补肾益气法通过PAR-1依赖的PI3-K/Akt途径的神经血管单元保护机制研究

Ischemic brain injury is a common pathological mechanism in the most of cerebrovascular diseases.But the mechanisms of neurovascular unit (NVU) damage induced by ischemia is still uncertain. In the anti-stroke network targets based on NVU,thrombin is involved in neurovascular unit damage and plays pleiotropic roles .Thrombin acts on the different components via different pathways mediated by PAR-1.Our project based on past study, a imaging probe labeled radionuclide is designed and injected in vivo ,and then the animals are imaged by small animal PET/CT. Then the immunofluorescence technique combines pharmacological agonists/antagonist study could to be revealed the dynamic localization and quantitative of proteolytic activity thrombin induced by cerebral ischemia and association between PAR-1 and NVU. Tonifying kidney and replenishing qi is the key therapeutic rule on stroke because kidney deficiency and qi deficiency are the root of the law. Therefor the rat with middle cerebral artery occlusion would to be treated by Bu-Shen-Yi-Qi-Fang so that the dynamic distribution and quantify of thrombin in model’s brain would be observed. And then, we probe to the NVU protection mechanism through the PAR-1 dependent PI3-K/Akt pathway. Finally, reliable experimental evidence for Bushen Yiqi recipe on stroke would be provided.

缺血性脑损伤是多种脑血管疾病过程的共同病理机制,导致的神经血管单元（NVU）损伤机制尚不明确。在基于NVU抗脑卒中的网络靶点中，凝血酶参与其中，并呈多效性特征。它对NVU不同组分的作用途径不同，常为蛋白酶激活受体PAR-1介导。本项目在既往研究基础上，设计基于PAR-1序列的特异性影像学分子探针，核素标记后植入活体动物模型，运用小动物PET/CT进行显像；采用免疫荧光技术，结合药理激动剂/拮抗剂的研究，揭示脑缺血激发的具有蛋白水解酶活性凝血酶的动态定位与定量，及其与NVU的相关性。“肾虚”、“气虚”为中风之本，补肾益气法为治本大法，本项目运用补肾益气方预处理大鼠大脑中动脉阻塞模型，观察对模型脑内活性凝血酶的动态分布与定量的影响，并通过PAR-1依赖的PI3-K/Akt途径探讨其神经血管单元保护机制。从而为补肾益气法治疗中风提供可靠的实验依据。

由于急性缺血性中风仍然是世界范围内致残和死亡的主要原因，有研究者建议研究神经保护剂联合溶栓药物或血管内取栓作为治疗急性缺血性脑卒中的新策略。在过去的十年中，诸多报道揭示凝血酶不仅对凝血系统，而且对神经元信号、发育和可塑性均有影响。我们既往研究提示补肾药生地具有对体外培养的凝血酶损伤的神经元具有保护作用，可减轻凝血酶导致的星形胶质细胞的凋亡；淫羊藿苷对皮质酮诱导凋亡的海马神经细胞具有保护作用。然而这些研究都是基于中药单药或单体针对体外的研究，而临床应用仍以复方制剂为多，鉴于中医的“整体观念”特点，结合补肾益气颗粒（BSYQG）对中风的较好临床疗效，观察BSYQG预处理对大脑中动脉阻塞（MCAO）大鼠的NVU保护作用，以及对脑内凝血酶的动态作用，对脑内的凝血酶活性的影响，进而对凝血酶受体PAR-1在MCAO中的作用及BSYQG预处理对PI3-K/Akt通路的影响做一初探。结果提示BSYQG对MCAO大鼠具NVU保护作用；具有较好的动态调节凝血酶表达的作用；其NVU保护作用是通过抑制凝血酶作用；可通过PI3-K/Akt通路对凝血酶抑制；可抑制活性脑内凝血酶。以上作用同阿加曲班相似。凝血酶损伤与PAR-1有关，并通过PI3-K/Akt通路起作用。