基于MI-RI大鼠心肌细胞代谢组学研究四逆汤中附子配伍甘草解毒增效机制

Study on attenuate and synergistic mechanism between Aconiti Lateralis Praeparata Radix and Glycyrrhizae Radix for toxicity reduction based on metabonomic of MI-RI mouse cardiomyocytes

四逆汤是经典《伤寒论》中的急救方,由附子、甘草(炙)、干姜组成,研究建立体外H9c2心肌细胞MI-RI模型,通过观察正常对照组、MI-RI模型组、四逆汤作用组和缺甘草四逆汤作用组细胞的形态学特征,测定各组细胞的存活率、LGH和CK活性。同时收集各组细胞培养液并采用GC-MS技术进行检测,对采集数据进行多元统计分析(PCA,PLS-DA,OPLSDA),得到17个代谢标志物(指认15个),并对其相对含量及代谢途径进行分析。研究结果显示四逆汤中附子配伍甘草解毒增效机制是通过调节糖酵解、脂质代谢、三羧酸循环和氨基酸代谢中氮的代谢等生物代谢途径得以实现的。研究也证明四逆汤与缺甘草四逆汤对MI-RI模型损伤H9c2心肌细胞均具有明显的保护作用,且四逆汤组保护作用更加显著。

Sini decoction（ SND）,a classical traditional Chinese medicine emergency formula recorded in Shanghan Lun（ Treatise on Febrile Diseases）,which is composed of Aconiti Lateralis Preparata Radix,Glycyrrhizae Preparata Radix and Zingiberis Rhizoma. Modern clinical and pharmacological researches have shown that SND can protect the myocardium effectively during myocardial ischemia reperfusion injury（ MI-RI）. A myocardial ischemia reperfusion injury model of H9c2 cardiomyocytes in vitro has been established. Four groups,control group,MI-RI Model group,SND group and SND without Glycyrrhizae Radix group,were arranged.The livability,the level of LDH and CK activity in H9c2 cardiomyocytes in different groups were tested. By combining with principal components analysis（ PCA）,partial least squares discriminant analysis（ PLS-DA）,orthogonal partial least squares projection of latent structures-discriminant analysis（ OPLS-DA）,17 biomarkers in extracellular fluid were identified and 15 of them were related to the pathway of biological processes. The results showed that the attenuate and synergistic mechanism between Aconiti Lateralis Praeparata Radix and Glycyrrhizae Radix for toxicity reduction was related with the glycolysis,lipid metabolism,citrate cycle and nitrogen metabolism of amino acids metabolism. The study proved the effect on H9c2 cardiomyocyte treated by MI-RI injury both SND group and SND without Glycyrrhizae Radix group,and compared with the SND without Glycyrrhizae Radix,the protective effect of myocardial ischemia reperfusion injury model of H9c2 cardiomyocyte from SND was stronger.