Intervention In The Notch Signaling Pathway Treats Hepatic Fibrosis In Rats

Background Hepatic fibrosis is a reversible wound-healing response characterized by the accumulation of extracellular matrix (ECM) in response to acute or chronic liver injury. The activation and proliferation of hepatic stellate cells (HSCs) has been identified as a critical event in the development of hepatic fibrosis. Activated HSCs are highly contractile and express a-smooth muscle actin (a-SMA) and ECM. They are a key target for anti-fibrotic therapies. The Notch signaling pathway is a highly conserved signal transduction mechanism. It is essential to normal embryonic development, cellular proliferation, specification, and differentiation. Notch receptors and ligands have been identified in mammals. Notch signaling is activated through an interaction of a Notch receptor with a ligand expressed on adjacent cells leading to proteolytic cleavages of Notch receptor. domain (NICD). The NICD then moves to the nucleus and Mastermind to activate transcription of downstream target genes. Increasing numbers of studies have reported that Notch signaling is involved in human fibrotic diseases. However, the role of Notch signaling in liver fibrosis has not been fully investigated. Epithelial-mesenchymal transition (EMT) is defined as a process whereby epithelial cells gradually lose their epithelial signatures while acquiring the characteristics of mesenchymal cells. Numerous reports have indicated a role for EMT in fibrosis and TGF-β1 is considered the most powerful inducer of EMT. The Notch signaling pathway was also found to contribute to EMT. In particular, recent advances have confirmed that myofibroblasts can be supplemented from cholangiocytes and hepatocytes by EMT during hepatic fibrosis. we hypothesize that Notch signaling might be involved in liver fibrogenesis.Objective To investigate whether Notch signaling is activated in Hepatic stellate cells (HSCs), and the effect of regulating Notch signaling on the activation of HSCs. rAAV2/1-Jaggedl-shRNA is established to inhibit Notch signaling in vivo for elucidating the effect of rAAV2/1-Jaggedl-shRNA on experimental hepatic fibrosis.Blocking Notch signaling by DAPT in vivo futher demonstrates that Notch signaling is related to hepatic fibrosis.Methods The expression of Notch signaling components in HSC-T6 cells treated or not with TGF-β1 was detected by Taqman Probe. The expression of myofibroblastic markerα-SMA and collagen I in HSC-T6 cells transfected with pcDNA3.1-Jagged lor Jaggedl siRNA were detected by western blot, respectively.Establish rAAV2/1-Jaggedl-shRNA and inject it in animal model of hepatic fibrosis. Transfection efficiency of RAAVwas observed by laser scanning confocal micro scope. The therapeutic effect was evaluated by expression of related protein of Notch signaling, ECM and EMT conmpared with the control group by Immunofluorescence,Immunohistochemistry and Western-blot.The DAPT was intraperit-oneally injected into model rat suffering from hepatic fibrosis.The same method was used to research the ralation beteen hepatic fibrosis and Notch signaling.Results Activated Notch signaling was observed in HSC-T6 and TGF-β1 treatment enhanced the remarkable expression of Notch signaling protein in HSC-T6.Over-expression of Jaggedl by plasmid transfection increased the expression ofα-SMA and collagen I in HSC-T6, while transient knockdown of Jaggedl by siRNA decreased the expression of these myofibroblastic marker. Moreover, the knockdown of Jaggedl antagonized activation of HSC-T6 induced by TGF-β1. The difference was statistically significant (P< 0.05). Hepatic fibrosis were effectively reversed by means of restraining EMT by rAAV2/1-Jagged1-shRNA injected in animal model of hepatic fibrosis. Compared with the control group, expression ofα-SMA,TGF-β1 and snail in treatment group was remarkably decreased. The difference was statistically significant (P<0.05). The DAPT had the similar therapeutic effect by its intraperitoneal injection in rat liver fibrosis model.Conclusions we hypothesize that Notch signaling might be involved in liver fibrogenesis. These results suggest that selective interruption of Notch signaling might be a novel anti-fibrotic strategy in hepatic fibrosis.