氧化应激在高糖诱导成骨细胞凋亡中的作用

Function of oxidative stress in apoptosis of osteoblast induced by high glucose

目的:探讨氧化应激在高糖诱导成骨细胞凋亡中的作用。方法:体外培养的MC3T3-E1细胞,分为正常对照组、N-乙酰半胱氨酸(N-acetyl-L-cysteine,NAC)组、11.0 mmol/L高糖组、11.0 mmol/L高糖+NAC组、22.0 mmol/L高糖组、22.0 mmol/L高糖+NAC组。应用Annexin V-FITC/PI处理不同组别细胞,流式细胞术检测细胞凋亡;Honchest33258染色观察细胞核形态;采用MTT法检测细胞增殖;采用DCFH-DA荧光探针检测细胞活性氧类(reactive oxygen species,ROS)的生成。结果:111.0 mmol/L高糖组较正常对照组MC3T3-E1细胞凋亡率显著增加(P=0.004);22.0 mmol/L高糖组较正常对照组和11.0 mmol/L高糖组MC3T3-E1细胞凋亡率明显增加(P=0.000;P=0.000)。211.0 mmol/L高糖组较正常对照组MC3T3-E1细胞增殖轻微下降(P=0.305);与正常对照组和11.0 mmol/L高糖组相比,22.0 mmol/L高糖组MC3T3-E1细胞增殖显著降低(P=0.001;P=0.009)。311.0 mmol/L和22.0mmol/L高糖组MC3T3-E1细胞胞内ROS水平均明显增加(P=0.000;P=0.000),但无明显浓度依赖性,抗氧化剂NAC可明显降低ROS水平,改善高糖所致的凋亡增加和增殖下降。结论:高糖可通过增加成骨细胞胞内ROS生成引起MC3T3-E1成骨细胞凋亡增加、增殖下降。

Objective：To investigate the function of oxidative stress in apoptosis and proliferation of MC3T3-E1 cells under high glucose. Methods：MC3T3-E1 cells cultured in vitro were divided into normal control group,N-acetyl-L-cysteine（NAC） group,11.0mmol/L high-glucose group,11.0 mmol/L high-glucose＋NAC group,22.0 mmol/L high-glucose group and 22.0 mmol/L high-glucose＋NAC group. Reactive oxygen species（ROS） production was measured with DCFH-DA fluorescent probe. Cell proliferation was measured by MTT analysis. Cells in different groups were stained with Annexin V-FITC/PI,then the apoptosis rates were detected by flow cytometry. Morphology of cell nucleus was observed with Honchest33258. Results：1Cell apoptosis of 11.0 mmol/L high-glucose increased dramatically compared with that of normal control group（P=0.004）. Cell apoptosis in 22.0 mmol/L high-glucose group increased significantly compared with that of normal control group and 11.0 mmol/L high-glucose group（P=0.000;P=0.000）. 2Proliferation in 11.0 mmol/L high-glucose slightly decreased compared with that of normal control group（P=0.305）. Compared with that of normal control and 11.0 mmol/L high-glucose group,proliferation in 22.0 mmol/L high-glucose group decreased significantly（P=0.001;P=0.009）. 3ROS production in osteoblasts remarkablely increased under high glucose（P=0.000;P=0.000）,but not in a dosedependent manner. NAC,as an antioxidant,could reduce ROS production obviously and ameliorate cell apoptosis and proliferation abnormality caused by high glucose. Conclusion ：High glucose can increase ROS production in osteoblasts and subsequently lead to the increase of apoptosis and decrease of proliferation.