二甲双胍抑制骨细胞ROS产生及激活AKT信号通路改善糖皮质激素性骨质疏松  

Metfomin inhibits cellular reactive oxygen species and activates AKT signaling pathway to improve glucocorticoid-induced osteoporosis

作　　者：黄颖 匡明杰 李云洁 赵僖格 钟馨桐 单敏慧 杨强[3] 赵艳红 HUANG Ying;KUANG Mingjie;LI Yunjie;ZHAO Xige;ZHONG Xintong;SHAN Minhui;YANG Qiang;ZHAO Yanhong(Department of Orthodontics,Stomatology Hospital of Tianjin Medical University,Tianjin,300070;Department of Orthopedics,General Hospital of Tianjin Medical University,Tianjin,300052;First Department of Spine,Tianjin Hospital of Tianjin University,Tianjin,300211,China)

机构地区：[1]天津医科大学口腔医学院正畸科,天津300070 [2]天津医科大学总医院骨科,天津300070 [3]天津大学天津医院脊柱一科,天津300070

出　　处：《第三军医大学学报》2019年第1期41-47,共7页Journal of Third Military Medical University

基　　金：国家自然科学基金面上项目(31470937)~~

摘　　要：目的探究二甲双胍改善糖皮质激素诱发骨质疏松(glucocorticoid induced osteoporosis,GIOP)的作用机制。方法分别用地塞米松(dexamethasone,DEX,10μmol/L),DEX(10μmol/L)+二甲双胍(1 mmol/L)处理MLO-Y4骨细胞,设置空白对照,分别用DCFH-DA探针测定骨细胞内ROS水平差异,Western blot检测AKT和Caspase-3蛋白表达差异,CCK-8实验检测细胞增殖活性,TUNEL染色评估细胞凋亡状态,RT-PCR检测SOD2和Cat相关mRNA表达。构建大鼠GIOP动物模型,通过micro-CT检测相关骨代谢参数值,评估二甲双胍对GIOP大鼠成骨作用的影响。结果细胞增殖毒性实验显示DEX可降低骨细胞增殖活性,与空白对照组比较差异有统计学意义(P <0. 05),DEX+二甲双胍组细胞增殖活性与空白对照组比较无明显差异。DCFH-DA探针免疫荧光染色显示,空白对照组和DEX+二甲双胍组细胞内ROS水平没有明显差异,而DEX组ROS水平明显高于其他两组,且RT-PCR显示SOD2和Cat mRNA的表达在DEX组中明显降低(P <0. 05),表明酶抗氧化系统的缺陷。TUNEL染色显示DEX组细胞凋亡数量明显增加,而DEX+二甲双胍组和空白对照组无明显差异,且DEX组的Caspase-3表达明显高于DEX+二甲双胍组和空白对照组,表明二甲双胍可以在DEX处理期间减少细胞凋亡数量。与DEX组相比,DEX+二甲双胍组中AKT蛋白高表达,说明二甲双胍可能激活AKT通路。与空白对照组相比,DEX组的显微结构参数值骨小梁厚度(TB. TH)、相对骨体积或骨体积分数(BV/TV)和骨小梁数量(TB. N)均显著降低(P <0. 05),参数骨小梁分离度(Tb. Sp)显著升高(P <0. 05),但DEX+二甲双胍组与空白对照组比较参数值无明显差异。结论二甲双胍可降低糖皮质激素引起的细胞ROS水平升高,激活AKT信号通路,抑制蛋白水解酶Caspase-3的活性,调控骨细胞增殖凋亡并改善糖皮质激素诱发骨质疏松骨代谢。Objective To clarify the underlying mechanism of metformin in improvement of glucocorticoid induced osteoporosis (GIOP). Methods Dexamethasone (DEX,10 μmol/L) alone or combined with metformin (1 mmol/L) was used to treat murine long bone osteocytes, Y4 (MLO-Y4), and the cells without any treatment served as blank control. Oxidant-sensing probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) was used to measure the production of reactive oxygen species (ROS), Western blotting was employed to detect the protein levels of AKT and Caspase-3, CCK-8 assay and TUNEL assay were conducted to study the cell viability and apoptosis, and realtime reverse transcriptase polymerase chain reaction (RT-PCR) was performed to test the mRNA levels of SOD2 and Cat. After the establishment of GIOP model in Sprague Dawley (SD) rats, micro-CT scanning was carried out to evaluate the effect of metformin on bone metabolism. Results CCK-8 assay demonstrated that DEX significantly reduced the proliferation of MLO-Y4 cells when compared with the blank control cells (P<0.05), while no obvious difference was seen between the DEX+metformin and control cells. DEX group had significantly higher ROS level than the DEX+metformin group and control group, but there was no difference between the latter 2 groups. RTPCR results showed that the mRNA levels of SOD2 and Cat were notably decreased in the DEX group (P<0.05), which indicating defects in enzymatic antioxidant defense system. TUNEL assay suggested the apoptosis was increased in the DEX group, but no difference was seen between the metformin group and control group. The results of Western blotting indicated that the DEX group had higher level of Caspase-3 than the other 2 groups, which suggested that metformin inhibits cell apoptosis induced by DEX, while, the AKT level was higher in the metformin group than the DEX group, showing that metformin activates the AKT signaling pathway. Compared with the blank control group, the microstructural parameters, including trabecular thickne

关 键 词：糖皮质激素诱发骨质疏松 二甲双胍 活性氧 AKT信号通路 

分 类 号：R681[医药卫生—骨科学] R963[医药卫生—外科学]