中国骨质疏松杂志

二甲双胍调控PI3K/Akt/mTOR通路减轻糖皮质激素诱导的成骨细胞凋亡

Metformin attenuates glucocorticoid-induced osteoblast apoptosis by regulating PI3K/Akt/mTOR pathway

DOI：10.3969/j.issn.1006-7108.2021.09.015

中文关键词: 二甲双胍糖皮质激素磷脂酰肌醇3激酶/蛋白激酶B/哺乳动物雷帕霉素靶蛋白通路成骨细胞细胞凋亡

英文关键词:Metformin glucocorticoid phosphatidylinositol 3 kinase/protein kinase B/mammalian target of rapamycin pathway osteoblasts apoptosis

基金项目:国家自然科学基金青年科学基金项目（81802128）

作者	单位
张丰姣毛雨何丽宋利革刘红梅许玲玉孙玲康志强* 王永魁	郑州大学附属郑州中心医院内分泌科, 河南郑州 450000

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中文摘要:

目的探讨二甲双胍(Met)调控磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(Akt)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路对糖皮质激素地塞米松(Dex)诱导的成骨细胞凋亡的影响。方法将体外培养的小鼠胚胎成骨细胞前体细胞MC3T3-E1分为对照组(正常培养）、Dex组(以Dex处理)、Met组(以Dex和Met共同处理)、Met+IGF-1组(以Dex、Met和PI3K/Akt/mTOR通路活化剂IGF-1共同处理)、Met+NVP-BEZ235组(以Dex、Met和PI3K/Akt/mTOR通路抑制剂NVP-BEZ235共同处理)，采用免疫印迹法(WB)检测MC3T3-E1细胞中PI3K、Akt、磷酸化(p)-Akt、mTOR和p-mTOR蛋白表达水平，通过噻唑蓝(MTT)法检测MC3T3-E1细胞存活率、流式细胞术检测MC3T3-E1细胞凋亡率、实时荧光定量PCR检测MC3T3-E1细胞中Bcl-2和Bax mRNA表达水平、Caspase-3活性测定试剂盒检测MC3T3-E1细胞Caspase-3活性、JC-1探针检测MC3T3-E1细胞线粒体膜电位变化。结果与对照组比较，Dex组细胞中PI3K、p-Akt、p-mTOR蛋白表达水平和细胞存活率、Bcl-2 mRNA表达水平以及线粒体膜电位均明显降低，而细胞凋亡率、Bax mRNA表达水平和Caspase-3活性均明显升高（P<0.05）；与Dex组比较，Met组细胞中PI3K、p-Akt、p-mTOR蛋白表达水平和细胞存活率、Bcl-2 mRNA表达水平以及线粒体膜电位均明显升高，而细胞凋亡率、Bax mRNA表达水平、Caspase-3活性明显降低（P<0.05）；给予IGF-1作用后Met对MC3T3-E1细胞的作用效果明显增强，而给予NVP-BEZ235作用后Met对MC3T3-E1细胞的作用效果明显减弱（P<0.05）。结论 Met可通过激活PI3K/Akt/mTOR通路抑制线粒体凋亡途径，减轻糖皮质激素Dex诱导的成骨细胞凋亡。

英文摘要:

Objective To investigate the effect of Metformin (Met) on dexamethasone (Dex) -induced apoptosis of osteoblasts by regulating phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway. Methods Embryonic osteoblast precursor cells MC3T3-E1 of mouse cultured in vitro were divided into control group (normal cultured), Dex group (treated with Dex), Met group (treated with Dex and Met), Met + IGF-1 group (treated with Dex, Met and PI3K/Akt/mTOR pathway activator IGF-1), Met + NVP-BEZ235 group (treated with Dex, Met and PI3K/Akt/mTOR pathway inhibitor NVP-BEZ235), the protein expression levels of PI3K, Akt, phosphorylated (P)-Akt, mTOR and p-mTOR in MC3T3-E1 cells were detected by Western blot (WB), MTT assay was used to detect the survival rate of MC3T3-E1 cells, the apoptosis rate of MC3T3-E1 cells was detected by flow cytometry, the mRNA expression levels of Bcl-2 and Bax in MC3T3-E1 cells were detected by real-time fluorescence quantitative PCR, Caspase-3 activity assay kit was used to detect caspase-3 activity in MC3T3-E1 cells, in addition, JC-1 probe was used to detect the changes of mitochondrial membrane potential in MC3T3-E1 cells. Results Compared with those in the control group, the protein expression levels of PI3K, p-Akt and p-mTOR, cell survival rate, mRNA expression level of Bcl-2, and mitochondrial membrane potential in Dex group were significantly lower, while the apoptosis rate, mRNA expression level of Bax and caspase-3 activity were significantly higher (P<0.05); compared with those in Dex group, the protein expression levels of PI3K, p-Akt and p-mTOR, cell survival rate, mRNA expression level of Bcl-2, and mitochondrial membrane potential in Dex group were significantly higher, while the apoptosis rate, mRNA expression level of Bax and caspase-3 activity were significantly lower (P<0.05); the effect of Met on MC3T3-E1 cells was significantly enhanced after IGF-1 treatment, while the effect of Met on MC3T3-E1 cells was significantly decreased after treatment with nvp-bez235 (P<0.05). Conclusion Met can inhibit mitochondrial apoptosis and reduce the glucocorticoid Dex-induced apoptosis of osteoblasts by activating PI3K/Akt/mTOR pathway.

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