ml,and 10μg/ml,the cell viability of activated HSC-T6 cells is significantly reduced.The qPCR results showed that the expression of α-SMA in activated HSC-T6 increased significantly,and the expression level of α-SMA in HSC-T6 cells after treatment decreased.Conclusion : 1.Compound monoammonium glycyrrhizinate and cysteine hydrochloride can improve the liver function and degree of liver fibrosis in CCl4-induced chronic liver fibrosis model.2.Compound ammonium glycyrrhizinate and cysteine hydrochloride can inhibit the activated hepatic stellate cells.3.The anti-hepatic fibrosis mechanism of compound monoammonium glycyrrhizinate and cysteine hydrochloride may be through up-regulating the Nrf2 pathway to resist oxidative stress.