从肝治心组方对氧糖剥夺/复灌H9c2心肌细胞模型铁代谢的调节作用

曾阳; 王瑾茜; 陈亚; 胡国恒; 张程程; 何飘; 朴美虹

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曾阳,王瑾茜,陈亚,胡国恒,张程程,何飘,朴美虹.从肝治心组方对氧糖剥夺/复灌H9c2心肌细胞模型铁代谢的调节作用[J].湖南中医药大学学报,2023,43(6):1021-1027[点击复制]

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从肝治心组方对氧糖剥夺/复灌H9c2心肌细胞模型铁代谢的调节作用

曾阳,王瑾茜,陈亚,胡国恒,张程程,何飘,朴美虹

(湖南中医药大学第一附属医院, 湖南长沙 410007;湖南中医药大学, 湖南长沙 410208)

摘要:

目的研究大鼠心肌缺血/再灌注（myocardial ischemia/reperfusion, MI/R）损伤后心肌细胞的铁代谢及从肝治心组方干预对铁死亡的调节作用。方法选择体外培养的大鼠H9c2心肌细胞，并采用氧糖剥夺/复灌（oxygen glucose deprivation/reperfusion, OGD/R）法建立体外MI/R模型。将细胞分为正常组、模型组、中药组、抑制剂组、激动剂组、联合组、空白血清组。除正常组外，其余各组均予以OGD/R诱导损伤，在复灌时分别给予完全培养基、含15%含药血清的高糖DMEM培养基、含铁死亡抑制剂Ferrostatin-1（Fer-1）（0.5 μmol/L）的完全培养基、含爱拉斯汀（Erastin）（10 μmol/L）的完全培养基、含Erastin（10 μmol/L）的15%含药血清培养基、含15%空白血清的高糖DMEM培养基。采用CCK-8检测细胞存活率，TMRE荧光探针检测线粒体膜电位，FerroOrange检测细胞内Fe²⁺水平，DCFH-DA荧光探针检测活性氧（reactive oxygen species, ROS）水平。结果与正常组比较，模型组与空白血清组细胞存活率及线粒体膜电位显著下降（P<0.01），Fe²⁺及ROS水平显著升高（P<0.01）。模型组与空白血清组线粒体膜电位、Fe²⁺水平差异无统计学意义（P>0.05）。与模型组比较，中药组及抑制剂组细胞存活率及线粒体膜电位显著升高（P<0.01），Fe²⁺及ROS水平显著降低（P<0.01）。中药组与抑制剂组线粒体膜电位、Fe²⁺及ROS水平差异无统计学意义（P>0.05）。与模型组相比，激动剂组细胞存活率及线粒体膜电位显著下降（P<0.01），Fe²⁺及ROS水平显著升高（P<0.01）。与激动剂组相比，联合组细胞存活率及线粒体膜电位显著升高（P<0.01），Fe²⁺及ROS水平显著降低（P<0.01）。结论从肝治心组方可以减少MI/R损伤，其机制可能与调节心肌细胞铁代谢、抑制铁死亡有关。

DOI：10.3969/j.issn.1674－070X.２０23.06.009

投稿时间：2022-12-26

基金项目:湖南省自然科学基金项目（2021JJ40418）；湖南省中医药科研项目（2021172）；长沙市科技计划项目（kq2014222）；湖南省卫生健康委员会科研计划项目（202103010864）；湖南中医药大学校级科研基金项目（2019XJJJ057）。

Regulation of iron metabolism in H9c2 cardiomyocyte model with oxygen glucose deprivation/reperfusion by Conggan Zhixin Compound Formula

ZENG Yang,WANG Jinxi,CHEN Ya,HU Guoheng,ZHANG Chengcheng,HE Piao,PIAO Meihong

(The First Hospital of Hunan University of Chinese Medicine, Changsha, Hunan 410007, China;Hunan University of Chinese Medicine, Changsha, Hunan 410208, China)

Abstract:

Objective To investigate the iron metabolism of cardiomyocyte after myocardial ischemia-reperfusion (MI/R) injury in rats and the regulatory effects of Conggan Zhixin Compound Formula on ferroptosis. Methods H9c2 cardiomyocytes of rats cultured in vitro were selected and an in vitro MI/R model was established by oxygen glucose deprivation/reperfusion (OGD/R) modeling. The cells were divided into normal group, model group, Chinese medicine group, inhibitor group, agonist group, joint group, and blank serum group. Except for the normal group, all other groups were subjected to OGD/R induced injury. During reperfusion, complete medium, high-glucose DMEM medium containing 15% drug-containing serum, complete medium containing Ferrostatin-1 (0.5 μmol/L), complete medium containing Erastin (10 μmol/L), 15% drug-containing serum medium containing Erastin (10 μmol/L), and high-glucose DMEM medium containing 15% blank serum were administered according to different groups. CCK-8 was used to measure cell survival rate, TMRE fluorescent probe was used to examine mitochondrial membrane potential, FerroOrange was used to determine intracellular Fe²⁺ level, and DCFH-DA fluorescent probe was used to identify reactive oxygen species (ROS) level in each group. Results Compared with the normal group, the cell survival rates and mitochondrial membrane potential in the model group and the blank serum group decreased significantly (P<0.01), and the levels of Fe²⁺ and ROS increased significantly (P<0.01). There was no significant difference in mitochondrial membrane potential and Fe²⁺ levels between the model group and the blank serum group (P>0.05). Compared with the model group, the cell survival rates and mitochondrial membrane potential in the Chinese medicine group and the inhibitor group increased significantly (P<0.01), and the levels of Fe²⁺ and ROS decreased significantly (P<0.01). There was no significant difference in mitochondrial membrane potential, Fe²⁺ and ROS levels between the Chinese medicine group and the inhibitor group (P>0.05). Compared with the model group, the cell survival rate and mitochondrial membrane potential in the agonist group decreased significantly (P<0.01), and the levels of Fe²⁺ and ROS increased significantly (P<0.01). Compared with the agonist group, the cell survival rate and mitochondrial membrane potential in the joint group were significantly higher (P<0.01), and the levels of Fe²⁺ and ROS were significantly lower (P<0.01). Conclusion Conggan Zhixin Compound Formula can reduce MI/R injury, and its mechanisms may be related to regulating iron metabolism of myocardial cells and inhibiting ferroptosis.

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