芪参益气方对H2O2损伤的心肌细胞线粒体结构和功能的保护作用及机制探讨

Protective effect and mechanism of Qishen Yiqi Formula on mitochondrial structure and function of cardiomyocytes injured by H_2O_2

目的制备H_2O_2诱导的心肌H9c2细胞损伤模型,通过线粒体结构、功能改变的多指标综合评价方法探讨芪参益气方对H9c2细胞的保护作用及作用机制。方法体外培养H9c2细胞,分为对照组、模型组(H_2O_2)、模型阳性对照羰基-氰-对-三氟甲氧基苯腙组(5μmol/L)、芪参益气方组(0.2 mg/mL),每组3个复孔,加入相应药物孵育24 h,H_2O_2诱导2 h制备心肌细胞损伤模型。采用Operetta高内涵成像系统评价H9c2细胞线粒体功能及形态纹理的改变;海马生物能量测定仪检测线粒体能量代谢的变化;流式细胞仪检测细胞的凋亡情况。结果芪参益气方能够明显降低H_2O_2损伤的H9c2细胞线粒体质量,增加线粒体膜电位和纹理的量化数值,同时增加线粒体基础耗氧率、ATP相关耗氧率、最大耗氧率以及储备能力,明显降低细胞早期及晚期凋亡率,增加活细胞率。结论基于细胞的线粒体结构和功能评价,证实芪参益气方通过改善线粒体功能及能量代谢减轻细胞凋亡保护H9c2细胞。

Objective To establish a cell-based multi-parameter mitochondrial structure and functional evaluation system for hydrogen peroxide（H2O2） injury in cardiomyocytes and investigate the protective effect of Qishen Yiqi Formula on H9 c2 cells and its mechanism. Methods For the in vitro myocardial cell injury model, H9 c2 cells were divided into control, model（H2O2）, positive control（carbonyl cyanide-cyanomethoxy basic hydrazine, 5 μmol/L）, and Qishen Yiqi Formula（0.2 mg/mL） groups, with three duplicates in each group, and were cultured in triplicate for 24 h with corresponding drugs, followed by 2 h H2O2 induction. H2O2 injury model was established with H9 c2 cell line. Mitochondrial function and morphological texture were evaluated by Operetta high content imaging system. Mitochondrial respiration and bioenergetic metabolism states were measured by Seahorse Bioscience XF extracellular flux analyzer. Cardiomyocyte apoptosis were quantified by flow cytometry. Results Qishen Yiqi Formula prevented the decrease of mitochondrial membrane potential and the increase of mitochondrial mass and improved mitochondrial morphological integrity. Functionally, Qishen Yiqi Formula increased basic oxygen consumption rate, ATP-linked oxygen consumption rate, maximum oxygen consumption, and the reserve capacity. It also reduced the apoptotic rate at early and end stage and increased the myocyte survival. Conclusion Based on the structure and functional evaluation of the mitochondria, results indicated that the compound Qishen Yiqi Formula protected the H9 c2 cells by improving the mitochondrial function and energy metabolism as well as reducing the apoptosis.