基于AMPK-mTOR-HIF-1α信号通路调控巨噬细胞极化探讨小檗碱治疗实验性关节炎的作用机制

M1 macrophages are of central importance in rheumatoid arthritis (RA) cytokines network environment, which contribute to facilitate joint inflammation and bone destruction. Anaerobic glycolysis is the main characteristic of the polarization and activation in M1 macrophages. It is may be a key molecular switch that up-regulate glycolysis in M1 macrophages by inhibiting the activation of AMPK. Our team found that berberine(BBR) reversed the polarization of M1 macrophages from AA rat spleen, down-regulated their glycolysis and up-regulated the activation of AMPK. However, it is unknown whether BBR promote M1 macrophages polarization to M2 through inhibiting the anaerobic glycolysis of macrophages by increasing the activation of AMPK. mTOR/HIF-1α are important signal molecules involved in energy metabolism in the downstream of AMPK, so it is worth to further research whether the roles are associated with promoting M1 macrophages polarization to M2 by regulating energy metabolism of macrophages through AMPK-mTOR-HIF-1α signal pathway.This project intends to carry out the research in CIA mouse, AA rats and RAW264.7 cells to discuss whether the anti-inflammatory mechanisms and the treatment of BBR in RA is associated with inhibiting mTOR-HIF-1α signal pathway to promote M1 polarization to M2 macrophage by increasing the activity of AMPK.

M1型巨噬细胞在类风湿关节炎(RA)的细胞因子网络环境中发挥了促进炎症反应和骨破坏的中心作用，有氧糖酵解是其极化和活化的主要特点。下调腺苷酸激活蛋白激酶(AMPK)活性，可能是促进巨噬细胞向M1型极化的关键分子开关。课题组前期发现小檗碱(BBR)可降低AA大鼠脾脏巨噬细胞M1/M2比值，抑制其糖酵解，上调细胞内AMPK活性。但BBR是否通过上调AMPK活性，抑制M1型巨噬细胞糖酵解，促进其向M2型极化，未见报道。mTOR/HIF-1α是AMPK下游参与能量代谢的重要信号分子，BBR是否通过AMPK-mTOR-HIF-1α信号，调控巨噬细胞能量代谢，促进巨噬细胞由M1型向M2型极化？值得进一步研究。本项目拟通过体内外实验，首次探讨BBR通过上调AMPK活性抑制mTOR/HIF-1α信号通路，促进M1型巨噬细胞向M2型极化，是其发挥抗炎作用和治疗RA的重要分子机制。

小檗碱(BBR)是治疗类风湿关节炎(RA)的经典方剂四妙丸和二妙散中的主要有效成分，课题组前期研究发现 BBR 对实验性关节炎具有良好的治疗作用，可减少巨噬细胞促炎症细胞因子产生，但具体作用机制未完全阐明。本项目以RA动物模型和小鼠RAW264.7巨噬细胞株为研究对象，深入探究了BBR对巨噬细胞极化和代谢的影响。研究结果发现，BBR可降低AA大鼠脾脏和关节滑膜组织中巨噬细胞M1/M2比值，上调腹腔巨噬细胞和滑膜巨噬细胞中腺苷酸激活蛋白激酶(AMPK)的表达、下调COX2，NF-κB，HIF-1α等重要炎症相关分子的表达。在抑制AMPK的CIA小鼠模型中，BBR改善CIA小鼠关节炎症状和抑制巨噬细胞的吞噬、迁移和促炎症因子的分泌功能的作用被取消，不能促进腹腔和脾脏巨噬细胞M1/M2平衡的恢复。进一步在RAW264.7-AMPKα-Null中研究显示，BBR不能促进LPS诱导的M1型巨噬细胞向M2型转化。并且，突变株细胞对LPS刺激更加敏感，促炎症细胞因子IL-1β、TNF-α的表达水平较正常细胞株相比显著增加，抗炎症细胞因子IL-10和TGF-β1显著减少。此外，在LPS刺激的突变细胞株中，BBR的抗炎作用被取消，并且mTORC1、HIF-1α的活化更为显著，BBR显著抑制正常细胞株中mTORC1、HIF-1α的活化，但对突变株没有影响。给予mTORC1的激活剂Leu后发现，BBR能降低Leu增强RAW264.7细胞的吞噬和迁移功能、促进巨噬细胞向M2型极化，但在AMPK突变细胞株，BBR没有明显作用。这些结果表明，AMPK可能是巨噬细胞极化的关键分子开关，BBR通过上调AMPK活性抑制mTORC1/HIF-1α信号通路，下调巨噬细胞糖酵解代谢，促进M1型巨噬细胞向M2型极化，是其发挥抗炎作用和治疗RA的重要分子机制。