Study On The Mechanism Of IFT20 In The Activation Of Hepatic Stellate Cells And The Development Of Hepatic Fibrosis

Research Background and Purpose:The occurrence and development of liver fibrosis are very complicated,which is a common pathological change of various chronic liver injuries and is also a necessary stage for most liver diseases to develop into cirrhosis.There are a large number of patients with chronic liver injury in China.Failure to intervene in time can lead to decompensated liver cirrhosis,and its end-stage complications can seriously affect the life safety of patients.The activation of hepatic stellate cells(HSC)is the central link in the occurrence and development of hepatic fibrosis.Cilia exist on the surface of eukaryotic cells.Recent studies have suggested that cilia loss is related to liver fibrosis,and intraflagellar transport(IFT)is the key to cilia formation and maintenance of normal function.It can transport various proteins and transmit related signal molecules,thus maintaining the stability of the internal and external environment of cells.The normal operation of intraflagellar transport depends on the cooperation of various intraflagellar transporters.IFT20 belongs to IFT-B complex and is expressed in large quantities in liver.At present,there is no study on the mechanism of IFT20 in the occurrence and development of liver fibrosis.Therefore,this study aims to investigate whether IFT20 participates in the activation of hepatic stellate cells and the occurrence and development of hepatic fibrosis.Method Carbon tetrachloride(CCL4)method was used to establish a mouse liver fibrosis model,and HE staining,Masson staining and Sirius Red staining methods were used to detect the modeling effect.Real-time PCR,Western blot,immunohistochemistry(IHC)and immunofluorescence(IF)were used to detect the expression and distribution of IFT20 in liver tissues of normal and liver fibrotic mouse models.Immunohistochemistry(IHC)and immunofluorescence(IF)were used to detect the expression and distribution of IFT20 in liver tissues of normal and fibrotic patients.Transforming growth factor TGF-β1 was used to stimulate human hepatic stellate cell LX-2 and mouse hepatic stellate cell HSC-T6.The expression changes of liver fibrosis related indexes such as α-SMA and IFT20 were detected by real-time PCR and Western blot.LX-2 was transfected with si RNA and IFT20 overexpression plasmid to construct IFT20 low expression and overexpression cell lines.real-time PCR and Western blot were used to detect the effect of IFT20 expression changes on HSCs activation.The CCK-8 cell proliferation experiment was used to detect whether the change of IFT20 expression level affected LX-2 cell proliferation ability,and the cell scratch experiment and Transwell cell experiment were used to detect whether the change of IFT20 expression level affected LX-2 migration ability.Real-time PCR and Western blot were used to detect whether the expression change of IFT20 was related to hedgehog signal pathway after LX-2 was further activated.hedgehog pathway inhibitor(cyclopamine)was used to treat LX-2.real-time PCR and Western blot were used to detect the expression change of IFT20.ResultIFT20 is expressed in hepatic stellate cells and mainly exists in hepatocytes.Compared with normal liver tissues,the expression of IFT20 in liver fibrosis patients and carbon tetrachloride-induced liver fibrosis mouse models is increased,and in liver fibrosis mouse models,the expression of IFT20 is proportional to liver fibrosis to some extent(p<0.05).After LX-2 and HSC-T6 were stimulated by TGF-β1,the expression of TGF-β1 and IFT20 increased significantly(p<0.05).After inhibiting the expression of IFT20 in LX-2 by si RNA,the expression of liver fibrosis related indexes is down-regulated,the expression of molecules downstream of hedgehog signaling pathway is down-regulated,and the proliferation and migration ability of LX-2 cell line are correspondingly weakened.On the contrary,overexpression of IFT20 plays the opposite role.After LX-2 was treated with hedgehog signaling pathway inhibitor,liver fibrosis related indexes and IFT20 expression were downregulated(P<0.05).ConclusionIFT20 promotes the activation of HSCs and the occurrence and development of liver fibrosis through hedgehog pathway,which provides a new idea for reversing the occurrence and development of liver fibrosis.