LOC339524调控小胶质细胞炎症反应对异氟烷后处理脑保护的影响与分子机制研究

Treatment and prevention of ischemia-reperfusion injury (IRI) in a stroke or cerebral resuscitation is still very difficult. Isoflurane postconditioning (IPC) can produce brain protection by inhibiting inflammation produced by cerebral ischemia-reperfusion injury, but the mechanisms need further study. LOC339524 is a new protein and separated and identified by applicant from IRI heart for the first time.Monoclonal antibodies of LOC339524 has already been prepared. Early studies have shown that hypoxia can induce high expression of LOC339524 in microglia, down-regulated expression of LOC339524 can significantly inhibit the expression of inflammatory cytokines induced by LPS in microglia. Isoflurane postconditioning can produce cerebral protection by inhibiting IRI-induced LOC339524 high expression in microglial cells.So we speculate theoretically that LOC339524 mediates the inflammatory reaction induced by IRI, IPC produce cerebral protection by regulating the expression of LOC339524. We plan to use gene over-expression and gene knockout technology, and study the molecular mechanisms for the effect of LOC339524 on regulation of microglial inflammation and cerebral protection produced by isoflurane postconditioning on model of oxygen glucose deprivation and focal cerebral ischemia. This project aims to elucidate the anti-inflammatory mechanism of IPC after cerebral ischemic damage and provide new targets for drug development of brain injury.

缺血再灌注损伤（IRI）的防治是脑中风、脑复苏等治疗中的难点，异氟烷后处理（IPC）可抑制脑IRI后炎症反应而产生保护作用，其机制有待深入研究。申请者前期从IRI心脏中分离、鉴定了LOC339524新蛋白并制备了单克隆抗体。前期研究显示，缺氧能诱导小胶质细胞高表达LOC339524，干扰该基因表达能明显拮抗LPS刺激诱导的小胶质细胞炎性因子表达，且IPC处理还能抑制IRI诱导的小胶质细胞LOC339524的高表达。由此推测LOC339524介导了IRI诱导的炎症反应，IPC通过对LOC339524的表达调控而产生保护效应。本项目拟在培养小胶质细胞氧糖剥夺和大鼠局灶性脑缺血模型上，采用基因过表达与基因敲除等技术，研究LOC339524对脑IRI中小胶质细胞炎症反应的调控及其在IPC介导的脑保护效应中的作用与分子机制，旨在进一步阐明IPC脑缺血损伤后的抗炎机制，为开发脑损伤治疗药物提供新靶点。

脑缺血/再灌注损伤（ischemia/reperfusion injury，IRI）是脑创伤、围术期脑卒中和心肺复苏后脑复苏等疾病或医疗事件的共同病理生理过程，对其的防治一直是脑损伤救治的重点和难点。脑IRI能通过诱导神经炎症并激活细胞内的信号通路引起神经细胞的损伤和死亡。异氟烷后处理（IPC）可抑制脑IRI后炎症反应而产生保护作用，其机制有待深入研究。本项目在前期研究的基础上，通过脂多糖（lipopolysaccharide，LPS）建立BV-2小胶质细胞的炎症损伤模型，构建了LOC339524高表达BV-2稳转细胞株、筛选了LOC339524的RNA干扰片段，研究了LOC339524调控小胶质细胞炎症反应及其分子机制。结果表明，小胶质细胞LOC339524的表达随LPS刺激浓度的增加和时间的延长显著上调，而上调的LOC339524能减少炎症因子和介质的表达；用RNA干扰LOC339524的表达，则炎症加重；采用定点突变技术和双荧光素酶实验，明确NF-κB可以结合到LOC339524启动子1509-1520区域并激活其表达；LOC339524可能是通过抑制Src-NF-κB和Src-p38MAPK信号通路来减轻炎症；此外，尽管LOC339524 RNA符合无义介导的RNA降解（nonsense-mediated RNA degradation，NMD）途径调控的经典结构，但通过上调或沉默NMD关键分子UPF1，发现LOC339524并不受NMD调控，其逃逸NMD调控可能是通过上调p-eIF2α来实现。本课题的研究结果为进一步阐明脑缺血损伤后的抗炎机制和开发脑损伤治疗药物提供新靶点。