诃子鞣酸对乌头碱致H9C2细胞损伤的保护作用及机制

Protective effect and mechanism of chebulagic acid on H9C2 cells injury induced by aconitine

目的通过研究诃子鞣酸对乌头碱致H9C2细胞损伤的保护作用,探讨蒙药诃子制草乌炮制减毒机制。方法以不同浓度的乌头碱、诃子鞣酸及乌头碱+诃子鞣酸分别作用于H9C2细胞,利用高内涵分析(HCA)技术检测并分析药物对H9C2细胞数量、细胞核面积、细胞核DNA含量、Ca^2+荧光强度及线粒体功能的影响。结果10、20μmol/L乌头碱作用于H9C2细胞24 h后,可导致细胞内Ca^2+超载;20μmol/L乌头碱可致H9C2细胞线粒体功能障碍;5、10、20μmol/L诃子鞣酸单独作用于H9C2细胞均无明显毒性;当10、20μmol/L诃子鞣酸与20μmol/L乌头碱共同作用于H9C2细胞24 h后,线粒体功能恢复,细胞内Ca^2+荧光强度降低;与对照组相比,无明显差异。结论乌头碱会引起H9C2细胞Ca^2+超载和线粒体功能障碍,可能是草乌致心肌细胞毒性的主要原因。诃子中的诃子鞣酸可明显抑制由乌头碱引起的Ca^2+超载,改善线粒体功能,起到解草乌毒的作用。

Objective To investigate the toxicity of aconitine to H9 C2 cells by HCA,and to further explore the effect of chebulagic acid,the active component of He Zi(medicine terminalia fruit,Fructus Chebulae),on the cardiotoxicity induced by aconitine,and to explore its mechanism in reducing the toxicity of He Zi(medicine terminalia fruit,Fructus Chebulae).Methods Different concentrations of aconitine,chebulagic acid and aconitine+chebulagic acid were used to act on H9 C2 cells respectively.Hoechst 33258 and other fluorescent probes were used to dye the cells.HCA technology was used to detect and analyze the effects of drugs of different concentrations on the number of H9 C2 cells,the area of nucleus,the DNA content of nucleus,the fluorescence intensity of Ca^2+and the influence on function of mitochondria.Results 10,20μmol/L aconitine caused Ca^2+overload in cardiomyocytes;20μmol/L aconitine caused mitochondrial dysfunction of cardiomyocytes in varying degrees after acting on cardiomyocytes for 24 hours;5,10,20μmol/L chebulagic acid had no obvious toxicity when acting on H9 C2 cells alone;10,20μmol/L of chebulagic acid combined with 20μmol/L aconitine restored the mitochondrial function and significantly reduced the fluorescence intensity of Ca^2+,compared with using aconitine alone.Compared with the control group,there was no significant difference between the two groups.Conclusions Aconitine causes calcium overload and mitochondrial dysfunction in cardiomyocytes,which may be the main cause of cardiotoxicity induced by aconitine.Chebulagic acid in He Zi(medicine terminalia fruit,Fructus Chebulae)seems to inhibit the Ca^2+overload induced by aconitine,improve the mitochondrial function,and detoxify aconitine.