The Molecular Mechanism Of HSCs Activation And Liver Fibrosis Mediated By ICOSL/ICOS Signaling Pathway In The ICOS-Tg Mice Infected With Schistosoma Japonicum

The main Pathogenesis of schistosomiasis is caused by schistosome eggs primarily in the liver and intestinal organs, which lead to hepatic fibrosis.Although the pathogenesis of schistosomiasis is not fully understood, some studies indicate the possible involvement of schistosomiasis effector cells and cytokines. A lot of studies have shown that the activated hepatic stellate cells are major effector cells of hepatic fibrosis.Schistosomiasis secreted a variety of cytokines in inflammatory processes,TGF-β1 is now recognized as one of the key pro-fibrotic cytokines.Smad protein are only TGF-β1 receptor messenger molecules,mediated TGF-β1 signals from cell membrane to cell nucleus,contributing to fibrosis gene expression, leading to extracellular matrix deposition in the liver and forming liver fibrosis.Recent studies have reportedmicro RNAs target one or more m RNA of TGF-β1/Smad signaling pathway, upregulation or inhibiting the activation and proliferation of HSCs, thereby affecting the liver fibrosis.Our previous studies have shown that, ICOSL / ICOS signal significantly increased the immune effects in ICOS-Tg miceinfected Schistosoma japonicum.ICOSL / ICOS signal not only mediate Th2 cell polarization, shifting the immune response to Th2, but also mediate Th17 polarization. The increase of ICOS signal ultimately promoted the formation of liver egg granuloma and liver fibrosis. In this study, ICOS-Tg miceand FVB / NJ mice were used as schistosomiasis experimental animal model to investigate how the co-stimulatory ICOSL / ICOS affect the HSCs activation and liver fibrosis signaling pathway, in order to find a new target to regulate the activation of HSCs and liver fibrosis, provide a new immunotherapy for the treatment of liver fibrosis. This study is divided into three parts: 1. The primary HSCs isolated,identified and cultured in mice infected with Schistosoma japonicumObjective:Isolate and identifymice infected with schistosoma japonicum and cultivate primary hepatic stellate cells, prepare the ground for subsequent experiments.Methods: Takingsitu liver perfusion and density gradient centrifugation, we separated ICOS-Tg miceand FVB / NJ mice primary HSCs of before infection(0W) and the early stages of infection(4W), acute infection of the lesion(7W, 9W). Trypan blue staining was applied to identify the survival rate of primary HSCs. Primary HSCs spontaneous blue-green fluorescence and its specific expression of glial fibrillary acidic protein(GFAP) were taken into consideration to identify the purity.Results: The freshly isolated mice primary HSCs reached the discovery rate of(2.34 ± 0.05) × 106 cells / mouse, the survival rate of 91%, and the purity of 90%. To observe primary HSCs under inverted fluorescence microscope at a wavelength of 328 nm UV excitation, freshly isolated HSCs canspontaneously radiate blue-green fluorescence.Primary HSCs by GFAP immunocytochemistry resulted in red fluorescence in cytoplasm.Conclusion:Viability and purity of primary HSCs isolatee and cultured from the mice infected with schistosoma japonicummeet the requirements of subsequent experiments.2. Effect of ICOSL/ICOS signaling on activation of HSCs and liver fibrosis in ICOS-Tg mice infected with Schistosoma japonicumObjective: To investigate how the regulation of co-stimulatory signal ICOSL / ICOS affect the activation of primary HSCs and the process of liver fibrosison ICOS-Tg miceand FVB / NJ miceinfected with Schistosoma japonicum.Methods:By applying Trizol Reagent,we extractedtotal RNA of 7 days primary HSCs. And real-time quantitative PCR(Real time fluorescence quantitative PCR,Real-time PCR) was used to detect primary HSCs α-SMA, Collagen-Ⅰ, Collagen-Ⅲ gene expression levels. Meanwhile, their dynamic changes were observed and compared.Results: With the course of lesion migration, primary HSCs of α-SMA, Collagen-1. The primary HSCs isolated,identified and cultured in mice Ⅰ, Collagen-Ⅲ gene expression levels gradually increase(1.403 ~ 5.207,1.051 ~ 5.347,1.313 ~ 3.614, gene / β-actin). primary HSCs of ICOS-Tg mice, α-SMA, Collagen-Ⅰ, Collagen-Ⅲ gene expression level(1.760 ~ 5.753,1.258 ~ 6.262,1.488 ~ 4.470, gene / β-actin) was significantly higher than the same period of FVB / NJ mice.Conclusion: The costimulation ICOSL / ICOS can promote the activation of primary HSCs and the development of liver fibrosis.3. Effect of ICOSL/ICOS signaling on the gene expression of TGF-β1/Smad signal molecular of liver fibrosis in ICOS-Tg mice infected with Schistosoma japonicumObjective: To investigate how the regulation of the co-stimulatory signal ICOSL / ICOS affect the signal pathway which lead tohepatic fibrosis on ICOS-Tg mice and FVB / NJ mice infected with Schistosoma japonicum.Methods:By applying Trizol Reagent,we extractedtotal RNA of 7 days primary HSCs, and by using Real-time PCR,we detected primary HSCs TGF-β1, Smad2, Smad3, Smad4, Smad7 gene expression level, we observed their dynamic changes. RNAiso for Small RNA was applied to extract total Small RNA of cultured 7 days primary HSCs, Real-time PCR was used to detect primary HSCs micro RNA-21, micro RNA-454 gene expression level. At the same time, the dynamic changes were analyzed and compared.Results: The mice infected with Schistosoma japonicum, primary HSCs of TGF-β1, Smad2, Smad3, mi R-21 gene expression began to rise from 4 weeks after infection,reached a relatively high level seven weeks after infection, decrease 9 weeks after infection, but remains at a high level; primary HSCs of ICOS-Tg mice, TGF-β1, Smad2, Smad3, mi R-21 gene expression was significantly higher than that in the same period of FVB / NJ mice. Furthermore, primary HSCs of Smad4 gene expression gradually increases; primary HSCs of ICOS-Tg mice, Smad4 gene expression was significantly higher than that in the same period of FVB / NJ mice. However, primary HSCs of mi R-454 and Smad7 gene expression level is gradually reducing, Smad7 gene expression level transient increase at 4 weeks after infection; primary HSCs of ICOS-Tg mice,Smad7 and mi R-454 gene expression level was significantly lower than the same period of FVB / NJ mice.Conclusion: The costimulation ICOSL / ICOS plays an important role in liver fibrosis on the mice infected with Schistosoma japonicum. The Costimulation ICOSL / ICOS promotes pro-fibrotic factor expression and inhibit anti-fibrotic factor expression, accelerating the progression of liver fibrosis.The results of this study show that the costimulation ICOSL / ICOS plays an important role in the activation of HSCs and the process of liver fibrosis.Upregulation of costimulatory signal ICOSL / ICOScan contribute to promote liver fibrosis cytokine expression and inhibit the expression of suppression fibrosis factor, thereby promoting the activation of HSCs, accelerateing the development of liver fibrosis.