高糖对成骨细胞MC3T3-E1凋亡和氧化应激的影响

Effects of high glucose on apoptosis and oxidative stress of osteoblast MC3T3-E1

目的评价高糖环境对成骨细胞MC3T3-E1凋亡和氧化应激的影响,为临床治疗糖尿病骨质疏松症提供实验基础。方法在细胞培养瓶中加入不同浓度高糖溶液(0、20、40、80 mmol/L)培养成骨细胞株MC3T3-E1,24 h后分别采用CCK-8测定其细胞存活率、细胞凋亡率,线粒体膜电位试剂盒测定其去极化程度,半胱天冬酶-3(Caspase-3)试剂盒检测其活性,DCFH-DA荧光探针检测ROS的生成。在80 mmol/L高糖环境暴露,MC3T3-E1细胞在抗氧化剂NAC作用下,采用上述方法测其线粒体膜电位和Caspase-3含量。结果与对照组相比,成骨细胞MC3T3-E1在高糖暴露下的存活率和线粒体膜电位下降,细胞凋亡率、细胞活性氧水平和Caspase-3活性增强,且都存在剂量依赖性;抗氧化剂NAC抑制高糖引起MC3T3-E1细胞线粒体膜电位降低和Caspase-3增高。结论高糖环境下,NAC通过抑制高糖引起的氧化应激,降低细胞凋亡率,保护成骨细胞MC3T3-E1。

Objective To evaluate the effects of high glucose on the apoptosis and oxidative stress of osteoblasts MC3T3-E1,and to provide a certain experimental basis for clinical treatment of diabetic osteoporosis. Methods Osteoblasts MC3T3-E1 were cultured in cell culture bottles with different concentrations of high glucose solutions( 0,20,40,80 mmol/L) for 24 hours. CCK-8 was used to determine the cell survival rate and apoptosis rate,mitochondrial membrane potential kit was used to measure the degree of depolarization,caspase-3 kit was used to detect its activity,and DCFH-DA fluorescence probe was used to detect the generation of ROS. The mitochondrial membrane potential and caspase-3 content of MC3T3-E1 cells were measured by the above method under the action of antioxidant NAC at 80 mmol/L high glucose exposure. Results Compared with the control group,osteoblasts MC3T3-E1 exposed to high glucose showed decreased cell survival rate and mitochondrial membrane potential,enhanced apoptosis rate,ROS level and caspase-3 activity,and dose-dependent tolerance. The antioxidant NAC inhibited the mitochondrial membrane potential decrease and caspase-3 increase of osteoblasts MC3T3-E1 induced by high glucose.Conclusion In high glucose environment,NAC can inhibit oxidative stress induced by high glucose,reduce apoptosis rate and protect osteoblasts MC3T3-E1.