NF-κB/miR-29b/HMGB1通路调控肝星状细胞自噬影响肝纤维化的机制研究

Activation and transdifferentiation of hepatic stellate cells (HSCs) caused by a variety of signaling transduction pathways triggered by inflammatory factors and cytokines are a key initiating event in the process of hepatic fibrosis. A variety of microRNAs (miRNAs) are involved in the regulation of signaling pathways in liver tissues, which regulate the expression of target genes after transcription or translation process, and influence the outcome of hepatic fibrosis. In our previous studies, we found hepatic cell apoptosis that are regulated by NF-κB/miR-503/bcl-2 signaling pathway in hepatic fibrosis, and Nrf2/Keap1/ARE signaling pathway regulates HSCs proliferation and activation by autophagy. Based on the results of previous studies, we suppose that the anti-fibrosis signaling molecule miR-29b which depends on the NF-κB signaling pathway may regulate the HMGB1 gene,and induce HSC autophagy and activation, which involved in the regulation of hepatic fibrosis. In this study, we will perform the experiments in vitro and in vivo.which will used to verify the mechanism of NF-κB/miR-29b/HMGB1 signaling pathway regulate HSC autophagy and induce hepatic fibrosis by cell transfection, miRNAs microarrays, RT-PCR and Western blotting. It will provide a new theoretical and experimental basis for further understand of molecular mechanisms of signaling pathway, miRNA, HSC autophagy and hepatic fibrosis in prevention and treatment of hepatic fibrosis.

细胞和炎症因子启动信号通路激活肝星状细胞（HSC)，是肝纤维化形成的关键环节。而肝组织中多种微小RNA（miRs)参与了信号通路的调控，并在转录后或翻译水平调控靶基因表达，影响肝纤维化的转归。在我们既往研究发现NF-κB/miR-503/bcl-2通路对肝细胞凋亡的调控作用，以及Nrf2/Keap1/ARE通路在自噬调控肝星状细胞增殖活化中作用的基础上，结合国内外有关研究成果，提出依赖NF-κB信号通路的抗纤维化信号分子miR-29b，可能通过调控HMGB1，诱导HSC自噬、活化，并参与肝纤维化的假说。本研究拟行体外、体内实验，采用细胞转染、miRNAs基因芯片、RT-PCR和免疫印迹等技术，验证NF-κB/miR-29b/HMGB1信号通路调控HSC自噬，影响肝纤维化的机制。为人们进一步认识信号通路、miRNA、HSC自噬和肝纤维化的分子关联机制及肝纤维化的防治提供新的理论和实验基础。

肝纤维化是肝损伤的常见结果，可发展为肝硬化甚至肝癌。肝星状细胞(HSC)的激活是肝纤维化的主要驱动因素，其中包括酒精性肝纤维化(ALF)。此外，前期研究发现细胞自噬有助于肝星状细胞的激活。本研究重点探讨长链非编码RNA XIST通过miR-29b/HMGB1调控肝星状细胞自噬和活化中的作用及机制。我们用100 mmol/L乙醇处理人HSC细胞(LX-2)，促使HSC活化。接着通过测定细胞活力和纤维化标志物α-平滑肌肌动蛋白(α-SMA)和1型胶原α1 (CoL1A1)的蛋白水平来评估HSC的活化。同时通过检测自噬标志物Beclin-1和LC3-II来评估自噬。使用荧光素酶报告法、qRT-PCR和western blot分析XIST、miR-29b和高迁移率族蛋白B1 (HMGB1)之间的相互调控作用。体内实验方面，以sh-XIST为靶点的慢病毒(LV-sh-XIST)通过尾静脉注射到ALF模型小鼠体内，以阐明XIST在ALF损伤中的体内作用。实验结果证实XIST在乙醇活化的LX-2细胞中表达上调。此外，XIST作为miR-29b的竞争性内源性RNA，促进HMGB1的表达，从而增强乙醇诱导的肝星状细胞自噬和激活。进一步的体内实验表明，LV-sh-XIST下调XIST可减轻ALF模型小鼠的ALF损伤。总之，XIST通过miR-29b/HMGB1轴增强乙醇诱导的肝星状细胞自噬和激活。以上研究结果为抗肝纤维化的靶点药物研发提供了部分实验依据。