Experimental Study Of Cannabidiol Attenuates Nonalcoholic Steatohepatitis Through Promoting Autophagic Flux

Chapter1 Cannabidiol attenuates methionine-choline deficient diet-induced nonalcoholic steatohepatitis in rats through promoting autophagic fluxObjective: The aim of this work was to test whether treatment with cannabidiol(CBD)attenuated nonalcoholic steatohepatitis(NASH)induced by methionine-choline-deficient diet(MCD)in rats and investigate the underlying molecular mechanism.Methods: Adult male rats were fed with MCD diet to induce NASH and treated with CBD(2mg/kg/day,i.p.)for 10 weeks.Liver/body weight,histological scores,transaminase in serum,hepatic cholesterol and triglyceride content,hepatic fibrosis and autophagic flux are compared between MCD diet with CBD treatment group and control group.Results: Treatment with CBD reduced ratio of liver/body weight,histological scores and fibrosis in livers,lowered activities of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)in serum,attenuated hepatic fat accumulation in rats fed with MCD diet.Furthermore,autophagic flux was impaired in livers of mice fed with MCD diet,evidenced by reduced ratio of LC3-II/LC3-I and increased protein expression of p62,which was reversed by treatment with CBD partly.Conclusion: Treatment with geraniol attenuated MCD-induced NASH in rats,at least in part,through promoting autophagic fluxChapter2 Cannabidiol attenuates palmitic acid-induced injury in cultured hepatocytesObjective: This work was designed to investigate the protection of cannabidiol(CBD)against palmitic acid(PA)-induced injury in cultured hepatocytes and the underlying mechanism associated with autophagic flux.Methods: Experiment 1: Primary cultured hepatocytes were stimulated with PA(800 ?mol/L)and treated with CBD(5 ?mol/L)and chloroquine(CQ,50 nmol/L)or not for 24 hours(1: control group;2: PA-stimulated group;3: PA-stimulated group treated with CBD;4: PA-stimulated group treated with CBD and CQ).Autophagic flux was evaluated by Western blot analysis.Apoptosis was measured by flow cytometry.The m RNA expression of genes involved in endoplasmic reticulum stress was determined by reverse transcription PCR.The mitochondrial function was determined by using fluorescent probe including Rh123 and lucigenin.Experiment 2: Primary cultured hepatocytes were treated with CBD alone for 24 h(1: control group;2: lower-dose CBD-treated group;3: higher-dose CBD-treated group).Then,the autophagic flux was evaluated by Western blot analysis.Results: When compared to control group,exposure to PA significantly led to impaired autopagic flux(evidenced by increased ratio of LC3-II/LC3-I and protein expression of p62),increased apoptosis,endoplasmic reticulum stress(evidenced by increased m RNA expression of C/EBP homologous protein,glucose-regulated protein 78,and X-box protein 1),and mitochondrial dysfunction(evidenced by reduced mitochondrial membrane potential and enhanced formation of mitochondrial reactive oxygen species).When compared to PA-stimulated group,CBD treatment significantly attenuated PA-induced impaired autophagic flux,apoptosis,endoplasmic reticulum stress,and mitochondrial dysfunction in cultured hepatocytes.The protection of CBD against PA was abolished by co-incubation with CQ.In addition,treatment with CBD alone had no significant effect on autophagic flux in cultured hepatocytes.Conclusion: Cannabidiol attenuates palmitic acid-induced impaired autophagic flux,apoptosis,endoplasmic reticulum stress,and mitochondrial dysfunction in cultured hepatocytes through promoting autophagic flux.Chapter3 Role of autophagy in methionine-choline deficient diet-induced advanced nonalcoholic steatohepatitis in mice.Objective: The aim of this study was to elucidate the role of autophagy in MCD-induced steatosis,fibrosis,inflammation,mitochondrial dysfunction,and endoplasmic reticulum(ER)stress in mice.Methods: Mice were fed with MCD diet and treated with rapamycin(an autophagy enhancer)or chloroquine(an autophagy inhibitor)for 10 weeks.Liver injury was evaluated biochemically and histologically together with hepatic gene expression analysis.Results: When compared to control group,the ratio of LC3-II/LC3-I was lower and p62 protein expression was higher in livers of mice fed with MCD diet.The ratio of liver/body weight,hepatic triglycerides levels,histological scores and fibrosis in livers,activities of alanine aminotransferase(ALT)in serum and Col1A1 m RNA levels were higher,mitochondrial dysfunction and ER stress were aggravated in rats fed with MCD diet.The results were reduced by treatment with rapamycin but enhanced by treatment with chloroquine.Conclusion: Autophagic flux was impaired in livers of mice fed with MCD diet,evidenced by reduced ratio of LC3-II/LC3-I and increased protein expression of p62.It was found that autophagy activation by rapamycin attenuated MCD-induced steatosis,fibrosis,inflammation,mitochondrial dysfunction,and ER stress.By contrast,MCD mice treated with chloroquine developed more liver injury.