从时序性差异调节角度研究药对川芎-当归干预缺血性脑损伤的分子机制

Because of the complicated pathological mechanism of ischemic brain injury, the integrated effect of intervention strategies in manner of dynamic regulation was paid more attention. HIF-1 alpha has a wide range of target gene clusters and plays an important role in ischemic stroke disease processes by regulating apoptosis and angiogenesis signaling pathways. The HIF-1α expression had a time ordered characteristic and was considered as a new therapeutic target. Our previous studies have shown that couplet medicines Chuanxiong-Angelica sinensis had clinical efficacy on ischemic stroke. In this study, a series of strategies including a combination of in vitro and in vivo experimental models, such as MCAO rats model, neurons and brain microvascular endothelial cells ischemia-reperfusion injury model will be used. Several analytical methodologies will be used to dissect the regulatory effect, molecular mechanisms and time ordered characteristic of couplet medicines Chuanxiong-Angelica sinensis by focusing on HIF-1α and its downstream signal pathway including VEGF/VEGFR pathway and BH3-only proteins/Caspases pathway. Pharmacodynamic mechanism differences between couplet medicines and HIF-1α degradation inhibitor will also be observed. This study will research drug targets and molecular mechanisms of couplet medicines Chuanxiong-Angelica sinensi in treating cerebral ischemia on the view angle of time ordered differential regulation, and provide basis for the clinical therapy.

缺血性脑损伤的病理机制复杂，干预策略在损伤后动态调节的整合效应日益受到重视。HIF-1α途径具有广泛的靶基因群，可同时调控凋亡和血管新生等信号通路，并具有时序性特征，在缺血性中风疾病进程中起重要作用，是治疗的新靶标。前期研究表明，养血活血、化瘀通络药对川芎-当归治疗缺血性中风临床有效，本研究拟以HIF-1α途径及其下游两条主要通路VEGF/VEGFR和BH3-only proteins/Caspases通路上功能性靶标群为研究主线，以MCAO大鼠模型及神经血管单元中的神经元和脑微血管内皮细胞损伤模型为研究对象，系统分析川芎-当归药对及主要单体成分对该研究主线上多个靶标群的调控效应、机制以及时序特征，与HIF-1α降解抑制物的药效机理差异以及对HIF-1α途径的依赖关系。从"时序性差异调节"角度系统研究药对川芎-当归治疗缺血性中风的作用靶点与分子机制，为养血活血化瘀通络药的临床应用提供依据。

缺血性脑损伤的病理机制复杂，干预策略在损伤后动态调节的整合效应日益受到重视。川芎-当归是中医治疗缺血性中风方剂中经典药对，但作用机制尚未完全明晰。HIF-1α具有广泛的靶基因群，通过调控凋亡和血管新生等信号通路，在疾病进程中起重要作用。本研究以HIF-1α途径为切入点，从“时序性差异调节”角度研究川芎-当归干预缺血性脑损伤的作用机制，获得以下结论：（1）以川芎-当归组成的中成药舒脑欣滴丸（SNX）对tMCAO大鼠脑I/R损伤具有保护效应，可减少梗死面积，减轻脑组织损伤，增加神经元存活，保护血脑屏障，抑制早期炎症反应；（2）川芎-当归经虚拟成分筛选出11个候选化合物，构建成分-靶点网络及中风病疾病-靶点网络，经PPI网络筛选获得234个核心靶点，KEGG分析富集得到HIF-1 pathway，以及PI3K-Akt pathway。（3）获得脑I/R损伤后HIF-1α表达的时空特征，6h~3d为第1时相，5d~7d为第2时相，在第1时相大量表达于半暗带皮质。SNX对HIF-1α表达干预具有时相差异，降低1d时表达，但增加3d时表达，该调控作用发生于蛋白水平而非mRNA水平。SNX可抑制第1时相BH3-only proteins/Caspases通路BNIP3、Nix、Noxa表达，降低Caspase3及细胞凋亡水平，增加第1时相VEGF/VEGFR通路VEGF、Flt-1、Flk-1表达，及7d时VEGF表达，促进血管新生。川芎-当归有效成分TMP、FA对OGD损伤PC12细胞具有保护作用，可剂量依赖地减低LDH、ROS、Caspase3水平，TMP:FA最佳配比为2:20。构建HIF-1α过表达质粒及siRNA/HIF-1α，明确了TMP和FA的保护作用依赖于抑制HIF-1α、BNIP3表达。明确了TMP、FA可增加缺氧/复氧及常氧时原代rBMECs的增殖及迁移。（4）采用iTRAQ蛋白组学技术获得SNX干预tMCAO模型大鼠不同时间的差异蛋白，经生物信息分析获得了急性期、亚急性期、慢性期蛋白组功能变化。聚类分析印证了SNX对HIF-1α pathway的时序性差异调节作用，经KEGG分析获得了神经修复再生相关的pathway。（5）明确了SNX可促进脑I/R损伤后神经修复，其作用机制为调控CXCR4/SDF -1轴CXCR4、SDF -1、ERK1/2、Akt表达。