基于宿主导向治疗（HDTs）研究穿心莲内酯对结核菌感染致炎症损伤的保护作用及机制

At present, the global tuberculosis control is facing the dilemma that a shortage of new drugs. Undoubtedly, antibiotics are the best choice for the effective clearance of the intracellular Mycobacterium tuberculosis. However, try this way more than 60 years cannot be truly effective control in Tuberculosis, so it is necessary to rethink a new strategy to solve this problem. This study based on the host-directed therapies (HDTs), focusing on the inflammatory injury induced by Mycobacterium tuberculosis infection, to explore the regulatory effect and the mechanism of andrographolide in Mycobacterium tuberculosis infected macrophage and further evaluate its effect on the whole animal level. To clarify the anti-inflammatory effects of andrographolide in Tuberculosis, the levels of inflammatory factors in vitro and in vivo after andrographolide intervention were analyzed. Furthermore, RNA interference, Western blot, Co-IP, fluorescence staining and immunohistochemistry were used to determine the correlation between the anti-inflammatory effect of andrographolide with the NLRP3 inflammasome, autophagy and TLR/NF-kB signaling pathway and define the exact target of andrographolide. Looking forward that the combination of andrographolide with clinical anti-TB drugs exerts synergistic therapeutic effect which keep an eye on both the bacteria and the host from the perspective of anti-bacterial and reduce the damage of inflammation. The discovery of such drugs will make a useful attempt to decrease the inflammatory damage, cut down the risk of drug resistance and death.

目前全球结核病控制面临缺乏新药的困境。对于结核菌这一胞内寄生菌的有效清除而言，抗生素无疑是最佳选择，但按此方式尝试60余年仍不能真正全面控制结核病，故需重新思考解决这一难题的新策略。本研究以宿主抗结核菌的免疫反应为导向(HDTs)，以结核菌感染诱发的炎症损伤为视角，探索穿心莲内酯在结核菌感染巨噬细胞水平的调控作用和机制，并在动物整体水平评价其药效。通过检测药物干预后体外、体内炎症因子的变化明确其在结核感染中的抗炎作用，进而通过RNA干扰、Western blot、Co-IP、荧光染色、免疫组化等手段明确药物抗炎作用的发挥与NLRP3炎性体、自噬、TLR/NF-kB等通路的相关性及作用靶标。期待其与临床抗结核药物联用，从抗菌及降低炎症损伤的角度针对细菌和机体两方面协同治疗。此类药物的发现将对减轻结核病机体炎症损伤，降低耐药性和死亡的发生几率做出有益尝试。

结核病HDTs治疗策略之一是降低过度炎症反应造成的组织损伤，为此本项目从自噬和NLRP3炎症小体两方面探讨穿心莲内酯调控结核菌感染诱导的炎症反应和机制。研究发现穿心莲内酯发挥抗炎作用的具体分子机制是通过调控NLRP3炎症小体活化和NF-κB信号通路来实现的。穿心莲内酯能够抑制NLRP3炎症小体活化，从而抑制过度炎症反应，包括降低结核菌感染诱导的caspase-1活化及促炎细胞因子IL-1β、IL-6和TNF-α的产生。穿心莲内酯不仅能降低LDH释放，还能抑制细胞死亡并阻止GSDMD-N的产生，显著抑制结核菌感染导致的细胞焦亡。穿心莲内酯抑制焦亡的机制除与NF-κB信号通路相关外，还与其抑制miR-155-5p的表达进而激活miR-155-5p靶基因Nrf2，促进下游抗氧化基因TRX2表达，从而抑制TNIXP与NLRP3蛋白的结合，抑制NLRP3炎症小体活化，最终发挥抑制细胞焦亡抗炎的作用。同时穿心莲内酯还具有良好的促自噬作用，增强的细胞自噬对胞内结核菌具有杀伤作用，穿心莲内酯诱导自噬的分子机制与抑制PI3K/Akt/mTOR 信号通路相关。增强的自噬也能通过降解NLRP3炎症小体复合物进一步抑制炎症。本项目的完成明确了穿心莲内酯具有结核病HDTs辅助治疗候选药物的潜在价值，为穿心莲内酯的临床新应用提供了实验依据。