Verapamil Induces Autophagy To Improve Liver Regeneration In NAFLD

This study is divided into three parts:the first part establishes the mouse model of non-alcoholic fatty liver with different degrees through feeding high-fat diet,then detects the basic autophagy state of each group after verifying the success of the model;the second part establishes the mouse liver regeneration model of different degrees of non-alcoholic fatty liver to detect the relationship between the postoperative autophagy and liver regeneration.The third part reveals the effects of verapamil on liver regeneration and the changes of postoperative autophagy in mice with the mild to moderate and severe fatty liver,and discusses the mechanism of verapamil’s effect on liver regeneration through autophagy.Part Ⅰ To establish a mouse model of non-alcoholic fatty liver disease(NAFLD)and detect the autophagy statusObjective:After establishing a stable and repeatable mouse model of C57BL/6 non-alcoholic fatty liver disease and observing its biochemical indexes and pathological features,the basic autophagy status of mice in each group was detected and compared.Methods:120 healthy male C57BL/6 mice were randomly divided into control group(n=30)and model group(n=90).The model of non-alcoholic fatty liver was established by feeding high-fat diet for different time.The body weight and oxidative activity of liver homogenate were detected at the 2nd and 4th month,and the changes of biochemical indexes such as blood glucose,serum glutamic pyruvic transaminase,glutamic oxaloacetic transaminase,triglyceride and cholesterol were observed.Pathological examination and transmission electron microscope examination were performed.The expression of autophagy-related proteins in liver tissue was detected by Western Blot to understand the basic autophagy status of each group,and the results were compared and analyzed.Results:The normal group was fed with control diet D12450B for 4 months,the mild to moderate and severe fatty liver groups were fed with D12490 for 2 months and 4 months respectively,and the steatohepatitis group was fed for 4 months with 20%carbon tetrachloride intraperitoneally for 2 weeks.There was no significant difference in blood glucose,glutamic pyruvic transaminase and glutamic oxaloacetic transaminase between the normal group and mild to moderate fatty liver group.The body weight of the fatty liver group was significantly higher than that of the normal group at the same time.When reaching severe fatty liver and steatohepatitis,the indexes of glutamic pyruvic transaminase,glutamic oxaloacetic transaminase and blood glucose increased significantly.The liver tissue sections of the fatty liver group showed different degrees of lipid droplet accumulation and the infiltration of inflammatory cells in the hepatocytes of the steatohepatitis group.Under transmission electron microscope,the ultrastructure of cells in fatty liver group swelled or deformed in different degrees,and some normal organelles disappeared.In the resting state,the level of autophagy in the severe fatty liver group was the strongest,the level of autophagy was different between the steatohepatitis groups,and the basic autophagy level was lower in the normal group.Conclusion:The mouse model of non-alcoholic fatty liver induced by high-fat diet combined with carbon tetrachloride is an ideal animal disease model with simple method and strong repeatability.It can provide a stable animal model for the study of the mechanism and drug treatment of non-alcoholic fatty liver.The level of autophagy in resting state is strongest with severe fatty liver.Part Ⅱ Study on the relationship between autophagy and liver regeneration in mice with different degrees of non-alcoholic fatty liver after partial hepatectomy(PH)Objective:The model of fatty liver regeneration was established to evaluate the changes and relationship between autophagy and liver regeneration after PH.Methods:After successful modeling,PH50%models of liver regeneration in mice were established according to the normal group,mild to moderate fatty liver group,severe fatty liver group and steatohepatitis group.Biochemical indexes were collected at each time point(0,6,12,24,36,48,72,120 and 168 h after an operation),liver regeneration rate,cell proliferation test and autophagy were evaluated,and the levels of signal pathway related proteins such as proliferation and autophagy were detected.Autophagosomes were observed by transmission electron microscope.Results:The survival rate of mice after liver regeneration in each group was more than 90%.In terms of liver regeneration after hepatectomy,the normal group and mild to moderate fatty liver group reached the peak of proliferation at 24-48 h after operation,and the mice with severe fatty liver and steatohepatitis reached the peak at 48-72 h.In terms of autophagy,the peak of autophagy was 48 hours after operation in normal group and mild to moderate fatty liver group,72 hours after operation in severe fatty liver group,and 12-24 hours in steatohepatitis group.Conclusion:There is a close relationship between autophagy and liver regeneration.The level of autophagy after hepatectomy increased with liver regeneration and decreased with the cessation of regeneration.The peak of autophagy was delayed and the ability of hepatocyte proliferation decreased in severe fatty liver group and steatohepatitis group after hepatectomy.Part Ⅲ Effect of verapamil on liver regeneration and autophagy mechanism in mice with mild to moderate and severe fatty liverObjective:To investigate the effect of verapamil on liver regeneration in mice with non-alcoholic fatty liver and the mechanism of autophagy.Methods:25mg/kg verapamil was injected intraperitoneally for 10 days before operation in the mild to moderate fatty liver group and severe fatty liver group.Before operation,the changes of body mass,liver biochemical indexes,oxidative stress active products and pathological morphology were observed,the changes of postoperative autophagy and the improvement of liver regeneration were detected,and the classical proteins of autophagy pathway were detected to explore the related mechanism.Results:For the mild to moderate and severe fatty liver group,the body weight,blood glucose and transaminase decreased,the expression of autophagy-related protein increased,autophagosome and lysosome fusion increased,and the expression of BrdU and PCNA increased after verapamil injection.There was no significant difference in the expression of m-TOR signal pathway classical protein between the verapamil group and normal saline group.Conclusion:Verapamil can induce autophagy,improve hepatocyte function and promote hepatocyte regeneration through the m-TOR independent signaling pathway,thus improving the process of liver regeneration after hepatectomy.