染色质重塑介导的针刺抗心肌缺血损伤机制研究

Angina pectoris (or angina) is a medical condition related to myocardial ischemia. Although acupuncture is widely accepted as an effective clinical treatment for angina cross the world, its mechanisms stay unclear. To today, epigenetic regulations of gene expression have been shown to be contributed to etiology and pathology of cardiovascular diseases, and modifications of epigenetic marks could be developed to be new targets for the treatment of these diseases. Our previous study showed that acupuncture regulated histone acetylation of VEGF gene and promoted angiogenesis in the ischemic myocardium in rats. We now propose that acupuncture could play anti-ischemic roles through regulation of histone remodeling at functional gene loci. To test this proposal, we first will carry out a clinical study to confirm the effects of acupuncture on angina pectoris patients, and determine gene expression profiles using RNA-seq and histone remodeling by ChIP-seq of H3K4me1，H3K4me2, H3k27ac and H3K27me3 marks. To verify the effects of acupuncture on histone remodeling, we generate conditional knockout mice in whose hearts the methyltransferase MLL4 controlling mono- and di-methylation of H3K4 is deleted. Using these mice, we will observe the protective effects of acupuncture on myocardial ischemia, and then determine the profiles of gene expression and histone remodeling by employing RNA-seq and ChIP-seq technologies. Our research will provide solid evidences that protection of acupuncture against myocardial ischemic injury is mediated by the regulation of histone remodeling, which could shed light on mechanistic studies and clinical applications of acupuncture.

针灸保护缺血心肌机理研究已深入基因水平，但尚未完全阐明，如针灸如何影响基因表达，介导基因转录，实现保护效应？组蛋白修饰介导的染色质重塑能有效调控目标基因表达，实现心肌保护。我们前期研究发现，针刺通过调节组蛋白乙酰化促进血管新生实现心肌保护。因此我们认为“针灸保护缺血心肌是通过调节组蛋白修饰和影响染色质重塑来实现的”。围绕该假说，本课题将从慢性稳定性心绞痛病例入手，采用高通量测序技术RNA-seq获得针刺前后基因表达谱的改变；用ChIP-seq对染色质重塑标记物H3K4me1、H3K4me2、H3k27ac和H3K27me3在基因上的结合进行系统生物学分析，证实针刺治疗该病与染色质重塑密切相关。为验证此发现，运用甲基化酶MLL4基因敲除小鼠，观察干扰H3K4甲基化后针灸对缺血心肌保护及基因表达谱影响，初步明确染色质重塑介导针刺保护缺血心肌作用机制，为针灸治疗缺血性心肌病临床运用提供科学依据。

本课题围绕染色质重塑介导针刺对缺血心肌所起的保护作用展开，揭示其中可能的调控机制。首先针对临床慢性稳定性心绞痛患者，探讨针刺内关穴对该疾病的治疗作用，明确了针刺内关穴能够有效减少患者心绞痛发作次数和硝酸甘油用量，降低患者VAS疼痛评分，改善患者与心绞痛严重程度相关的中性粒细胞与淋巴细胞比率；通过高通量测序中性粒细胞的全基因表达谱，发现针刺内关穴导致表达改变的基因与炎症、免疫、能量代谢等信号通路相关，特别是IL8等与免疫调节有关的基因。.随后，通过针刺干预心肌缺血的小鼠模型，发现针刺内关导致小鼠心脏组织组蛋白H3K4甲基化和H3K27乙酰化水平增加，同时针刺之后心肌梗死面积减少，血管新生标志物CD31和VEGF表达升高，提示针刺可能增加了缺血心肌的血管新生。进一步通过Mll4心肌细胞条件性敲除转基因小鼠模型，发现Mll4心肌细胞敲除后，针刺使缺血心脏组织组蛋白H3K4甲基化增高的效应被减弱，同时针刺增加VEGF表达的效应也接近丧失，提示Mll4可能参与介导了针刺保护心肌缺血损伤的效应。为深入研究Mll4在心肌缺血动物模型中发挥作用的分子机制，采用高通量测序Mll4条件敲除后缺血心肌组织中的基因表达谱，发现MLL4基因参与了心梗后细胞增殖分裂、纤维化以及低氧应答反应HIF1a等相关信号通路。总之，本课题基本阐明了针刺内关穴对心脏缺血损伤的保护作用，其发生作用的分子机制可能是由于针刺内关穴促进心脏缺血区的血管新生，缓解心肌细胞缺氧损伤。血管新生的关键因子VEGF表达在针刺时增加，可能是由于心肌细胞内Mll4介导的组蛋白甲基化代表的染色质重塑改变引起的。