电针治疗缺血性中风的新机制--激活δ阿片受体-BDNF/TrkB通路而抑制小胶质细胞的过度活化

Microglial overactivation is the committed step in inflammatory injury of penumbra in the subacute phase of ischemic stroke. We have previously reported that DOR activation up-regulates BDNF-TrkB signaling thereby decreasing TNF-α level in the ischemic/hypoxic brain protection. DOR plays an important role in the electro-acupuncture (EA) induced brain protection against ischemic injury, therefore, we hypothesized that EA exerted neuroprotective effects against ischemic insult by modulating the local inflammation in rats with DOR-BDNF/TrkB pathway. To further explore the mechanism of DOR in EA brain protection, rats were subjected to middle cerebral artery occlusion and reperfusion model. Brain function was assessed by neurological deficits scores. Infarct volume was measured using triphenyltetrazolium chloride. Microglia activation, the level of pro-inflammatory factors and the activity of a key inflammation regulatory factor NF-κB were detected with immunohistofluorescence, western blot, Elisa as well as electrophoretic mobility shift assay (EMSA) analysis. Neuronal apoptosis were detected by TUNEL staining. Analyze the hypothesis from cellular, cytokine, and transcription factor levels that "EA exerts ant-ischemic stroke via activate DOR-BDNF/TrkB pathway suppress over-activated microglia in the subacute stage and save the neuron in the ischemic penumbra”. This project not only deepens the previous research, but also provides new thoughts to explore the mechanism underlying the efficacy of EA on ischemic stroke.

小胶质细胞过度活化是脑缺血亚急性期半影区炎性损伤的关键因素，课题组以往研究提示，激活DOR可上调BDNF/TrkB信号，从而抑制小胶质细胞过度活化及促炎症因子TNF-α的释放，进而实现脑缺血缺氧保护。既然DOR是电针抗脑缺血损伤的重要机制，电针治疗脑缺血可能与DOR-BDNF/TrkB抗炎作用有关。为深入探讨其机制，本项目拟建立MCAO/再灌大鼠模型，观察电针及DOR阻断后的脑缺血反应。拟采用神经行为评分检测脑功能，TTC检测脑梗死体积，免疫荧光、WB、Elisa及EMSA检测小胶质细胞的活化、炎症因子含量及炎症通路关键因子NF-κB的活性，TUNEL染色检测神经元凋亡。分别从细胞、胞内因子及转录水平，论证"电针可能通过激活DOR-BDNF/TrkB信号通路抑制亚急性期小胶质细胞过度活化，挽救半影区神经元治疗脑缺血"的假说。本项目既是对以往研究深入，也为阐明电针治疗缺血性中风提供新的思路。

DOR受体已经证实具有神经保护作用，电针水沟(GV26)和内关(PC6)对脑卒中后的保护作用与DOR激活密切相关。然而，DOR激活和电针治疗的神经保护作用的潜在作用机制尚不明确，本研究探讨了DOR激活和电针介导的I/R损伤的神经保护的潜在抗炎机制。.方法：建立氧-糖剥夺/复氧(OGD/R)细胞培养和大脑中动脉阻塞/再灌注(MCAO/R)大鼠来诱导体内外缺血损伤。体外实验给药DOR激动剂/拮抗剂/siRNA来研究DOR激活是否可以通过抗炎来发挥神经保护，体内实验通过电针水沟（GV26）和内关（PC6）激活DOR发挥神经保护作用。通过形态学改变、CCK-8、乳酸脱氢酶（LDH）漏出率检测细胞生长、增殖，通过TUNEL染色试剂盒来检测细胞凋亡。采用实时定量逆转录聚合酶链式反应（RT-qPCR）检测DOR、促炎和抗炎细胞因子的mRNA水平，采用Western blot检测DOR的蛋白表达和酶联免疫吸附法（ELISA）检测BDNF蛋白表达。焦油紫（CV）染色检测脑梗死体积大小，神经功能评分评估神经功能恢复情况，免疫荧光检测大脑中促炎和抗炎细胞因子的表达情况。.结果：DOR激活显著减少OGD/R诱导的PC12/BV2小胶质细胞的形态学损伤，减少乳酸脱氢酶漏出率和细胞凋亡，增加细胞活力。DOR激活逆转OGD/R诱导的DOR mRNA和蛋白下调，以及BDNF在上清和细胞内的蛋白表达下调。DOR激活也减少OGD/R模型诱导的促炎细胞因子包括TNF-α、IL-1β和IL-6的基因表达，同时增加抗炎细胞因子IL-4和IL-10的基因表达。而DOR拮抗剂Nanltrindole及DOR siRNA阻断DOR激动剂诱导的保护作用。电针治疗可显著降低MCAO/R诱导的脑梗死体积，并且降低神经功能缺损评分。电针治疗明显增加了IL-10的蛋白表达和减少了IL-1β的蛋白表达，而假电针治疗在MCAO/R大鼠模型并没有保护作用。在电针治疗前给予Nanltrindole/DOR siRNA，逆转了电针的保护作用。.结论：我们的结果表明，DOR激活在OGD/R模型中的神经保护作用可能是通过BDNF/TrkB信号通路抑制炎症反应实现的。电针水沟穴（GV26）和内关穴（PC6）对脑缺血再灌注损伤的神经保护作用可能与通过DOR-BDNF/TrkB途径抑制亚急性期小胶质细胞过度活化，挽救半影区神经元相关。