Targeting CIAPs Attenuates CCl₄-induced Liver Fibrosis By Increasing MMP9 Expression Derived From Neutrophils

BackgroundLiver fibrosis(liver fibrosis)is the common pathological basis of chronic liver disease caused by viral hepatitis,alcohol abuse,non-alcoholic fatty liver disease,cholestasis,autoimmune hepatitis,drugs,genetic and other factors,and is the early stage of the formation of cirrhosis and the necessary pathological stage.Current studies have shown that liver fibrosis is a reversible process,and early intervention in liver fibrosis can slow down,stop or reverse the fibrosis process,control the disease progression and improve the prognosis.In recent years,with the emergence of a large number of fibrosis related studies,we have made great progress in the understanding of the pathogenesis of liver fibrosis.Many attempts have been made in the experimental and clinical trials of new anti-fibrosis treatments,but the basic problems have not been solved,and effective treatment methods have not been found.Therefore,it is of great clinical significance to explore the molecular mechanism of liver fibrosis reversal and to develop effective anti-liver fibrosis drugs for the treatment of chronic liver disease.Inhibitor of apoptosis proteins(IAPs)are a class of anti-apoptotic proteins,which can block the activation of caspases and then exert an anti-apoptotic effect.Common IAPs include cellular inhibitor of apoptosis protein 1(cIAP1),cIAP2 and XIAP,which are all expressed in liver cells.Current studies have shown that IAPS molecule can be a potential therapeutic target for idiopathic pulmonary fibrosis.However,the role of IAPs in liver fibrosis/cirrhosis have not been studied.Objective and ValueThis study aims to investigate the role of IAPs in the occurrence and development of liver fibrosis/cirrhosis,verify the anti-fibrosis effect of targeting cIAPs in mouse liver fibrosis model,and explore the related molecular mechanism.The results of this study further clarify the molecular mechanism of liver fibrosis and the anti-fibrosis effect of targeting IAPs,suggesting that IAPs may be a potential target of liver fibrosis.MethodsIn this study,we firstly analyzed the gene expression profile of liver tissues of 83 patients with chronic hepatitis B by microarrays,found the correlation between the mRNA expression level of IAPs(cIAP1,cIAP2,XIAP)and the severity of liver fibrosis by Scheuer staging score.The protein levels of cIAP1 and cIAP2 in cirrhosis liver tissues were analyzed by immunohistochemical staining by using human liver tissue microarrays.The liver fibrosis model was established in C57BL/6J mice to analyze the mRNA and protein levels of IAPs molecules.Next,the anti-fibrosis effect was observed in the CCl4-induced mouse liver fibrosis model by means of hepatocellular specific gene knockout and small molecule drug targeting inhibition of IAPs molecule,and the molecular mechanism of fibrosis formation and reduction was further explored.Finally,matrix metalloprotein 9(MMP9)inhibiting,neutrophils immune depletion,and C-C Motif Chemokine Ligand 5(CCL5)gene knockout were used for further verification.ReslultsMicroarray analysis revealed that the mRNA expression of cIAP1 and cIAP2 in the liver tissues of patients with chronic hepatitis B was positively correlated with the Scheuer-S score of liver fibrosis stage,while XIAP was not.The protein levels of cIAP 1 and cIAP2 in cirrhosis liver tissues were higher than those in normal liver tissues.We established a mouse liver fibrosis model,and the expression of cIAP2 was significantly up-regulated in the liver tissues of mouse liver fibrosis.Next,in the CCl4induced mouse liver fibrosis model,the hepatocyte specific knockdown of cIAP2 or the targeted inhibition of cIAPs by the small molecule drug APG-1387 effectively reduced collagen fiber formation and improved liver fibrosis,which was manifested by reduced collagen fiber staining and collagen area in the liver.Further mechanism research found that targeting cIAPs inhibiting intrahepatic cIAPs molecules,not directly promoted apoptosis of hepatic stellate cells,but induced hepatocytes to secrete chemokines CCL5,recruiting neutrophils infiltration in liver,upregulated the intrahepatic MMP9 expression,reversed MMP9/TIMP1 imbalances,led to degradation of extracellular matrix,inhibition of hepatic stellate cell activation,and decreased collagen fiber formation,result in attenuating liver fibrosis.Moreover,in mouse models,the anti-fibrosis effect of APG-1387 can be blocked by inhibition of MMP9 enzyme activity by CTT peptide,neutrophils immune deleption by anti-LY6G,and CCL5 gene knockout.ConclusionsThese data suggest that cIAPs play a novel role in the pathogenesis of liver fibrosis,and targeting cIAPs possesses the potential to be a promising treatment for liver fibrosis by increasing MMP9 derived from CCL5 chemotactic neutrophils.