栝楼桂枝汤调控miR-155抑制缺血脑卒中后炎症反应的机制研究

The pathology of ischemic stroke include neuroinflammation, mediated by microglia, contributing to muscular spasticity followed by brain damage. Among various miRNA, miR-155, one of specific miRNAs in brain, has been described as being up-regulated and related with NF-kB signaling pathway in microglia upon their activation in post-ischemia. Many miR-155 targets are pro- or anti-infammatory genes and its expression leads modification of inflammation contributing to brain damage. Gua Lou Gui Zhi Decoction (GLGZD) has long been used in China to clinically treat dysfunction after stroke such as muscular spasticity, but the precise mechanisms are largely unknown. We have confirmed the neuroprotective and anti-spasticity activities of GLGZD Using a focal cerebral ischemia/reperfusion injured rat model, and it significantly reduced LPS-induced expression of cytokines and NF-kB activation in microglial cells, which demonstrated that the induction of miR-155 expression may be one of mechanisms of GLGZD treatment. Therefore, using Real-time PCR, Western-blotting, immunohistochemical staining and laser scanning confocal microscope after setting up MCAO animal and inflammtory cell model, we will further explore the mechanism of neuroinflammation after ischemic stroke regulated by Gualou-Guizhi decoction based on miR-155 regulation, and finding the target of GLGZD by cell transfection with miR-155 expression plasmid, providing theoretic foundation for clinical application of GLGZD.

缺血脑卒中后炎症是造成脑组织继发损伤引起脊髓反射亢进诱发痉挛的重要机制。在卒中后炎症反应中，miR-155是调节炎症相关基因及NF-kB炎症信号通路的关键脑特异性miRNA。课题组临床研究和动物实验已证实栝楼桂枝汤治疗卒中后痉挛有确切疗效，且前期研究表明该方可抑制小胶质细胞激活后炎症因子的产生及炎症信号通路的活化，提示栝楼桂枝汤的治疗作用可能与miR-155调节炎症基因及其信号通路有关。本项目在此基础上通过制备大鼠MCAO模型及体外细胞炎症模型，应用Real-time PCR、Western blot、免疫组织化学、激光共聚焦等方法探讨栝楼桂枝汤调控miR-155及其靶基因和NF-kB炎症信号通路的作用机制，并通过细胞转染miR-155过表达质粒验证该方的作用靶点，阐明栝楼桂枝汤调节miR-155抑制卒中后炎症、减轻神经细胞损伤缓解痉挛的分子机制，为临床用药提供基础理论依据。

缺血脑卒中后炎症是造成脑组织继发损伤引起脊髓反射亢进诱发痉挛的重要机制。在卒中后炎症反应中，miR-155是调节炎症相关基因及NF-kB炎症信号通路的关键脑特异性miRNA。课题组临床研究和动物实验已证实栝楼桂枝汤治疗卒中后痉挛有确切疗效，且前期研究表明该方可抑制小胶质细胞激活后炎症因子的产生及炎症信号通路的活化，提示栝楼桂枝汤的治疗作用可能与miR-155调节炎症基因及其信号通路有关。项目研究结果提示，栝楼桂枝汤能够通过NF-kB信号通路降低MCAO大鼠中缺血脑组织中炎症细胞的浸润及炎症因子、炎症相关基因的表达，这种调控作用可能与miR-155表达有关。同时，栝楼桂枝汤可抑制体外活化小胶质细胞中炎症因子及炎症相关基因的表达，这种抑制作用可能与NF-kB信号通路相关，并通过细胞转染过表达miR-155验证了该方的作用靶点。项目结果证实了栝楼桂枝汤调节miR-155抑制卒中后炎症、减轻神经细胞损伤缓解痉挛的分子机制，为临床用药提供基础理论依据。