Effect of glycitin on osteoblast apoptosis induced by dexamethasone
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[目的]探讨黄豆黄苷(glycitin,GL)对地塞米松(dexamethasone,DEX)诱导的成骨细胞凋亡和氧化应激损伤的保护作用,并探讨其可能的分子机制.[方法]应用DEX刺激MC3T3-E1细胞模拟激素性股骨头坏死(glucocorticoids-induced os-teonecrosis of the femoral head,GC-ONFH)体内的激素环境,根据细胞处理不同分为3组,对照组(blank control,BC):培养基中加入等量的 PBS;DEX组:培养基中加入浓度为 100 μmol/L 的 DEX;DEX+GL 组(dexamethasone+glycitin,DEX+GL):培养基中加入浓度为100µmol/L的DEX以及浓度为15 μmol/L的GL,干预时间为24 h,不分时间组.MC3T3-E1细胞传代常规培养48 h后,将细胞培养基更换为成骨诱导培养基.[结果]流式细胞检测方面,与BC组比较,DEX组细胞凋亡率显著增加,与DEX 组相比,DEX+GL 组细胞凋亡率显著降低[(9.8±1.5)％vs(17.7±1.4)％vs(13.6±0.4)％,P＜0.001].RT-qPCR 检测方面,BC 组,DEX 组和 DEX+GL组的基因表达水平分别为,Collagen I[(1.0±0.0)vs(0.5±0.3)w(1.0±0.2),P=0.011],Runx-2[(1.0±0.0)vs(0.6±0.1)vs(1.1±0.0),P＜0.001],Cleaved Caspase 3[(1.0±0.0)vs(1.3±1.3)vs(0.9±0.0),P=0.002]、Bax[(1.0±0.0)vs(1.4±0.3)vs(0.8±0.1),P=0.008],差异均有统计学意义.Western blot 检测方面,与 BC 组相比,DEX 组 ALP、Collagen Ⅰ、Runx-2、Bcl-2、Wnt3a、β-catenin的蛋白表达水平显著下降(P＜0.05);与DEX组相比,DEX+GL组上述蛋白表达水平显著增加(P＜0.05).与BC组相比,DEX组Cleaved Caspase 3、Bax的蛋白表达水平显著增加(P＜0.05),与DEX组相比,DEX+GL组上述两指标的蛋白表达水平显著减少(P＜0.05).与BC组相比,DEX组的细胞绿色荧光显著增强(P＜0.05),与DEX组相比,DEX+GL组绿色荧光显著减弱(P＜0.05).[结论]GL能够逆转DEX介导的成骨细胞成骨抑制、改善DEX介导的成骨细胞凋亡增加,保护DEX介导的成骨细胞氧化应激损伤,其机制可能与GL激活Wnt3a/β-Catenin信号通路有关.
[Objective]To investigate the protective effect of glycitin(GL)on apoptosis and oxidative stress injury of osteoblasts in-duced by dexamethasone(DEX),and to explore its possible molecular mechanism.[Methods]MC3T3-E1 cells were stimulated with DEX to simulate the hormonal environment in vivo in glucocorticoids-induced osteonecrosis of the femoral head(GC-ONFH).According to different treatments,the cells divided into 3 groups,including blank control group(BC)with the same amount of PBS added into the medium,the DEX group with DEX in 100 μmol/L added into the medium,and the DEX+GL group with DEX in 100 μmol/L and GLin 15 p.mol/L added to the medium,and the intervention time of 24 h.The MC3T3-E1 cells were routinely cultured for 48 h,and the cell medium was changed to osteo-genic induction medium.[Results]In term of flow cytometry,the apoptosis rate of DEX group was significantly increased compared with that of the BC group,while which of DXM+GL group was significantly decreased compared with the DEX group[(9.8±1.5)％vs(17.7±1.4)％vs(13.6±0.4)％,P＜0.001].In term of RT-qPCR detection,the gene expression levels of BC,DEX and DXM+GL groups were as follows:Colla-gen I[(1.0±0.0)vs(0.5±0.3)vs(1.0±0.2),P=0.011].Runx-2[(1.0±0.0)vs(0.6±0.1)vs(1.1±0.0),P＜0.001].cleaved Caspase 3[(1.0±0.0)vs(1.3±1.3)vs(0.9±0.0),P=0.002],BAX[(1.0±0.0)vs(1.4±0.3)vs(0.8±0.1),P=0.008]respectively,with statistically significant differences among the 3 groups.In term of western blot assay,protein expression levels of ALP,Collagen Ⅰ,Runx-2,Bcl-2,Wnt3a and β-catenin in DEX group were significantly decreased compared with those in BC group(P＜0.05),while which in DXM+GL group were significantly in-creased compared with those in the DEX group(P＜0.05).Howerver,the protein expression levels of Cleaved Caspase 3 and BAX in the DEX group were significantly increased compared with the BC group(P＜0.05),while which were significantly decreased in the DXM+GL group compared with those in DEX group(P＜0.05).The green fluorescence of DEX group was significantly enhanced compared with that in the BC group(P＜0.05),whereas which in DXM+GL group was significantly weakened compared with that in the DEX group(P＜0.05).[Conclu-sion]GL does reverse DEX-mediated osteoblast inhibition,improve DEX-mediated osteoblast apoptosis,and protect Dex-mediated osteo-blast oxidative stress injury,which may be related to the activation of Wnt3a/β-Catenin signaling pathway by GL.
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