The Role Of Resveratrol In Nonalcoholic Steatohepatitis Related Hepatocellular Carcinoma And Molecular Mechanism

With the development of economy and the change of people’s lifestyle,nonalcoholic fatty liver disease(NAFLD),which is related to obesity and metabolic syndrome,has become the largest liver disease in China.The spectrum of NAFLD includes nonalcoholic fatty liver(NAFL),nonalcoholic steatohepatitis(NASH),liver fibrosis/cirrhosis and hepatocellular carcinoma(HCC).To date,NASH related HCC has been generally recognized,and become the main pathogeny of HCC in Europe and the United States.In the future,these diseases will be major ones in our country as well.It is important to explore the mechanism and look for effective interventions.In this study,we fed mice with high fat high cholesterol(HFHC)diet combined with streptozotocin(STZ)to establish an NASH related HCC(NASH-HCC)animal model and identified the model;the intervention in the NASH-HCC mice was conducted with the resveratrol(RES).We confirmed that resveratrol could reduce the incidence of carcinogenesis.By detecting bile acids profiles and the change of gut microbiota and analyzing the correlation,we spectulated that it was one of the important mechanism of resveratrol to change the components of bile acids by regulating the gut microbiota and the receptor of bile acids in NASH-HCC.Part 1 The Establishment and Identification of the Animal Model of NASH-HCC Objective To establish and identify the mice model of NASH-HCC.Methods The C57BL/6J male mice were injected with STZ 200 ug at 4 days after birth and were fed with HFHC diet to establish NASH-HCC model.Results The C57BL/6J mice developed typical NASH features at week 9,severe inflammatory lesions at week 12.Most of them appeared early HCC at week 16 and all of them developed HCC at week 20.Conclusion The HFHC diet combined with STZ could establish accurate,effective animal model and simulate the tumor natural history of human fatty liver.Part 2 Pharmacodynamics Study of Resveratrol in NASH-HCC Objective To investigate the pharmacodynamics of resveratrol in NASH-HCC.Methods 4-week male C57BL/6J mice were divided into 3 groups: STZ+SC+Saline(Saline gavege/d,Control,n=10),STZ+HFHC+Saline(Saline gavege/d,Model,n=10),STZ+HFHC+RES(RES gavege 400 mg/kg/d,n=10),mice were sacrificed at the end of week9,12 and 16 for samples.Results Mice treated with resveratrol had lower liver function,blood sugar,blood lipid and inflammation at 12 week which had the most inflammatory indices.At 16 week,resveratrol significantly improved liver function,blood sugar,blood lipid and liver index.Resveratrol lowered the steatosis,lobular inflammation,TNF-α,MCP-1,CD68,F4/80,Collagen I,TGF-β,MMP-9,GPC3 level and reduced the incidence of HCC.Conclusion Resveratrol alleviated steatosis,inflammation and fibrosis in the NASH-HCC process and reduced the occurrence of HCC.Part 3 The Mechanism of Resveratrol in NASH-HCC Objective To investigate the mechanism of resveratrol in NASH-HCC.Methods 16 S rDNA gene sequences assay of gut microbiome in mice of different groups was performed.Extracted the serum 20 ul from the control group,model group and RES intervention group(5-6 mice)to detect quantitatively the absolute concentration of bile acids by UPLC-MS/MS.The correlation of bile acids composition and gut microbiota were analyzed.Results Resveratrol could change the structure of gut microbiota and lowered the abundance of Firmicutes and it’s subordinates,Prevotella,increased the abundance of Bacteroides,Proteobacteria,Lactococcus,Lactobacillus and Bifidobacterium.Resveratrol reduced the toxic bile acids,such as CA,CDCA,GCA,TCA,DCA,TDCA,TCDCA,TLCA,TβMCA and total bile acid level,and increased bile acid receptor FXR and downstream target genes SHP,BSEP level.Resveratrol promoted intrahepatic bile acids excretion and inhibitedβ-catenin activation;By Correlation analysis,we found that resveratrol could influence bile acids metabolism by changing the structure of gut microbiota.Conclusion The mechanism of resveratrol in NASH-HCC might be associated with the reduction of bile acid components by remodeling gut microbiota,promoting the excretion of bile acids in the liver and inhibiting the activation of β-catenin.