褪黑素通过Nrf-2途径抑制高铁诱导成骨细胞的凋亡

Melatonin inhibits the apoptosis induced by excess iron via activation of Nrf-2 signal pathway in osteoblasts

目的探讨Nrf-2途径在褪黑素抑制高铁诱导成骨细胞凋亡中的作用。方法用细胞贴壁法培养成骨细胞(h FOB1.19)后,将枸橼酸铁铵(400μmol/L)和褪黑素(100μmol/L)加入细胞培养基,培养24 h后,Annexin V-FITC/PI流式细胞学检测细胞凋亡水平;DCFH-DA荧光探针检测细胞内ROS水平;Western blot检测凋亡相关蛋白线粒体内Bax和caspase-3,以及Nrf-2途径相关蛋白Nrf-2和HO-1。结果高浓度枸橼酸铁铵干预成骨细胞后,与对照组相比,细胞内ROS水平增高,枸橼酸铁铵处理后凋亡增加,凋亡相关蛋白,线粒体内Bax表达增加,caspase-3表达增加,同时,Nrf-2途径相关蛋白Nrf-2及HO-1表达增加。而褪黑素可以进一步增加细胞内的Nrf-2及HO-1水平,同时降低细胞内ROS水平,细胞凋亡率和凋亡相关蛋白表达明显降低。结论褪黑素可以通过Nrf-2途径抑制成骨细胞的氧化应激水平,从而降低高铁引起的成骨细胞凋亡。

Objective To investigate the effect of melatonin on the apoptosis of h FOB1. 19 cells induced by excess iron.Methods hFOB1. 19 cells were treated with ferric ammonium citrate（ 400μmol / L） and melatonin（ 100μmol / L） for 24 h. The apoptosis rate and the level of ROS were detected using flowcytometry. Proteins associated with apoptosis like Bax and caspase-3and associated with Nrf-2 signal pathway like Nrf-2 and HO-1 were determined using Western blotting. Results The level of ROS and apoptosis rate increased after intervention with excess iron. The levels of Bax in the mitochondria and cleaved caspase-3 in the cytosol increased. However,after the pretreatment with melatonin,the level of ROS,apoptosis rate,and expression of apoptosis associated proteins decreased,and the expression of Nrf-2 and HO-1 increased. Conclusion Melatonin inhibits the level of oxidation in osteoblasts via Nrf-2 signal pathway,resulting in the reduction of apoptosis induced by excess iron.