基于NF-KB/MAPK信号通路探寻延龄草甾体皂苷类抗心肌缺血再灌注损伤的“有效成分-可识别指纹图谱-候选药物-效应靶点”的分子机制研究

Ischemic heart disease(IHD) of myocardial cell damage or apoptosis is caused by myocardial ischemia-reperfusion injury(MIRI),studying the treatment drug of IHD from the anti-MIRI is very important significance.This group of preliminary pharmacodynamic study clarify the effective part and effective components of Trillium tschonoskii steroidal saponins in anti-myocardial ischemia,and determine the 45 steroidal saponins(4 new compounds) of effective part,three effective components at lower doses than DIAOXINXUEKANG has the significant effect of anti-myocardial infarction,playing a protective role in anti-MIRI,but the pharmacodynamic material and molecular mechanism of Trillium tschonoskii anti-MIRI in NF-KB/MAPK signal pathway is not clear,which needs to be further studied.On this basis,this project builds coronary artery ligation in rats after reperfusion damage model and myocardial cells H9c2 hypoxia/reoxygenation model,applicating serum of pharmaceutical chemistry combined with chemical synthesis,UPLC-Q-TOF-MS, molecular biology technical methods to study the Trillium tschonoskii steroidal saponins effective components and their migration into the blood components and metabolites on anti-MIRI candidate drugs,interpretation “Effective components- Identification of fingerprint-Candidate drugs-Effect target”relevance research, reveal the target compositions and molecular mechanism in NF-KB/MAPK signal pathway about anti-MIRI candidate drugs of Trillium tschonoskii.Laying foundations for the comprehensive development utilization of Trillium tschonoskii and providing a new idea to study on creating new drugs of anti-IHD.

心肌缺血再灌注损伤（MIRI）是引起缺血性心脏病（IHD）心肌细胞损伤或凋亡的主要原因，从抗MIRI方面开展治疗IHD药物的研究具有非常重要意义。前期本课题组明确延龄草甾体皂苷类抗IHD有效部位和有效成分，从中分离鉴定45个甾体皂苷类成分（4个新化），其中3个有效成分单体在较低剂量比地奥心血康治疗心肌梗死效果显著，对MIRI有保护作用，其作用于NF-KB/MAPK信号通路的分子机制不清楚，有待深入研究。在此基础上，本项目拟在整体和细胞药理学层面上，应用血清药物化学结合化学合成、UPLC-Q-TOF-MS和分子生物学等技术方法开展延龄草甾体皂苷类有效成分及其移行入血成分以及代谢产物抗MIRI的候选药物研究，诠释“有效成分-可识别指纹图谱-候选药物-效应靶点”的内涵，阐明候选药物抗MIRI作用于NF-KB/MAPK信号通路的分子机制，为延龄草综合开发利用奠定基础,为抗IHD新药创制提供新思路。

本课题研究结果表明偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→2)-β-D-吡喃葡萄糖苷（简称Y2T）、偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-吡喃葡萄糖苷（简称Y3T）和偏诺皂苷元-3-O-α-L-吡喃鼠李糖基(1→4)-α-L-吡喃鼠李糖基(1→4)-[α-L-吡喃鼠李糖基(1→2)]-β-D-吡喃葡萄糖苷（简称Y4T）单体均能显著降低因冠脉结扎所致心肌缺血模型大鼠ST段和T波的异常抬高，抑制血清中CK、LDH的活力，减少心肌缺血大鼠的心肌梗塞范围，确定为治疗心肌缺血的有效成分，其中Y3T疗效最优。在抗缺氧/复氧诱导的H9c2心肌细胞凋亡的试验中，结果证实Y2T、Y3T和Y4T及其代谢产物偏诺皂苷元-3-O-β-D-吡喃葡萄糖苷（简称Y1T）、偏诺皂苷元（简称YT）通过降低IL-6、IL-1β、TNF-α等心肌细胞炎症因子水平，减轻心肌细胞肿胀和炎性细胞浸润，促进凋亡抑制蛋白Bcl-2的表达和抑制促炎症蛋白和凋亡蛋白NF-κB、TNF-α、COX-2、iNOS、Caspase-3和Bax蛋白的表达发挥抗心肌细胞炎症或凋亡，上述成分主要通过iNOS-NO-NF-κB/MAPK信号通路的相关蛋白NF-κB、p53、C-myc、Fas及其下游底物分子（主要为NF-κB、TNF-α、COX-2、iNOS、Caspase、C-Jun、C-fos、Bcl-2/Bax等）发挥抗心肌缺血再灌注损伤作用；初步诠释“移行入血Y2T和Y3T有效成分-可识别指纹图谱-效应机制（代谢产物YT和Y1T有效成分）-靶点（iNOS-NO-NF-κB/MAPK）”的同一结构母核偏诺皂苷协同作用于NF-κB/MAPK信号通路的相关性科学内涵，为开发治疗缺血性心脏病自主知识产权候选新药的研制奠定坚实基础。