Molecular Mechanism Of HSCs Activation Related Signaling Pathway Regulated By ICOSL/ICOS Mediated Liver Fibrosis In Mice Infected With Schistosoma Japonicum

Schistosoma japonicum infection caused by cumulative liver granuloma inflammation and subsequently fibrosis is the central part of the disease,and hepatic stellate cells are major effector cells of hepatic fibrosis.Activation and proliferation of HSCs play an important role in the development of liver fibrosis.Studies have shown that the activation of HSCs is very complex and involves multiple cytokines and signal transduction pathways.Transforming growth factor β1 /Smad pathway,mitogen-activated proteinkinase(MAPK)pathway,phosphatidyl inositol-3-kinase(PI-3K)pathway,nuclear factor the NF-kappa B pathway,Janus kinase/signal transduction and transcriptional activation(JAK/STAT)pathway are associated with HSCs proliferation and activation of signal transduction pathways.These signaling pathways have been studied wide in recent years.However,the mechanism of these signal transduction pathways in the process of hepatic fibrosis induced by S.japonicum remains to be further elucidated.Our previous studies have shown that,the increase of ICOS signal ultimately promoted the formation of liver egg granuloma and the activation of HSCs.In this study,we established ICOSL-KO and wild-type C57BL/6J mice as experimental animal models for schistosomiasis.To explore the role of co-stimulatory signal ICOSL/ICOS in the activation of HSCs related signaling pathway in mice infected with S.japonicum.The pathogenesis of HSCs can be further elucidized to provide a new way to regulate the development of liver fibrosis.Part one.The primary HSCs isolated,identified and cultured in mice infected with S.japonicumObjective: Isolate and identify mice infected with S.japonicum and cultivate primary hepatic stellate cells,to prepare for the subsequent experiments.Methods: By taking situ liver perfusion and density gradient centrifugation,we separated ICOSL-KO mice and C57BL/6J 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 intracytoplasm specific expression of glial fibrillary acidic protein(GFAP)were taken into consideration to identify the purity.Results: The mice primary HSCs isolated freshly,it reached the discovery rate of(1.81 ± 0.2)× 106 cells / mouse,the survival rate of 90%.To observe fresh isolated primary HSCs under inverted fluorescence microscope at a wavelength of 328 nm UV excitation,it can spontaneously blue-green fluorescence.Primary HSCs by GFAP immunocytochemistry resulted in red fluorescence in cytoplasm and the purity of 90%.During the primary culture of HSCs,the growth and status of the cells were good.Conclusion: Viability and purity of primary HSCs isolated and cultured from the mice infected with S.japonicum meet the requirements of subsequent experiments.Part two.The dynamic expression of pro-fibrotic molecules in HSCs from mice infected with S.japonicum and detection of molecular genes related to activation and proliferation signal pathwaysObjective: To investigate the activation of primary HSCs in the chronic pathogenesis of C57BL/6J mice infected S.japonicum and the dynamic expression of their genes involved in the activation and proliferation signal pathways.Methods: By applying Trizol Reagent,we extracted total RNA of 7 days primary HSCs.And real-time quantitative PCR(Real time fluorescence quantitative PCR,Real-time PCR)was used to detect α-SMA,Collagen-I,Collagen-III,TGF-β1 and their activation-related pathway molecules gene expression level in primary HSCs of C57BL/6J mice.Meanwhile,compared the dynamic changes of each gene during schistosomiasis.Results: With the progress of the disease,α-SMA,Collagen-Ⅰ,Collagen-Ⅲ,TGF-β1,Smad4 gene expression levels gradually increase in primary HSCs.Smad2,Smad3,ERK1,ERK2,PI-3K,AKT,JNK,NF-κB,JAK1,STAT3,STAT5 gene expression levels began to rise from 4 weeks after infection,peaked at 7 weeks,decreased at 9 weeks in primary HSCs.In the process of schistosomiasis,the expression level of Smad7 in the primary HSCs was briefly elevated at 4 weeks after infection andthen decreased.But STAT1 gene kept continuous declination after infection.Conclusion: In the chronic pathogenesis of S.japonicum,HSCs have been activated.The TGF-β1/Smad signaling pathway plays a key role in liver fibrosis on the mice infected with S.japonicum.The related molecular genes of ERK,JNK,PI-3K/AKT,NF-κB,and JAK/STAT signaling pathways were positively correlated with the progression of hepatic fibrosis caused by S.japonicum,but STAT1 were negatively correlated.The results of this paper show that the related genes