柚皮素对氧化应激所致成骨细胞凋亡的影响及机制研究

Effects of naringenin on apoptosis of osteoblasts mediated by oxidative stress and its action mechanism

目的观察柚皮素对氧化应激导致的成骨细胞凋亡的影响,并探讨其机制。方法体外分离培养成骨细胞,细胞分空白对照组、单纯H_2O_2作用组、H_2O_2+0.1μmol/L柚皮素组、H_2O_2+1μmol/L柚皮素组、H_2O_2+10μmol/L柚皮素组。MTT法检测成骨细胞活力;流式细胞术检测成骨细胞凋亡率;荧光显微镜观察活性氧(ROS)含量;比色法检测丙二醛(MDA)含量;Real time-PCR和Western-blot检测凋亡相关蛋白Bcl-2、Bax、Caspase-3表达情况。结果与空白对照组比较,单纯H_2O_2处理组细胞活性明显降低,凋亡率及ROS、MDA含量明显增加;同时Bcl-2 mRNA和蛋白表达明显下调,Bax、Caspase-3 mRNA和蛋白表达明显上调(P<0.05)。与单纯H_2O_2处理组比较,柚皮素预处理24 h能够明显增强细胞活性,降低细胞凋亡率及ROS、MDA含量,上调Bcl-2 mRNA和蛋白表达,下调Bax、Caspase-3 mRNA和蛋白表达,呈剂量依赖性(P<0.05)。结论柚皮素在氧化应激条件下能够促进成骨细胞增殖,抑制成骨细胞凋亡,其机制与上调Bcl-2表达,下调Bax、Caspase-3表达有关。

Objective To observe the effects of naringenin on apoptosis of osteoblasts mediated by oxidative stress,and to explore its action mechanism. Methods The osteoblasts were separated and cultured in vitro. The experiment was divided into 5 groups： blank control group,simple H2O2 group,H2O2＋ naringenin low-does group（ 0. 1μmol / L）,H2O2＋naringenin medium-dose group（ 1μmol / L） and H2O2＋ naringenin high-dose group（ 10μmol / L）. The cell viabilities of osteoblasts were detected by MTT assay. The cell apoptosis rates were detected by flow cytometry. The levels of reactive oxygen species（ ROS） were observed by fluorescence microscopy. MDA contents were measured by colorimetric technique. The expression levels of Bcl-2,Bax and Caspase-3 mRNA and protein were detected by Real time-PCR and Western Blot,respectively. Results As compared with those in blank control group,the cell viabilities in simple H2O2 group were obviously decreased,however,the cell apoptosis rates and the levels of ROS and MDA were significantly increased,meanwhile,the expression levels of Bcl-2 mRNA and protein were obviously down-regulated,however,the expression levels of Bax and Caspase-3 mRNA and protein were significantly up-regulated（ P〈0. 05）. As compared with those in simple H2O2 group,the cell viabilities in naringenin groups pretreated for 24 hours were obviously increased,and the cell apoptosis rates and the levels of ROS and MDA were significantly decreased,moreover,the expression levels of Bcl-2 mRNA and protein were obviously up-regulated,however,the expression levels of Bax and Caspase-3 mRNA and protein were significantly downregulated,with a dose-dependent manner（ P〈0. 05）. Conclusion Naringenin can promote proliferation of osteoblasts and inhibit apoptosis of osteoblasts mediated by oxidative stress,and its action mechanism may be correlated to up-regulating the expressions of Bcl-2 and down-regulating the expressions of Bax and Caspase-3.