基于“成分-网络靶点-效应”的荭草抗心肌缺血损伤有效成分群及作用机制研究

Polygonum orientale L. is a characteristic ethnic drug in Guizhou province, commonly used for treating cardiovascular diseases with exact efficacy. In our previous study, P. orientale was systematically studied including chemical compositions, quality process, pharmacological effects, serum pharmacochemistry, serum pharmacology, pharmacokinetics and PK-PD, result in that flavonoids, phenolic acids and alkaloids may be its pharmacodynamic material basis. However, the active ingredients that really plays a therapeutic role among these components and its mechanism are still unclear. Previous studies found that P. orientale can inhibit apoptosis, oxidative damage and regulate energy metabolism, suggesting the treatment of myocardial ischemia of P. orientale via multi-pathological approach and targets. In this study, the molecular targets of P. orientale are screened by network pharmacology based on its chemical constituents. Molecule docking is used to find effective components, and its efficacy is verified through H9c2 cells hypoxia injury model and myocardial ischemia injury model in rats. The effect of active components on regulation of key proteins is proved with protein inhibitors and agonists in vivo and in vitro by applying western blot, immunofluorescence and RT-qPCR are applied to confirm the active ingredients and mechanism of action of P. orientale for anti-myocardial ischemia, which will provide a theoretical basis for the development and application of P. orientale.

荭草是贵州治疗心肌缺血性疾病的特色品种，其疗效确切，具有较大的开发潜力。课题组前期研究了其化学成分、质量工艺、药理活性、血清药化、血清药理、体内过程和PK-PD相关性等，初步明确了黄酮类、酚酸类和生物碱类可能是其药效物质。但是在这众多成分中真正发挥治疗作用的有效成分群还不明确，其作用机制也不清楚。前期研究发现荭草能抗细胞凋亡、抗氧化损伤和调节能量代谢，提示荭草抗心肌缺血可能是通过多环节发挥作用的。因此，本课题基于前期研究基础，应用网络药理学预测荭草多途径作用靶标，利用分子对接方法遴选荭草活性成分群，通过H9c2缺氧和大鼠心肌缺血损伤模型验证荭草活性成分群抗心肌缺血的效应；结合抑制剂和激动剂，应用western blot、immunofluorescence、RT-qPCR等技术验证荭草活性成分群对关键蛋白的调节作用，阐释荭草抗心肌缺血的有效成分和作用机制，为荭草深度开发和应用提供科学依据。

荭草是贵州治疗心肌缺血性疾病的特色品种之一，具有较大的开发潜力。课题组前期的研究初步明确了黄酮类、酚酸类和苯丙素类可能是其药效物质。但是在这众多成分中真正发挥治疗作用的有效成分群不明确，其作用机制也不清楚，限制了荭草的深入开发和利用。本项目在前期研究基础上，结合网络药理学和分子对接技术遴选荭草的活性成分群，并利用小鼠冠状动脉结扎诱导的心肌缺血模型和H2O2诱导的H9c2细胞损伤模型分别验证了荭草和荭草活性成分群的生物效应，明确了荭草抗心肌缺血的主要活性成分群为原儿茶酸、荭草素、异荭草素、牡荆素、山柰素-3-O-β-D-葡萄糖苷、槲皮素、N-反式-对羟基苯乙基阿魏酰酪胺和N-P-香豆酰酪胺。针对这些成分，利用网络药理学方法预测荭草活性成分的主要作用机制，结果表明与之密切相关的通路主要包括PI3K/Akt、TNF、MAPK、FoxO、HIF-1、Toll-like receptor等。再将这些活性成分群与主要核心作用蛋白进行分子对接，结果发现这些成分与AR、PPARG、SOD2这三类靶点的潜在相互作用较强，并且三者中与SOD2有较好的结合作用，说明SOD2极有可能为这些成分的作用靶点。然后分别在小鼠心肌缺血模型和H9c2细胞模型中对荭草调控信号通路PI3K/Akt、MAPK、SIRT3/SOD2和Toll-like receptor的作用进行验证，结果发现荭草的确能调控PI3K/Akt信号通路、MAPK信号通路、SIRT3/SOD2和Toll-like receptor信号通路，具有抗细胞凋亡、抗氧化损伤、保护线粒体和抗炎等作用，最后发挥保护心肌细胞的功能。本项目研究结果表明荭草能通过调节多条通路MAPK、PI3K/Akt、SIRT3/SOD2和Toll-like受体发挥保护心肌细胞的作用，最终阐释了荭草活性成分抗心肌缺血的作用机制，可为深度开发应用荭草资源奠定基础。