基于CX3CR1介导的小胶质细胞激活研究异钩藤碱抗脑缺血再灌注损伤的作用及机制

There is no ideal drug for the treatment of cerebral ischemia-reperfusion injury (IRI). Microglia-mediated inflammatory response throughout the process of IRI. Therefore, intervention of inflammatory injury mediated by over-activation of microglia is one of the strategies to treat IRI. Chemokine CX3C receptor 1 (CX3CR1) was involved in the activation of microglia after IRI. Isorhynchophylline (IRN) is one of the main components of Uncaria rhynchophylla. It have been proved that IRN can alleviate neuronal damage induced by glucose deprivation, but direct evidence is still unavailable till now. Our pre-experimental results demonstrated that IRN could improve the neurological deficits of IRI rats, reduce the volume of cerebral infarction, inhibit CX3CR1-mediated microglia activation. Therefore, this project is designed to observe the effect of IRN on IRI in ischemia-reperfusion injury mouse model induced by the intraluminal suture method and the action of IRN on the activation of the microglia. Then, the underlying molecular mechanism of the effect of IRN on IRI is further studied by using CX3CR1 knockout mice. On the basis of this, the molecular mechanism of IRN against IRI is further detected in mouse microglia BV2 cells subjected to oxygen-glucose deprivation and with the help of gene overexpression and interference technique, which will provide the basis for application of IRN into the treatment of IRI.

目前尚无理想的脑缺血再灌注损伤(IRI)治疗药物。小胶质细胞介导的炎症反应贯穿IRI过程，干预小胶质细胞过度激活所介导的炎症损伤成为治疗IRI的策略之一。趋化因子CX3C受体1(CX3CR1)参与了IRI后小胶质细胞的活化。异钩藤碱(IRN)是我省道地中药材钩藤主要成分之一，IRN可缓解氧糖剥夺诱导的神经元损伤，但尚无治疗IRI的直接证据。预实验发现，IRN能够改善IRI大鼠的神经功能缺陷，减少脑梗死体积，抑制CX3CR1介导的小胶质细胞激活。因此，本项目拟通过线栓法致IRI小鼠模型，首先确证IRN对该模型的疗效，并观察IRN对小胶质细胞激活状态的影响，进一步采用CX3CR1基因敲除小鼠分析IRN抗IRI作用与CX3CR1的关系。在此基础上，通过缺氧缺糖恢复诱导小鼠小胶质细胞株BV2细胞激活模型，借助基因过表达及干扰技术，深入研究IRN抗IRI的分子机制，为IRN用于治疗IRI提供依据。

目前尚无理想的脑缺血再灌注损伤(IRI)治疗药物。小胶质细胞介导的炎症反应贯穿IRI过程，干预小胶质细胞过度激活所介导的炎症损伤成为治疗IRI的策略之一。我省道地中药材钩藤在中医上被用于治疗肝阳上亢型脑血管疾病，而异钩藤碱(IRN)作为钩藤主要成分之一可缓解氧糖剥夺诱导的神经元损伤。然而，尚无IRN治疗脑缺血再灌注损伤的直接证据。本项目首先通过线栓法制备脑缺血模型，并在缺血2h后行再灌注诱导脑缺血再灌注损伤，发现IRN治疗对脑缺血再灌注损伤所致的神经功能损伤，脑梗死，神经元病变及脑水肿均有缓解作用；同时我们观察了IRN对模型动物脑缺血再灌注后小胶质细胞形态、表型和功能的影响，结果发现IRN可抑制IRI导致的小胶质细胞促炎型激活,降低IL-1β、TNF-α，IL-6的表达，升高抗炎型标志物YM1/2的表达。同时，我们证实趋化因子CX3C受体1 (CX3CR1)参与了IRI后小胶质细胞的活化，而IRN对脑缺血再灌注损伤主要通过抑制CX3CR1介导的炎症反应实现。在此基础上，我们进一步通过缺氧缺糖恢复诱导小鼠小胶质细胞株BV2细胞激活模型，在细胞水平确证了IRN对小胶质细胞炎性激活的抑制作用。此外，我们利用RNA干扰技术使CX3CR1低表达，证实IRN的抗脑缺血再灌注损伤所致的小胶质细胞激活主要通过抑制CX3CR1实现。综上所述，本项目从传统中医理论和实践出发，采用现代药理学方法对钩藤的单体化合物IRN进行研究，阐明了其确切的药理作用，为中药钩藤及IRN用于治疗脑缺血再灌注提供基础药理学依据。这既是通过现代研究手段结合中医理论对中药药效的诠释，也有助于钩藤的合理开发利用，以带动地方经济发展。