腺苷A3受体调控小胶质细胞M1\M2极性转化参与SAH后早期脑损伤的作用及机制研究

Inflammation response mediated by microglia activation is one of the main pathophysiological processes involved in the early brain injury after subarachnoid hemorrhage(SAH). In this process, recent studies gain more and more attentions into transformation of microglial cells from M1 polarisation(classic activation) to M2 polarisation(substitution activation), which could inhibit pro-inflammatory effects of microgila while strengthening their anti-inflammation and repairing effects. Our previous study showed that endogenous adenosine A3 receptor (A3R) activation could exhibit neuroprotective effects toward early brain injury after SAH through regulation of microglia M1/M2 type polarity conversion. However, the specific mechanism is still out of clue. Based on the evidences above, we propose that adenosine released after SAH could enhance interferon regulatory factor 4(IRF4) gene transcription to induce microglial M2 polarisation, via activating the A3R on microglial surfaces and downstream PI3K-STAT3 signal pathway, and finally lead to amelioration of early brain injuy and better outcomes. In the current project, by using immunohistochemistry, quantitative PCR, reporter gene, EMSA and ChIP techniques, we intend to take microglial cells as reseach subjects on both in vivo and in vitro SAH models, and try to uncover the hidden misteries about how adenosine A3R regulates microglia M1/M2 type polarity conversion. We expect the current study could lay a theoretical foundation for the development of the adenosine A3R as a new therapeutic target of SAH.

小胶质细胞活化介导的炎症反应在蛛网膜下腔出血（SAH）后早期脑损伤中发挥重要作用。我们前一个基金课题发现SAH后小胶质细胞内源性腺苷A3受体（A3R）激活可发挥神经保护作用，但具体机制尚不清楚。最新研究证实，小胶质细胞免疫功能表型（M1\M2）转化可能是在不同病理状态下发挥炎性损害/神经保护作用的关键。结合文献报道，我们提出SAH后小胶质细胞表面A3R活化，激活PI3K-STAT3信号,增强IRF4基因表达，诱导其M2样极化，发挥抑炎及神经保护作用的假说。本研究拟建立大鼠SAH在体和离体模型，以小胶质细胞为研究对象，采用免疫组化、定量PCR、报告基因、EMSA和ChIP等技术，阐明腺苷A3R通过调控小胶质细胞M1/M2极性转化产生抗炎特性进而发挥神经保护作用的具体分子调控机制，为制定以腺苷A3R为靶点，诱导小胶质细胞朝抗炎特性分化以获得更佳神经保护的SAH治疗新策略奠定理论基础。

蛛网膜下腔出血（Subarachnoid hemorrhage，SAH）是一种具有高致残、高致死率的严重疾病，发病多见于中青年，严重威胁人类健康和生命。小胶质细胞活化介导的炎症反应在蛛网膜下腔出血(SAH)后早期脑损伤中发挥重要作用。小胶质细胞免疫功能表型(M1\M2) 转化可能是在不同病理状态下发挥炎性损害/神经保护作用的关键。调控小胶质细胞的表型转换，促进小胶质细胞M2型极化，抑制小胶质细胞M1型极化，可能减轻SAH后小胶质细胞活化介导的炎性损伤，保护神经功能。结合文献报道，我们提出SAH 后小胶质细胞表面 A3R 活化，诱导其向 M2 样极化，发挥抑炎及神经保护作用的假说。因此本研究主要针对激活或干预小胶质细胞腺苷A3受体EPO受体和通路，观察SAH后小胶质细胞表型转换以及对中枢炎症水平的影响。我们基因转染、沉默、Western Blot及免疫荧光染色等技术取得下列研究成果： 1. 阐明了腺苷A3受体活化调控小胶质细胞M1向M2样极化在SAH后早期脑损伤中的抗炎作用及分子机制。发现腺苷A3受体活化可以减轻SAH后炎症因子的表达释放、参与小胶质细胞M1/M2极性转化的调控可能通过JAK2-STAT3通路发挥抗炎作用。2．阐明了EPO促进小胶质细胞M1向M2样极化在SAH后早期脑损伤中的抗炎作用及分子机制。发现EPO激活JAK2-STAT3信号通路促进SAH后小鼠脑内小胶质细胞表型M2样极化并减轻脑内促炎反应。改善脑细胞凋亡、神经元损伤、脑组织水肿及脑组织白蛋白渗出量。体外实验证实EPO通过EPOR/JAK2-STAT3信号通路促进小胶质细胞表型的M2样极化。3. 另外发现BPV(pic)能够改善SAH后神经元凋亡，并改善神经功能、减轻血脑屏障破坏和脑水肿。可能通过抑制PTEN蛋白表达和PTEN的磷酸肌醇3磷酸酶活性调节谷氨酸 AMPA 受体亚基表达有关。