目的: 在体外细胞水平研究纳米氧化铈(cerium oxide nanoparticles, CeNPs)对阿霉素(doxorubicin, DOX)心肌细胞损伤的影响及可能的作用机制。方法: 利用不同浓度DOX培养大鼠心肌细胞(H9C2)24 h,确定体外模拟DOX心肌损伤的最佳浓度。以此为基础,加入粒径为20 nm的不同浓度的CeNPs与DOX共处理24 h,同时设立正常对照组。采用细胞计数盒(CCK8)法测定心肌细胞活力,用流式细胞仪测定不同组活性氧(reactive oxygen species,ROS)表达量,同时用蛋白免疫印迹法(Western-blot)测定不同组凋亡相关蛋白Bcl-2、Bax的表达量。结果: 选择1 μM DOX处理24 h作为造模浓度;与正常对照组相比,模型组心肌细胞存活率下降、ROS表达量增加、抗凋亡蛋白Bcl-2表达量下降、而促凋亡蛋白Bax的表达量升高(P＜0.05)。与1 μM DOX单独处理组相比,低浓度CeNPs(10 μg/mL、20 μg/mL、50 μg/mL)与1 μM DOX共处理后心肌细胞存活率上升、ROS表达减少、抗凋亡蛋白Bcl-2表达量上调并且促凋亡蛋白Bax的表达量下调(P＜0.05);而高浓度CeNPs(100 μg/mL)与1 μM DOX共处理后,相较于1 μM DOX单独处理组,细胞存活率下降更明显(P＜0.05)、ROS表达量增加(P＜0.05),而凋亡相关蛋白的表达量没有明显变化(P＞0.05)。结论: 粒径为20 nm的低浓度CeNPs(10 μg/mL、20 μg/mL、50 μg/mL)可以通过缓解氧化应激水平,减少细胞凋亡保护DOX诱导的心肌细胞损伤;而高浓度CeNPs(100 μg/mL)对DOX心肌损伤没有保护作用。
Objective: To investigate the effect of cerium oxide nanoparticles (CeNPs) on doxorubicin (DOX) -induced myocardial cell injury and its possible mechanism in vitro. Methods: H9C2 cardiomyocytes were cultured with different concentrations of DOX for 24h to determine the optimal concentration to simulate DOX myocardial injury in vitro. On this basis, different concentrations of CeNPs with particle size of 20 nm and DOX were added for 24 h, and a normal control group was set up. CCK8 assay was used to measure the viability of cardiomyocytes, flow cytometry was used to measure the expression of reactive oxygen species (ROS), and Western-blot was used to measure the expression of apoptosis-related proteins (Bcl-2, Bax). Results: DOX was treated with 1 μM for 24 h as the molding concentration. Compared with normal control group, the survival rate of myocardial cells in model group decreased, ROS expression increased, anti-apoptotic protein Bcl-2 expression decreased, and pro-apoptotic protein Bax expression increased (P＜0.05). Compared with the 1 μM DOX separate treatment group, the survival rate of myocardial cells increased, ROS expression decreased, the expression of anti-apoptotic protein Bcl-2 was up-regulated and the expression of pro-apoptotic protein Bax was down-regulated after co-treatment with low concentration CeNPs (10 μg/mL、20 μg/mL、50 μg/mL) and 1μM DOX treatment (P＜0.05);Co-treatment of CeNPs at a high concentration (100 μg/mL) and 1 μM DOX significantly decreased the cell survival rate (P＜0.05) and increased the expression of ROS (P＜0.05), while the expression of apoptosis-related proteins did not change significantly (P＞0.05). Conclusion: Low concentration CeNPs (10, 20, 50 μg/mL) with particle size of 20 nm can alleviate oxidative stress and reduce apoptosis to protect myocardial cell damage induced by DOX. High concentration of CeNPs (100 μg/mL) has no protective effect on DOX myocardial injury.
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