Molecular Mechanism Of HSCs Activating TGF-β1/Smad Signaling Regulated By ICOS-Tg Mice Infected With Schistosoma Japonicum

Hepatic fibrosis is a chronic liver disease caused by a variety of causes,including viral infection,alcoholic liver disease,non-alcoholic steatohepatitis and Schistosoma japonicum infection.It is characterized by abundant secretion of pro-inflammatory and pro-fibrotic cytokines cause excessive deposition of extracellular matrix in the liver,which gradually leads to hepatic fibrosis.The main pathogenic mechanism of schistosomiasis hepatic fibrosis is the formation of egg granuloma and secondary hepatic fibrosis caused by eggs in liver and intestine.Hepatic stellate cells(HSCs)are the cytological basis of hepatic fibrosis and the main effector cells of hepatic fibrosis.Their activation and proliferation are the initial conditions for the formation of hepatic fibrosis.Activation and proliferation of HSCs play a significant role in the development of schistosomiasis hepatic fibrosis.Studies have shown that the activated process of HSCs involves a large number of cytokines and cell signal transduction pathways,of which TGF-β1/Smad signaling pathway is one of the most’ classical pathway in the activated process of HSCs.In recent years,there are more intensive studies have been carried out.However,the detailed mechanism of TGF-β1/Smad signaling pathway in hepatic fibrosis induced by Schistosoma japonicum remains to be further elucidated.Our previous studies had shown that ICOSL/ICOS signaling can regulate the activation of HSCs by TGF-β1/Smad signaling pathway and mediate the development of liver fibrosis at the mRNA level.In this study,ICOS-Tg transgenic mice(ICOS-Tg)and its wild-type FVB/NJ mice were used to build experimental animal models of schistosomiasis japonicum hepatic fibrosis.At the protein level,the effects of TGF-β1/Smad signaling pathway on the activation of HSCs in ICOS-Tg mice with schistosomiasis japonicum hepatic fibrosis were investigated,and the roles of various signaling molecules in this pathway were elucidated,and the regulation of HSCs activition was clarified.The key target protein molecule of HSCs can provide scientific basis for searching for immunologic checkpoints which can regulate the activation of HSCs and control the occurrence and development of liver fibrosis.Part Ⅰ.The primary HSCs were extracted,identified and cultured in mice infected with S.japonicumObjective:In order to prepare for the subsequent experiments,we conducted to extract,identify and cultivate the primary hepatic stellate cells from mice infected with S.japonicum.Methods:Firstly,ICOS-Tg mice and its wild-type FVB/NJ mice were used to build experimental animal models of schistosomiasis japonicum hepatic fibrosis.Then primary HSCs were extracted from four different stages of mice:control group(OW),early granuloma formation stage(4W),acute granuloma stage(7W)and early hepatic fibrosis formation stage(9W)by using liver situ enzyme perfusion and OptiPrep density gradient centrifugation.Finally,primary HSCs were counted with neubauer counting board and their survival rate was determined by trypan blue staining.The purity of primary HSCs was identified by two other methods,one is fluorescence of primary HSCs was observed under 328 nm ultraviolet excitation light;another is glial fibrillary acidic protein(GFAP)immunocytochemistry was performed on primary HSCs after 3 days of culture.Results:(1)The primary HSCs of mice grew well and deformed obviously in the course of culture.The number of primary HSCs freshly extracted was(2.45±0.3)×106 per mouse,and the survival rate was more than 95%.(2)By using inverted fluorescence microscopy,the primary HSCs of mice were observed under ultraviolet excitation at 328 nm,and blue-green fluorescence could be seen in the cytoplasm.