气血并治方有效组分对缺氧/复氧损伤心肌保护作用的机制研究  

Effects of Hypoxia/Reoxygenation on Myocardial Cells: Protected by Components of Water Extract from Qi-Xue Bing-Zhi Recipe

作　　者：于永慧 张佩 刘剑刚[1] 李澎[1] 张大武[1] 王承龙[1] 

机构地区：[1]中国中医科学院西苑医院心血管病中心,北京100091

出　　处：《世界科学技术-中医药现代化》2017年第12期2017-2023,共7页Modernization of Traditional Chinese Medicine and Materia Medica-World Science and Technology

基　　金：国家自然科学基金面上资助项目:活血益气中药干预动脉粥样硬化缺血心肌巨噬细胞-线粒体关键环节的机制研究(81273934);负责人:王承龙

摘　　要：目的:观察气血并治方组分配伍(Components of water extract from Qixue Bingzhi Recipe,CWQB)对缺氧/复氧(Hypoxia/Reoxygenation,H/R)损伤心肌细胞的保护作用,探讨其改善心肌能量代谢从而防止细胞凋亡的相关性机制。方法:分离、提取、培养出生1-2天的SD乳鼠原代心肌细胞,缺氧3 h复氧2 h制作H/R模型,分为Control组(正常氧组)、H/R组(缺氧/复氧模型)、TMZ组(缺氧/复氧模型+100μmol/L曲美他嗪(Trimetazidine,TMZ))和CWQB组(缺氧/复氧模型+1mmol/L气血并治方有效组分)。采用高效液相色谱分析法(HighPerformance Liquid Chromato-graph,HPLC)测定各组细胞高能磷酸化合物-一磷酸腺苷(Adenosine mono-phosphate,AMP)、二磷酸腺苷(Adenosine diphosphate,ADP)、三磷酸腺苷(Adenosine triphosphate,ATP)的含量并计算总腺苷酸量(Total adenylic acid,TAN)、能荷(Energy charge,EC)水平;酶联免疫吸附法(Enzyme linkedimmuno sorbent assay,ELISA)法检测心肌细胞损伤标志物肌酸激酶同工酶(Creatine kinase-MB,CK-MB)、肌钙蛋白T(Troponin,c Tn T)水平,流式细胞术检测心肌细胞早期凋亡率。Pearson相关性检验分析心肌损伤标志物、早期凋亡率与高能磷酸化合物含量之间的关系。结果:与H/R组比较,TMZ组、CWQB组AMP含量下降,ADP、ATP含量和TAN、EC水平均有所升高,其中TMZ组ADP、ATP、TAN升高程度较CWQB组更为显著(p<0.05);TMZ组、CWQB组CK-MB、c Tn T含量显著下降,其中CWQB组c Tn T含量下降更为显著(p<0.05);TMZ组、CWQB组早期凋亡率有所下降,TMZ组下降更为显著(p<0.05)。相关性分析显示,CK-MB水平与ADP浓度呈显著负相关;早期凋亡率与AMP浓度呈显著正相关,与ADP、ATP浓度呈负相关(P<0.01)。结论:CWQB能够在降低H/R心肌细胞AMP浓度,升高ADP、ATP浓度和TAN、EC水平的同时,减轻心肌损伤标志物CK-MB、c Tn T含量,抑制心肌细胞的早期凋亡率,从而发挥对H/R损伤心肌细胞的保护作用。This study was aimed to observe the protective effects of components of water extract from Qi-Xue Bing-Zhi recipe（CWQB） on myocardial cells from hypoxia/reoxygenation（H/R） injury, by optimizing the metabolism of high-energy phosphates and then preventing cell damage and early apoptosis correlatively. Myocardial cells were separatedand extracted from newborn SD rats. And then, H/R models were made by depriving oxygen for 3 hours and thenregaining for 2 hours. Cardiomyocytes were divided into four groups, which were the control（normal oxygen） group, H/R（H/R model） group, TMZ（H/R model ＋ 100 μmol/L Trimetazidine, TMZ） group, and CWQB（H/R model ＋ 1 mmol/LCWQB） group. High-performance liquid chromatography（HPLC） was used in the content determination of adenosinemonophosphate（AMP）, adenosine diphosphate（ADP）, adenosine triphosphate（ATP） in each group for the calculation oftotal adenylic acid（TAN） and energy charge（EC）. Enzyme linked immunosorbent assay（ELISA） was used in thedetection of myocardial damage markers, such as creatine kinase-MB（CK-MB） and cardiac troponin T（c Tn T）. The early apoptosis of myocardial cells were assessed by flow cytometry（FCM）. The results showed that compared with H/R group,contents of AMP in the TMZ group and CWQB group were decreased, while contents of ADP, ATP, TAN and EC were allincreased in both groups. The increasing degrees of ADP, ATP and TAN in TMZ group were higher than those of theCWQB group（P〈0.05）. Contents of CK-MB and c Tn T were significantly decreased in the TMZ group and the CWQBgroup. Content of c Tn T decreased more significantly in the CWQB group（P〈0.05）. In the TMZ group and the CWQBgroup, early apoptosis rate was decreased. The decreasing in the TH group was more significant（P〈0.05）. Thecorrelation analysis showed that the level of CK-MB and concentration of ADP had significant negative correlation. Theearly apoptosis rate and AMP had significant positive correlation. The early apopt

关 键 词：气血并治方 有效组分 缺氧/复氧心肌细胞 保护作用 高能磷酸化合物 心肌损伤标志物 早期凋亡率 

分 类 号：R285[医药卫生—中药学]