MEF2对TGF-β的转录调控在异氟醚后处理抗脑缺血再灌注损伤中的作用

Cerebral ischemia-reperfusion injury plays a key role in neuronal dysfunction, and effective treatments are still limited. Isoflurane is a common clinic volatile anesthetic. Numerous studies established that isoflurane postconditioning can induce neuroprotective effect. However, mechanism of it remains unclear. Our previous studies demonstrated isoflurane postconditioning could reduce cerebral ischemia-reperfusion injury via modulating the TGF-β1/Smad-MAPKs signaling pathway in rats' hippocampal. Nevertheless, mechanism of transcriptional regulation in TGF-β signaling pathway is still unclear. Most research articles indicate MEF2 is often functionally expressed during process of ischemia-reperfusion in rats' brain. In addition, MEF2 is vital for transcriptional regulation in TGF-β signaling pathway. We assume that neuroprotective function of isoflurane postconditioning in cerebral ischemia-reperfusion is regulated by MEF2 through TGF-β signaling pathway and inflammatory pathway, which will activate the related signaling proteins’ expression by signaling crosstalk. They may be the new mechanisms of isoflurane in the aspect at non-anesthetic effect. To prove this hypothesis, ischemia-reperfusion experimental model will be established in vivo and in vitro of rats brain; measures of molecular biology, immune fluorescence technology and chromatin immunoprecipitation will be taken to observe the regulative effects of MEF2 through TGF-β signaling pathways in isoflurane postconditioning in rats. To testify this new mechanism of isoflurane, technology of etholoy, functional proteomics and cytology will be adopted. We anticipate results of our researches will provide the new theoretical basis for precaution of cerebral ischemia-reperfusion injury.

脑缺血再灌注损伤是导致神经功能障碍的重要原因,但其有效的治疗措施十分有限。异氟醚是临床上常用吸入麻醉药物,以往研究提示其后处理可对脑缺血再灌注损伤产生神经保护作用,然而其作用机制尚不清楚。本课题组研究发现异氟醚后处理可以通过调节TGF-β1/Smad-MAPKs信号通路减轻大鼠海马区的神经损伤,然而,异氟醚后处理后,TGF-β的转录调控机制尚且未知。研究提示在大鼠脑缺血再灌注损伤后MEF2转录因子有功能表达,且MEF2可调控TGF-β转录水平,因此我们提出假说:异氟醚后处理大鼠脑缺血再灌注损伤时,可通过MEF2上调TGF-β转录水平,通过抑制炎症反应,产生神经保护作用。本课题拟通过建立大鼠模型,采用分子克隆和基因沉默技术、电生理技术、免疫荧光、ChIP等技术,阐述MEF2对TGF-β的转录调控在异氟醚后处理对抗脑缺血再灌注损伤中的作用,为异氟醚后处理诱导的脑保护作用找到新的作用机制。

脑缺血再灌注损伤常导致神经细胞的炎性损伤级联反应，引起神经细胞结构和功能的变化，且发生率高，目前尚无有效的治疗手段。研究发现MEF2 作为TGF-β信号通路下游重要的转录因子，调控异氟醚后处理中TGF-β信号通路对TLR4 炎性通路的转录作用，但MEF2转录因子作用于异氟醚和TGF-β信号通路的具体机制尚不清楚，也是我们本项目研究所关注的重点。在本项目研究中，我们通过建立大脑中动脉栓塞（MCAO）模型和离体脑片氧糖剥夺（OGD）模型，选用吸入麻醉药异氟醚进行后处理治疗，成功构建了AAV·MEF2D·RNAi，并通过脑立体定位靶向注射进行脑内顶叶皮层、海马区域的MEF2基因精准敲低，通过神经行为学研究、神经电生理技术、免疫荧光标记技术及分子生物学等技术，分别从离体和在体实验层面，以神经元、小胶质细胞的结构功能变化为切入点，深入探讨了MEF2 对TGF-β信号通路转录调控在异氟醚后处理脑缺血再灌注损伤中关键作用。研究结果发现：TGF-β3/Smad3信号通路及MEF2C在异氟醚后处理诱导脑保护中的作用机制。异氟醚通过激活MEF2D调控焦亡信号通路下游分子IL-18的表达水平减轻神经元焦亡现象。异氟醚后处理调控MEF2D进一步转录Activin A/P38信号通路减轻脑缺血再灌注损伤后内质网应激。异氟醚亦可激活AMPK/ULK1信号通路上调自噬抑制NLRP3炎症小体释放IL-1β、IL-18炎症因子减轻CIRI大鼠海马神经元损伤并改善大鼠神经功能及认知学习记忆功能。MEF2D靶向转录调控HMGB1/TLR4炎症通路促进大鼠海马小胶质细胞向抗炎表型转化对抗神经炎症，减轻大鼠卒中后抑郁样神经行为。整体研究紧密围绕肌细胞增强因子MEF2这一研究目标，为揭示MEF2的生物学功能参与吸入麻醉剂异氟醚发挥抗缺血缺氧性脑损伤保护作用的中枢作用机制提供了实验依据。拓展了临床麻醉学内涵，为围术期合理使用吸入麻醉药、围术期脑卒中的麻醉治疗角度提供了新的的参考价值。