(3)GFAP immunocytochemical staining showed red fluorescence in the cytoplasm with purity of over 90%.Conclusion:The survival rate and purity of primary HSCs from mice were over 90%.High quality cell specimens were obtained successfully.Part Ⅱ.The dynamic changes of pro-fibrotic molecules mRNA and protein expression in HSCs of ICOS-Tg mice infected with Schistosoma Japonicum during chronic pathogenic periodObjective:To investigate the activation of primary HSCs in ICOS-Tg mice and FVB/NJ mice and the dynamic changes of collagen secretion-related genes and protein expression in liver fibrosis during chronic fibrosis in host infected with Schistosoma japonicum.Methods:The primary HSCs were collected and cultured for 7 days in different infection stages.Total RNA and total protein in HSCs were extracted.The expression levels of a-SMA,Collagen type Ⅰ and Collagen type Ⅲ in primary HSCs of ICOS-Tg mice and its wild-type FVB/NJ mice were detected by fluorescence quantitative PCR and Western Blotting respectively.The dynamic changes of three target molecules in the chronic pathogenesis of schistosomiasis japonicum hepatic fibrosis were analyzed and compared.Results:With the prolongation of the course of infection,the expression levels of a-SMA,Collagen type I and Collagen type III in primary HSCs kept increasing.The level of mRNA expression began to increase in the early stage of granuloma formation(4W),and increased sharply in acute granuloma stage(7W)and early stage of hepatic fibrosis(9W).The expression of protein began to increase at the early stage of granuloma formation(4W),and increased slowly in acute granuloma stage(7W)and early stage of hepatic fibrosis(9W).Compared with FVB/NJ mice in the same period,the expression levels of a-SMA,Collagen type I and Collagen type III in primary HSCs of ICOS-Tg mice were significantly increased(P<0.05 or P<0.01 or P<0.001),and the change trend of transcriptional level of a-SMA,Collagen type I and Collagen type III was consistent with that of translational level of protein.Conclusion:During the chronic pathogenic period of schistosomiasis japonicum hepatic fibrosis,HSCs remained active.Compared with the wild type,ICOSL/ICOS signal was significantly up-regulated in ICOS-Tg mice infected with Schistosoma japonicum,which promoted the activation of HSCs and the production of collagen in liver.Among them,the level of mRNA and protein of a-SMA molecules were the most obviously up-regulation(P<0.05 or P<0.001),which suggested that by interfering ICOSL/ICOS signal may be an important immune checkpoint to regulate the occurrence and development of hepatic fibrosis.Part Ⅲ.Molecular Mechanisms of HSCs activating TGF-β1/Smad signaling pathway regulated by ICOSL/ICOS mediated liver fibrosisObjective:To investigate the effect of up-regulation of co-stimulatory signal ICOSL/ICOS on protein molecules in primary HSCs activated by TGF-β1/Smad signaling pathway in ICOS-Tg mice and its wild-type FVB/NJ mice infected with Schistosoma japonicum.Methods:The total proteins in primary HSCs were extracted by Beiber total protein extraction kit after 7 days of culturing.The expression levels of TGF-β1,pSmad2/3,Smad2/3,Smad4 and Smad7 proteins in primary HSCs of ICOS-Tg mice and its wild-type FVB/NJ mice were detected by Western Blotting.The dynamic changes of the expression levels of each protein in these two mice were analyzed and compared.Results:After being infected with Schistosoma japonicum,the expression level of mice of TGF-β1 protein in TGF-β1/Smad signaling pathway began to rise in the early stage of granuloma formation(4W).With the development of the disease,its expression increased gradually in acute granuloma stage(7W)and early stage of hepatic fibrosis(9W).The expression level of TGF-β1 protein in primary HSCs of ICOS-Tg mice was significantly higher than that of FVB/NJ mice in the same period<0.05 or P<0.001).The expression levels of pSmad2/3,Smad2/3 and Smad4 protein increased in the early stage of granuloma formation(4W),peaked at acute granuloma stage(7W)and decreased slightly at early stage of hepatic fibrosis(9W).The expression levels of pSmad2/3,Smad2/3 and Smad4 protein in primary HSCs of ICOS-Tg mice were significantly higher than those of control FVB/NJ mice at the same time(P<0.01 or P<0.001).In the course of disease progression,the expression level of Smad7 protein in primary HSCs increased only in the early stage of granuloma formation(4W),and then started to decrease in acute granuloma stage(7W)and early stage of hepatic fibrosis(9W).Compared with FVB/NJ mice in the same period,the expression level of Smad7 protein in primary HSCs of ICOS-Tg mice decreased significantly(P<0.01 or P<0.001).Conclusion:In the experimental model of schistosomiasis japonicum in ICOS-Tg mice,the protein expression of TGF-β1 and Smad2/3 in the TGF-β1/Smad signaling pathway was significantly increased,while the protein expression of Smad7 was significantly decreased,which suggested that the effect of ICOSL/ICOS signaling on the progress of hepatic fibrosis in mice may be mediated by regulating TGF-β1/Smad signaling to activate the promotion/suppression of fibrosis factors in HSCs.