基于巨噬细胞内质网应激-自噬-凋亡的调控探讨补阳还五汤稳定动脉粥样硬化易损斑块的机制

How to improve the atherosclerosis (AS) stability of vulnerable plaques is the key to the prevention of acute coronary syndrome . Excessiveendoplasmic reticulum stress (ERS) in plaque’s macrophages caused excessive autophagy, eventually leading to promote plaque instability of apoptosis process, PERK/eIF2a/CHOP ，IRE1a/TRAF2/ASK1 pathway induced ERS-autophagy-apoptosis . Haemorrheological nature, and qi stagnation and blood stasis resistance to heart of collaterals is related to the pathogenesis of vulnerable plaques. Prophase project is supported by the national natural fund found yiqi huoxue by Buyanghuangwu decoction also narrow area of vulnerable plaques, and enhances the stability of plaque macrophages and moderate regulation autophagy and inhibited cell apoptosis. The project is proposed on the basis of these result, make vulnerable plaques in ApoE - / - mice and macrophage ERS model, the combination of different level to explore ultimately also within Buyanghuangwu decoction enhances the stability of plaque and plaque macrophages ERS - regulatory mechanism of autophagy and apoptosis, related to PERK/eIF2a/CHOP ，IRE1a/TRAF2/ASK1 pathway ,reveals the maintain macrophage function and mechanism of the steady state, provide new ideas and new targets for treatment of the AS.

如何提高动脉粥样硬化(AS)易损斑块的稳定性是预防急性冠脉综合征猝死的关键。斑块内巨噬细胞过度内质网应激（ERS）引起过度自噬，最终导致细胞凋亡过程共同导致易损斑块的不稳定，而ERS引起细胞自噬—凋亡与PERK/eIF2a/CHOP、IRE1a/TRAF2/ASK1通路密切相关。中医认为寒凝、气滞、血瘀阻于心之络脉是易损斑块的病机，前期国家自然基金资助项目发现益气活血通络经方补阳还五汤缩小斑块面积、提高易损斑块稳定性与适度调控巨噬细胞自噬及抑制细胞凋亡有关。本项目拟在前期基础上，通过ApoE-/-小鼠易损斑块与巨噬细胞不同程度ERS模型相结合，探讨补阳还五汤提高斑块稳定性与抑制斑块内巨噬细胞过度ERS引起过度自噬导致巨噬细胞凋亡的调控机制与PERK/eIF2a/CHOP、IRE1a/TRAF2/ASK1通路的关系，揭示其维持巨噬细胞稳态作用及机制，为治疗AS提供新思路、新靶点.

在ApoE-/-小鼠复制的动脉粥样硬化易损斑块模型基础上腹腔注射衣霉素0.9 mg/kg，造成内质网应激模型。与正常组相比，模型组AS病变明显，易损指数升高，血清白介素IL-1β和 IL-6 含量均显著升高（P<0.05）；模型组血管组织中炎性巨噬细胞较多，细胞凋亡率明显升高，模型组血管组织中GRP78、IRE1、P-IRE1、XBP1、PERK、EIF2α、CHOP、Bcl-2、BAX蛋白表达明显升高，ATF6蛋白表达明显降低，补阳还五汤与内质网应激抑制剂4-PBA组对GRP78、IRE1、P-IRE1、XBP1、PERK、EIF2α、CHOP有降低作用（P<0.05）模型组的凋亡相关蛋白BAX的表达水平明显上升伴随Bcl-2的表达水平下降，4-PBA与补阳还五汤中、高剂量都可降低BAX，升高Bcl-2表达水平（P<0.05）。.在培养的巨噬细胞（中，5ug·ml-1 LPS浓度引起巨噬细胞轻度ERS，40ug·ml-1 LPS浓度引起巨噬细胞重度ERS，同时诱发巨噬细胞显著凋亡。LPS诱发巨噬细胞ERS与IRE1-XBP1、PERK-eIF-2a-ATF-4、ATF-6信号通路激活有关；补阳还五汤含药血清抑制巨噬细胞ERS-细胞凋亡与调控IRE1-XBP1、PERK-eIF-2a-CHO、、Caspase12、Bcl-2、Fas-Fasl途径相关。补阳还五汤含药血清升高轻重度ERS时巨噬细胞内自噬水平，改善受到阻碍的自噬流，促进细胞自噬，减轻内质网的蛋白负荷，抑制ERS的过度激活及细胞过度凋亡。沉默巨噬细胞CHOP基因后，补阳还五汤仍可通过抑制巨噬细胞内质网应激诱发的炎症，其作用机制与调控GRP78、IRE1-XBP1、PERK-eIF-2α-ATF4-CHOP通路蛋白表达有关。.本课题研究证实补阳还五汤提高不稳定斑块的稳定性与调控巨噬细胞自噬-内质网应激-凋亡密切相关。