Ursodeoxycholic Acid Combined With Double-modified Graphene Oxide Improves Liver Injury In Cholestatic Hepatocytes And Bile Duct Ligation Mice Through Caspase-3 Signaling Pathway

Objective: To establish cell and animal models of cholestasis,and to explore whether ursodeoxycholic acid(UDCA)loaded with double modified graphene oxide(NGO-PEG-PEI)can enhance the efficacy of UDCA in cholestasis model and the mechanism.Methods: The cholestasis model of human liver cell line L-02 was established by using deoxycholic acid(DCA).The growth rate,proliferation status,mitochondrial membrane potential,DNA damage and apoptosis of L-02 cells were observed after treatment with different drugs.The effect and potential mechanism of UDCA combined with NGO-PEG-PEI on L-02 cells were explored.The mice model of acute cholestasis was established by bile duct ligation(BDL).The changes of serum results,gross and pathological changes of liver tissue and ultrastructure of cells were detected after 14 days of administration.In order to explore the potential mechanism,we studied the changes of apoptosis pathway related proteins in the liver of mice after BDL modeling.Results: 1.In cholestasis cell model and cholestasis BDL mouse model,oxidative stress plays an important role in cell and liver damage.2.In cholestasis cell model,UDCA combined with NGO-PEG-PEI can improve cell growth rate,protect cell proliferation activity,protect mitochondrial membrane potential,reduce DNA damage,and reduce cell apoptosis rate.In addition,NGO-PEG-PEI has a certain concentration effect.3.In BDL mice model of cholestasis,UDCA combined with NGO-PEG-PEI improved the pathological changes of liver tissue necrosis and fibrosis,the damage of serum results and mitochondria in liver ultrastructure,and the combination outperformed monotherapy of UDCA.4.In the BDL mice model of cholestasis,the expression of Caspase-3,Bcl-2 and Bax in the UDCA combined with NGO-PEG-PEI group were significantly decreased compared with the model control group and the blank control group;the expression of NQO1 and HO-1 in the UDCA combined with NGO-PEG-PEI group were significantly increased compared with the blank control group,while significantly decreased compared with the model control group.And the expression of autophagy related proteins LC3,p62 and p-P62 were down regulated.Conclusion: Cell and mouse models of cholestasis were successfully established in this study.It was found that oxidative stress played an important role in the damage of cells and liver tissues.The protective effect of UDCA loaded with NGO-PEG-PEI on cell and liver injury in cholestasis model was studied.Cholestasis caused mitochondrial related oxidative stress injury in cells and tissues,while the combination of NGO-PEG-PEI and UDCA could alleviate the oxidative stress injury in cells and BDL mice,increase the upstream protein Bcl-2 and decrease Bax,down regulate Caspase-3,HO-1 and NQO1,and down regulate the expression of autophagy related proteins LC3,p62 and p-P62.These results indicate that the combined use of NGO-PEG-PEI and UDCA in improving cholestasis model may have its therapeutic effect through Caspase-3 apoptosis pathway.