活性氧与铁死亡通路相互作用在丙酮醛引起的小鼠胚胎成骨细胞损伤中起重要作用

Role of interaction between reactive oxygen species and ferroptosis pathway in methylglyoxal-induced injury in mouse embryonic osteoblasts

目的探讨活性氧和铁死亡在丙酮醛诱导小鼠胚胎成骨细胞(MC3T3-E1)损伤中是否存在相互作用。方法应用丙酮醛损伤MC3T3-E1细胞建立模拟糖尿病骨质损伤的细胞模型。应用细胞计数试剂盒-8(CCK-8)测定MC3T3-E1细胞的存活率;罗丹明123染色荧光显微镜照相法测定线粒体膜电位;双氯荧光素(DCFH-DA)荧光显微镜照相法检测胞内活性氧水平;碱性磷酸酶试剂盒检测定碱性磷酸酶活性;茜素红染色观察成骨细胞晚期标志物-矿化结节的形成;铁离子试剂盒检测铁离子水平;Western blot检测成骨细胞的抑制铁死亡的标志蛋白谷胱甘肽过氧化物酶4(GPX4)的表达水平。结果0.6 mmol/L丙酮醛处理MC3T3-E1细胞24 h可明显减少GPX4的表达(P<0.001),同时可使胞内铁离子浓度升高,细胞存活率降低,线粒体膜电位丢失,胞内活性氧水平升高,碱性磷酸酶活性降低和矿化结节减少(P<0.001)。应用2 mmol/L活性氧清除剂N-乙酰半胱氨酸和丙酮醛共处理MC3T3-E1细胞24 h可增加GPX4的表达(P<0.01),应用4μmol/L铁死亡抑制剂铁抑素-1与丙酮醛共处理成骨细胞24 h可使胞内活性氧水平降低(P<0.001);应用N-乙酰半胱氨酸或铁抑素-1与丙酮醛共处理成骨细胞24 h均能对抗丙酮醛引起的上述其他细胞损伤(P<0.001)。结论活性氧与铁死亡通路相互作用在丙酮醛引起MC3T3-E1成骨细胞损伤中起重要的作用。

Objective To explore the interaction between reactive oxygen species(ROS)and ferroptosis in methylglyoxal-induced injury of mouse embryonic osteoblasts(MC3T3-E1 cells).Methods MC3T3-E1 cells were treated with methylglyoxal to establish a cell model of diabetic osteoporosis.CCK-8 assay was used to detect the viability of MC3T3-E1 cells.Rhodamine 123 staining followed by photofluorography was used to examine mitochondrial membrane potential(MMP).The intracellular ROS level was detected by 2',7'-dichlorodihydrofluorescein diacetate staining with photofluorograph.Alkaline phosphatase(ALP)activity in the cells was detected using an ALP kit,the number of mineralized nodules was determined with alizarin red S staining,and the level of iron ions was detected using a detection kit.The expression level of glutathione peroxidase 4(GPX4,a marker protein that inhibits ferroptosis)in the osteoblasts was determined using Western blotting.Results Treatment of MC3T3-E1 cells with 0.6 mmol/L methylglyoxal for 24 h significantly inhibited the expression level of GPX4(P<0.001),increased intracellular iron ion concentration,decreased the cell viability,increased the loss of MMP and intracellular ROS level,decreased both ALP activity and the number of mineralized nodules in the cells(P<0.001).Co-treatment of MC3T3-E1 cells with 2 mmol/L N-acetylcysteine(NAC,a ROS scavenger)and methylglyoxal significantly increased the expression level of GPX4(P<0.01);co-treatment with 4 mmo/L FER-1(a ferroptosis inhibitor)and methylglyoxal obviously decreased the intracellular ROS level(P<0.001).Co-treatment of the cells either with NAC and methylglyoxal or with FER-1 and methylglyoxal attenuated methylglyoxal-induced injuries in the osteoblasts(P<0.001).Conclusion The interaction between ROS and ferroptosis pathway plays an important role in methylglyoxal-induced injury of mouse embryonic osteoblasts.