氧化应激和DNA损伤在小剂量电离辐射诱导血管内皮细胞损伤中的作用研究

Study on the role of oxidative stress and DNA damage in vascular endothelial cell injury induced by low dose ionizing radiation

目的:评估氧化应激和DNA损伤在小剂量X射线对EA.hy926人脐静脉内皮细胞(HUVEC)(简称EA.hy926细胞)损伤中的作用。方法:将EA.hy926细胞进行X射线照射,根据照射剂量将其分为对照组(0 mGy)、187.5 mGy组、375 mGy组和750 mGy组。X射线照射后使用2,7-二氯荧光素二乙酸酯(DCFH-DA)荧光探针检测细胞内活性氧(ROS)水平,并用比色法测定细胞内脂质过氧化产物丙二醛(MDA)含量和超氧化物歧化酶(SOD)活性。利用蛋白免疫印迹法(Westernblot)检测4组X射线照射后的生物蛋白γ-H2AX及磷酸化P65蛋白(p-P65蛋白)表达变化。结果:X射线照射EA.hy926细胞后1 h和24 h,187.5 mGy组、375 mGy组和750 mGy组均较对照组细胞内2’,7’-二氯荧光素(DCF)荧光强度显著增加,差异有统计学意义(F=107.5,F=20.60;P<0.05)。X射线照射后24 h,EA.hy926细胞内MDA含量较对照组显著升高,而SOD活性较对照组显著下降,差异均有统计学意义(F=52.12,F=506.40;P<0.05)。照射后1 h,γ-H2AX蛋白表达在呈剂量依赖性显著增加,p-P65蛋白表达显著升高,与对照组相比差异均有统计学意义(F=176.00,F=105.50;P<0.05);照射后24 h,375 mGy组和750 mGy组γ-H2AX蛋白和p-P65蛋白表达量与对照组相比显著升高,差异均有统计学意义(F=623.50,F=232.60;P<0.05)。结论:电离辐射可诱导内皮细胞发生氧化应激和DNA损伤,并可伴随NF-κB信号通路的激活,引起EA.hy926细胞损伤。

Objective: To evaluate the role of oxidative stress and DNA damage in EA.hy926 human umbilical vein endothelial cells(HUVEC)(referred to as EA.hy926 cells) injury induced by low-dose X-ray. Methods: EA.hy926cells were irradiated with X-rays divided into control group(0 mGy), 187.5 mGy group, 375 mGy group and 750mGy group according to the irradiation dose. After X-ray irradiation, intracellular reactive oxygen species(ROS)levels were measured by the 2,7-dichlorofluorescein diacetate(DCFH-DA) fluorescent probe, and intracellular lipid peroxidation product malondialdehyde(MDA) content and superoxide dismutase(SOD) activity were measured by colorimetric. The changes of γ-H2AX and p-P65 protein expression in the four groups after X-ray irradiation were detected by Western blot. Results: At 1h and 24h after X-ray irradiation of EA.hy926 cells, compared with the control group, the 2’,7’-Dichlorofluorescein(DCF) fluorescence intensity increased significantly in the 187.5mGy, 375mGy and 750mGy groups, the difference was statistically significant(F=107.5, F=20.60;P<0.05). At 24h after X-ray irradiation, the malondialdehyde(MDA) content in EA.hy926 cells was significantly higher than that in the control group, and superoxide dismutase(SOD) activity was significantly lower than that in the control group, the difference was statistically significant(F=52.12, F=506.40;P<0.05). At 1h after irradiation, the expression of γ-H2AX protein was increased significantly in a dose-dependent manner, the expression of p-P65 protein was increased significantly,compared with the control group, the difference was statistically significant(F=176.00, F=105.50;P<0.05);at 24h after irradiation, compared with the control group, the expression of γ-H2AX protein and p-P65 protein in the 375mGy and 750mGy groups was increased significantly, the difference was statistically significant(F=623.50, F=232.60;P<0.05). Conclusion: Ionizing radiation can induce oxidative stress and DNA damage in endothelial cells, which can be accompanied by the activation of NF-κB signaling pathway and cause EA.hy926 cell damage.