雌激素对小鼠成骨细胞 MC3T3-E1 氧化应激损伤的保护作用及机制探究
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武汉市第四医院,武汉 430000

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Protective effect and associated mechanism of action of estrogen on oxidative stress injury of MC3T3-E1 osteoblasts in mice
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Wuhan Fourth Hospital,Wuhan 430000, China

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    摘要:

    目的 探讨雌激素对小鼠成骨细胞 MC3T3-E1 氧化应激损伤的保护作用及机制。 方法 将体外培养 MC3T3-E1 细胞分为空白对照组、H2O2 组(300 μmol / L)、H2O2+ 雌激素 0. 1 μmol / L 组、H2O2 + 雌激素 1 μmol / L 组、H2O2+ 雌激素 10 μmol / L 组和 H2O2+ NAC 1 mmol / L 组,分别与相应浓度的药物进行孵育。 CCK-8 检测各组细 胞增殖活性,流式细胞仪检测细胞凋亡率和线粒体膜电位;试剂盒检测丙二醛(MDA)和超氧化物歧化酶( SOD)水平;荧光探针法检测细胞活性氧(ROS)水平;Western Blot 检测 Smad5、Runx2、Bax、Bcl-2、cleaved Caspase-3 蛋白表达。 结果 与空白对照组比较,H2O2 组小鼠成骨细胞的增殖活性明显下降(P< 0. 05),细胞凋亡率和 JC-1 阳性细胞率明显升高(P< 0. 05),MDA 和 ROS 水平明显升高(P< 0. 05),SOD 活性明显下降(P< 0. 05),Smad5、Runx2 和 Bcl-2 蛋白表达明显下降(P< 0. 05),Bax 和 cleaved Caspase-3 蛋白表达明显升高(P< 0. 05);与 H2O2 组比较,H2O2 + 雌激素 1 μmol / L 组、 H2O2 + 雌激素 10 μmol / L 组和 H2O2 + NAC 1 mmol / L 组细胞的增殖活性明显升高 (P< 0. 05),细胞凋亡率和 JC-1 阳性细胞率明显下降(P< 0. 05),MDA 和 ROS 水平明显下降(P< 0. 05),SOD 活性明显升高(P< 0. 05),Smad5、Runx2 和 Bcl-2 蛋白表达明显升高(P< 0. 05),Bax 和 cleaved Caspase-3 蛋白表达明显下降(P< 0. 05)。 结论 雌激素可能通过激活 Smad5 / Runx2 信号轴的表达,降低氧化应激损伤 MC3T3-E1 细胞的 ROS 水平,提高细胞的分化能力。

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    Objective To explore the protective effect and associated mechanism of action of estrogen on oxidative stress injury of mouse MC3T3-E1 osteoblasts (OB). Methods MC3T3-E1 cells cultured in vitro were divided into a blank control group, H2O2 group (300 μmol / L), H2O2+ 0. 1 μmol / L estrogen group, H2O2+ 1 μmol / L estrogen group, H2O2+ 10 μmol / L estrogen group, and H2O2+ 1 mmol / L NAC group, which were incubated with the corresponding concentrations of the drugs. The proliferative activities of cells in each group were determined by CCK-8. The apoptosis rate and mitochondrial membrane potential (MMP) were determined by flow cytometry. The levels of malondialdehyde (MDA) and superoxide dismutase (SOD) were determined using kits. The level of reactive oxygen species (ROS) was determined by a fluorescent probe method . The expression of Smad5, Runx2, Bax, Bcl-2, and cleaved Caspase-3 proteins was determined by western blotting. Results Compared with those in the blank control group, the proliferative activities of cells were significantly decreased (P< 0. 05), apoptosis rate and positive rate of JC-1 cells were significantly increased (P< 0. 05), levels of MDA and ROS were significantly increased (P< 0. 05), SOD activity was significantly decreased (P< 0. 05), expression of Smad5, Runx2, and Bcl-2 proteins was significantly decreased ( P < 0. 05), and expression of Bax and cleaved Caspase-3 proteins was significantly increased in the H2O2 group (P< 0. 05). Compared with those in the H2O2 group, proliferative activities of cells were significantly increased (P< 0. 05), apoptosis rate and positive rate of JC-1 cells were significantly decreased (P< 0. 05), levels of MDA and ROS were significantly decreased (P< 0. 05), SOD activity was significantly increased (P< 0. 05), expression of Smad5, Runx2, and Bcl-2 proteins was significantly increased (P< 0. 05), and expression of Bax and cleaved Caspase-3 proteins was significantly decreased in the H2O2+ 1 μmol / L estrogen group, H2O2+ 10 μmol / L estrogen group, and H2O2 + 1 mmol / L NAC group (P< 0. 05). Conclusions Estrogen may reduce the level of ROS in MC3T3-E1 cells damaged by oxidative stress and improve the differentiation ability of cells by activating the expression of the Smad5 / Runx2 signal axis.

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徐昊.雌激素对小鼠成骨细胞 MC3T3-E1 氧化应激损伤的保护作用及机制探究[J].中国实验动物学报,2020,28(6):824~830.

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  • 收稿日期:2020-05-19
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  • 在线发布日期: 2021-02-05
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