目的 建立大鼠H9c2心肌细胞缺氧/复氧（H/R）损伤模型，考察圣草次苷改善心肌缺血再灌注损伤的作用机制。方法 利用无糖无血清培养基结合厌氧（94%N₂、5%CO₂、1%O₂）处理H9c2心肌细胞4h后，更换新鲜完全培养基再放入正常孵箱复氧24h，制备H/R损伤模型。在造模前12h给予圣草次苷（5、10、20μg·mL^-1），细胞活力及乳酸脱氢酶（LDH）检测实验中选择灯盏乙素（20μg·mL^-1）作为阳性药，对照组及模型组给予等体积DMSO。MTT法测定细胞存活率；试剂盒检测细胞培养上清液中LDH水平；试剂盒检测细胞内丙二醛（MDA）水平和超氧化物歧化酶（SOD）、过氧化氢酶（CAT）、谷胱甘肽过氧化物酶（GSH-Px）活力；TUNEL染色检测细胞凋亡；DCFH-DA和JC-1探针分别检测细胞内活性氧（ROS）和线粒体膜电位改变；Western blotting检测核蛋白中转录因子（NF）-E2相关因子2（Nrf2）和总蛋白中血红素氧合酶1（HO-1）、γ-谷氨酰半胱氨酸连接酶（GCL）水平。结果 与对照组比较，H/R损伤诱导的模型组细胞存活率明显下降，凋亡明显增加，LDH水平明显升高，细胞内MDA水平明显升高，SOD、CAT、GSH-Px活力明显降低，ROS释放明显增多，线粒体膜电位明显降低，差异均有统计学意义（P＜0.01）；与模型组比较，圣草次苷可剂量相关性地改善上述变化，其中10、20μg·mL^-1组均差异显著（P＜0.01）。Western blotting结果显示，与对照组比较，模型组细胞Nrf2核转位以及HO-1、GCL表达水平无显著变化；与模型组比较，圣草次苷10、20μg·mL^-1显著增加Nrf2核转位及HO-1、GCL表达水平（P＜0.01）。结论 圣草次苷能够保护H/R诱导的H9c2心肌细胞损伤，其可能通过激活Nrf2抗氧化信号通路，增加细胞内源性抗氧化能力，抑制氧化应激损伤，保护线粒体功能以阻止细胞凋亡的发生。

Objective The hypoxia/reoxygenation (H/R) injury model of rat H9c2 cardiomyocytes was established to investigate the mechanism of Eriocitrin (ERI) in improving myocardial ischemia-reperfusion injury.Methods H9c2 cardiomyocytes were treated with sugar-free serum-free medium combined with anaerobic (94% N₂, 5% CO₂, 1% O₂) for 4 h, and then replaced with fresh complete medium and reoxygenated in normal incubator for 24 h to prepare H/R injury model. ERI (5, 10, and 20 μg·mL^-1) treatment from 12 h before modeling. Breviscapine (20 μg·mL^-1) was selected as the positive drug for cell viability and lactate dehydrogenase (LDH) detection. The control group and model group were treated with equal volume DMSO. Cell viability was measured by MTT assay. The levels of LDH, malondialdehyde (MDA) and the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) were detected by the kit. Cell apoptosis were detected by TUNEL staining. DCFH-DA and JC-1 probes were used to detect the changes of intracellular reactive oxygen species (ROS) and mitochondrial membrane potential (MMP). The expression of nuclear protein NF-E2-related factor 2 (Nrf2) and expression of heme oxygenase-1 (HO-1) and glutamate cysteine ligase (GCL) expression in total protein was detected by western blotting.Results Compared with control group, H/R decreased cell viability and increased apoptosis rate and LDH level significantly (P <0.01). Besides, H/R decreased MMP and increased ROS production and the level of MDA (P <0.01). The activities of SOD, CAT and GSH-Px decreased significantly (P <0.01). ERI could improve these changes in a dose-dependent manner compared with model group, and there were significant differences in 10 and 20 μg·mL^-1 groups (P <0.01). Western blotting results showed that compared with control group, the nuclear translocation of Nrf2 and the expression levels of HO-1 and GCL in the model group had no significant changes. Compared with model group, ERI 10 and 20 μg·mL^-1 significantly increased Nrf2 nuclear translocation and the expression levels of HO-1 and GCL (P <0.01).Conclusions ERI protected against H/R induced H9c2 injury and apoptosis by increasing the endogenous antioxidant capacity of cells, inhibiting oxidative stress damage, and protecting mitochondrial function, which may be mediated by Nrf2 signaling pathway.

R285.5

中国医学科学院医学与健康科技创新工程(2021-I2M-1-031)