丹川红及其吸收成分急性心脏保护的离体分子机理

Traditional Chinese medicine(TCM) formula Guanxin II cantained Salvia miltiorrhiza Bge., Ligusticum chuanxiong Hort., Paeonia lactiflora P., Carthamus tinctorius L. and Dalbergiae odoriferae lignum. The former three constitutes DCH (Danchuanhong). The two formulae's accumulations in our previous study involved in follows two points:1)their cardioprotective absorbed compounds(ACs) are FTA (ferulic acid, tanshinol and hydroxyl safflor yellow A); 2)two formulae and FTA following a single oral administration to rat with acute myocardial infarction (AMI) to them acutely and simultaneously induced similar increase in first coronary flow(CF) and then myocardial flow (MF) in ischemic area, and subsequent decrease in cardiomyocyte apoptosis(CA) without chronotropic, inotropic and hypertension in coronary circulation, hinting coronary vasodilatation whose direct evidence is unavailable in references. However, above in-vivo pharmacology is elusive and let alone in-vitro molecular mechanism. The latter methodology in which crude extract with ACs and non-ACs is directly added to in-vitro medium resulted in false positive or negative datum.The present application designed protocol in which ACs similar to DCH in acute cardioprotection is directly added in-vitro medium, suggesting to demystify molecular pharmacology by TCM formula. The experimental procedure includes:1)rapid coronary vasodilatation and indirect anti-apoptosis will is available following a high dose of DCH or FTA are once gavaged to mice with health or apoE-/-+ high fat diet before AMI operation; 2)in in-vitro model including thoracic aorta rings, several cardiomyocyte apoptosis, the changes in vasomotion, Src-Akt- eNOS signal, proliferation of vessel smooth cell, oxidative stress, inflammation response and caspase/apoptosis，et al are compared and analyzed pre- and post-adding FTA in escalating dose which contained their concentration in circulatory blood of mice, and their parent formula DCH or ACs-contained serum following gavaged to mice with DCH. All together, the conclusion from above experiment will highlight in-vitro molecular pharmacology involved in TCM formula via elucidating CF/MF-coronary vasodilatation-indirect anti-apoptosis in cardiomyocyte.

我们证实：冠心II号(丹参/川芎/红花/赤芍/降香)与丹川红(前三味)一次灌胃，均通过吸收成分(ACs)FTA(阿魏酸、羟基红花黄色素A与丹参素)急性心脏保护，不影响冠脉循环的变时/力/压,增加冠脉流量(CF)间接抗凋亡,提示系冠脉舒张所致，但缺乏证据且分子机理远不清楚。目前草药粗制剂直接加入离体实验导致伪性结果。本项目采用代表丹川红疗效的ACs加入离体实验可破解难题进而阐明该方舒血管离体分子机制。健康/apoE-/-+高脂小鼠心梗前一次灌胃该方：体内FTA分析并确定舒血管间接抗凋亡；用血管环/多种心肌凋亡细胞离体模型，以血管张力/Src/Akt/eNOS信号/平滑肌增殖与细胞氧化应激/炎性反应/caspase-凋亡为指标，观察浓度梯度FAT(含其血药浓度范围)加入离体系统对上述指标的影响并与粗制剂/含药血清对照，揭示CF↑-冠脉舒张-间接抗凋亡关系。藉此开创一条方剂离体分子机制研究新途径

中药冠心II号(丹参、川芎、红花、赤芍、降香)与丹川红(前三味)一次灌胃，均通过吸收成分(ABCs)FTA(阿魏酸、羟基红花黄色素A与丹参素)起急性心脏保护，不影响冠脉循环的变时、力、压,增加冠脉流量(CF)间接抗凋亡，提示系冠脉舒张所致，但缺乏证据且分子机理远不清楚。方法 在BAP(生物方剂分析药理)策略的指导下，建立LC-MS/MS方法对DCH中的丹酚酸A 、丹酚酸B、迷迭香酸、羟A、丹参素、原儿茶酸、阿魏酸和紫草酸8个成分进行体外定量分析并结合吸收入血、入脑等部位的3个主要成分阿魏酸、丹参素和羟A的体内定性分析来确定丹川红的ABCs；利用口服DCH入血成分的药物代谢动力学实验，确定了血药浓度的药时曲线，从而确定了模型鼠的灌胃取材时间；利用能够代表丹川红疗效的ABCs加入离体实验以破解普遍离体方法中粗制剂、阳离子等干扰从而准确的阐明离体分子机制。同时建立慢性ApoE-/-高脂模型小鼠，固定时间灌胃6周后取材，比较丹川红吸收成分与母方疗效的差异，并进一步研究FAT中F抗动脉粥样硬化的机理。结果 丹川红中8个成分具有良好的线性关系(r>0.990)，血浆中5成分也具有良好的线性关系(r>0.994)且获得了血浆中最高浓度Cmax和相应的时间Tmax；冠心II号与丹川红的离体方法学实验，内皮完整下分为空白对照(KH)、GII汤剂、空白血清、含药血清组、FTA剂量等于含药血清中FTA浓度组、 F=FTA比值中F含量组和F=T组进行张力的对比，得出血浆中FTA最高浓度，并完成了除含药血清(易起泡)以外的剂量依赖性曲线；ApoE-/-高脂模型小鼠的行为学和体重变化符合抑郁模型的变化趋势，同时病理学检测结果证明了给药后主动脉中斑块的减少，血管张力Src、Akt、eNOS信号、平滑肌增殖与细胞氧化应激、炎性反应、caspase-凋亡等指标有所下降。结论 建立的方法操作简便、灵敏度高、分析速度快，可同时测定丹川红中多个成分的含量和吸收成分，有助于快速进行该方的多成分质量控制和体内药物分析；离体方法学实验排除了粗制剂或血清体积上，离体剂量依赖性不一致、药效物质不清楚、存在的内源性外源性干扰，严格按照ABCs离体实验剂量等于血药浓度(或血比值)准确地反应方剂离体药效机制，藉此开创一条方剂离体分子机制研究新途径。