in ERK,PI-3K /AKT and JAK/STAT signaling pathway have a significant change in liver fibrosis progression and also play an important role.It may be a new way to regulate the occurrence and development of hepatic fibrosis through intervention of the above-mentioned related signal pathways.Part three.Effect of ICOSL/ICOS signaling on the expression of pro-fibrotic molecules genes and genes related to activation and proliferation signal pathways in HSCs from mice infected with S.japonicumObjective: To investigate the effect of down-regulation of co-stimulatory signal ICOSL/ICOS on the activation of primary HSCs in ICOSL-KO mice and C57BL/6J mice infected with S.japonicum,as well as the genes involved in their activation and proliferation signal pathways.Methods: By applying Trizol Reagent,we extracted total RNA of 7 days primary HSCs.And real-time quantitative PCR(Real time fluorescence quantitative PCR,Real-time PCR)was used to detect α-SMA,Collagen-I,Collagen-III,TGF-β1 and their activation-related pathway molecules gene expression level in primary HSCs of ICOSL-KO mice and C57BL/6J mice infected with S.japonicum.Meanwhile,the dynamic changes were analyzed and compared.Results: 1.The mice infected with S.japonicum,primary HSCs of α-SMA,Collagen-Ⅰ,Collagen-Ⅲ gene expression gradually increased;primary HSCs of ICOSL-KO mice,α-SMA,Collagen-Ⅰ,Collagen-Ⅲ gene expression was significantly lower than that in the same period of C57BL/6J mice.2.The expression levels of TGF-β1 and Smad4 in the TGF-β1/Smad signaling pathway were gradually increased;primary HSCs of ICOSL-KO mice,TGF-β1,Smad4 gene expression was significantly lower than that in the same period of C57BL/6J mice.Smad2,Smad3 gene expression began to rise from 4 weeks after infection,peaked at 7weeks after infection,decreased at 9 weeks after infection;primary HSCs of ICOSL-KO mice,Smad2,Smad3 gene expression was significantly lower than that in the same period of C57BL/6J mice.In the process of schistosomiasis,the expression level of Smad7 was briefly elevated at 4 weeks after infection and then decreased;primary HSCs of ICOSL-KO mice,Smad7 gene expression was significantly higher than that in the same period of C57BL/6J mice.3.MAPK pathway is another intracellular signal transduction pathway in HSC,MAPK protein family including extracellular signal-regulated kinases(ERK),c-Jun N-terminal kinase(JNK)and so on.Primary HSCs of ERK1,ERK2,JNK gene expression began to rise from 4 weeks after infection,peaked at 7 weeks after infection,decreased at 9 weeks after infection,but remains at a high level;primary HSCs of ICOSL-KO mice,ERK1,ERK2,JNK gene expression was significantly lower than that in the same period of C57BL/6J mice.4.The C57BL/6J mice infected with S.japonicum,primary HSCs of PI-3K and AKT gene expression began to rise from 4 weeks after infection,peaked at 7 weeks after infection,decreased at 9 weeks after infection,however primary HSCs of AKT gene expression was gradually increased after infection;primary HSCs of ICOSL-KO mice,PI-3K,AKT gene expression was significantly lower than that in the same period of C57BL/6J mice.5.The expression levels of NF-κB began to rise from 4 weeks after infection,peaked at 7 weeks after infection,decreased at 9 weeks after infection,but remains at a high level on the C57BL/6J mice infected with S.japonicum.In ICOSL-KO mice,the expression level was slightly increased after infection,but the overall trend was not obvious;primary HSCs of ICOSL-KO mice,NF-κB gene expression was significantly lower than that in the same period of C57BL/6J mice.6.Primary HSCs of JAK1,STAT3,STAT5 gene expression began to rise from 4weeks after infection,peaked at 7 weeks after infection,decreased at 9 weeks after infection,but remains at a high level,however STAT5 gene expression in ICOSL-KO mice has been increasing and higher than C57BL/6J mice at 9 weeks after infection.The expression levels of STAT1 was gradually decreased after infection,primary HSCs of ICOSL-KO mice,STAT1 gene expression was significantly lower than C57BL/6J mice,only higher than C57BL/6J mice at 9 weeks after infection.Conclusion: In ICOSL-KO mice infected with S.japonicum,liver fibrosis wasalleviated to some extent.The down-regulation of the co-stimulatory signal ICOSL/ICOS can reduce activation of HSCs,it can promote pro-fibrotic gene expression and inhibit anti-fibrotic gene expression in the related signal pathways of HSCs activation.The results of this paper show,the expression of related genes in PI-3K/AKT and JAK/STAT signaling pathway was more significantly down-regulated,which may be a potential regulatory target for anti-fibrosis.