基于Hippo信号通路抑制星形胶质细胞激活介导电针脑缺血耐受机制

Inhibiting the activation of astrocyte by electroacupuncture plays an important role in the treatment of stroke. But the mechanism of how to inhibit astrocyte by electroacupuncture is unknown. YAP and MST1 are both key proteins of Hippo pathway. Nuclear YAP of astrocyte plays an important role in inhibiting astrocyte activation, while p-MST is an important protein that prevents YAP from entering the nucleus. Previous studies have found that MCAO mice had increased p-MST and p-YAP, decreased intranuclear YAP, and activated Hippo pathway and astrocyte, while electroacupuncture could reverse these effects induced by cerebral ischemia reperfusion. It shows that cerebral ischemic tolerance by electroacupuncture, inhibition of astrocyte activation and the Hippo pathway are closely linked. Therefore,the research group propose a scientific hypothesis: Electroacupuncture can inhibit the activation of the hippo pathway in the penumbra zone of cerebral infarction, and reduce the phosphorylation of MST1, induce the dephosphorylation of YAP, and then promote the entry of YAP into the nucleus to inhibit the activation of astrocyte , thereby achieving the effect of cerebral ischemic tolerance. In this study, MCAO mice and astrocyte OGD/Rep model are established. The changes of the pathway proteins and astrocyte activation are observed by using western blot, immunofluorescence respectively. From the aspects of animal, cell and molecule, providing the proof that Hippo pathway inhibits astrocyte activation in mediating cerebral ischemia tolerance of electroacupuncture.This study will provide the scientific basis for the treatment of cerebral infarction with acupuncture.

电针抑制星形胶质细胞（AS）激活对治疗脑梗塞有重要作用。但电针如何抑制AS，机制不明。YAP和MST1是Hippo通路的关键蛋白。核内YAP可抑制AS激活，p-MST是阻止YAP入核的重要蛋白。我们发现MCAO小鼠AS中的p-MST增多、核内YAP减少,AS被激活，而电针可减少AS中的p-MST、增加核内YAP，抑制AS激活。提示电针脑缺血耐受、抑制AS激活、Hippo通路三者有密切联系。因此，我们提出科学假说：电针通过抑制脑梗死半暗带区AS中hippo通路，抑制MST1磷酸化，进而抑制YAP磷酸化，促使YAP进入细胞核，从而抑制AS激活，产生脑缺血耐受。本研究拟建立小鼠MCAO和原代AS的OGD/Rep模型，分别运用WB、免疫荧光等技术，观察通路蛋白、AS激活等指标，从整体、细胞、分子等不同层面，论证Hippo通路抑制AS激活介导电针脑缺血耐受。本研究将为针刺治疗脑梗塞提供依据。

研究背景：脑梗塞是引起成人高致残率和死亡率的神经系统疾病。电针预处理百会穴可以改善脑缺血再灌注引起的脑损伤，但具体机制不明确。有研究表明电针抑制星形胶质细胞激活对治疗脑梗塞有重要作用。但电针是否通过抑制Hippo通路，增加细胞核内YAP表达来抑制星形胶质细胞激活达到脑保护的效应，目前未阐明。本研究通过建立MCAO模型和原代星形胶质细胞的OGD/R模型，研究Hippo通路在抑制星形胶质细胞激活介导电针脑缺血耐受中的作用。.主要研究内容：.第一部分、通过腺病毒转染沉默YAP表达，电针预处理，建立MCAO模型，观察神经行为学评分，再采用TTC等方法，论证YAP表达沉默可以逆转电针抑制星形胶质细胞激活和电针脑缺血耐受作用。.第二部分、提取原代星形胶质细胞，运用Verteporfin抑制细胞核内YAP发挥生物学作用，行OGD/R造模，采用Western-blot技术等技术，论证抑制细胞核YAP加重炎症反应。.第三部分、通过注射腺相关病毒转染过表达MST1和MST抑制剂抑制MST1的磷酸化，建立MCAO模型和OGD/R模型，采用TTC、CCK-8等技术，从在体动物和离体细胞模型两个方面论证抑制MST1磷酸化是电针促使YAP核表达，抑制星形胶质细胞激活，发挥脑保护作用的必要条件。 .结果：脑缺血再灌注损伤后，电针预处理可以增加脑缺血半暗带星形胶质细胞核内YAP的表达，沉默YAP表达逆转电针的神经保护和减轻星形胶质细胞激活的作用，抑制细胞核YAP会加重神经炎症反应。过表达MST1减弱电针的神经保护作用，抑制MST1的磷酸化可以模拟电针预处理产生YAP核表达增加，神经保护和减轻星形胶质细胞激活的作用。总之，电针预处理通过抑制Hippo信号通路核心蛋白MST1磷酸化，促进YAP核表达，从而抑制星形胶质细胞激活，发挥脑缺血保护作用。.科学意义：本研究通过在体与离体实验，通过药理学与遗传学方法来阐明了Hippo通路通过抑制星形胶质细胞激活介导了电针的脑缺血耐受效应。本研究的成果将有利于推动电针治疗脑梗塞的机制研究，并进一步完善电针治疗缺血性脑损伤的理论基础和科学依据。