基于“生新祛瘀”理论的川芎嗪联合黄芪甲苷促缺血心肌血管新生作用机制的研究

The subject to the "theory of traditional Chinese medicine for removing blood stasis and new law" as the guide, from “Promote the circulation of Qi and blood , dredging of the old new" two levels, Selecting "Ligustrazine and astragaloside", Using the chicken chorioallantoic membrane (CAM) model, and the uniform design method of "2 factors and 4 levels" grouping design, The degree of angiogenesis in chorioallantoic membrane as screening index, to obtain" the best formula" by regression analysis; To establish the model of myocardial ischemia of rats, to observe the effect of Ligustrazine and astragaloside in angiogenesis and growth factors; Through the hypoxia on hypoxic injury of endothelial cells induced by the factor -1 (HIF-1 α) study, investigate the angiogenesis study the mechanism of Ligustrazine and astragaloside; From the PI3K/AKT signal transduction pathway mediated by VEGF on the combination of the two to promote effect of angiogenesis in ischemic myocardium; The purpose of this study is to clarify the connotation of science of Ligustrazine and astragaloside in promoting angiogenesis of ischemic myocardium, and provide theoretical and experimental support for promoting blood circulation and removing blood stasis and supplementing qi and activating blood circulation drugs reasonably, and to further elucidate the stasis has important significance in promoting new remove stasis of myocardial ischemia.

本课题以“生新为祛瘀之法”的中医理论为指导，从“疏其气血、化旧生新”两个层面，选择临床常用的活血化瘀药-川芎嗪和益气活血药-黄芪甲苷两种有效成分为代表，运用鸡胚绒毛尿囊膜（CAM）模型，采用均匀设计法“2因素4水平”分组设计，以尿囊膜的血管增生程度为筛选指标，经均匀设计回归分析获得“最佳配方”；建立心肌缺血大鼠模型，从整体水平观察川芎嗪和黄芪甲苷血管新生作用及相关生长因子影响；通过对缺氧损伤的内皮细胞中低氧诱导因子-1（HIF-1α）的考察，从细胞水平考察川芎嗪和黄芪甲苷促新生血管机制；从VEGF所介导的PI3K/AKT信号转导通路分子水平探讨二药联合促缺血心肌血管新生作用机制。本课题旨在科学阐明川芎嗪和黄芪甲苷促缺血心肌新生血管的内涵，为活血化瘀与益气活血药合理配伍提供理论和实验支撑，并对进一步阐明祛瘀生新法治疗缺血性心脏病具有重要意义。

本课题以“生新为祛瘀之法”的中医理论为指导，从“疏其气血、化旧生新”两个层面，选用活血化瘀药及益气活血药代表中药的两种有效成分-川芎嗪和黄芪甲苷，探讨川芎嗪和黄芪甲苷促缺血心肌新生血管抗缺血性心脏病内涵。运用鸡胚绒毛尿囊膜（CAM）模型，采用方差分析方法，以尿囊膜的血管增生程度为筛选指标，获得“最佳配方”；以人脐静脉内皮细胞（HUVEC）缺氧损伤为模型，考察川芎嗪与黄芪甲苷配伍对人脐静脉内皮细胞血管生成的作用，结果显示川芎嗪与黄芪甲苷均能提高细胞存活率，川芎嗪（80µg/mL）剂量组+黄芪甲苷（40µg/mL）组具有极显著差异，说明川芎嗪与黄芪甲苷配伍可协同增效；川芎嗪黄芪甲苷配伍可能通过增加血管生成靶向因子血管内皮生长因子（VEGF）和血管生成素-2（Ang-2）的表达发挥促进血管生成的作用。以H2O2诱导HUVEC损伤模型，考察川芎嗪与黄芪甲苷配伍对过氧化氢诱导的人脐静脉内皮细胞氧化损伤的保护作用，结果显示川芎嗪（80µg/mL）+黄芪甲苷（40µg/mL）对 H2O2诱导的 HUVECs氧化损伤有保护作用，能缓解氧化应激诱导的 HUVECs 凋亡，其机制与 PI3K/Akt 信号通路有关。川芎嗪与黄芪甲苷对左冠状动脉狭窄致心肌缺血大鼠血管生成作用表明川芎嗪与黄芪甲苷协同作用于TGF-β1，从而减轻缺血心肌纤维化,提高梗死区域CD31，促进血管新生，进行治疗性干预可协同保护心脏功能，减轻MI后期不良心室重塑。川芎嗪和黄芪甲苷诱导的心脏保护机制涉及这两种化合物的抗纤维化和促血管生成活性，这可能是通过Shh途径介导的。黄芪为传统的补气药，川芎为血中气药，专行血中之气，黄芪伍川芎补气行气、化瘀通络，针对气虚血瘀之胸痹标本同治。本课题科学阐明川芎嗪和黄芪甲苷配伍协同增效促缺血心肌新生血管的机制，体现中医“生新祛瘀”治疗缺血心脏病的内涵，为活血化瘀与益气活血药合理配伍提供理论和实验支撑，并对进一步阐明祛瘀生新法治疗心肌缺血具有重要意义。