| 【BACKROUND & OBJECTIVE】 Non-alcoholic fatty liver disease(NAFLD) is a manifestation of the metabolic syndrome in the liver., charactered by insulin resistance(insulin resistance, IR) and genetic susceptibility. Similar to alcoholic liver disease(ALD), the Pathology of NAFLD presents liver steatosis without excessive alcohol drinking. NAFLD can be classified into several types, which are non-alcoholic simple fatty liver( NAFL), non-alcoholic steatohepatitis(NASH), cirrhosis and hepatocellular carcinoma. With the development of society,the obesity and metabolic syndrome become more common in the world, in the past 20 years, the prevalence of NAFLD has grown rapidly in Asian countries and in developed areas of China, such as Shanghai, Guangzhou and Hong Kong the prevalence of NAFLD is even about 15%. However, the etiology and pathogenesis of NAFLD remains unclear. In present, China still has a high incidence of viral hepatitis infection,which are mainly chronic HBV and HCV. With the prevalence of NAFLD, the incidence of chronic viral hepatitis with NAFLD has gradually increased, so more attention should be paied on the combination of these two diseases Currently, the explicit researches on NAFLD patients with chronic viral hepatitis focused mainly on HCV. NAFLD can promote HCV patients to develop into cirrhosis and liver cancer, and in turn, HCV can lead the steatosis of hepatic cells,which accelerate the progress of NAFLD. In contrast, the study in HBV with NAFLD focus mainly on the observation of clinic phenomenon, and the conclusion is also not consistent. Some studies believe that fatty liver promotes the clearance of hepatitis B virus; some studies have shown that fatty liver has no effect on replication of hepatitis B virus; and other studies even prove that fatty liver can promote the replication of hepatitis B virus and reduce chronic hepatitis B patients antiviral efficacy,thus may increase the risk of cirrhosis and liver cancer. Similar to hepatitis C virus, the hepatitis B virus can also induce the hepatic steatosis? And lead to lipid metabolism disorders? Current researchs are studied mainly by the use of transfected HBV plasmid into HBV-related liver cancer cell models and clinical patient samples. Patients are diagnosed with fatty liver mainly by clinical ultrasound diagnosis and liver biopsy, but there are some limitation for doing so, which are the limited number of samples and the compliance of patient. Animal models can avoid the two defect mentioned above. But now, the animal models of hepatitis B with NAFLD are mainly transgenic mice, fully immuned and continuous replication of hepatitis B virus mouse model is lacked. The animal model adopted in this study can eliminate these limitations. The objectives of this study are as follows:1 To successfully establish a mouse model of hepatitis B viral persistent replication combined with NAFLD; 2. To investigate the effects of NAFLD on HBV replication and the dynamic observation of HBV replication on lipid metabolism, mainly on the synthesis and metabolism of cholesterol.【METHOD】 1. We used hydrodynamic injection to introduce HBV genotype B DNA plasmid(p GEM4 Z / HBV1.3) into the hepatocytes of male FVB / N mice, and the next day collect eye canthus venous blood to detect the serum HBe Ag level,the HBe Ag positive mice were fed with 60% high fatty diet(HFD) for 14 weeks,the changes in the general conditions,body weight,and Serum biochemical index such as ALT、AST、Glucose、HDL、LDL、TG、TC、Insulin and the morphology of liver(HE and Oil Red) are observed continuously. 2. In order to observe the changes in HBV replication, HBc Ag in liver was detected by Immunohistochemic, HBe Ag in serum and HBs Ag in liver were measured by immune chemiluminescence, quantitative PCR and Southern blot was used to detected HBV DNA in liver tissue. Triglycerides and cholesterol was measured in homogenized liver,RT-PCR was used to detect the changes of cholesterol synthesis and metabolism-related genes,so we can observe the effect of persistent HBV replication on cholesterol metabolism. 3. The liver lipid oxidation product MDA was detected by MDA-TBA【RESULT】1. The mouse model of persistent HBV replication with NAFLD was established successfully. The next day after injection,about 85% mice was HBe Ag positive. Feeding with HFD for 14 weeks, FVB/N became sedentary,obese, greasy haired and gained body weight.and about 70%mice was HBe Ag positive. The serum biochemical index(ALT、AST、Glucose、HDL、LDL、TG、TC、Insulin)and HOMA-IR in HFD group increased significantly(p<0.05).Compared with control,the liver weight is also increased and charactered with yellowish color, rounded edges, the greasy surface, fragile touch.A number of lipid droplets deposited in the hepatocytes seen in the HE staining and Oil Red staining. 2. Immunohistochemistry showed that HBc Ag was expressed in hepatocytes of HBV group and HBV with NAFLD group, the number of positive cells in HPF was significantly lower in the HBV with NAFLD group(P <0.05) as well as in southernblot. HBe Ag in serum and HBs Ag in liver were measured by immune chemiluminescence and found in the HBV with NAFLD group,the level was significantly decreased,respectively.Also, quantitative PCR showed that the HBV DNA was also decreased in HBV with NAFLD group(p<0.05);and a positive correlation between the HBV DNA level and the serum HBe Ag level, HBs Ag and HBc Ag level in liver was found. 3. Triglycerides was measured in homogenized liver and found that in HFD significantly increased(p<0.05).Feeding HFD for 4 weeks, the cholesterol in homogenized liver and the related genes(SREBP2、HMGCR、LDLR、CYP7a)did not show any significant difference in four groups.while in 6 weeks,the cholesterol and the related genes was significantly increased in HBV groups compared with HBV with NAFLD,NAFLD and controls(p<0.05).In 10 weeks and 12 weeks,the phenomenon remains the same and finally in 14 weeks,the cholesterol level did not have any siginificant difference in 4 groups,and the related genes in HFD was significant decreased(p<0.05). 4. Compared with HBV with NAFLD,NAFLD and control,the expression level of MDA in HBV group was significantly increased(p<0.05).【CONCLUSION】 1. We have successfully established the persistent HBV replication with NAFLD mouse model,and provide a foundation for the study of the relationship between NAFLD and HBV infection. 2. NAFLD can suppress the HBV replication and reduce the peripheral HBe Ag, HBs Ag and HBc Ag level in liver tissue. 3. HBV replication have an influence on the expression of cholesterol metabolism-related gene. 4. HBV promote the lipid oxidation in hepatic cells and strengthen liver tissue oxidative stress. |
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