The Role Of TOPK In The Activation Of Hepatic Stellate Cells And Liver Fibrosis During The Murine Schistosomiasis

Objectives: Liver fibrosis is the major pathology in Schistosoma japonicum infection due to the granuloma caused by eggs deposition in the liver,hepatic stellate cells(HSCs)have been identified as the major cellular contributors to the development of granulomas and fibrosis.T-LAK cell-originated protein kinase(TOPK)is a recently identified serine-threonine protein kinase and a member of the MAPKK(MAPK kinase)family that has an essential role in a variety of biological processes,such as the development and malignant transformation of tumor,cell cycle,cell proliferation and apoptosis.However,the role and mechanism of TOPK in S.japonicum infection and the activation of HSCs have not been reported.In this study,we investigated the role of TOPK in the activation of HSCs and liver fibrosis during the murine schistosomiasis.Methods: Anesthetized eight-week-old female BALB/c mice were percutaneouslly infected with 25±1 cercariae of S.japonicum.After four weeks of infection,mice were treated with HI-TOPK-032(TOPK inhibitor)for two weeks by tail intravenous injection at an interval of three days.Massion staining was used to determine the liver pathology,the expression of ?-SMA and the accumulation of Collagen ?1 were measured by Western blot.Moreover,primary hepatic stellate cells were isolated to detect the expression of TOPK.Human hepatic stellate cell line LX-2 was cultured in vitro and was treated with indicated concentration of HI-TOPK-032 for 12,24,36 and 48 hours,the cell viability was detected by CCK8 measurement.LX-2 cells were treated with 2 ?M HI-TOPK-032 for 36 h and followed by flow cytometry to detect the proliferation and apoptosis of the cells in vitro.Real-Time PCR and immunofluorescence staining were used to measure the expression of ?-SMA and Collagen ?1 in LX-2 cells.The expression of ?-SMA,Collagen ?1,ERK1/2,JNK1/2 and p38 MAPK and the phosphorylation of ERK1/2,JNK1/2 and p38 MAPK were detected by Western blot.Results: The expression of TOPK increased in liver tissue from mice infected with S.japonicum for six weeks analysised with Western blot,immunofluorescence staining showed that the fluorescence intensity of TOPK was enhanced in mice infected S.japonicum.The isolated primary hepatic stellate cells from mice infected for six weeks also expressed higher TOPK.Masson staining showed that the collagen deposition and the degree of liver fibrosis were significantly reduced,the expression of ?-SMA and Collagen ?1 were also decreased after TOPK inhibition.The cell viability of LX-2 cells was remarkably suppressed in vitro when TOPK was inhibited,and the inhibiting effect is dose-and time-dependent.Flow cytometry showed that the proliferation of cells was suppressed,the ratio of cells in the S phase decreased,and the proportion of apoptofic cells increased.In addition,the inhibition of TOPK also resulted in lower expression of ?-SMA and Collagen ?1 at both transcription and protein levels of LX-2 cells,and the fluorescence intensity of ?-SMA and Collagen ?1 also weakened.The expression of ERK1/2,JNK1/2 and p38 MAPK in Lx-2 cells was not shown to be different after TOPK inhibition,but the phosphorylation was remarkablely decreased.Conclusion: S.japonicum infection induced higher expression of TOPK kinase in liver tissue,especially in the activated HSCs.When TOPK was functional suppressed,the pathology of schistosomiasis in liver was markedly alleviated due to the reduced activation and proliferation of HSCs,while more cell apoptosis was induced.The expression of ?-SMA and Col1a1 in liver tissues and hepatic stellate cells was also reduced after TOPK inhibiton.The role of TOPK in promoting the activation,proliferation and differentiation of HSCs may be achieved through intracellular MAPK signaling pathway which was significantly characterised by the increased phosphorylation of ERK1/2,JNK1/2 and p38 MAPK molecules.In conclusion,our study proved that TOPK protein kinases are involved in the activation of HSCs caused by schistosome infection,and the TOPK can be considered as a potential target for the therapy or improvement of hepatic fibrosis in liver diseases.