基于AMPK/SIRT1/NF-κB信号通路对翼首草治疗“黑真布病”的作用机制研究

“Black Zhenbu disease” is a characteristic and dominant disease in Tibetan medicine, similar to rheumatoid arthritis (RA). “Black Zhenbu disease” is treated by cold-cool herbs “Clear away Chiba heat” in Tibetan medicine. Pterocephali herba is a commonly used for the treatment of “Black Zhenbu disease”, and has the characteristics of bitter and cold. Early studied showed that Pterocephali herba has anti-RA effect by regulating the expression of NF-κB in synovial membranes and improving energy metabolism disorders (NSFC81274193). And energy metabolism is closely related to the AMPK/SIRT1 pathway. According to the pathological features of joint inflammation and energy metabolism disorder during the pathogenesis of “Black Zhenbu disease”, a hypothesis is put forward: “Black Zhenbu disease” is treated Pterocephali herba, which inhibits inflammatory response and regulates energy metabolism by regulating AMPK/SIRT1/NF-κB signaling pathway. In the study, CIA rat with heat pattern model and TNF-α induced synovial fibroblasts are adopted, AMPK agonist AICAR and SIRT1 inhibitor NAM as a control. Using molecular biology techniques such as Elisa, Western Blot, and RT-PCR. From the perspective of energy regulation and inflammatory response, the mechanism of AMPK/SIRT1/NF-κB signaling pathway in the treatment of “Black Zhenbu disease” is revealed, and reveal the scientific connotation of cold-cool herbs “Clear away Chiba heat”.

“黑真布病”为藏医药治疗的特色优势病种，类似于类风湿性关节炎。藏医治疗“黑真布病”以寒凉药物“清赤巴热”为治则。翼首草，味苦、性寒，为治疗“黑真布病”的常用藏药，前期发现其具有调控关节滑膜NF-κB表达、改善能量代谢紊乱的作用（NSFC81274193），而能量代谢又与AMPK/SIRT1密切相关。本课题针对“黑真布病”发病过程中关节炎症反应、能量代谢紊乱等病理特征，提出“翼首草可介导AMPK/SIRT1/NF-κB信号通路抑制炎症反应、调节能量代谢，达到治疗‘黑真布病’作用”的假说。拟采用CIA大鼠热证模型和TNF-α诱导滑膜成纤维细胞，以AMPK激动剂AICAR、SIRT1抑制剂NAM为对照，运用Elisa、WB、RT-PCR等分子生物学技术，从能量调控、炎症反应的角度，揭示翼首草介导AMPK/SIRT1/NF-κB通路治疗“黑真布病”的作用机制，诠释寒凉药物“清赤巴热”的科学内涵。

翼首草是具有抗类风湿性关节炎（类似于藏医“黑真布病”）作用的常用藏药，所含成分复杂。前期在翼首草的化学成分、药效评价等方面做了大量研究，然而在研究过程中深入的作用机制往往没有给予足够重视。针对此类问题，项目以翼首草为研究对象，筛选主要的抗类风湿性关节炎活性成分，从能量调控、炎症反应为切入点，揭示翼首草介导AMPK/SIRT1/NF-κB信号通路发挥治疗“黑真布病”的作用机制。研究发现，翼首草中主要含有三萜皂苷、单环烯醚萜苷、双环烯醚萜苷、酚酸类等成分，从正丁醇提取物中分离纯化得到了11个化合物，其中1个新的环烯醚萜四聚体（Pterocephanoside A）。随后从代谢的角度探讨翼首草提取物对完全弗氏佐剂诱导的佐剂性关节炎模型大鼠的影响，发现翼首草提取物可以调节代谢紊乱，促进失常的新陈代谢表型向正常水平回归，从缬氨酸、亮氨酸和异亮氨酸降解、花生四烯酸代谢等8条代谢途径对佐剂性关节炎大鼠进行治疗。最后，以翼首草提取物中含量较高的吴茱萸苷、马钱苷、獐牙菜苷和大花双参苷为研究对象，以LPS诱导的小鼠单核巨噬细胞构建体外细胞炎症模型，进行活性筛选，发现吴茱萸苷可显著降低炎性介质的表达，具有良好的抗炎活性。随后进行了系列验证实验，吴茱萸苷可通过使类风湿性关节炎成纤维样滑膜细胞内Ca2+和ROS超载而促进线粒体通透性转换孔持续开放和线粒体膜电位耗散，引起线粒体功能下调；同时使线粒体呼吸和糖酵解速率降低而抑制其能量代谢，由此激活AMPK/SIRT1/NF-κB通路，发挥改善炎症反应并促进细胞凋亡的作用，达到治疗“黑真布病”的目的。本项目以能量调控、炎症反应为切入点，探索基于AMPK/SIRT1/NF-κB信号通路揭示翼首草治疗“黑真布病”科学内涵的新模式，为藏族寒凉药物治疗“黑真布病”的研究提供了新的思路。本项目按照预期计划开展研究，发表学术论文7篇，其中SCI论文6篇，中文核心期刊论文1篇，协助培养硕士研究生1人，申请发明专利1项。