丹参酮ⅡA微乳通过调控组蛋白乙酰化保护脑缺血再灌注损伤的分子机制

Acute ischemic stroke is a leading cause of death, disability, and dependence in worldwide, the effective methods of neuroprotection and nerve repair have remained unsolved.Tanshinone ⅡA against ischemia reperfusion brain injury on oxidative stress, inflammatory reaction and apoptosis inhibition, but the control mechanism and regulation of tanshinone ⅡA are not clear. Histone acetylation plays an important role in neuroprotection and nerve repair, and the epigenetic effect of tanshinone ⅡA is also discovered. While, whether the tanshinone ⅡA has the neuroprotective effect through a special pathway of the epigenetic mechanism is still unknown. The applicant engaged in the effect of Chinese medicinal herb against cerebral ischemia reperfusion injury. Our previous study showed tanshinone ⅡA microemulsion can permeate through the blood brain barrier and significantly improve nerve protective effect. In present study, with transient focal cerebral ischemia model induced by MCAO, the neuroprotection of tanshinone ⅡA microemulsion is evaluated by ELISA, Western blot and CHIP-PCR method, to explore tanshinone ⅡA microemulsion against cell apoptosis, toxicity of inflammatory cytokines, excitement, and nerve protection effect through regulating histone acetylation. In addition, the establishment of hippocampal neurons OGD/R model, further verify the hypothesis at cellular level, and provide a new target and mechanism for future clinic application.

缺血性脑卒中发病、致死、致残率高，缺血后再灌注引起严重的迟发性神经元损伤，临床上尚无有效神经保护与修复方法。丹参酮ⅡA对缺血再灌注脑损伤的氧化应激、炎性反应及细胞凋亡等环节具有抑制作用，但其调控机制不清。组蛋白乙酰化在神经保护与修复中具有重要意义，丹参酮ⅡA等丹参活性物已被发现有相关调控作用，但其是否与上述神经保护作用有特异通路关联尚未见报道。申请者前期研究发现丹参酮ⅡA制成微乳后能快速透过血脑屏障，进一步将其应用于大鼠脑缺血再灌注脑损伤模型，结果表明与丹参酮IIA溶液比较，丹参酮IIA微乳能够显著增强神经保护作用。本课题拟继续将丹参酮ⅡA微乳应用于大鼠缺血再灌注脑损伤模型，探讨丹参酮ⅡA微乳通过调控组蛋白乙酰化，参与抑制细胞凋亡、炎性细胞因子、兴奋毒性，进而起到神经保护作用。此外，建立海马神经元OGD/R模型，在细胞水平进一步验证假设，为丹参酮ⅡA微乳临床应用阐明新的靶点机制与适应症。

本研究建立了大鼠 MCAO 缺血/再灌注损伤模型，采用 ELISA法检测大鼠脑组织中HDAC活性，双重免疫荧光染色H3K18ac/MAP-2和H4K8ac/MAP-2，Western blot分析H3K18ac、H4K8ac、NMDAR1、Caspase-3及MAP-2的表达，RT-PCR分析了NMDAR1、Caspase-3的基因表达情况。原代培养海马神经元，建立OGD/R模型，MTT法检测细胞毒性，LDH法检测细胞膜完整性，TUNEL法检测细胞凋亡，双重免疫荧光染色H3K18ac/MAP-2和H4K8ac/MAP-2，Western blot分析H3K18ac、H4K8ac、NMDAR1、Caspase-3及MAP-2的表达，RT-PCR分析了NMDAR1、Caspase-3的基因表达情况。在动物与细胞水平验证了TanⅡA ME对缺血再灌注损伤的神经保护作用，明确了TanⅡA ME通过调控组蛋白 H3K18、H4K8 乙酰化，参与抑制 NMDAR1和Caspase-3的表达以及增加MAP-2的表达，进而起到脑缺血再灌注损伤的神经保护作用。