目的 研究橙皮苷（Hsd）减轻H9c2心肌细胞低氧损伤的作用。方法 将H9c2心肌细胞分为三组,即常氧对照组(normoxia)、低氧组(hypoxia)、低氧+Hsd组(H+Hsd),探索缓解H9c2心肌细胞损伤靶点时再设置低氧+Hsd+JNK激动剂茴香霉素组（H+Hsd+Ani）,在37℃和5%CO2浓度条件下培养常氧对照组,H+Hsd组用不同浓度的Hsd(5、10、15、20、40、50、80 µmol/L)提前常氧干预12 h,后与低氧组一起置于低氧工作站低氧(0.5%O2) 48 h。CCK8实验检测细胞活力;流式细胞仪分析检测细胞凋亡和活性氧;试剂盒法检测细胞中丙二醛(malondialdehyde,MDA)和超氧化物歧化酶（superoxide dismutase, SOD)活性;酶联免疫吸附试验(ELISA)检测肌酸激酶同工酶MB（creatine kinase myocardial band, CKMB)和心肌肌钙蛋白I(cardiac troponin I, cTn-I)浓度;乳酸脱氢酶（lactate dehydrogenase,LDH）测定试剂盒检测LDH活性;蛋白免疫印迹检测相应蛋白水平;细胞免疫荧光检测p65入核情况。结果 与常氧对照组相比,低氧组细胞活力和SOD活性降低(P<0.01),凋亡率、活性氧和MDA增加(P<0.01),心肌损伤标志物活力升高(P<0.05),JNK、JUN、p65磷酸化水平升高(P<0.05),p65入核增多,IKBα降解(P<0.05);Hsd干预可以明显减小凋亡率(P<0.05),促进增殖(P<0.05),减轻氧化应激和心肌损伤(P<0.05),降低JNK、JUN、p65磷酸化水平(P<0.05),减少p65入核量,增加IKBα表达(P<0.05)。结论 Hsd可以保护低氧诱导的H9c2细胞损伤,其作用机制可能为抑制JNK-JUN-NF-kB信号通路,降低细胞凋亡比例,对抗氧化应激。
Abstract
Objective To investigate the role of hesperidin（Hsd）in attenuating hypoxic injury in H9c2 cells. Methods H9c2 cells divided into three groups: the normoxia group, the hypoxia group, and the hypoxia + Hsd group (H+Hsd). In addition, a hypoxic +Hsd+JNK agonist anisomycin group (designated as H+Hsd+Ani) was established to investigate the mechanism underlying the action of Hsd on H9c2 cells. The normoxia group group was maintained at 37°C with 5% CO2. The H + Hsd group received pre-treatment with varying concentrations of Hsd (5, 10, 15, 20, 40, 50, and 80 µmol/L) under normoxic conditions for 12 hours before being transferred to a hypoxic workstation for exposure to low oxygen levels (0.5% O2) for a duration of 48 hours. Cell viability was assessed using the CCK8 assay. Apoptosis and reactive oxygen species levels were evaluated by flow cytometry. The content of malondialdehyde (MDA) and superoxide dismutase (SOD) activity within cells was measured using corresponding kits. Creatine kinase MB isoenzyme (CKMB) and cardiac troponin I (cTn-I) levels were quantified via enzyme-linked immunosorbent assay (ELISA). Lactate dehydrogenase (LDH) activity was determined using an LDH assay kit. Corresponding protein levels were analyzed by western blotting techniques, while cellular immunofluorescence assay was employed to detect p65 nuclear translocation. Results Compared to the normoxia group, the hypoxia group exhibited reduced proliferation capacity (P<0.05), increased apoptosis rate and oxidative stress (P<0.01). Additionally, phosphorylation levels of JNK, JUN, and p65 were elevated (P<0.05), nucleation of p65 was enhanced. Meanwhile, the degradation of IKB alpha was observed (P<0.05). Conclusion Hsd has the potential to protect H9c2 cells against the damages induced by hypoxia. The underlying mechanism is associated with the JNK-JUN-NF-kB signaling pathway, by reducing the rate of apoptosis, mitigating oxidative stress.
关键词
橙皮苷 /
低氧 /
心肌细胞 /
心肌损伤
Key words
hesperidin /
hypoxia /
myocardial cell /
myocardial damage
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