Effects And Mechanism Of SPINK1 On The Rat Hepatic Stellate Cells HSC-T6

Liver fibrosis,an abnormal proliferation of connective tissue in the liver,caused by a various of pathogenic factors,such as virus,alcohol,fat and other factors.Any liver injury has a pathophysiological process of liver fibrosis during the liver repairing and healing.Liver fibrosis is the intermediate pathological process in which chronic liver disease deteriorates into cirrhosis.It can also be restored to health by eliminating the pathogenic factors.The development of liver fibrosis and its potential molecular mechanisms are of great value in the treatment of liver diseases.Studies have shown that the development of liver fibrosis is the result of the combined action of various cells and cytokines,among which the most critical step is the activation of hepatic stellate cells（HSC）.Therefore,the removal or inactivation of activated HSCs from the liver may be an important means for the liver to recover from liver fibrosis.As an important regulator of protease activity,Serine protease inhibitor kazal-type 1（SPINK1）was first found in the pancreas.The spatial structure of SPINK1 is very similar to that of the epidermal growth factor（EGF）.Studies have shown that SPINK1 could bind to the epidermal growth factor receptor（EGFR）of EGF and regulate the proliferation of hepatocytes and hepatocellular carcinoma cells,and participate in the occurrence of inflammation,cell proliferation and the occurrence and development of tumors.In addition,SPINK1 was highly expressed in a variety of liver disease models.In the early study of our laboratory,Rat Genome 230 2.0 was used to detect the gene expression profile of CCl₄-induced rat liver fibrosis model.The results showed that the expression of SPINK1 was significantly up-regulated at 3w,6w and 9w in the model.However,the effect and mechanism of SPINK1 on liver fibrosis have not been reported yet.Therefore,this study intends to explore the effect and mechanism.of SPINK1 on HSC-T6,a key cell in the process of liver fibrosis.In this study,a CCl₄-induced liver fibrosis model was constructed in rats,and HE staining and liver function indexes were used for identification.Immunohistochemical examination showed that the expression levels ofα-SMA and SPINK1 were highly expressed compared with the control group.In addition,similar changes consistent with the above were observed in a cell model of HSC-T6 activation induced by TGF-β-1 in vitro,suggested that SPINK1 might be associated with HSC-T6 activation and liver fibrosis.To further explore the function of SPINK1 on the biological behavior in HSC-T6,this article used the method of gene addition/treatment with recombinant protein,upregulated the expression of both endogenous and exogenous SPINK1 in HSC-T6 cells,then MTT,flow cytometry method,EdU and cell scratch test of HSC-T6 cells was used for detecting the cell vitality,cell proliferation,cell cycle and the ability of migrating.Respectively,qRT-PCR and Western blot was used to detect the expression of related pathway genes/proteins and speculate the possible mechanism of SPINK1 in regulating HSC-T6 cells.The results showed that the activity,proliferation,cell cycle and migration of HSC-T6 cells were significantly improved after endogenous and exogenous SPINK1 levels were increased by gene addition and treatment with recombinant protein.Phosphorylation of proteins in the PI3K/AKT and JAK/STAT signaling pathways suggests that SPINK1 may regulate the proliferation of HSC-T6 through these two signaling pathways.In addition,bothα-SMA and COL1A1,markers of HSC-T6 cell activation,were highly expressed,and phosphorylation of SMAD3 in the TGF-βsignaling pathway confirmed that SPINK1 was associated with HSC-T6 cell activation and liver fibrosis.To further clarify the mechanism by which SPINK1 regulates the activation of HSC-T6 cells through the TGF-βsignaling pathway,the above conclusions were verified by the addition of the TGF-βsignaling pathway inhibitor SB431542.