高糖通过提高ROS水平和钙超载诱导小鼠MC3T3-E1成骨细胞凋亡

目的: 探讨钙离子(Ca²⁺)超载在高糖诱导成骨细胞株MC3T3-E1凋亡中的作用以及活性氧簇(reactive oxygen species, ROS)的影响。方法: 培养小鼠颅骨源性成骨细胞株MC3T3-E1,给予高浓度D-葡萄糖诱导细胞凋亡。采用MTT法检测不同浓度D-葡萄糖处理24和48 h后MC3T3-E1细胞的增殖情况; D-葡萄糖(35 mmol/L)处理24 h后,Annexin V-FITC/PI检测细胞凋亡;Fura-2/AM荧光探针检测细胞内Ca²⁺浓度; DCFH-DA荧光探针检测ROS的生成。结果: MC3T3-E1高糖处理后,细胞增殖明显抑制呈剂量效应关系,早期凋亡率和总死亡率分别增加至(24.16±3.53)%和(63.74±4.32)%。高糖处理后细胞内ROS水平明显增加,抗氧化剂N-乙酰半胱氨酸可以在降低ROS水平的同时抑制高糖所引起的成骨细胞凋亡。细胞内游离Ca²⁺水平也明显增加,并且通过膜通透性Ca²⁺螯合剂BAPTA-AM处理后,细胞内Ca²⁺浓度明显降低,同时成骨细胞凋亡率也明显降低。加入镧离子(La³⁺)离子抑制储量操纵性钙通道(store-operated Ca²⁺ channels,SOCC)可以降低细胞内Ca²⁺浓度,减低高糖所引起的成骨细胞凋亡。在高糖诱导成骨细胞凋亡中,细胞内ROS和Ca²⁺的增加呈相互促进的关系。结论: 高糖可以通过增加细胞内ROS水平,引起钙离子通过SOCC通道释放,造成细胞内Ca²⁺超载,从而诱导成骨细胞凋亡。

Calcium overload and reactive oxygen species mediate high glucose-induced apoptosis of mouse osteoblast MC3T3-E1 cells

AIM: To investigate the role of reactive oxygen species(ROS) and calcium overload in the apoptosis of MC3T3-E1 cells induced by high glucose.METHODS: Cultured mouse skull bone-derived osteoblast cell line MC3T3-E1 was treated with high concentration of D-glucose to induce apoptosis.The proliferation of MC3T3-E1 cells was detected by MTT assay after treated with different concentrations of D-glucose for 24 h and 48 h.The apoptotic rate and the intracellular levels of calcium and ROS were also measured after the cells were treated with high glucose(35 mmol/L) for 24 h.RESULTS: After high glucose treatment,the cell proliferation was inhibited.The early apoptosis and total cell death increased to(24.16±3.53)% and(63.74±4.32)%,respectively.High glucose treatment significantly increased intracellular levels of ROS and Ca2+.The increased apoptotic rate was reduced by addition of antioxidant N-acetylcysteine and calcium chelator BAPTA-AM.Inhibition of store-operated Ca2+ channels by La3+ also decreased the intracellular level of Ca2+ and cell apoptosis induced by high glucose.CONCLUSION: High glucose increases intracellular ROS level and the release of Ca2+ through the store-operated Ca2+ channels,thus resulting in intracellular Ca2+ overload and leading to apoptosis of osteoblasts.