基于线粒体自噬调控NLRP3炎症小体活化探讨真武汤治疗慢性肾小球肾炎的作用机制

Chronic glomerulonephritis (CGN) is a multiple and intractable disease which seriously threats human health. A classic prescription Zhen-wu-tang was confirmed efficacy on curing CGN both in clinic and experiment researches. The main pathological basis of CGN is podocyte lesion caused by immune inflammation. NLRP3 inflammasome activation may cause serious podocyte lesion. Meanwhile, it has been proved that mitophagy plays a critical role in NLRP3 inflammasome inactivation and podocyte protection. Further, our previous study found that Zhen-wu-tang could induce autophagy in kidney via PI3K/AKT pathways. Based on the above mentioned, we put forward a hypothesis that Zhen-wu-tang could induce mitophagy through suppressing PI3K/AKT/mTOR pathways, which would further inactivate NLRP3 inflammasome and reduce podocyte lesion, subsequently cure CGN. The present investigation is designed to confirm the relationship between NLRP3 inflammasome activation and podocyte lesion in CGN, analyze the regulation of mitophagy to NLRP3 inflammasome inactivation, and investigate the regulatory mechanism of Zhen-wu-tang on PI3K/AKT/mTOR pathways in CGN. All these experiments are conducted on CGN rat model and LPS-treated podocyte cell model by means of small interfering RNA, laser confocal microscopy, flow cytometry, RT-PCR. To sum up, the ultimate aim of this study is to explore the effect of Zhen-wu-tang on NLRP3 inflammasome inactivation with the modulatory function of mitophagy through PI3K/AKT/mTOR pathways, which will eventually reveal the mechanism of Zhen-wu-tang on podocyte lesion and provide a reference for the treatment of CGN.

慢性肾小球肾炎（CGN）为临床多发难治疾病，严重威胁人类健康，经方真武汤治疗CGN有确切疗效。免疫炎症导致的足细胞损伤是CGN的核心病理基础，NLRP3炎症小体活化是足细胞重要损伤机制。课题组发现真武汤可通过抑制PI3K/AKT通路促进肾脏自噬，基于线粒体自噬在炎症小体活化及足细胞功能中的关键调节作用，我们提出“真武汤可能通过PI3K/AKT/mTOR诱导线粒体自噬，进而抑制NLRP3炎症小体活化，减少足细胞损伤治疗CGN”。本课题拟采用基因沉默、激光共聚焦、流式细胞技术、荧光定量PCR等手段，从动物及细胞模型探讨NLRP3炎症小体在CGN中的作用，研究线粒体自噬对NLRP3炎症小体活化的调控作用，明确CGN的治疗新靶点。结合真武汤对PI3K/AKT/mTOR自噬通路的干预，探讨真武汤对CGN线粒体自噬及NLRP3炎症小体活化的影响，阐明其治疗CGN的机制，为慢性肾脏疾病治疗提供科学依据。

项目背景：慢性肾小球肾炎是由免疫炎症介导的难治性疾病，为导致终末期肾衰竭的主要原因，严重威胁着人类健康。因此探讨CGN发病机制，寻找安全有效的CGN治疗药物具有重要意义。真武汤作为《伤寒论》温阳利水法的代表方剂，临床和实验研究证实真武汤对CGN有确切疗效。NLRP3炎症小体为近年免疫炎症研究热点，与CGN发病关系密切。已有研究表明线粒体自噬可通过调节线粒体功能，抑制NLRP3炎症小体活化。本项目旨在探究线粒体自噬对NLRP3炎症小体在CGN中的调控作用，以揭示CGN的发病机制，并探讨真武汤治疗CGN的作用机理。.主要研究内容：①、真武汤对CGN模型大鼠的治疗作用；②、真武汤对NLRP3炎症小体及足细胞损伤的影响；③、真武汤对线粒体自噬NLRP3炎症小体的调节作用；④、真武汤对PI3K/AKT/mTOR信号通路的调控作用。.结果：真武汤对C-BSA诱导的CGN模型大鼠有良好的肾脏保护作用，其含药血清可改善足细胞损伤，抑制NLRP3炎症小体活化；线粒体自噬抑制足细胞 NLRP3 炎症小体活化，减少线粒体ROS积累，真武汤可提高线粒体自噬水平，并能改善线粒体功能，抑制线粒体ROS生成。真武汤抑制 CGN 大鼠及足细胞 NLRP3 炎症小体活化与其抑制 PI3K/AKT/mTOR 信号通路的有关。.科学意义：①、基于NLRP3炎症小体在免疫炎症的核心调控作用，且免疫炎症损伤在CGN发病中发挥关键作用，本项目深入探讨NLRP3炎症小体在CGN发病中的调节机制，为CGN的防治提供药物作用的新靶点。②、基于前期实验证实真武汤对CGN模型大鼠的抗炎作用，本研究从PI3K/AKT/mTOR介导线粒体自噬调控NLRP3炎症小体，深入探讨真武汤改善CGN的作用机制，为真武汤临床治疗CGN提供理论和实验依据。