Establishment Of A Hepatic Steatosis Murine Model Induced By Chronic Viral Hepatitis

Research background and proposalViral hepatitis B and C are a worldwide disease with over8%of HBV carrier and about5%of HCV infection in China.Cirrhosis and hepatocellular cancer can all occur as a result of chronic viral hepatitis. Recently,some patients with chronic viral hepatitis had hepatic steatosis accroding to the liver imaging and pathological findings.Even up to50%-72%of patients accompanied by HCV infection sufferred from hepatic adipose infiltration.Hepatic steatosis was a very key factor in the progress of liver fibrosis and antiviral treatment. Animal models have made a tremendous contributions to the disease research for human.The establishment of animal models of various diseases has been regarded as the key link to the research of pathogenesis and corresponding therapeutic methods.The underline mechanism for the development of hepatic steatosis induced by chronic viral hepatitis is not fully understood.In the context of present situation which chronic viral hepatitis model has some limications that was absent of pathogen replication in vivo and significantly different from the clinical situation,meanwhile, knowing the absent of an ideal representative hepatic steatosis model induced by chronic viral hepatitis. MHV (mouse hepatitis virus) is a coronavirus which was found in1949.As a kind of RNA virus, MHV could Lead to the diseases of liver, central nervous system and immune dysfunction severely. These of MHV-A59are known as hepatotrophic viruses and neural tissue,which can be the animal model of copying the nervous demyelinating diseases and outbreaking hepatitis.There are some researchses that establishment of cell models based on of MHV-A59,the kinds of mice and the infective dose of MHV-A59for chronic viral hepatitis. MHV-A59as an important pathogenic factor of chronic viral hepatitis, we dedicated to establish and characterize a chronic viral hepatitis and hepatic steatosis murine models in C57BL/6mice induced by MHV-A59.Based on this animal model of hepatic steatosis induced by chronic viral hepatitis of MHV-A59,part of the mechanism were discussed in order to provide the ideal animal model and theoretical basis in understanding the mechanism of a hepatic steatosis induced by chronic viral hepatitis.MethodsC57BL/6mice at the age of6-8weeks were used in my experment. We prepared MHV-A59at the concentrations of10-3、10-4、10-5、10-6and10-7.30C57BL/6mice were randomly divided into5groups(6mice in each group).The mice in5different groups were injected0.5ml MHV-A59at different concentrations via abdomen.We observed the number of the death mice in two weeks and calculated LD50in two weeks of MHV-A59mice by statistical software.We determined the proper concentrations of MHV-A59and established the mice models which hepatic steatosis induced by chronic viral hepatitis of MHV-A59. Then120mice were randomly divided into4groups:①controlled group(fed standard diet),②high fat group (fed high fat diet),③viruses group(infected with MHV-A59and fed standard diet),④high fat and viruses group(infected with MHV-A59and fed high fat diet), the proportioning of High lipid feed:87.6%Normal diet+2%cholesterol+10%lard+ 0.4%sodium cholate.All the mice were injected0.5ml MHV-A59in concentrations of10-6via abdomen in1d and30d. Then we observed their general state of health and weight, and then collected serum and liver tissues of mice in the13weeks. The serum were selected to detect MHV antibodies (ELISA method), serum lipide and serum transaminase(wet chemistry methods), frozen section of liver was measured by Oil Red O staining, paraffin section of liver was measured by HE stain.Results1、 C57BL/6mice at the age of6-8weeks received0.5ml in different concentrations of MHV-A59intraperitoneally and were observed the signs of toxicity, the peak time of death was3-5d.We calculated LD50in two weeks of MHV-A59to mice by statistical software was10-5.5according to formula of Reed-Mueneh.So we took the establishment of mice models in C57BL/6mice which hepatic steatosis induced by chronic viral hepatitis of MHV-A5910-6(All the mice were injected0.5ml MHV-A59in concentrations of10’6via abdomen).2、The general state and weight change of mice:the wice were observed the signs of bad appetite and reducing body weights which was serverly for the mice in high fat and viruses group(infected with MHV-A59and fed high fat diet).3、MHV-A59antibody detection showed that the reference OD value of mice in high fat and viruses group, viruses group were strong positive, the reference OD value of mice in controlled group and high fat group were negative.4、The serum lipide indexes showed that there were no significant difference among every group according to TG and HDL-C; TC in high fat,in high fat and viruses group were higher than that in controlled group,there was satistical difference (P<0.05),TC in viruses group was also higher than that in controlled group,but there was no significant difference (P>0.05); HDL-C in high fat,in high fat and viruses group were higher than that in controlled group,there was satistical difference (P<0.01), HDL-C in high fat and viruses group was higher than that in high fat group,there was satistical difference (P<0.05)5、The serum transaminase indexes showed that the levels of serum ALT and AST in three groups were higher than than in controlled group, there was satistical difference (P<0.01)6、HE stain of liver showed that Hepatic lobules disorganised,hepatocyte swelling, focal、fragment and bridging necrosis,lymphocytic infiltrate in portal tract were found in viruses group and in high fat and viruses group;but hepatocyte ballooning could be found in HE stain of high fat and viruses group which suggested hepatic steatosis.7、Oil Red O stain of liver showed that big hepatocellular macrovesicular red lipid droplets in high fat group, small and less lipid droplets were found in viruses group, lipid droplets in high fat and viruses group were more intensive and bigger than that in high fat group.With the help of Motic Med software, liver lipid contents(LLC) in viruses group,in high fat group,in high fat and viruses group were higher than that in controlled group, there was satistical difference (P<0.05); and LLC in high fat and viruses group was higher than that in high fat group (P<0.05).The results suggested that viruses infection and high fat diet could lead to lipid accumulation in liver,meanwhile lipid accumulation in liver because of high fat diet could be aggravated by viruses infection.ConclusionWe established and characterize a chronic viral hepatitis and hepatic steatosis murine models in C57BL/6mice induced by MHV-A59combined high fat diet.The hepatic index of the model were similar to the hanppen and development of chronic viral hepatitis in human.The model we established could be applied in studies towards understanding the mechanism and drug screening of hepatic steatosis induced by chronic viral hepatitis, which have an important real-world implication.