The Role Of ADAM9 In Alcoholic Liver Fibrosis And The Molecular Mechanism Of Regulation

Objective In this study,CRISPR/Cas9 technology was used to edit ADAM9 sequence,and aims to study the role of ADAM9 in alcoholic liver fibrosis and its molecular mechanism.Methods(1)ADAM9-sg RNA3 transfects rat hepatic stellate cells HSC-T6:The active ADAM9-sg RNA3 plasmid previously screened in our laboratory is transfected into rat liver stellate cells HSC-T6,after purine enzyme screening,the transfected cell DNA was extracted for PCR amplification,electrophoresis,gel recovery,and then sequenced to verify the effective sg RNA3 protein.(2)In vitro experiments: culture rat hepatic stellate cells HSC-T6,divided into four groups,normal HSC-T6 control group: no treatment;normal HSC-T6 + alcohol group: direct induction culture with alcohol;ADAM9-sg RNA3 + Alcohol group: Stable transfection of effective sg RNA3 was given alcohol-induced culture;JNK inhibitor + alcohol group: JNK inhibitor SP600125 and culture solution were mixed and added to the cells for 24 h,and then induced with cell culture.Expression of related factors by immunoblotting: disintegrin-metalloproteinase ADAM9,c-Jun amino terminal kinase(JNK)signaling pathway phosphorylation protein pc-Jun,smooth muscle actin α-SMA,proliferation Nuclear antigen PCNA,apoptosis-associated protein Caspase-3.(3)In vivo experiments: 220 healthy and clean C57 BL / 6J male mice were randomly divided into 4 groups: Group A: normal control group(10): fed with control Lieber-De Carli feed TP4030C;group B: normal saline + alcohol group(70): tail vein injection of normal saline;Group C: ADAM9-sg RNA3 + alcohol group(70): tail vein injection of effective ADAM9-sg RNA3 plasmid;group D: JNK inhibitor + alcohol group(70): tail vein injection of JNK inhibitor SP600125;Groups B,C,and D were fed with Lieber-De Carli alcohol feed TP4030 A for 4 weeks,and then combined with intraperitoneal injection of 5% CCl4 olive oil solution 2ml / kg,2 times / week,until the 8th week,an animal model of alcoholic liver fibrosis in mice was established and killed at 8 weeks.Remove eyeball to take blood,separate mouse serum and detect AST and ALT.After liver treatment,paraffin section hematoxylin-eosin staining(HE staining)was performed to detect liver injury in mice;Sirius red staining was used to detect mouse liver fibrosis;Hoechst33258 apoptosis staining Detection of apoptosis of liver cells;ADAM9,PCNA,vascular endothelial growth factor VEGF,apoptosis-associated protein Bax,stress protein HSP70,key metabolic enzyme cells Pigment P4502E1(CYP2E1),tumor necrosis factor TNF-α,α-SMA,p-c-Jun.Result DNA sequencing results showed that the gene sequences of sg RNA3 groups were significantly different from the normal gene sequences.western blot results showed that the expression of ADAM9 protein in HSC-T6 cells transfected with ADAM9-sg RNA3 plasmid was significantly lower than that in normal HSC-T6+alcohol group(P<0.01),and the difference was statistically significant.therefore,sg RNA3 was determined to be an effective guide RNA.In vitro experiments:1.Western blotting results: Compared with Normal + alcohol group,ADAM9-sg RNA3 +alcohol group and JNK inhibitor + alcohol group cells ADAM9,p-c-Jun,α-SMA,PCNA protein expression were significantly reduced(P<0.01),The protein expression of Caspase-3 increased significantly(P<0.05 or P<0.01).In vivo experiments: 1.Changes in serum transaminase: serum AST and ALT levels in mice in the saline +alcohol group,ADAM9-sg RNA3 + alcohol group,and JNK inhibitor + alcohol group were significantly higher than those in the normal group(P<0.01 or P<0.05)The AST and ALT levels in serum of ADAM9-sg RNA3 + alcohol group and JNK inhibitor +alcohol group were significantly lower than those in saline + alcohol group(P<0.01 or P<0.05).Compared with the ADAM9-sg RNA3 + alcohol group,the ALT level was significantly reduced(P<0.05).2.Hematoxylin-eosin staining: Compared with Normal group,mice in the salin + alcohol group,ADAM9-sg RNA3 + alcohol group,and JNK inhibitor + alcohol group had significant liver damage(P<0.01);compared with salin +alcohol Compared with ADAM9-sg RNA3 + alcohol group and JNK inhibitor +alcohol group,hepatocyte necrosis was significantly reduced(P<0.01).Compared with ADAM9-sg RNA3 + alcohol group,hepatocytes in JNK inhibitor + alcohol group were significantly reduced.The reduction of necrosis was more significant(P<0.05).3.Picric acid-Sirius red staining results: Compared with mice in the Normal group,mice in the other three groups had significant liver fibrosis(P<0.01 or P<0.05),compared with normal saline + alcoholic mice,ADAM9-sg RNA3 + alcoholic group and JNK inhibitor + alcoholic group mice had more significant reduction of liver fibrosis(P<0.01);compared with ADAM9-sg RNA3 + alcoholic group,JNK inhibitor +Hepatic fibrosis was alleviated more significantly in the alcohol group(P<0.05).4.Hoechst33528 staining results: Compared with mice in Normal group,mice in the saline + alcohol group,ADAM9-sg RNA3 + alcohol group,and JNK inhibitor +alcohol group all had significant hepatocyte apoptosis(P<0.01);Compared with the mice in the + alcohol group,the number of hepatocyte apoptosis in the ADAM9-sg RNA3 + alcohol group and the JNK inhibitor + alcohol group was reduced(P<0.05 or P<0.01);compared with the ADAM9-sg RNA3 + alcohol group,In comparison,the number of liver cell apoptosis in the JNK inhibitor + alcohol group was more significant(P<0.05).5.Western blot test results: Compared with normal saline+alcohol group,ADAM9-sg RNA3+ alcohol group and JNK inhibitor+alcohol group were injected intravenously into liver CYP2E1,Bax,TNF-α,ADAM9,α-SMA,pc-Jun The protein expression level of VEGF,PCNA,and HSP70 was significantly increased(P<0.05 or P<0.01),while the protein expression levels of VEGF,PCNA,and HSP70 were significantly increased(P<0.05 or P<0.01).Conclusion In mouse alcoholic fibrosis,ADAM9 promotes liver fibrosis,and ADAM9 promotes alcoholic liver fibrosis in mice by activating the JNK signaling pathway.