Feng Y, Yang D, Zhi X, Deng H, Zhang W, Wang R, Wu W
Second School of Clinical Medicine, Southern Medical University, Guangzhou 510515, China.
Department of Endocrinology, East Ward of Guangdong Geriatric Institute, Guangdong Provincial People's Hospital, Guangdong Academy of Medical Sciences, Guangzhou 510080, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2022 Jan 20;42(1):108-115. doi: 10.12122/j.issn.1673-4254.2022.01.13.
To explore the interaction between reactive oxygen species (ROS) and ferroptosis in methylglyoxalinduced injury of mouse embryonic osteoblasts (MC3T3-E1 cells).
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.
Treatment of MC3T3-E1 cells with 0.6 mmol/L methylglyoxal for 24 h significantly inhibited the expression level of GPX4 ( < 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 ( < 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 ( < 0.01); co-treatment with 4 mmo/L FER-1 (a ferroptosis inhibitor) and methylglyoxal obviously decreased the intracellular ROS level ( < 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 ( < 0.001).
The interaction between ROS and ferroptosis pathway plays an important role in methylglyoxal-induced injury of mouse embryonic osteoblasts.
探讨活性氧（ROS）与铁死亡在甲基乙二醛诱导的小鼠胚胎成骨细胞（MC3T3-E1细胞）损伤中的相互作用。
用甲基乙二醛处理MC3T3-E1细胞以建立糖尿病骨质疏松细胞模型。采用CCK-8法检测MC3T3-E1细胞的活力。用罗丹明123染色后进行荧光摄影检测线粒体膜电位（MMP）。用2',7'-二氯二氢荧光素二乙酸酯染色结合荧光摄影检测细胞内ROS水平。使用碱性磷酸酶（ALP）试剂盒检测细胞中的ALP活性，用茜素红S染色测定矿化结节数量，并用检测试剂盒检测铁离子水平。采用蛋白质免疫印迹法测定成骨细胞中谷胱甘肽过氧化物酶4（GPX4，一种抑制铁死亡的标志物蛋白）的表达水平。
用0.6 mmol/L甲基乙二醛处理MC3T3-E1细胞24小时后，显著抑制了GPX4的表达水平（<0.001），增加了细胞内铁离子浓度，降低了细胞活力，增加了MMP的丧失和细胞内ROS水平，降低了细胞中的ALP活性和矿化结节数量（<0.001）。用2 mmol/L N-乙酰半胱氨酸（NAC，一种ROS清除剂）与甲基乙二醛共同处理MC3T3-E1细胞，显著增加了GPX4的表达水平（<0.01）；用4 mmo/L FER-1（一种铁死亡抑制剂）与甲基乙二醛共同处理，明显降低了细胞内ROS水平（<0.001）。用NAC和甲基乙二醛或FER-1和甲基乙二醛共同处理细胞，减轻了甲基乙二醛诱导的成骨细胞损伤（<0.001）。
ROS与铁死亡途径之间的相互作用在甲基乙二醛诱导的小鼠胚胎成骨细胞损伤中起重要作用。
