Pathogenesis Of Hepatocytes Necrosis/apoptosis In MHV-3 Induced Fulminant Hepatitis And Correlative Genes Intervention

【BACKGROUND&OBJECTIVE】In developing countries and the Asia-Pacific region, HBV infection is seriously epidemic,the incidence rate of HBV infection is high as 9~10%。In china severe hepatitis caused by HBV infection become one of the common infectious diseases. The hallmark of this severe disease is the extreme rapidity of the necromicroinflammatory process, resulting in widespread or total hepatocellular necrosis in weeks or even days, and occurrence of hepatic encephalopathy. The mechanisms for virus-induced severe hepatitis are still not fully understood. Due to the lack of specific and effective clinical treatment, unless an emergency liver transplant, the majority of patients are with poor prognosis. Therefore investigating the mechanism of hepatocytes death in severe hepatitis, and developing gene therapeutic means with which we can block the pathological process of this servere disease has become a serious issue in this field.Fibroleukin/fibringogen-like protein 2(fgl2) or fgl2 prothrombinase which was found recently belongs to profibrinogen superfamily. fgl2 expressed in activated macrophage and vascular endothelial cell induces prothrobin to thrombin, and activates the coagulation process. Recently we and our colleges demonstrated that fgl2 expression was closely related to the development of severe hepatitis from studies with both severe hepatitis patients and animal models.Target gene silence can be applied to evaluate the role and importance of the target gene(s) in the pathogenesis of interested disease. This technology also provides us a novel means for clinical intervention of disease development. However efficient transformation of foreign gene(s) into liver remains a"bottleneck"and limits the application of this modern technique. Currently high levels of gene transfer to mouse liver could be achieved by tail vein hydrodynamic injection of plasmid DNA solution in a large volume. It has been proposed that the injected DNA solution accumulates mainly in the liver because of its flexible structure and blood flow cease induced by transient heart failure, which can accommodate large volume of solution, and the hydrostatic pressure forces plasimid DNA into the liver cells before it is mixed with blood. This technology has been extensively used in gene therapy for mouse studies.Hepatocytes apoptosis occurred in the liver plays an important role in the process of many liver diseases, especially hepatic failure caused by various reasons. Actually LPS toxicity is due to serious apoptosis that further induce liver injury and destruction in the FHF model induced by LPS/D-GalN. In the process of severe hepatitis, Fas and TNF system activated, Bcl-2 protein family imbalances and other factors will affect the severity of hepatocytes apoptosis. Previous studies have demonstrated that mfgl2 plays important role in the development of hepatocytes apoptosis. IFN-gamma and TNF-alpha induced hepatocytes apoptosis in the mice liver are dependent on the expression of mfgl2. However the contribution of hepatocytes apoptosis to the development of murine hepatitis virus strain 3 (MHV-3) induced hepatic failure in Balb/cJ mice has never been explored, which in turn might be one of the important aspects for understanding the pathogenesis of hepatocytes injury.Therefore the purposes of this study are as the follows:1. To establish a simple and reliable model of fulminant viral hepatitis in mice, for studying the pathogenesis and treatment of fulminant viral hepatitis.2. To construct a mfgl2 antisense plasmid which can inhibit the expression of mfgl2 in Raw264.7 cells, a mouse macrophage derived cell line.3. To investigate the effect of mfgl2 antisense plasmid on mfgl2 expression in vivo and the disease progress in MHV-3 induced fulminant hepatitis mice model.4. To investigate the expression of apoptosis-related proteins and their correlation with hepatocytes apoptosis in MHV-3 induced fulminant hepatitis mice model. 【METHODS】1. Fulminant viral hepatitis animal model was established by MHV-3 infection of Balb/cJ. HE staining was performed to observe the pathological change of live tissue for evaluation of HAI scores. The expression of mfgl2 and fibrin deposition was observed by immunohistochemistry staining. mfgl2 antisense plasmid and mfgl2 sense plasmid as a control were constructed. Raw 264.7 cells were transfected with mfgl2 antisense, intervention effect of mfgl2 antisense in vitro was detected by Real-time PCR, Western-blot. And functional PCA assay. Target gene was introduced into mice liver by hydrodynamic injections, and expression efficiency of target gene was detected; After hydrodynamic injection of mfgl2 antisense plasmid, the survival rate of mice, hepatic pathological change and serum biochemical disorder were examined and compared between mice with/without mfgl2 antisense plasmid intervention. The expression of mfgl2 was detected by Real-time PCR, immunohistochemistry staining, and in-situ hybridization.2. The TUNEL method was used to detect hepatocytes apoptosis in MHV-3 induced fulminant hepatitis and then AI (apoptotic index) was evaluated3. The expression of Fas and TNFR1 in hepatocytes was observed by immunohistochemistry staining. Western-blot assay was performed for measurement of Bax and Bcl-2 expression, and ratio of Bax/Bcl-2 was calculated.【RESULTS】1. Massive inflammatory cells infiltration and hepatocytes death were evident in the hepatic lobulbe in MHV-3 induced fulminant hepatitis in Balb/cJ mice. HAI scores gradually increased, with the highest score of 16.0±1.4 at 72 hours post MHV-3 infection. The level of ALT, AST, and TBil gradually increased, with the highest level of 8192.3±224.4 IU/L, 9410.3±954.4 IU/L and 11.6±0.75 umol/L at 72 hours post MHV-3 infection,while serum ALB gradually decreased, with the lowest level of 19.6±1.25 g/L at 72 hours post MHV-3 infection.2. The mfgl2 antisense plasmid and mfgl2 sense plasmid was successfully constructed as evidenced by the restriction enzyme mapping as shown and further confirmed by sequence analysis. Target gene was introduced into mice liver by hydrodynamic injections, and repeated injection can improve the efficiency of expression up to 40% 24 hours later. A dose dependent inhibitory effect of mfgl2 expression by mfgl2 antisense plasmid was observed in IFN-gamma treated Raw246.7 cells. By hydrodynamic delivery, mfgl2 anti-sense plasmid significantly reduced mfgl2 expression in vivo, markedly ameliorates inflammatory infiltration, fibrin deposition and hepatocytes death, prolonged the survival time period and elevated the survival rate from 0 up to 33.3% in Balb/cJ mice with MHV-3 induced fulminant hepatitis.3. Compared with the control group (A/J mice with MHV-3 infection), a large number of hepatocytes apoptosis were emerged, and AI increased gradually, with the highest level of 79.8±7.4% at 72 hours post MHV-3 infection of Balb/cJ mice. The upregulated expression of TNFR1, Fas and Bax and down regulated expression of Bcl-2 (an increasing ratio of Bax/Bcl-2) was observed in mice with MHV-3 infection.【CONCLUSION】1. In this study we have successfully re-established the fulminant viral hepatitis model induced by MHV-3 and demonstrated that hydrodynamic tail vein injections efficiently introduced target gene into mice liver, which provide fundamental techniques for further studies.2. Efficient and specific mfgl2 gene silence targeted by the constructed mfgl2 antisense plasmid sheds light on the future investigation of gene therapeutic strategies for patients with fulminant viral hepatitis and disease such as acute rejection of xeno- or allograft transplantation and SLE, which fgl2 gene has been shown to play a key role in the disease development.3. Hepatocytes apoptosis was also first evidenced in MHV-3 induced fulminant viral hepatitis model, thus the therapeutic targets should also cover the key genes involved in the process of hepatocytes apoptosis.