Nrf2对NASH肝脏纤维化的保护作用及机制研究

Non-alcoholic steatohepatitis (NASH) is a common chronic liver disease in China, and fibrosis is an important symbol of its progress, and oxidative stress is one of the important mechanisms of its development. Rencent studies have shown that Nrf2 is a key regulator of the cellular response to oxidative stress and can improve the inflammation of NASH. Therefore, it can be speculated that Nrf2 may has protective effects on liver fibrosis of NASH. However, there are not enough studies on the protective effects and mechanisms of Nrf2 on liver fibrosis of NASH. Our preliminary studies have proved that Nrf2 inhibits hepatic stellate cells (HSC) activation and extracellular matrix deposition induced by oxidative stress. In this project, the protective effects and partly mechanisms of Nrf2 on liver fibrosis of NASH will be studied. It will be dynamically observed that the associations of oxidative stress and inflammation, HSC activation and fibrosis degree in NASH rats, and the effects of Nrf2 on these indexes are also to be explored. The effects of Nrf2 on activation, proliferation, apoptosis and function of HSC induced by oxidative stress will be extensively studied in vitro. The molecular mechanisms of PI3K/AKT pathway involved Nrf2 regulation on HSC will be observed using different channel blockers. This project will heplful to clarify the protective effects and molecular mechanism of Nrf2 on NASH fibrosis,and is of great significance to identify new NASH control strategies.

非酒精性脂肪性肝炎（NASH）是我国常见慢性肝病，纤维化是其进展的重要标志，氧化应激是其重要发病机制。新近证实Nrf2是细胞抗氧化应激反应的中枢调节者，能够改善NASH炎症，推测有助于抑制纤维化进展。但是，Nrf2对NASH纤维化保护作用及机制如何，目前报道不多。我们前期研究表明Nrf2能够抑制氧化应激引起的肝星状细胞（HSC）活化及细胞外基质分泌。在此基础上，本课题研究Nrf2对NASH纤维化的保护作用及部分分子机制。动态观察NASH大鼠肝脏氧化应激与炎症、HSC活化及纤维化程度的相关性，探讨Nrf2对其影响；建立HSC氧化应激细胞模型，体外实验深入研究Nrf2对HSC活化、增殖、凋亡及功能的调控作用；采取阻断剂干扰方法，观察PI3K/AKT信号通路在Nrf2调节HSC中的分子机制。完成本课题将为阐明Nrf2对NASH纤维化的保护作用及分子机制，对确定新的NASH防治策略具有重要意义。

背景：Nrf2在非酒精性脂肪性肝炎（NASH）具有保护作用，可能有其抗氧化能力相关，但精确机制不明。.方法：通过慢病毒构建上调或下调Nrf2的稳转HSC-T6和BRL-3A细胞株，应用葡萄糖氧化酶构建氧化应激模型。MTT检测细胞存活率，流式细胞仪检测ROS，Elisa检测MDA、LDH和SOD水平。RT-PCR和蛋白印记检测Nrf、p66Shc、IQGAP1等表达情况。最后，应用Nrf2敲除转基因鼠，通过高脂饮食构建NASH模型，体内实验探讨Nrf2在氧化应激及抗肝纤维化中的作用及机制。.结果：RT-PCR和蛋白印记证实上调或下调Nrf2的HSC-T6和BRL-3A稳转细胞株的成功构建。氧化模型中，在两种细胞株上调Nrf2可显著降低MDA、LDH和ROS水平，同时提高细胞存活率和SOD水平，而下调Nrf2可得到相反的结果。上调Nrf2可下调p66Shc的表达增加IQGAP1的表达，而下调Nrf2则得到相反的结果。最后，高脂饮食Nrf2敲除转基因鼠模型显示，Nrf2敲除可以增加肝脏的炎症反应、上调MDA、并上调MDA、TNFα、IL6、Collagen I和Collagen III的水平，下调GSH水平，同时激活组织中NF-κB、上调p66Shc并下调IQGAP1。.结论：Nrf2可通过抗氧化应激、调控炎症和对抗纤维化发挥抗NASH的作用，可能和下调p66Shc和上调IQGAP1表达有关，为Nrf2在NASH中预防及作为治疗靶点提供了数据和研究基础。