| Liver fibrosis is a wound-healing process in response to chronic liver injury, which is characterized by an excessive deposition of extracellular matrix (ECM), especially type I and III collagen. Therefore, it is well accepted that activation of HSCs is a pivotal event in the progress of liver fibrosis. And inhibition of activated HSCs proliferation contributes to the reversal of liver fibrosis. Interferon regulatory factor 3 (IRF3), one member of the interferon regulatory factor (IRF) family, is an essential part of immune regulation. And recent research has revealed IRF3 play a crucial role in liver diseases, such as hepatic steatosis, liver inflammation and alcoholic liver injury. However, the understanding of the function of IRF3 in the regulation of hepatic fibrosis, especially on the activation and proliferation of HSC, has not been reported. So in this study, we focus on the abnormal expression of IRF3 and its related molecular mechanisms in HSC proliferation and fibrotic tissue liver tissue The main content is contained as follows:1. IRF3 expression in human fibrotic liver tissues.Human liver fibrotic tissue samples were obtained from patients undergoing partial liver resection at the Department of Surgery, the First Hospital of Anhui Medical University and Anhui Provincial Hospital. The degree of fibrosis was classified as normal liver and mild to moderate fibrosis (usually 2 cm beyond cancer tissue)according to the Liver Cancer Study Group of Japan. We use Immunohistochemistry, RT-qPCR and Western blot to measure the expression of IRF3 protein and mRNA. The results indicated that IRF3 mRNA and protein were significantly increased in human fibrotic liver tissue compared to the normal liver, and mainly was expressed in HSC. And we also use immunosuppressive double staining method to observe localization of IRF3 in liver. Double immunofluorescence results in human samples demonstrated that IRF3 (green) was highly expressed in a-SMA (red) positive cells in the fibrotic area, suggesting HSC may be one of main cell types which express IRF32. Effect of IRF3 on TGF-pl-induced LX-2 cells in vitroIn order to validate that alteration of IRF3 expression during HSC activation also occurs in vitro, we detected the expressions of IRF3 mRNA and protein in cultured human LX-2 cells. Here, we showed a significant increase in IRF3 expression as the increased concentrations of TGF-β1 (from 0 to 10ng/ml) for 24 h, indicating that TGF-β1 promoted IRF3 expression in a dose-dependent manner Moreover, the expression of IRF3 was analyzed at 0,6,12,24 and 48 h in TGF-β1-induced LX-2 cells. It was significantly increased with the time prolonging, the highest being at 48 h in TGF-β1-induced HSCs. Next, we investigated the effects of IRF3 on TGF-β1-induced LX-2 activation and ECM protein production. The cells transfection with IRF3-siRNA significantly decreased IRF3 expression, in comparison to the cells transfected with scrambled-siRNA. And western blot and Real time qPCR results show that inhibition of IRF3 significantly down-regulated a-SMA and Collal expression compared to scrambled siRNA transfected TGF-β1-treated LX-2 cells. Consistent with this result, we determined that overexpression of IRF3 by transfecting them with pcDNA3-IRF3. RT-qPCR and western blot analysis of IRF3 levels was significantly increased in LX-2 cells compared with control. Moreover, it was found that a-SMA and Collal expression were dramatically enhanced.3. IRF3 modulates cell proliferation and apoptosis of LX-2 cells via AKT pathwayIn order to investigate the effect of IRF3 on proliferation and apoptosis of LX-2 cells, we use MTT assay and FCS in this study. MTT assay found that cell proliferation was significantly suppressed in TGF-β1-induced LX-2 cells transfected with IRF3-siRNA compared with control at 24 h. And the flow cytometry analyses showed that the proportion of apoptotic cells induced by transfection of IRF3-siRNA was greater than that induced by transfection of the control siRNA. In addition, we found inhibition of IRF3 expression caused significant decrease of p-AKT level, suggesting that knockdown of IRF3 induced apoptosis in TGF-β1-induced LX-2 cells, at least in part, by regulating AKT signaling pathway... |
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