黄豆黄苷对地塞米松诱导成骨细胞凋亡的影响△（开放获取）

Effect of glycitin on osteoblast apoptosis induced by dexamethasone

摘要:

[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100 μmol/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) 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]，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。

Abstract:

[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 tosimulate the hormonal environment in vivo in glucocorticoids-induced osteonecrosis of the femoral head (GC-ONFH). According to differenttreatments, the cells divided into 3 groups, including blank control group (BC) with the same amount of PBS added into the medium, the DEXgroup with DEX in 100 μmol/L added into the medium, and the DEX+GL group with DEX in 100 μmol/L and GLin 15 μmol/L added to themedium, 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 thatof 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 differencesamong 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 DEXgroup were significantly increased compared with the BC group (P＜0.05), while which were significantly decreased in the DXM+GL groupcompared with those in DEX group (P＜0.05). The green fluorescence of DEX group was significantly enhanced compared with that in the BCgroup (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.