miR-29调控星形胶质细胞AQP4在脑缺血再灌注损伤中的作用及机制研究

The brain edema induced by cerebral ischemia/reperfusion injury(CIRI) is a common and lethal complication, but its mechanism is not well understood. Up to present, there are lots of limitations to prevent and cure brain edema during ischemic injury . AQP4 plays a pivotal role in maintaining water balance in the central nervous system(CNS) and in the development of brain edema post CIRI.Recent studies showed that miRNAs are involved in the pathophysiological process of ischemic brain injury. In our pilot study, we have found that miR-29 can directly bind to AQP4 mRNA3'-UTR at post-transcriptional level and regulate AQP4 expression in rat astrocytes; we also found the expression of AQP4 and miR-29 showed contrary tendency in astrocytes subjected to oxygen glucose deprevation (OGD),and the miR-29 expression is different in the cerebral ischemic core and penumbra in mice CIRI model. However, the function of miR-29 on brain edema remains unknown. In this study, by using vitro and vivo CIRI model, we investigate the role of miR-29 on the AQP4 expression and the effect of miR-29 on the development of CIRI, the results would provide new methods and potential clinical treatment for the basic research and ischemic cerebrovascular diseases.

脑缺血再灌注损伤（CIRI）继发的脑水肿是卒中常见且致命性的并发症，其机制未清，现有防治措施尚存在诸多局限。脑内水通道蛋白-4(AQP4)是主要表达在星形胶质细胞足突上负责水分子转运的蛋白，其功能与CIRI后脑水肿进展密切相关。最新研究显示，miRNAs参与缺血性脑损伤的病理生理过程，为探索CIRI后脑水肿机制提供新的契机。我们前期预实验发现，miR-29可直接与星形胶质细胞AQP4 mRNA3'-UTR区结合，在转录后水平调控AQP4表达；在星形胶质细胞OGD模型中AQP4与miR-29表达趋势相反，小鼠MCAO模型中亦显示缺血中心区与半暗带区miR-29表达不同。然而，miR-29在缺血性脑损伤脑水肿中的作用及机制尚不清楚。本研究拟通过体内外CIRI模型，探讨miR-29对星形胶质细胞AQP4基因表达的调控及对CIRI的保护作用，为缺血性脑血管病相关基础研究和临床治疗提供理论依据。

本研究首先采用Dual-Luciferase双荧光素酶报告基因检测系统，明确 LOOP区单核苷酸多态性影响microRNAs的加工处理和功能， 并且确定了AQP4可能是miR-29a及miR-130b的靶点。然后，本研究采用原代培养的星形胶质细胞糖氧剥夺/复氧（OGD/RX）模型和大脑中动脉阻塞模型（MCAO）及分子生物学、细胞生物学、流式细胞仪等手段，发现：1）脑缺血再灌注损伤（CIRI）后星形胶质细胞受损，脑水肿发生，AQP4表达上调，同时miR-29a表达下调，miR-29a mimic保护星形胶质细胞并有效抑制AQP4表达，而miR-29a inhibitor促进AQP4表达并进一步损伤细胞， miR-29a通过调控AQP4表达而具有脑保护作用。2）miR-130b对OGD/RX星形胶质细胞具有保护效应，与其下调AQP4表达有关。3）Probenecid缺血后给药方案具有减轻脑水肿的脑保护效应，此效应与促炎因子HMGB1的抑制及AQP4的下调相关。上述结果证实了我们的假设，我们可以从一个基于非编码RNA调控脑缺血再灌注后脑水肿反应的方向研究脑水肿的发生机制。在完成以上研究的基础上，课题组展开了脑缺血再灌注损伤与炎症-免疫机制相关的延伸研究。首次发现甘草素（Glycyrrhizin）缺血后给药，具有改善小鼠CIRI后3天神经行为学功能、脑水肿发生，减少缺血区脑组织神经元坏死及炎性细胞、T细胞浸润的脑保护效应，其机制与HMGB1介导的T细胞抑制相关。