丹参酮ⅡA靶向ET-1抗缺血性脑损伤的内源干细胞修复及蛋白组学机制研究

Previous studies have shown that tanshinoneIIA (Tan IIA) has neuroprotective effects on rodents after focal cerebral ischemia, but its exact roles and mechanisms are still largely not clear. Based on our previous findings, after tMCAO, transgenic mice with overexpression of astrocytic ET-1 (GET-1) show more severe brain damage with larger infarct area, brain edema and BBB breakdown. The increased brain damage in the GET-1 mice results from activation of ETA receptors and increased in oxidative stress. Recent pharmacological studies have shown that tanshinone Ⅱ A can down-regulate the expression of receptor ETA receptor, regulate ET-1-induced brain microvascular endothelial function. Our preliminary studies have shown that ET-1, as a mitotic factor, affects the process of differentiation and differentiation of endogenous stem cells after cerebral ischemia. Therefore, the present study hypotheses that tanshinone IIA targets ET-1 and regulates stem cell repairment against ischemic brain injury. Through the proteomic study, the mechanism and the protective effect of tanshinone ⅡA on cerebral ischemic injury will be explored. By comparing the transgenic mice to that of wild-type mice after tMCAO, the present study will also shed light on the role of tanshinone ⅡA on the survival, proliferation, differentiation and nerve repairment of stem cells and other aspects. In a nutshell, the present study is to elucidate the mechanism of tanshinone ⅡA in promoting neuronal repairment after ischemic brain injury, and to develop new methods of stem cell therapy and potential therapeutic drugs for treating ischemic stroke.

丹参酮ⅡA（tanshinone IIA）对局灶性脑缺血损伤大鼠具有神经保护作用，但是其作用靶点尚不明确，本课题组基于星型胶质细胞ET-1过表达鼠（GET-1）脑缺血再灌注损伤模型，发现星型胶质细胞ET-1过表达以及ETA受体的激活增加了脑缺血损伤后过氧化损伤，引起更严重的神经功能缺损，现代药理研究显示丹参酮ⅡA能够下调受体ETA受体的水平，减少ET-1产物来调节脑微血管内皮功能。我们还发现ET-1影响脑缺血后内源性干细胞的增值与分化过程，因此本项目提出丹参酮ⅡA 靶向ET-1抗缺血性脑损伤的内源干细胞修复及蛋白组学机制研究，探讨丹参酮ⅡA对脑缺血损伤的保护作用及干细胞存活、增殖、分化和神经修复等方面的影响，并通过对疾病组织和正常组织，用药前后比较，得到特定疾病的标志性蛋白和关键蛋白，阐明丹参酮ⅡA促进缺血性脑损伤后神经修复的位点，为干细胞治疗和药物开发新的靶点提供策略。

缺血性脑血管疾病是临床上常见的疾病，短暂的缺血造成的神经元的损伤是可逆性的，但持久的缺血必然造成脑神经元坏死或凋亡，及早通过药物以及介入等恢复脑血流灌注尤为重要。丹参酮ⅡA（tanshinone IIA）对局灶性脑缺血损伤大鼠具有神经保护作用，但是其作用靶点尚不明确。星型胶质细胞释放的物质内皮素 ET-1（endothelin-1）能够增加血脑屏障的通透性，在脑损伤中发挥着重要的作用。本课题组通过优化PCR 扩增条件和琼脂糖凝胶检测条件，筛选出纯合子的GET-1转基因小鼠开展动物实验。通过构建星形胶质细胞（C6）和神经干细胞（NSCs）共培养体系，建立氧糖剥夺再灌注（OGD/R）模型，发现丹参酮ⅡA可促进正常及OGD/R损伤的NSCs的增殖；还可促进共培养体系中NSCs增殖个数和神经球的直径。此外，丹参酮ⅡA可促进共培养体系中NSCs分化为神经元。通过制备星型胶质细胞ET-1过表达鼠（GET-1）脑缺血再灌注损伤模型，研究丹参酮ⅡA对缺血性脑损伤恢复期神经再生的调节作用，发现丹参酮ⅡA治疗可以缓解tMCAO损伤导致的小鼠体重降低，减少梗塞面积，还可显著改善神经功能，转角测试中右转率降低，转棒测试中棒上停留时间延长，同时，在踏空测试中踏空率显著降低。并且，可以减轻梗塞区域神经元的凋亡；增加小鼠海马NSCs的数目和分布范围，促进NSCs分化为神经元。蛋白组学研究发现其差异蛋白表达与调控脂质代谢及铁代谢等多个通路作用有关。本项目阐明丹参酮ⅡA 促进缺血性脑损伤后神经修复的位点，对提高脑缺血患者生存质量有着十分重要的科学意义和临床应用价值。