羚角钩藤汤加减联合肢体缺血预适应治疗中风脑水肿的分子机制研究

Brain edema is a hot and difficult problem in research of stroke. Modified Ling Jiao Gou Teng formula developed by our team for the treatment of stroke, has demonstrated positive therapeutic effect clinically. But the mechanism remains unclear. According to the theory of preventive treatment of traditional Chinese medicine and new findings of the limb ischemia preconditioning protection of vital organs, we hypothesized that Modified Ling Jiao Gou Teng formula combined application of limb ischemia preconditioning is likely to be a cure for stroke throughout its regulatory effect of blood brain barrier (BBB) function. We optimize the best parameters of limb ischemia preconditioning. Brain tissue pathological changes were evaluated by TTC staining, MRI technology, electron microscope examination. Aided with immunohistochemical, Westernblot, qRT-PCR techniques, expression of Occludin, Claudin-5, MMP-2, MMP-9 and AQP-4 in blood brain barrier were detected to investigate the mechanism of reducing brain edema in the early brain injury, which were treated by Modified Ling Jiao Gou Teng formula combined application of limb ischemia preconditioning. Through this project research, we explore the core mechanism of treatment stroke in acute phase by Modified Ling Jiao Gou Teng formula, and evaluate the feasibility of limb ischemia preconditioning as adjuvant therapy of traditional Chinese medicine. Besides, this project can provide evidence for reasonable clinical administration of drugs and guidance for Chinese medicine treatment of stroke.

脑水肿是中风领域研究的热点和难题。课题组前期以羚角钩藤汤加减治疗中风急性期脑水肿疗效显著，但作用机制仍不清楚。本研究根据中医“治未病”的诊疗理念，结合肢体缺血预适应保护远隔重要脏器的最新认识，提出假说：羚角钩藤汤加减联合肢体缺血预适应，通过综合调整血脑屏障功能治疗中风急性期脑水肿。拟优选肢体缺血预适应相关参数，采用TTC染色、MRI技术、电镜检查等手段评价脑组织病理学变化，并通过免疫组化、Westernblot、qRT-PCR等技术检测血脑屏障Occludin、Claudin-5、MMP-2、MMP-9和AQP-4等关键靶点表达，在组织和分子水平阐明羚角钩藤汤加减联合肢体缺血预适应治疗缺血性脑中风急性期脑水肿的作用机制。通过本项目研究，探索羚角钩藤汤加减治疗中风急性期脑水肿的核心机制，评价肢体缺血预适应作为中医药辅助疗法的可行性，为临床合理用药提供依据，对于指导中医药治疗中风具有重要意义。

脑水肿是中风领域研究的热点和难题。课题组前期以羚角钩藤汤加减治疗中风急性期脑水肿疗效显著，但作用机制仍不清楚。本研究根据中医“治未病”的诊疗理念，结合肢体缺血预适应保护远隔重要脏器的最新认识，提出假说：羚角钩藤汤加减联合肢体缺血预适应，通过综合调整血脑屏障功能治疗中风急性期脑水肿。采用MRI技术、神经功能评分，优选肢体缺血预适应相关参数，采用HE染色、电镜检查等手段评价脑组织病理学变化，采用免疫组化、Westernblot、qRT-PCR等检测血脑屏障Occludin、Claudin-5、MMP-2、MMP-9和AQP-4等关键靶点表达，探讨羚角钩藤汤加减联合肢体缺血预适应治疗缺血性脑中风急性期脑水肿的作用机制。肢体缺血预适应5min较肢体缺血预适应10 min、15 min组有较好的降低脑水肿率、脑梗死率，降低神经损伤功能评分，对大鼠中动脉栓塞再灌注引起的脑损伤具有更好的保护作用，选用肢体缺血预适应5min用于后续实验研究。缺血预适应、中药组、预适应+中药组可显著降低脑缺血再灌注24h、72h后脑水肿率、梗死率，降低脑损伤神经评分，神经细胞缺血性改变明显减轻，空泡化改变减轻，血管周围间隙和神经细胞周围间隙增宽明显减轻，存在较多的存活细胞，细胞排列较整齐，细胞质较饱满丰富，血管内皮细胞连续性完整，细胞紧密连接较完整。免疫组化、Western blot、qRT-PCR检测结果表明，缺血预适应、中药组、预适应+中药组可不同程度提高脑缺血再灌注24h、72h后脑缺血半影区Occludin、Claudin-5、ZO-1、TIMP-1的表达，降低MMP-2、MMP-9、AQP-4的表达，推测缺血预适应、中药羚角钩藤汤加减通过保护血脑屏障紧密连接，降低基底膜的损伤，降低水通道蛋白4的活性，发挥保护血脑屏障，减轻脑水肿，减轻神经功能损伤的作用。羚角钩藤汤加减方与肢体缺血预适应联合应用作用较二者单独使用有一定的增强，可以发挥更好脑缺血再灌注的保护作用，“肢体缺血预适应预防+中药急性期治疗”的新型诊疗模式，在治疗中风急性期方面具有较好的临床应用前景。

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