回药木香油方抗脑缺血再灌注损伤的物质基础及对NLRP3信号网络的影响

Ischemic stroke is the most common disease and is associated with high mortality and morbidity, has seriously threatened the public health. "HuiHui prescription" is the direct original version on Hui medicine research, which documented the treatment of ischemic with MU Yangyou prescription. Our previous research showed that Mu Xiangyou prescription has a protective effect against focal cerebral ischemic injury by attenuating the mitochondrial apoptosis pathway. In this study, the hypothesis " Mu Xiangyou prescription's protective effect against focal cerebral ischemic injury relating to the chemical constituents that migrating to blood and tissue, the mechanism of this chemical constituents mainly by inhibiting the ROS-NLRP3-caspase 1 and TLR4-NFκB-NLRP3 pathways" was put forward.In this sdudy , the method of determination of the chemical constituents that migrating to blood and tissue of Mu Xiangyou prescription by GC-MS and HPLC-MS. The models of hippocampal slices and hippocampal neurons after oxygen –glucose deprivation /reperfusion injury to be used for screening protective effect of Mu Xiangyou prescription. The methods of immunohistochemistry, Western blot, RT-PCR will be used to test the oxidative stress factors, inflammatory factors and apoptosis factors in NLRP3 pathway. To reveal whether the chemical constituents that migrating to blood and tissue of Mu Xiangyou prescription could attenuate focal cerebral ischemia-reperfusion injury in rats through regulation of NLRP3 pathway.

缺血性卒中发病率、致残率高，危害大。我们前期实验表明，收载于《回回药方》的木香油方可以通过抗细胞凋亡对脑缺血再灌注损伤起保护作用。但木香油方中哪些成分起作用，药效成分具体作用机制是什么亟待研究。本研究提出“木香油方抗脑缺血再灌注损伤作用主要与其入血/组织成分有关;药效成分机制是通过抑制ROS-NLRP3-Caspase1和TLR4-NFκB-NLRP3两个通路所体现”的假说。本项目拟首先采用GC-MS/HPLC-MS分析木香油方的化学成分及入血/组织成分及代谢成分；其次，对入血/组织成分及代谢成分分别采用膜片钳和细胞培养方法对氧糖剥夺损伤的大鼠海马脑片和海马神经元进行药效作用筛选；最后，采用免疫组化、Western blot及RT-PCR等方法和手段，以NLRP3信号网络的两个通路相关因子为指标，对筛选出的有效成分采用以上两个体外筛选方法和大鼠MCAO再灌注损伤模型进行机制研究。

缺血性卒中发病率、致残率高，危害大。我们前期实验表明，木香油方对脑缺血再灌注损伤起保护作用。但木香油方中哪些成分起作用，药效成分具体作用机制是什么亟待研究。本项目首先采用GC-MS/HPLC-MS分析木香油方的化学成分及入血成分；体外成分分析并标定去氢木香内酯、花生四烯酸甲基酯、甲基丁香酚、胡椒碱、石竹烯、木香烃内酯、芝麻素、肉豆蔻醚8个成分。入血成分标定了35个成分。对木香油方、去氢木香内酯和木香烃内酯分别采用氧糖剥夺损伤的大鼠海马脑片和PC12细胞进行药效作用研究，结果说明，木香油方和去氢木香内酯和木香烃内酯对海马脑片和PC12细胞损伤均有保护作用。采用免疫荧光、Western blot等方法和手段，观察木香油方、去氢木香内酯和木香烃内酯的抗凋亡及抑制自噬的作用，结果说明，木香油方、去氢木香内酯和木香烃内酯的抗凋亡作用与线粒体介导的caspase3通路有关，抑制自噬与AMPK/mTOR通路有关。其结果为临床应用提供了实验支持。在本项目经费支持下基本完成了项目预期内容，发表期刊论文3篇，培养硕士研究生3名，本科生3名。