| [BACKGROUND&OBJECTIVE]Non-alcoholic fatty liver disease (NAFLD) is the demonstration of the metabolic syndrome in the liver, and is closely related to diet and lifestyle. NAFLD includes simple steatosis, non-alcholic steatohepatitis (NASH), NASH induced fibrosis/cirrhosis, liver cancer. With the improvement of living standards, the incidence of NAFLD in developing countries increased significantly. In China, the incidence of NAFLD has nearly doubled in the past10years, and is close to a quarter in some areas. There is a high incidence of viral hepatitis infection in China, with about100million HBV infection and40million HCV infected, and the patients combined with NAFLD tend to increase, which attracted the attention of researchers.The present study concentrated in NAFLD combined with chronic hepatitis C (CHC). NAFLD can increase the risk of liver cirrhosis and hepatocellular carcinoma (HCC) in patients with CHC, and reduce the efficacy of antiviral therapy. However, hepatitis C virus (HCV) itself can induce liver steatosis; therefore, conclusions derived from NAFLD combined CHC may not applicate extensively. As to the impact of NAFLD on chronic hepatitis B (CHB), the current findings were not consistent, and differ with the phenomenon seen in CHC study. Some studies believe that liver steatosis has a protective effect on CHB:NAFLD patients with a lower hepatitis B virus (HBV) viral load, but a higher hepatitis B surface antigen (HBsAg) clearance. On the other hand, some studies show that NAFLD may increase the risk of cirrhosis and HCC, reduce efficacy of entecavir antiviral therapy in HBV infected population. Some other studies suggest that NAFLD had no effect on HBV viral load or antiviral therapy of interferon. In fact, there are many difficulties for researches in this area, which may attribute to these contradictory conclusions. First, the definition of liver steatosis is that the lipid deposition in the liver exceeds5%. The fatty liver was diagnosed by liver ultrasound in the studies, rather than iver biopsy, which was refused bu the patients. The liver ultrasound can only detect the moderate fatty liver with a fat deposition of more than30%, but not the mild fatty liver (liver fat deposition was5-30%). As a result, a mild fatty liver patient may be recruited in the control group. Second, it is difficult to ensure the consistency of metabolism factors, viral factors and treatment between the subjects. Third, NAFLD is often accompanied by obesity, high cholesterol, high blood sugar, insulin resistance and other metabolic syndrome manifestations, population-based study can not rule out the impact of these factors on the course and outcome of CHB, the liver was observed in liver tissue is difficult to obtain immunity, metabolism changes in other aspects. Third, population-based study can not rule out the impact of these co-factors on the course and outcome of CHB, or observe the immunity and metabolism changes in the liver. Therefore, the impact NAFLD of chronic viral hepatitis is still remained to be elucidated.An animal model NAFLD combined with viral hepatitis is necessary for the research of the impact of NAFLD on viral hepatitis, which can overcome the above deficiencies in clinical study. However, there is no acknowledged animal model of NAFLD combined with viral hepatitis currently. In this study, we used high fatty diet (HFD) and MHV-3to induce a mouse model of NAFLD combined with viral hepatitis, and investigated the impact of NAFLD on the viral hepatitis in the established mouse model. Therefore, the purposes of this study are as the follows:1. To establish a C3H mouse model of NAFLD combined with viral hepatitis;2. To investigate the hepatitis virus replication and hepatic injury in this mouse model; 3. To investigate the immunological mechanisms underlaying the impact of NAFLD on viral hepatitis.[METHODS]1. The C3H/HeN mice were fed with60%high fatty diet (HFD) for12weeks. Tthe changes in the general condition, body weight, alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG), total cholesterol (TC), High density lipoprotein (HDL), Low density lipoprotein (LDL), glucose (GLU), insulin, Homeostasis Model Assessment-Insulin Resistance (HOMA-IR), and hepatic pathology were observed continuously..2. NAFLD mice were intraperitoneally injection with murine hepatitis virus-3(MHV-3). General condition and survival of mice were observed; serum biochemistry parameters and hepatic pathological demonstrations were checked; Reverse transcription-polymerase chain reaction (RT-PCR) was performed to detect MHV-3in mice liver.3. NAFLD mice and normal diet (ND) mice were intraperitoneally injection with MHV-3. The survival rates between the two groups were compared; serum biochemistry parameters and hepatic pathological demonstrations were investigated and compared; real-time PCR was performed to compare MHV-3viral load in mice liver between the two groups.4. The proportions of hepatic CD3+CD8+CD69+T cells, CD3+CD8+PD-1+T cells and CD4+CD25+Foxp3+Treg were detected, and were compared between the two groups; the hepatic expressions of IL-1β, IL-6, IL-10, IL-17A, IL-22, IL-33, TNF-a and Fg12were compared by real-time PCR.[RESULTS]1. The C3H mouse model of NAFLD was established successfully. Feeding with HFD for12weeks, C3H/HeN mice became obese, greasy haired, sedentary and gained greater body weight than the control group. There was no significant increase in ALT or AST. TG, TC, HDL, LDL, Glucose, insulin levels and HOMA-IR in HFD group increased significantly; the livers in HFD mice were larger and weighed more than ND mice. A great number of lipid droplets deposited in the hepatocytes seen in the HE staining and Oil red O staining.2. The C3H mouse model of NAFLD combined with viral hepatitis was established successfully. MHV-3infected NAFLD mice died on day3to day12with a mortality of20%. The sick mice appeared apathetic, dull haired, erected and curled body, trembling, and teetering. The serum ALT and AST levels increased significantly in infected NAFLD mice. The liver became pallid with diffused pinpoint bleeding. In addition to the formation of lipid droplets in liver cells, ballooning degeneration, necrosis, lymphocyte infiltration could also be seen in HE staining. MHV-3viral replication could be detected by PCR.3. No significant difference in survival rate was observed between the infected HFD mice and infected ND mice. MHV-3replicated more actively in infected mice with significant difference on day4(P=0.002). The increase in ALT and AST was greater in infected NAFLD mice than that in the infected ND mice. The hepatic inflammation and necrosis were more serious in NAFLD mice.4. Expression of intrahepatic IL-1β, IL-6, IL-10, IL-17A, IL-33, TNF-a, Fg12increased after MHV-3infection, and the increase was more obvious in NAFLD mice; expression of intrahepatic IL-22reduced, and the reduce was more obvious in NAFLD mice.5. Intrahepatic CD4+CD25+Foxp3+Treg cells reduced in early MHV-3infection, and became more than that in ND mice on day16; the intrahepatic CD3+CD8+CD69+T and CD3+CD8+PD1+T cells increased in early MHV-3infection, and the increase was more obvious in NAFLD mice, and the proportion of cells kept higher than that in ND mice.[CONCLUSION]1. The C3H mose model of NAFLD was successfully established by feeding HFD for12weeks in this study. The C3H mose model of NAFLD combined with viral hepatitis was established by intraperitoneal infection with MHV-3on the basis of NAFLD mice.2. NAFLD can promote the MHV-3replication and aggravate hepatic inflammation.3. NAFLD can promote the release of cytokines and increase the hepatic proportion of CD3+CD8+CD69+T, decrease the proportion of CD4+CD25+Foxp3+Treg and lead to an aggravated liver inflammation and necrosis in early MHV-3infection. In the late infection,the increased proportion of CD4+CD25+Foxp3+Treg cells and CD3+CD8+PD1+T cells may hinder viral clearance. |
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