Mechanism Research Of IgG78 Regulates The Progress Of Liver Fibrosis By Targeting TEM1 Through GAS6/AXL Signal Pathway

Liver fibrosis is one of the major diseases threatening human life and health,especially in developing countries.Liver fibrosis is a pathophysiological process of liver connective tissue hyperplasia caused by various pathogenic factors such as alcohol abuse,obesity,and viruses.It is mainly manifested as the production of liver fibrous scars and collagen deposition.If it is not effectively inhibited,liver fibrosis will develop into liver cirrhosis,and may further develop into liver cancer.Therefore,it is urgent to explore the mechanism of the occurrence and development of liver fibrosis and to develop therapeutic strategies to inhibit liver fibrosis.Tumor endothelial marker1(TEM1)is a membrane protein that is specifically and highly expressed in fibrotic diseases,and its high expression in fibroblasts can promote the occurrence and development of fibrotic diseases.Growth arrest-specific gene 6(GAS6)plays an important role in the progression of liver fibrosis,but its expression regulation mechanism is unclear.In this study,we intend to explore the molecular mechanism how TEM1 regulates the expression of GAS6 and its role in the progression of liver fibrosis,and to examine the effect of TEM1-specific antibody on the biological characteristics of fibroblasts and in the treatment of liver fibrosis in a mouse model,and provide potential targets and strategies for the diagnosis and treatment of liver fibrosis.Methods:(1)By using tissue microarray that includes liver tissues from normal human,hepatitis,and liver cirrhosis patients,detect the expression of TEM1 by immunohistochemical staining,and analyze its correlation with the degree of liver disease.By using a liver fibrosis mouse model which was established by intraperitoneal injection of CCl4,examine the expression of TEM1 in liver tissues by qRT-PCR and Western blot,and examine whether TEM1 mainly expressed in hepatic stellate cells by double immunofluorescence staining.(2)Knockdown TEM1 expression in fibroblasts,evaluate whether TEM1 can regulate the expression of STAT6 and GAS6 at m RNA and protein levels by using qRT-PCR and Western blot analysis.Knockdown STAT6 expression in fibroblasts,evaluate its regulation on the expression of GAS6.Evaluate whether STAT6 binds to the promoter of GAS6 and induces its expression by using dual-luciferase reporter gene experiments.Overexpress or knockdown TEM1 expression in fibroblasts,evaluate its regulation on the expression of α-SMA,to clarify the function of TEM1 in liver fibrosis.Knockdown the expression of TEM1 or GAS6 in fibroblasts,evaluate its effect on cell migration through Transwell assay.(3)Treat fibroblasts by fully human antibody IgG78,examine cell proliferation by CCK8 staining,examine cell migration by Transwell assay,examine cell apoptosis and cell cycle by flow cytometry,and examine the expression of TEM1,GAS6,and fibrosis-related proteins α-SMA and COL1A1 by Western blot.(4)Treat mouse models of liver fibrosis with IgG78,examine the level of collagen fibers by Sirius red staining and Masson staining,examine the expression of STAT6,GAS6,α-SMA,and COL1A1 by immunohistochemical staining.In addition,examine the serum GAS6 level by ELISA,analyze the inhibitory effect of IgG78 on liver fibrosis and the possible molecular mechanism.Results:(1)The expression level of TEM1 in human and mouse liver fibrosis tissues was higher than that in normal liver tissues,and the positive expression of TEM1 was positively correlated with the severity of liver fibrosis,suggesting that TEM1 is an important marker of liver fibrosis.Results of double immunofluorescence staining showed that TEM1 was co-localized with GFAP,α-SMA,and vimentin,which suggested that TEM1 was mainly expressed in hepatic stellate cells.(2)When the expression of TEM1 was downregulated in fibroblasts,the expression of STAT6 and GAS6 was also downregulated.STAT6 could directly bind on the promoter region of GAS6 and promote its transcription.Downregulate the expression of STAT6 and downregulate GAS6 expression.Overexpress or knockdown the expression of TEM1 in fibroblasts,the expression of α-SMA was correspondingly upregulated or downregulated.Downregulation of the expression of TEM1 or GAS6 in fibroblasts can significantly inhibit cell migration.(3)Treat fibroblasts with IgG78 can inhibit cell proliferation and migration,induce cell apoptosis,block cell cycle in G1 phase,and inhibit the expression of TEM1,GAS6,α-SMA,and COL1A1.(4)After IgG78 treatment,the degree of liver fibrosis in mice was significantly reduced;immunohistochemical staining results showed that IgG78 treatment could effectively inhibit the expression of STAT6,GAS6,α-SMA,and COL1A1 in liver tissue.In addition,the serum level of GAS6 in mice treated with IgG78 also significantly decreased.Conclusion:The expression of TEM1 is gradually increased in the process of liver fibrosis,and TEM1 is mainly expressed in hepatic stellate cells,indicating that TEM1 is an important regulatory molecule in the occurrence and development of liver fibrosis.TEM1 can indirectly regulate the expression of GAS6 by regulating the expression of STAT6 in hepatic stellate cells,thereby activating the GAS6/AXL pathway,therefore promoting the proliferation,migration,and activation of fibroblasts.TEM1 antibody IgG78 can down-regulate the expression of TEM1 in fibroblasts,inhibit the proliferation and migration of fibroblasts,induce apoptosis,block cell cycle at G1 phase,and inhibit the expression of fibrosis-related proteins α-SMA and COL1A1.IgG78 could inhibit mouse liver fibrosis,inhibit the expression of STAT6,GAS6,α-SMA,and COL1A1 in liver tissue,and inhibit the level of GAS6 in serum.These results further confirm that TEM1 can be used as an effective therapeutic target for liver fibrosis,and TEM1 antibody IgG78 can be used as a potential drug candidate for the treatment of liver fibrosis.