黄芩苷与栀子苷配伍抑制小胶质细胞过度活化诱导5-lox酶抗脑缺血恢复期炎性损伤的机制研究

During cerebral ischemic recovery stage，it is not clear that microgial overactive induce cerebralinflammatory injury.We have found that compatibility of Baicalin and Geniposide has proctive effect by inhibiting 5-LOX / CysLTs pathway on brain of rats based on .So we establishment cerebral ischemic injury model by tie the bilateral carotid, using Wester-blot methods and applied 5-LOX blockers( zileuton) and CysLTsR blockers( montelukast), to detect the 5-LOX/CysLTsR inflammatory pathway changes in ischemic brain, explore cerebral ischemia caused by the recovery path changes microglia activation and 5-LOX in ischemic brain tissue recovery high expression sites and laws , and to explore the relationship between the two. Further pretreated with drugs and blocker on microglia .Meanwhile using transfection methods make 5-Lox and OX-42 gene overexpression and silencing, from the molecular level to explore excessive activation of microglia and 5-LOX receptor expression induced change of 5-LOX/CysLTsR pathway,thus to clarify the mechanisms of cerebral ischemic inflammatory injury though 5-LOX/CysLTs pathway induced by excessive activatate microglial ,and provide experimental evidence and theoretical basis for the prevention and treatment of cerebral ischemic injury antipyretic and drug development .

脑缺血疾病恢复期，小胶质细胞过度活化介导脑缺血炎性损伤的机制不明确，在前期研究发现栀子苷配伍黄芩苷通过抑制5-LOX /CysLTs受体通路抗脑缺血炎性损伤基础上，本项目复制脑缺血模型，采用Wester-blot等方法，应用5-LOX阻断剂齐留通和CysLTs受体阻断剂孟鲁司特，研究脑缺血炎性损伤的5-LOX/CysLTsR 通路变化，探索脑缺血恢复期引起该通路变化的小胶质细胞活化过程及5-LOX在缺血恢复期脑组织表达规律。进一步用药物和阻断剂预处理小胶质细胞，转染法使5-LOX和OX-42基因过表达和基因沉默，从分子层次研究小胶质细胞过度活化与5-LOX表达的相互影响，诱导5-LOX/CysLTsR 通路变化过程，力求阐明小胶质细胞过度活化促进5-LOX/CysLTs 通路变化是脑缺血恢复期炎性损伤的机制，为清热药有效成分防治脑缺血损伤及新药研发奠定基础。

脑缺血疾病恢复期，小胶质细胞过度活化介导脑缺血炎性损伤的机制不明确，清热功效方药治疗脑缺血恢复期的生物学基础不清楚。本项目在中医药配伍理论指导下，采用现代网络药理与实验研究技术，从整体、细胞和分子层次开展了脑缺血疾病恢复期的生物学基础和分子机制研究，探讨清热药有效成分黄芩苷、栀子苷（BC/GD）及其配伍的作用靶点和机制。BC/GD配伍后可以持续降低脑缺血大鼠恢复期脑组织CysLTs含量，该效果在1-21d更为显著，BC/GD配伍均能对5-LOX/CysLTs/CysLT通路产生抑制作用；1-21d天中，小胶质细胞激活增加，5-LOX表达增高，BC/GD配伍各组均可以抑制小胶质细胞激活，降低5-LOX表达；细胞实验发现BC/GD配伍抑制小胶质细胞分泌促炎因子TNF-a和IL-lβ，促进小胶质细胞IL-10和TGF-β抗炎因子和神经保护因子的分泌；诱导小胶质细胞由M1型向M2型极化状态的转变；抑制5-LOX/CysLTs通路，下调5-LOX、CysLT1、CysLT2蛋白的表达；网络药理学研究发现黄芩苷、栀子苷抗脑缺血损伤相关靶点45个，KEGG数据库提取脑缺血相关通路26条，与脑缺血及其再灌注损伤密切相关的生物功能6个，其中与靶点连接度高的功能是抗炎作用。以上结果揭示缺血后脑炎性损伤的5-LOX/CysLTsR 通路变化是脑缺血恢复期病理表现的生物学基础；阐明小胶质细胞过度活化促进5-LOX/CysLTs 通路变化是脑缺血恢复期炎性损伤的机制，发现5-LOX、CysLTs、CysLTsR是脑缺血恢复期炎性损伤的作用靶点之一，BC/GD配伍可以通过抑制5-LOX/CysLTs通路，下调5-LOX、CysLT1、CysLT2蛋白的表达治疗脑缺血，为清热药有效成分防治脑缺血恢复期损伤及药物研究开发奠定基础。