基于组蛋白H3K9ace表观遗传修饰的电针抗缺血性脑损伤机制研究

Acupuncture promotes recovery of neurological function by the overall improvement of brain ischemic injury. It has been regarded as a potential rehabilitative treatment for stroke. However, its mechanism has not fully elucidated, such as acupuncture how to achieve synergistic neuroprotective effects of multiple targets. Histone H3 acetylation plays an important role in the development, maturation and damage repair of central neural system by regulated genes transcript activity and expression. Our previous studies showed that acupuncture treatment could reverse 30% to 50% cerebral ischemia-reperfusion-induced gene expression abnormalities, and increased acetylation of histone 3 lysine 9 (H3K9ace) significantly. Additionally, we found that acupuncture enhanced H3K9ace modification in the promoter region of target gene, and enhanced gene transcription activity. Based on these findings, we propose that acupuncture may achieve anti-brain injury effect by regulating the acetylation of H3K9ace. To confirm this hypothesis, neurobiological, histomorphological and molecular techniques, such as ChIP assay, will be applied to explore the role of H3K9ace in the neuroprotective mechanism of acupuncture on ischemic stroke. This study will provide scientific evidences at molecular and epigenetic level for acupuncture treatment, which is an effective therapeutic approach for brain injury.

针刺促脑中风后康复机制研究已深入到基因水平，尚未完全阐明，如针刺如何实现多靶点协同调节效应？最新研究显示，组蛋白H3乙酰化介导的表观遗传修饰能全面影响基因转录和表达，在神经系统损伤修复中具有重要地位。我们预实验结果显示针刺可逆向调节30%~50%由脑缺血再灌注所诱发的异常表达基因；同时组蛋白3第9位赖氨酸乙酰化（H3K9ace）修饰亦明显增加。因此，我们认为“针刺通过增强靶基因启动子区H3K9ace修饰提高其转录活性，实现多靶点抗脑损伤效应”。围绕该假说，本课题基于针灸整体调节理论，以缺血性脑中风模型为载体，在前期筛选明确针灸抗脑损伤靶基因基础上，运用神经生物学、组织形态学及染色质免疫沉淀分析（ChIP assay）等分子生物学技术，从H3K9ace修饰调节基因转录活性角度阐明其抗脑损伤的多靶点调控机制；以期为针刺抗脑损伤机制研究提供新的思路，为针刺抗脑损伤的临床运用提供科学的实验依据。

大量研究显示组蛋白H3乙酰化介导的表观遗传修饰在神经系统的发育、成熟及损伤修复中具有重要地位，本课题组前期研究证实针刺通过增强靶基因启动子区H3K9ace修饰提高靶基因转录活性，因此我们认为针刺可能是通过调节组蛋白乙酰化修饰实现多靶点抗脑损伤作用。本研究通过选取百会穴作为治疗穴位，以缺血性脑中风为研究对象，首先利用组织形态学、神经生物学技术检测各组大鼠血脑屏障致密性、脑缺血损伤体积、神经细胞再生及大鼠神经功能损伤情况，验证了针刺治疗可以显著降低血脑屏障通透性、减少缺血后损伤体积与细胞凋亡，同时促进神经细胞再生与神经功能的恢复，对抗缺血再灌注对大鼠脑组织所造成的损伤作用；随后通过检测中风动物针刺治疗前后脑内HDAC与HAT活性变化，各类HDACs在脑内表达的改变以及组蛋白3表观遗传修饰蛋白的变化，再结合H3K9ace 、H3K27ace在TIMP1、TIMP2、MMP9 及Caspase-3、bax、bcl-2 基因启动子区域的富集程度与相关因子mRNA表达水平变化的关系，证实针刺治疗可以在下调缺血后脑组织内 HDAC活性的同时上调HAT酶活性，增加H3K9、H3K27乙酰化修饰水平，从而直接调控DNA 靶序列转录活性以达到抗脑损伤作用；从组蛋白表观遗传修饰角度阐明针刺多靶点抗脑损伤的调控机制；将针刺抗脑损伤作用的分子生物学机理研究推进到表观遗传学水平；并为针刺抗脑损伤的临床运用提供科学的实验数据。