乳酸链球菌素诱导氧化应激促进人骨肉瘤MG63细胞凋亡

Nisin induces apoptosis of human osteosarcoma MG63 cells via oxidative stress

目的:探讨乳酸链球菌素(nisin)对人骨肉瘤MG63细胞凋亡及其相关的氧化应激机制。方法:乳酸链球菌素单独或联合抗氧化剂N-乙酰-L-半胱氨酸(N-acetyl-L-cysteine,NAC)处理MG63细胞后,采用CCK-8法检测细胞活力;annexin-V/PI双染法检测细胞凋亡;2',7'-二氯荧光素二乙酸酯(2',7'-dichlorofluorescein diacetate,DCFH-DA)荧光探针检测细胞内活性氧簇(reactive oxygen species,ROS)含量;5,5',6,6'-四氯-1,1',3,3'四乙基苯并咪唑羰花青碘化物(5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl benzimidazolyl carbocyanine iodide,JC-1)荧光探针检测线粒体膜电位(mitochondrial membrane potential,MMP)的变化,通过Western blot法检测凋亡相关蛋白Bax、Bcl-2和cleaved caspase-3的水平。结果:乳酸链球菌素呈浓度依赖性降低MG63细胞活力,促进细胞凋亡,上调cleaved caspase-3蛋白水平,增加Bax/Bcl-2蛋白表达的比值,并可诱导细胞内ROS大量生成,降低MMP(P<0.05)。抗氧化剂NAC能显著抑制乳酸链球菌素诱导的MG63细胞凋亡,下调cleaved caspase-3蛋白水平,降低Bax/Bcl-2蛋白表达的比值,并降低细胞内ROS水平,恢复MMP(P<0.05)。结论:乳酸链球菌素可能通过诱导人骨肉瘤细胞氧化应激,进而激活线粒体凋亡通路促进细胞凋亡。

AIM:To investigate the effect of nisin on apoptosis of human osteosarcoma MG63 cells and its related oxidative stress mechanism.METHODS:The MG63 cells were cultured in the medium containing different concentrations of nisin with or without antioxidant N-acetyl-L-cysteine(NAC).The cell viability was measured by CCK-8 assay.Apoptosis was analyzed by flow cytometry with annexin-V/PI staining.The production of intracellular reactive oxygen species(ROS)was detected by redox-sensitive dye 2',7'-dichlorofluorescein diacetate(DCFH-DA).The 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl benzimidazolyl carbocyanine iodide(JC-1)was used to detect the changes of mitochondrial membrane potential(MMP).The protein levels of apoptosis-associated molecules Bax,Bcl-2 and cleaved caspase-3 were determined by Western blot.RESULTS:Nisin decreased the viability of MG63 cells and promoted the apoptosis in a dose-dependent manner.It also up-regulated the protein level of cleaved caspase-3,increased the protein expression ratio of Bax/Bcl-2,triggered a large amount of intracellular ROS generation and reduced the MMP(P<0.05).Moreover,antioxidant NAC significantly inhibited nisin-induced apoptosis of MG63 cells,down-regulated the protein level of cleaved caspase-3,decreased Bax/Bcl-2 ratio,reduced intracellular ROS level,and restored the MMP(P<0.05).CONCLUSION:Nisin may promote oxidative stress in human osteosarcoma cells,activate mitochondrial apoptosis pathway,and eventually induce apoptosis.