Expression And Role Of Activin A And Its Signaling Protein In Acute Liver Injury Of Mouse Induced By Con A

Activin belongs to the transforming growth factor-beta (TGF-β) superfamily and hasextensive biological functions. In the nervus aspect, It can maintain neuron survival andneurite growth; In the endocrine regulation aspect, not only can promote the secretion offollicle-stimulating hormone (FSH), but prevent the release of adreno corticotropic hormone(ACTH); In addition, Activin has immune adjustment function, involving in the acute phasereaction, tissue damage and healing process. In liver, Activin can restrain DNA synthesis ofliver cells; induce liver cell apoptosis and promote hepatic stellate cell (HSC) to produceextracellular matrix. Actinvin A injected via mouse tail vein can induce acute liver injury.Activin mainly contains Activin A, Activin B, Activin AB and et al molecule forms, amongwhich the role of Activin A has a clearer research and close relation with liver diseases. Inthe process of hepatitis, liver cirrhosis, liver failure, the abnormal expression of Activin Acan be found.In order to discuss the expression and role of Activin A and its signaling protein inacute liver injury, this study chose Con A-induced acute liver injury of C57BL/6mousemodel as the experimental animal model to analyse the expressions of Activin A, Activintype IIA receptor (ActRIIA) and Activin receptor interacting protein (ARIP2) in acute liverinjury by the enzyme linked immunosorbent assay (ELISA), immunohistochemical stainingand reverse transcription PCR (RT-PCR) and explore the protection effect of ARIP2in liverinjury by injection of ARIP2gene overexpression and gene silence plasmid via tail vein.Preparation of Con A-induced acute liver injury of mouse modelIn this experiment, Con A(15mg/kg) was injected by one time via tail vein of C57BL/6mice. At1d,3d,5d,7d after the treatment, we killed the mice; collected the sera and livertissues to detect the related indexs. The results showed that the liver injury was most seriousat1d, recovered gradually at3d,5d to normal level at7d through observation of liver generalspecimens, detection of serum alanine transaminase (ALT) and aspertate aminotransferase(AST) levals, and pathological analysis of HE staining. The above data indicated that thisstudy prepared successfully the ConA-induced acute liver injury of mouse model.The expressions of Activin A, ActRIIA and ARIP2in Con A-induced acute liverinjury of mouse model Activin A mRNA expression remained at a higher level during the liver injury. At1d,the expression of Activin A significantly increased in liver injury slices byimmunohistochemical staining. The levels of Activin A in sera of acute liver injury of mousemodel increased significantly at3d and5d. The expressions of ActRIIA mRNA and proteinwere consistent with the expressions of Activin A mRNA and protein. RT-PCR andimmunohistochemical staining results revealed that ARIP2expression in liver injury repairprocess elevated, and was still at a high leval at5d,7d. The above data suggested that in ConA-induced acute liver injury process, Activin A-ActRIIA signaling pathway maybe mediateliver injury; by contrast, the raised expression of Activin A signaling negative regulatoryprotein ARIP2maybe avail the repair of liver injury.The effect of ARIP2gene overexpression on liver injuryIn order to determine the role of ARIP2in liver injury, The study adopted theestablished gene therapy method in the laboratory to further analyse the effect of ARIP2gene overexpression on liver injury. The study established pcDNA3-ARIP2, pcDNA3control groups, Con A+ARIP2experimental group and Con A+pcDNA3experimentalcontrol group. Overnight after the injection of plasmid via mouse tail vein, Con A wasinjected by the same method to induce mouse liver injury. At1d after Con A injection,ALT、AST level changes were detected; Liver tissue pathological injury degree wasobserved by HE staining. The results showed that at1d after Con A injection, the ALT andAST levels in sera of Con A+ARIP2group were significantly lower than the ConA+pcDNA3control group, and Statistical difference was significant (p<0.01). The ALT、AST levals in sera and pathological results between ARIP2and pcDNA3control groupsshowed no significant differences compared to normal liver tissues. There were large livernecrosis, serious liver cell cable damagement, liver cell dissolving disappearance in injuryarea of Con A+pcDNA3control group.Pathological injury degree of the Con A+ARIP2group was significantly lighter than Con A+pcDNA3group, and liver index was highercompared to the Con A+pcDNA3group. The above data suggested that ARIP2geneoverexpression could reduce liver injury. The mechanism might be that ARIP2overexpression made the effect of Activin A inhibiting liver cell proliferation and inducingliver cell apoptosis in liver been restrained. In addition, ARIP2overexpression might reducethe combination of Activin A and its receptor by promoting the endocytosis of ActRIIA toblock the pathogenic role of Activin A-ActRIIA signaling pathway in Con A-induced liverinjury. The effect of ARIP2gene silence on liver injuryTo further validate the protection effect of ARIP2on liver injury, The study asloadopted ARIP2gene silence method to detect the effect of ARIP2on the liver injury, usingthe methods before to set up experimental groups and establish animal model of gene silenceby injection of ARIP2shRNA expression plasmid via tail vein. At1d after Con A treatment,the serum ALT and AST level changes were detected. The results showed that the ALT andAST levels of Con A+ARIP2shRNA group were obviously higher than ConA+pGCsi-U6/Neo experimental control group, and Statistical difference wassignificant(p<0.05); Pathological results showed that in Con A+ARIP2shRNA group therewere larger liver necrosis and more necrosis areas compared to Con A+pGCsi-U6/Neo group.And liver index was significantly lower than the Con A+pGCsi-U6/Neo experimentalcontrol group. The above data indicated that ARIP2gene silence could aggravate liver injurydegree, whose mechanism might be that ARIP2shRNA made mouse own produced ARIP2reduce and blocked the negative regulatory effect of ARIP2on ActivinA signaling pathway,thus aggravated liver injury mediated by Activin A.