从内质网应激放大TLR4炎症信号通路研究竹节参总皂苷抑制非酒精性脂肪性肝炎的分子机制

In recent years the increasing prevalence of Non-alcoholic steatohepatitis (NASH) has caused serious harm to human health in China. There is still a lack of effective intervention strategy for the NASH. The research showed that the TLR4 signaling activation to release lots of inflammatory factors and the disorder of lipid metabolism induced endoplasmic reticulum stress (ERS) are acknowledged as having an important role in initiating NASH. ERS sensitizes TLR4 signal pathway to increase production of inflammatory cytokines is the newly discovered key factor for the inflammatory diseases. Our previous study found that total saponins of Panax japonicus effectively prevented NASH process through inhibition of the inflammatory reaction. The pre-experiment results showed that Panax japonicus saponins regulated the ERS signal molecular GRP78 and CHOP, and inhibited TNF-α and IL-1β cytokine expression. These experimental results suggest that total saponins of Panax japonicus may prevent the inflammation of NASH through the intervention of ERS signaling molecules to TLR4 signal pathway. Based on the hypothesis, the effect of total saponins of Panax japonicus on the prevention of NASH will be studied in the high fat induced NASH model. Using recombinant virus, immunofluorescence, and siRNA techniques, the molecular mechanism of total saponins of Panax japonicus will be investigated on the regulation of ERS signaling molecules which amplifies TLR4 inflammatory signaling. This research will provide new ideas and new targets for the prevention and treatment of NASH.

非酒精性脂肪性肝炎 (NASH) 发病率逐年上升，严重危害人类健康，目前仍缺乏有效干预措施。有研究显示，TLR4活化释放大量炎性因子和代谢紊乱诱导的内质网应激 (ERS) 反应是启动NASH的重要因素。新近研究发现，ERS通过放大TLR4信号通路增加炎性因子释放是炎症性疾病的关键环节。我们前期研究发现，竹节参总皂苷通过抑制炎性因子表达能有效干预NASH进程；预实验结果显示，竹节参总皂苷可调控ERS信号分子GRP78和CHOP，抑制TNF-α和IL-1β的表达，提示竹节参总皂苷可能通过干预ERS信号分子，进而阻断ERS对TLR4信号的放大效应，干预NASH的肝脏炎症反应。基于此，我们应用高脂诱导NASH模型，采用重组病毒、免疫荧光、siRNA等技术，研究竹节参总皂苷干预NASH炎症反应的分子机制是否与ERS信号分子介导的TLR4炎症信号放大效应密切相关，为NASH的防治提供新思路和新靶点。

随着现代生活方式及饮食结构的改变，非酒精性脂肪性肝炎&nbsp;（non-alcoholic steatohepatitis, NASH）的发病率逐年上升，严重危害人类健康，目前仍缺乏有效干预措施，而内质网应激（ERS）通过放大toll样受体4（TLR4）信号通路增加炎性因子释放是炎症性疾病的关键环节。因此本项目进行如下研究：一）在细胞水平建立肝细胞脂毒性炎症模型：游离饱和脂肪酸棕榈酸（PA）和/或内质网应激激动剂（TG）可诱导内质网应激放大TLR4信号介导的炎症细胞模型。p-IRE1α蛋白、TLR4蛋白、炎性因子IL-6的分泌量显著升高，NF-κB蛋白进入细胞核；竹节参皂苷Ⅳa可改善这些炎症反应。这些结果提示ERS信号分子可以通过放大TLR4信号通路增加炎性因子的释放，而竹节参皂苷Ⅳa可减少ERS信号分子介导的TLR4炎症信号放大效应。二）在动物水平建立了短期诱导NASH动物模型（高脂+DSS），为药物筛选及NASH的分子机制研究提供了实验基础。三）竹节参总皂苷改善NASH的发生发展。1）应用高脂饮食诱导小鼠NASH模型，结果显示竹节参总皂苷干预后可明显改善模型组小鼠体质量、肝质量指数及血清ALT、肝组织TG水平，肝组织病理学变化明显改善，脂质代谢相关基因和炎症相关基因表达水平显著下降。2）另外我们发现竹节参总皂苷可以通过调节miRNA，包括miR-34a （miR-181 a）靶向PPARα， miR199 -5p靶向HGF/c-Met，及 miＲ-17-5p靶向MFN2等信号通路改善NASH中脂质代谢和炎症反应，为NASH的药物干预分子靶点提供新思路。 3）应用高脂饮食联合腹腔注射CCl4建立脂肪性肝纤维化模型，竹节参皂苷IVa干预后，明显改善小鼠肝脏的脂质沉积、炎性浸润、纤维化。另外免疫荧光染色发现CD36和Nlrp3的蛋白表达主要共定位于肝窦内皮细胞。因此我们建立体外细胞培养内皮细胞，应用PA诱导内皮细胞炎症模型，结果显示竹节参皂苷IVa干预后，CD36，Nlrp3的表达水平明显降低（p<0.05）。提示高脂诱导NASH的肝纤维化小鼠模型中，代谢性炎症反应相关基因CD36和Nlrp3表达升高，并且主要定位于肝窦内皮细胞，而竹节参皂苷IVa可通过CD36-NLRP3信号通路降低脂质沉积与改善肝脏炎症，为防治NASH的药物筛选提供新思路和新靶点。