| Part I Construction and identification of liver fibrosis of mice with Schistosoma JaponicaObjective: To construct mice models with Schistosoma Japonica, reliable and resemble to human liver fibrosis,and to observe the progress of liver fibrosis, and to provide for further investigation on pathogenesy and treatment of liver fibrosis of Schistosomiasis Japonica. Methods: Mice were infected with Schistosoma Japonicum cercariae percutaneously, and liver biopsies were done at different time point after infection(2 wk,4 wk,6 wk,8 wk,12 wk,16 wk,20 wk,24 wk). Liver lesions were evaluated using HE staining and transmission electron microscopy. The dynamic changes of liver collagen location and content as well as liver fibrosis degree at different time point after infection were evaluated by Van Gieson staining.Results: All Mice infected with Schistosoma Japonica were confirmed by HE and VG staining, and all animals had been constructed for models of hepatic fibrosis. Granulomae were found in liver at 6 weeks after infection, and achieved the peak of the quantity and size at 8 weeks after infection, and fewer and smaller because of transforming to tubercle-like node from then on. During 20 to 24 weeks after infection, the typical argilla-pipe hepatic fibrosis was observed. A small quantity of liver collagen was detected in and around granulomae. From then on, collagen content and degree of liver fibrosis increased or aggravated progressively with course of diseases.Conclusion: Hepatic fibrosis models of mice with Schistosoma Japonica can show reliably the progress of human hepatic fibrosis of Schistosomiasis Japonica, which lays the foundation for further study on pathogenesy and treatment of liver fibrosis of Schistosomiasis Japonica.PartⅡThe experimental studies on dynamic changes of OB-Rb mRNA and protein expression and their significance in liver fibrosis of mice with Schistosoma JaponicaObjective: To observe the dynamic changes in the long form of leptin receptor(OB-Rb) mRNA and protein expression in mice liver by Schistosoma Japonica infection at different period, and to investigate the roles of leptin and its receptor in liver fibrosis of mice with Schistosoma Japonicum.Methods: Mice infected with Schistosoma Japonica cercariae percutaneously were served as animal models of liver fibrosis, and liver biopsies were done at different time point after infection. Liver lesions were evaluated using HE and VG staining. Immunohistochemistry for OB-Rb andα-smooth muscle actin(α-SMA) as an activated hepatic stellate cell(HSC) marker was performed to observe their dynamic changes by SP technique. OB-Rb mRNA dynamic expression was measured by RT-PCR.Results:Theα-SMA and OB-Rb positive cells increased gradually with course of diseases. Co-localization of OB-Rb andα-SMA in activated HSCs was in the localization of collagen fibers. OB-Rb mRNA expression in model group began at the 4th week after infection and increased progressively and persisted at the 24th week after infection. There was a positive correlation betweenα-SMA protein expression and collagen content(r=0.956, P<0.01) or degree of liver fibrosis(r=0.804, P<0.01). Similarly, there was a positive correlation between OB-Rb protein or mRNA expression and collagen content(r=0.965, P<0.01; r=0.945, P<0.01) or degree of liver fibrosis(r=0.823, P<0.01; r=0.880, P<0.01).Conclusion: The close relationships exist between leptin, HSC and liver fibrosis by Schistosomiasis Japonica. HSC is a key cell and leptin is a novel profibrogenic cytokine in the development of liver fibrosis by Schistosomiasis Japonica.PartⅢThe experimental studies on the molecular mechanisms of leptin in liver fibrosis of mice with Schistosoma JaponicaObjective: To observe the dynamic changes in leptin and phosphatidylinositol 3-kinase(PI3K) protein expression as well asα1(I) procollagen mRNA expression in mice liver by Schistosoma Japonica infection at different period, and to investigate the roles of PI3K pathway in liver fibrosis of mice with Schistosomiasis Japonica by leptin. Methods: Mice infected with Schistosoma Japonica cercariae percutaneously were served as animal models of liver fibrosis, and liver biopsies were done at different time point after infection. Liver lesions were evaluated using HE and VG staining. Immunohistochemistry for leptin and PI3K was performed to observe their dynamic changes by SP technique. PI3K protein dynamic expression was detected by Western Blot, andα1(I) procollagen mRNA dynamic expression was measured by RT-PCR.Results:The leptin and PI3K positive cells increased gradually with course of diseases. Co-localization of leptin and PI3K in activated HSCs was in the localization of collagen fibers. PI3K protein expression began at the 4th week after infection and increased progressively and persisted at the 24th week after infection. There was a positive correlation between leptin protein expression and collagen content(r=0.758, P<0.01) orα1(I) procollagen mRNA(r=0.832, P<0.01) or degree of liver fibrosis(r=0.823, P<0.01). Similarly, there was a positive correlation between PI3K protein and leptin(r=0.882, P<0.01) or collagen content(r=0.889, P<0.01) orα1(I) procollagen mRNA(r=0.708, P<0.01) or degree of liver fibrosis(r=0.807, P<0.01). There was a positive correlation betweenα1(I) procollagen mRNA and collagen content(r=0.824, P<0.01) or degree of liver fibrosis(r=0.612, P<0.01).Conclusion: Leptin promotedα1(I) procollagen mRNA expression and protein production by stimulating PI3K phosphorylation in the progress of liver fibrogenesis of mice with Schistosomiasis Japonica, and PI3K pathway has an important effect in the process.PartⅣThe experimental studies on therapeutic effect of vitamin E and its mechanisms on liver fibrosis of mice with Schistosoma JaponicaObjective: To observe the effect of vitamin E(VitE) onα-SMA, leptin and PI3K protein expression as well asα1(I) procollagen mRNA expression, and to investigate the therapeutic roles of VitE and its mechanisms in liver fibrosis of mice with Schistosoma Japonica. Methods: Mice were infected with Schistosoma Japonicum cercariae percutaneously. That liver fibrosis reached gradeⅡor above was considered to be a marker of therapeutic start.Mice were randomly divided into five groups: normal control group, model group, and intervention groups which were treated with three different doses of VitE, including 150 mg/kg, 50 mg/kg, and 5 mg/kg. The mice were killed at the end of the eighth week. Liver lesions were evaluated using HE and VG staining as well as transmission electron microscopy. The malondialdehyde(MDA) content and superoxide dismutase(SOD) activity in liver tissue were determined by spectrophotometric method. Immunohistochemistry forα-SMA as an HSC marker and leptin was performed by SP technique. PI3K protein expression was detected by Western Blot, and theα1(I) procollagen mRNA expression was measured by RT-PCR.Results:VitE reduced MDA content(P<0.01) and increased SOD activity(P<0.01) in the liver in model group in a dose-dependent manner. Besides, VitE decreased the number ofα-SMA(P<0.01) and leptin(P<0.01) positive cells in a dose-dependent manner. Further, VitE diminished increased PI3K protein expression(P<0.01), collagen content(P<0.01) and inhibited increasedα1(I) procollagen mRNA expression(P<0.01) in the liver in model group in a dose-dependent manner.Conclusion: VitE has evident therapeutic effects on liver fibrosis produced in mice by Schistosoma Japonicum infection, and the mechanisms are associated with VitE opposing lipid peroxidation, inhibiting HSC activation and proliferation, inhibiting leptin protein expression, blocking PI3K pathway, and reducingα1(I) procollagen mRNA expression and collagen production. |
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