咖啡酸苯乙酯通过Nrf-2途径抑制地塞米松诱导的成骨细胞凋亡

目的 探讨Nrf-2途径在咖啡酸苯乙酯(CAPE)抑制地塞米松(DEX)诱导成骨细胞凋亡中的作用.方法 用细胞贴壁法培养小鼠颅顶前骨细胞(MC3T3-E1),采用10μmol/L DEX建立细胞损伤模型,以不同浓度CAPE(0.05、0.25、1μmol/L)预处理细胞2h后加入地塞米松共孵育24h;细胞增殖-毒性检测试剂盒(CCK-8)检测细胞增殖活性;依据CCK-8检测结果确定药物浓度后将实验分为对照组、咖啡酸苯乙酯组(CAPE组)、地塞米松组(DEX组),咖啡酸苯乙酯+地塞米松组(CAPE+DEX组);DCFH-DA荧光探针检测细胞内活性氧(reactive oxygen species,ROS)水平;Caspase-3活性检测试剂盒检测Caspase-3酶活性,Western blot法检测Nrf-2 途径相关蛋白Nrf-2和血红素氧合酶1(heme oxygenase,HO-1)蛋白表达水平;流式细胞仪检测细胞凋亡率.结果 10μmol/L DEX作用下细胞形态发生明显变化,损伤作用明显.与对照组相比,DEX组内细胞存活率显著下降(P＜0.01),而细胞内 ROS水平、细胞凋亡率及Caspase-3酶活性显著增加(P＜0.01);同时,Nrf-2途径相关蛋白 Nrf-2及HO-1表达量减少,差异有统计学意义(P＜0.01).与DEX组相比,CAPE+DEX组细胞存活率显著上升(P＜0.01),Nrf-2途径相关蛋白 Nrf-2及HO-1表达明显增加(P＜0.01);此外,CAPE+DEX组细胞内 ROS 水平、细胞凋亡率及Caspase-3酶活显著降低,差异有统计学意义(P＜0.01).结论 咖啡酸苯乙酯可以通过Nrf-2途径降低细胞内ROS水平进而减轻地塞米松诱导的氧化应激所致细胞损伤及凋亡.

Caffeic acid phenethyl ester inhibits the apoptosis induced by dexamethasone via the regulation of Nrf-2 pathway in osteoblasts

Objective To explore the effect of caffeic acid phenethyl ester(CAPE)on the apoptosis of MC3T3-E1 preosteoblasts induced by dexamethasone(DEX).Methods MC3T3-E1 cells were exposed to DEX(10 μmol/L)to establish a model of osteoblast injury.The incubated MC3T3-E1 cells were pretreated with various concentrations of CAPE for 2 hours,and then cultured in combination with 10 μmol/L DEX for 24 hours.Cell proliferation was evaluated by cell count kit(CCK-8).The final concentration of drug was determined by the results of CCK-8,then cells randomly divided into four groups: Control group,CAPE group,DEX group,and CAPE+DEX group.Cell apoptosis rate was detected by Annexin V-FITC/PI staining and flow cytometry in each group.Caspase-3 activity was monitored by Caspase-3 activity assay kit;Intracelluar ROS was detected by DCFH-DA fluorescence staining.The expressions of Nrf-2 and HO-1 were examined by Western blot.Results Dexamethasone could induced MC3T3-E1 cell injury with overt morphological and cell activity changes at 24 hours,especially the 10 μmol/L DEX.Compared with control group,and the cell activi-ty was decreased significantly(P＜0.01),while the level of intracellular ROS,cell apoptosis rate and Caspase-3 activity increased significantly(P＜0.01).Moreover,the Nrf-2 and HO-1 was decreased significantly(P＜0.01).Compare with DEX group,expression of Nrf-2 and HO-1(P＜0.01 vs DEX group)in CAPE+DEX group were significantly increased(P＜0.01).In addition,the levels of ROS,apoptosis and Caspase-3 activity were significantly lower in the CAPE+DEX group(P＜0.01).Conclusion CAPE inhibits the level of ROS in osteoblasts via regulating Nrf-2 pathway,resulting in the reduction of apoptosis induced by dexamethasone.