牛膝活性成分β-蜕皮甾酮通过Akt信号干预地塞米松诱导的骨细胞凋亡

β-Ecdysone protects osteocytes from glucocorticoid-induced apoptosis via Akt signal pathway in vitro

目的探讨牛膝活性成分β-蜕皮甾酮对地塞米松诱导MLO-Y4骨样细胞凋亡的抑制作用及其可能的作用机制。方法 10μmol/L地塞米松(dexamethasone,Dex)作用于MLO-Y4骨样细胞,以PI3K/Akt抑制剂LY294002为对照,β-蜕皮甾酮(β-Ecdysone,β-Ecd)干预,分为6组:①正常组;②10μmol/L Dex模型组;③10μmol/L Dex+10μmol/Lβ-Ecd组;④10μmol/L Dex+10μmol/Lβ-Ecd+50μmol/L LY294002组;⑤10μmol/L Dex+0.1μmol/Lβ-Ecd组;⑥10μmol/L Dex+0.1μmol/Lβ-Ecd+50μmol/L LY294002组。作用48 h后,Annexin V-FITC/PI法检测细胞早期凋亡率,Western-Blot检测Akt1、Phospho-Akt(Thr308)、Connexin43(CX43)、Caspase9、Bad、Phospho-Bad蛋白表达。结果与正常组比较,Dex组细胞凋亡率升高,Caspase9、Bad蛋白表达升高,Akt1、CX43蛋白表达降低;与造模组比较,β-Ecd减弱Caspase9蛋白表达,使Akt1、CX43蛋白表达增强;LY294002使Akt1、CX43蛋白表达明显减弱,而β-Ecd并不能使LY294002降低的Akt1蛋白表达升高。结论 0.1μmol/L、10μmol/Lβ-蜕皮甾酮可能通过Akt信号途径干预地塞米松诱导的骨细胞凋亡。

Objective To investigate the effects of β-ecdysone on glucocorticoid-induced apoptosis by MLO-Y4 osteocyte-like cells and the possible mechanism. Methods MLO-Y4 cells were treated with 10 μM dexamethasone(Dex) and divided into 6 groups:(1) Control group;(2) 10 μmol/L Dex group;(3) 10 μmol/L Dex + 10 μmol/L β-Ecd group;(4) 10 μmol/L Dex +10 μmol/L β-Ecd + 50 μmol/L LY294002 group;(5) 10 μmol/L Dex +0.1 μmol/L β-Ecd group;and(6) 10 μmol/L Dex +0.1 μmol/L β-Ecd + 50 μmol/L LY294002 group. After 48 hours, the cell apoptosis was measured by Annexin V-FITC/PI assay. The protein expressions of Caspase9, Bad, Phospho-Bad, Akt1, Phospho-Akt(Thr308) and Connexin43(CX43) were examined with Western blotting. Results Comparing to those in control group, apoptosis rate, protein expressions of Caspase9 and Bad increased, and Akt1 and CX43 decreased in Dex group. Comparing to those in model groups, β-ecdysone inhibited Caspase9 expression, and increased the expressions of Akt1 and CX43. LY294002 significantly reduced the protein expressions of Akt1 and CX43. β-Ecdysone could not rescue the decreased Akt1 expression by LY294002. Conclusion Ecdysone of 0.1 μmol/L and 10 μmol/L may prevent the osteocyte apoptosis induced by glucocorticoid through Akt signal pathway.