开窍药调控RNA结合蛋白产生的应激颗粒抗脑缺血损伤的机制研究

The key of neuronal repair is how to activate Stress Granule (SG) in stroke, and the RNA-binding protein just is the important molecule in regulating SG. Under the theory of resuscitation in traditional Chinese medicine, XING NAOJING, as the famous prescription in emergency resuscitation，was found to induce the SG and up-regulate the RNA-binding motif protein 3 (RBM3). So, we provided a hypothesis that the effective ingredients of resuscitation selectively act on RBM3, which induce the Stress Granule, have the integrated function to resist the neuron damage in cerebral ischemia. Taken the cell induced with free-serum medium as the SG cell model, we construct the RBM3 promoter luciferase reporter gene system to illuminate the effective cluster response element of resuscitation medicines and target binding proteins with RBM3 gene. And we build the regulating technique of RNA-binding protein targeted to SG mRNA, and use RNAi methods to block RBM3 gene express, or transfect RBM3 gene into the SG cell model to illustrate the resistant apoptosis mechanism of RNA-binding proteins regulated by resuscitation effective ingredient acting on Stress Granule. In the cerebral ischemic animal model, we further to confirmed the integrate mechanism of effective ingredient targeting RNA-binding protein and Stress Granule to resist against the apoptosis. Our research will promote the resuscitation theory development, and provide the foundation for resuscitation theory and an innovation in drugs on treatment of stroke.

如何促进应激颗粒(Stress Granule, SG)是中风修复的关键问题, RNA结合蛋白为调控SG关键分子。在中医开窍理论指导下，我们发现，开窍急救名方醒脑静及其配伍开窍药促进SG形成和RNA结合模序蛋白3（RBM3）表达上调。因此，提出“开窍有效成分选择性作用于RBM3所产生的SG，抗脑缺血损伤的整合作用"假说。以无血清诱导细胞应激颗粒为模型，构建RBM3启动子驱动的荧光素酶报告基因系统，阐明RBM3对开窍有效成分反应元件，与RBM3启动子作用的靶蛋白。建立RBM3调控 SG靶mRNA的关键技术，采用RNAi技术封闭RBM3，或转染RBM3到 SG细胞模型中，阐明开窍有效成分调控RBM3影响 SG形成，抗细胞凋亡损伤分子机理。在脑缺血动物模型进一步阐明开窍影响RBM3和SG，抗脑缺血凋亡机制。本项目促进中医开窍理论发展，为开窍在临床防治中风提供理论依据，为新一代抗中风新药奠定基础。

急性缺血性中风是当今致死率和致残率最高的急危重症，因此如何降低缺血性中风损伤，是影响中风患者治疗和恢复效果的关键问题。前期研究发现促进应激颗粒(Stress Granule, SG)形成是可以显著降低脑缺血损伤,而RNA结合蛋白是调控SG的关键分子。通过构建MCAO大鼠模型，筛选出脑缺血特异性表达的microRNAs。大鼠MCAO模型免疫组化结果显示,TIA1表达在再灌注6h时明显上升，在24h水平时降低。构建无血清刺激SG细胞模型，发现无血清刺激18h后的SG产生最为明显。在无血清刺激模型中加入开窍药物有效成分进行干预，通过qRT-PCR和流式细胞法实验发现，左旋樟脑组和甲基丁香酚组的RBM3表达和TIA1表达显著增加。对PC12细胞中的RBM3进行沉默，发现在无血清刺激条件下SG生成减少，细胞凋亡率增加。RBM3免疫沉淀实验和LC-MS/MS质谱分析结果发现，RBM3与G3BP1和多种核转运蛋白相互结合。RNA免疫共沉淀技术（RIP）发现，RBM3与miR-335的加工成熟密切相关。构建PC12细胞的糖氧剥离刺激模型（OGD），采用免疫共沉淀实验和免疫荧光实验发现RBM3与METTL3和G3BP1的相互结合。构建PC12细胞的RBM3和METTL3的基因敲除模型和过表达模型，发现RBM3和METTL3促进 OGD刺激下的SG生成和成熟体miR-335增加。通过左旋樟脑药物干预，发现左旋樟脑降低大鼠MCAO模型的凋亡水平，升高大鼠MCAO模型中的METTL3表达、m6A水平、miR-335生成和SG生成水平。以上结果证实本课题“开窍有效成分选择性作用于RBM3所产生的SG，抗脑缺血损伤的整合作用”的假说，为中医开窍理论发展和开窍中药的临床应用提供理论依据，为研发新一代抗中风新药奠定基础。研究成果已发表SCI收录论文6篇，完成2名博士学位论文,3名硕士学位论文。