黄豆黄苷对地塞米松诱导成骨细胞凋亡的影响  

Effect of glycitin on osteoblast apoptosis induced by dexamethasone

作　　者：李润泽 刘启彬 陈长军 马小杰 宫傲 张磊[1] LI Run-ze;LIU Qi-bin;CHEN Chang-jun;MA Xiaojie;GONG Ao;ZHANG Lei(Department of Orthopedics,The First Affiliated Hospital,Shandong First Medical University,Jinan,Shan-dong 250000,China;Shandong University of Traditional Chinese Medicine,Jinan,Shandong 250000,China)

机构地区：[1]山东第一医科大学第一附属医院(山东省千佛山医院)骨外科,山东济南250000 [2]山东中医药大学,山东济南250000

出　　处：《中国矫形外科杂志》2024年第24期2264-2270,共7页Orthopedic Journal of China

基　　金：山东省重点研发计划项目(编号:2019GSF107057);山东省自然科学基金项目(编号:ZR2020MH087,ZR2023QH517);济南市临床医学科技创新计划项目(编号:202328059)。

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 osteonecrosis 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100μmol/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15μmol/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。[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 100μmol/L added into the medium,and the DEX+GL group with DEX in 100μmol/L and GLin 15μmol/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 andβ-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 en

关 键 词：黄豆黄苷 成骨细胞 成骨分化 细胞凋亡 ROS WNT/Β-CATENIN 

分 类 号：R318[医药卫生—生物医学工程]