基于花生四烯酸CYP450代谢网络的复方丹参方抗心肌缺血再灌注损伤的物质基础及机制研究

Coronary heart disease including acute myocardial infarction (AMI) has been the major cause of death in many countries. Early and effective reperfusion of the myocardium is the definitive treatment for acute myocardial infarction to reduce the size of infarction and improve the clinical outcome. However, the restoration of coronary blood flow after prolonged ischemia can paradoxically exacerbate myocardial damage, which is termed myocardial ischemia-reperfusion injury (MIRI). In recent years, it is recognized that the CYP 450 pathways of arachidonic acid (AA) is involved in MIRI, and the drugs which could inhibit the CYP2C/4A/4F/sET pathways or induce CYP2J pathway will have good effects on MIRI. Compound Danshen is an important Chinese medicinal formula widely used for the treatment of cardiovascular disease, and its protection on MIRI has been generally confirmed. However, its pharmacological mechanisms have not been well elucidated. The objective of this study is to investigate the absorbed ingredients of Compound Danshen on the CYP450 metabolism network of AA with the thoughts systems biology and network pharmacology, as well as to elucidate its underlying protecting mechanisms on MIRI both in vivo and in vitro. This research will be a good example for pharmacological mechanism exploring of herbal medicines with multiple-compounds and targets.

冠心病急性心肌梗死（AMI）是危害人类健康和生命的一大类疾病。早期和有效的心肌再灌注是治疗AMI、改善预后的有效手段，但是心肌缺血再灌注损伤（MIRI）是治疗这类疾病时常遇到的现象，严重影响了治疗效果和预后。近年来研究表明，花生四烯酸的CYP450代谢网络参与介导MIRI，调控该网络中关键酶的活性是防治MIRI的新靶点。复方丹参方是治疗冠心病的良方，其抗MIRI作用已被大量研究证实，但是其作用机制至今仍未完全阐明。本课题将首先从复方丹参方抗MIRI的物质基础研究着手，利用现代先进的仪器分析技术，找出其发挥效应的入血成分群；然后在整体、细胞、亚细胞水平研究这些成分对花生四烯酸CYP450代谢网络各个关键酶的抑制及调控机制，阐明其发挥作用的准确效应靶点及相应物质基础，为阐明中药多组分、多靶点的作用机制提供良好的理论和实验基础。

花生四烯酸（AA）是生物体内含量丰富的不饱和脂肪酸，是机体内许多重要生物活性物质的前体，间接影响着生物体内许多重要的生物学功能。近年来，大量研究表明AA的CYP450代谢产物EETs、DHETs和 20-HETE在人体的心血管疾病特别是心肌缺血再灌注损伤（MIRI）中发挥着重要的功能。复方丹参方（CDP）作为具有较好临床应用价值的传统名方，在防治MIRI 方面已具有一定的药理研究基础，但其机制尚未完全阐明。本课题围绕CDP的MIRI保护作用是否与影响AA CYP450代谢途径有关这一科学问题在整体动物、细胞及亚细胞水平展开了一系列的研究。结果发现：1）CDP给药后，可以上调大鼠体内代谢生成具有心血管保护作用的EETs的表氧化酶如CYP2J和CYP2C的表达、而下调sEH（使EETs失活）的表达，因此在心肌缺血再灌后，CDP给药组大鼠体内EETs的浓度大幅升高；2）复方丹参方中丹参酮IIA、隐丹参酮、二氢丹参酮I、丹参酮I、丹酚酸A、丹酚酸B、丹酚酸C和紫草酸对CYP2C8/9、CYP2J2、CYP4A11、CYP4F2、CYP4F3A和sEH存在不同程度的可逆性抑制作用，但是除了二氢丹参酮I对CYP4A11和CYP4F3A、丹参酮I对CYP4A11的抑制作用在nM浓度起效外，绝大多数的抑制作用需要达μM浓度才能发挥；3）CDP中的酚酸类成分如丹酚酸A可以上调HepG2细胞中CYP2J2的表达。因此，本课题通过研究初步证实影响AA的 CYP450代谢途径确实是CDP抗MIRI的机制之一，且这一作用主要是通过影响AACYP450代谢酶的表达、进而增加EETs的浓度来发挥。