白藜芦醇改善多囊卵巢综合征模型大鼠卵巢颗粒细胞线粒体功能和抑制凋亡的作用

Effects of resveratrol on improving mitochondrial function and inhibiting cell apoptosis in ovarian granulosa cells in a rat model of polycystic ovary syndrome

目的:观察白藜芦醇对多囊卵巢综合征(polycystic ovary syndrome,PCOS)模型大鼠卵巢颗粒细胞增殖和凋亡的影响以及线粒体的形态和功能的改变,并探讨其可能的保护机制。方法:32只雌性SD大鼠幼鼠随机分为2组,PCOS模型组(n=24)和模型对照组(n=8)。成功提取PCOS组颗粒细胞并体外培养,然后用不同浓度的白藜芦醇处理颗粒细胞,CCK-8法检测细胞活力的变化;Mito Tracker Deep Red染色观察细胞线粒体的形态变化;JC-1检测线粒体膜电位的变化;ATP检测试剂盒检测ATP的变化;分光光度计检测与凋亡密切相关casppase-3和caspase-9酶活性变化;最后,蛋白免疫印迹法检测颗粒细胞内caspase-3和caspase-9蛋白水平的变化。结果:白藜芦醇处理PCOS模型大鼠卵巢颗粒细胞后,颗粒细胞的增殖活性明显增强(P=0.000);Mito Tracker Deep Read染色结果显示线粒体质量明显增加,形状呈管状或长条状;颗粒细胞内ATP产量和线粒体膜电位也明显增加(P=0.000、P=0.000)。另外,白藜芦醇也降低了caspase-3和caspase-9酶活性和蛋白水平的表达(P=0.000、P=0.000)。结论:白藜芦醇抑制了PCOS模型大鼠卵巢颗粒细胞的凋亡,也促进了细胞的增殖,其机制与白藜芦醇改善颗粒细胞线粒体的形态和功能有关。

Objective:To observe the effects of resveratrol on the proliferation and apoptosis of ovarian granulosa cells(OGCs) and the changes in the morphology and function of mitochondria in a rat model of polycystic ovary syndrome(PCOS),and explore its possible protective mechanism.Methods:Thirty-two young female Sprague-Dawley rats were randomly divided into two groups:polycystic ovary syndrome model group(n=24) and model control group(n=8).Granulosa cells were extracted from the PCOS group and cultured in vitro,and treated with different concentrations of resveratrol.CCK-8 assay was used to measure the changes in cell activity;Mito Tracker Deep Red staining was used to observe the morphological changes in cell mitochondria;JC-1 was used to measure the changes in mitochondrial membrane potential;ATP kits were used to determine the changes in ATP content;spectrophotometry was used to measure the changes in enzymatic activity of caspase-3 and caspase-9,which are closely associated with apoptosis;finally,Western blot was used to measure the changes in the levels of caspase-3 and caspase-9 proteins.Results:After treatment with resveratrol,the OGCs in the rat model of PCOS showed a significant increase in proliferation activity(P=0.000).Mito Tracker Deep Red staining showed a significant increase in mitochondrial mass,with tubular or elongated shapes observed.ATP production and mitochondrial membrane potential in granulosa cells also significantly increased(both P=0.000).In addition,resveratrol reduced the enzymatic activity and protein expression of caspase-3 and caspase-9(both P=0.000).Conclusion:Resveratrol inhibits the apoptosis of OGCs and,ated with the improvement of mitochondrial morphology and function of granulosa cells by resveratrol.