circ-ATF6.28治疗缺血性脑卒中溶栓治疗后再灌注损伤的分子机制研究

Ischemia-Reperfusion Injury (IR Injury) is usually induced by thrombolytic therapy with brain stroke, which caused neuronal apoptosis. And endoplasmic reticulum stress (ERS), as one of mechanisms of endogenous apoptosis, has been proven to involve in the occurrence and development of brain stoke. However, the molecular mechanism is unclear now. In the preliminary works of the project, we found the abnormal expression of miR-29b-3p and the potential target gene GRP78/Bip in neuronal OGD models and hypothesized that the maker transmembrane protein GRP78/Bip in ERS might be the research target in Ischemia-Reperfusion Injury. The biological function of GRP78/Bip was reactivated by exogenous circ-AFB6.28 competitively combined with miR-29b-3p and ERS was suppressed. The apoptosis of nerve cells caused by Ischemia-Reperfusion Injury was reversed. In order to prove this hypothesis, the project studies their mechanism of action among circ-ATF6.28, miR-29b-3p and GRP78/Bip from external and internal environment and observes changes in Brain stroke. We hope the research can provide new ideas and the theoretical basis for clinic on Ischemia-Reperfusion Injury (IR Injury) caused by thrombolytic therapy with brain stroke.

通过溶栓来治疗缺血性脑卒中常会引发再灌注损伤，引起神经细胞凋亡，而内质网应激（ERS）作为内源性凋亡的机制之一，已被证实参与缺血性脑卒中的发生和发展，但其分子机制尚不清楚。本项目前期工作在神经细胞缺糖缺氧模型中发现异常表达的miR-29b-3p和潜在靶基因GRP78/Bip，进而假设ERS的标志性蛋白GRP78/Bip可能作为再灌注损伤的研究靶点，通过外源性circ-ATF6.28竞争性结合miR-29b-3p导致GRP78/Bip生物学功能的重新激活，抑制ERS，从而逆转再灌注损伤导致的神经细胞凋亡。为证实此假想，本项目分别从体内外环境对circ-ATF6.28、miR-29b-3p和GRP78/Bip间的作用机制进行研究并通过大鼠脑部切片来观察脑卒中的改变程度。希望本研究能够为缺血性脑卒中溶栓治疗后再灌注损伤的临床治疗提供新思路和理论依据。

缺血性脑卒中患者细胞外囊泡中的 miR-143-3p 表达水平明显高于健康对照组，说明 miR-143-3p 表达水平与脑卒中有一定的相关性。前期研究显示脑卒中细胞模型中，miR-143-3p 表达水平明显高于正常对照组，然而 miR-143-3p 下调具体机制尚不清楚。大量数据显示 circRNA-miRNA-mRNA 调控网络失衡会通过调节星型胶质细胞自噬影响脑卒中的发展。本研究以生物信息学分析为基础，筛选出 miR-143-3p 相结合的 circRNA 及mRNA，构建 circRNA-miRNA-mRNA 调控网络。然后以 A172 和 SK-N-AS 细胞模型和 C57BL/6J 大鼠动物模型为研究对象，观察 circRNA-miRNA-mRNA 调控网络在细胞和动物模型中的表达变化，同时通过慢病毒调控 circRNA-miRNA-mRNA 调控网络表达，探讨其对脑卒中疾病的影响。同时通过双荧光素酶报告基因检测circRNA-miRNA ， miRNA-mRNA 靶向结合 。 最 后 在 细 胞 水 平 上 探 讨circRNA-miRNA-mRNA 调控网络影响脑卒中相关机制。本课题证实 circRNA0025984 通过 miR-143-3p/TET1/ORP150 通路保护神经元，从而改善缺血性脑卒中损伤。