基于PI3K/Akt/mTOR信号通路研究丹参酮IIA对脑梗死后自噬激活的调控机制

Cerebral infarction is still lack of effective treatments except for the super early thrombolysis. Excessive autophagy activation after cerebral infarction results in cell death, which is a key target for the treatment of ischemic stroke. PI3K/Akt/mTOR is the upstream signaling pathways inducing autophagy. If the PI3K/Akt pathway blocked, mTOR activity was suppressed leading to autophagy. But the exact regulatory mechanisms of excessive autophagy activation after cerebral infarction is still unknown. Tanshinone IIA has nerve protective effect against cerebral infarction, but its regulatory mechanism is remain unclear. Our previous researches found that cerebral infarction along with PI3K/Akt inhibition. Moreover, tanshinone IIA can activate PI3K/Akt pathway. It is reasonable to speculate that Tanshinone IIA may activate the PI3K/Akt/mTOR signal and restrain its downstream autophagy and then have protective effects. Based on these above problems, this proposal intends to investigate the autophagy activation mechanisms mediated by PI3K/Akt/mTOR pathway after cerebral infarction, and tanshinone IIA regulation function on autophagy by building MCAO model and neurovascular unit. Cerebral infarction volume, cell vitality, a series of autophagy related proteins such as PI3K、mTOR、LC3 and their mRNA level will be examined. We anticipate that this project will provide experimental basis, new ideas and new methods for traditional Chinese medicine prevention and treatment of cerebral infarction.

脑梗死除超早期溶栓外尚缺乏有效治疗手段。脑梗死后自噬过度激活可致细胞死亡，是其治疗的关键环节；PI3K/Akt/mTOR是自噬上游信号通路，当PI3K/Akt抑制时，mTOR活性被抑制，诱导下游自噬。但脑梗死后自噬过度激活的调控机制尚不清楚。丹参酮IIA对脑梗死有神经保护作用，但其调控机制不明。前期研究发现，脑梗死后伴PI3K/Akt抑制，而丹参酮IIA可激活PI3K/Akt。因此有理由推测：丹参酮IIA可能激活PI3K/Akt/mTOR信号而抑制其下游自噬产生神经保护作用。本课题拟在大鼠MCAO模型与神经血管单元模型上，检测脑梗死体积，细胞活力，PI3K、mTOR、LC3等自噬相关蛋白及其mRNA水平等，从体内、体外两个层面，多角度诠释脑梗死后PI3K/Akt/mTOR通路介导的自噬激活机制及丹参酮IIA的调控作用，可望为脑梗死临床治疗提供实验依据和新思路，为中医药防治脑梗死提供新方法。

PI3K/Akt/m-TOR信号通路是调节细胞增殖、生长、分化、生存的重要通路，同时又是调节自噬的上游通路。既往研究发现脑梗死后伴自噬上游信号通路PI3K/Akt的抑制，而丹参酮IIA可激活PI3K/Akt，但丹参酮IIA能否因此而抑制其下游自噬尚需进一步研究。本项目将在研究过程中首先进行鼠海马细胞株的培养，进而构建鼠海马神经细胞OGD模型。我们通过观察普通培养以及缺氧缺糖培养后细胞的增殖变化，研究脑梗塞后对细胞的增殖影响，使用不同浓度的丹参酮IIA处理OGD模型中的神经细胞，研究丹参酮IIA对于细胞的神经保护作用（包括细胞增殖，氧化损伤，线粒体膜通透性，自噬作用）。基于PI3K/Akt/m-TOR信号通路探讨脑梗死后自噬过度激活机制及丹参酮IIA对脑梗死后自噬的调控机制，可望为脑梗死临床治疗提供实验依据和新思路，为中医药防治脑梗死提供新方法。