SIRT1对H2O2诱导的人卵巢颗粒细胞氧化应激损伤的影响

Effect of SIRT1 on H2O2-induced oxidative damage in human ovarian granulosa cells

目的·探讨沉默信息调节因子1(silent information regulator 1,SIRT1)在H2O2诱导的人卵巢颗粒细胞氧化应激损伤中的作用及可能机制。方法·分别用0、100、250、500、1000μmol/L H2O2处理人卵巢颗粒细胞SVOG 4、8、12、24 h,CCK-8法检测细胞活力,选择合适的H2O2浓度和处理时间用于建立颗粒细胞体外氧化应激损伤模型。荧光显微镜观察H2O2处理后细胞核形态变化,试剂盒检测细胞丙二醛(malondialdehyde,MDA)含量和超氧化物歧化酶(superoxide dismutase,SOD)活性;实时定量PCR、Western blotting分析分别检测SIRT1、衔接蛋白P66SHC(the 66 kDa Src homology 2 domain containing isoform)及B淋巴细胞瘤-2因子(B-cell lymphoma-2,BCL-2)mRNA和蛋白的表达水平;脂质体转染SIRT1过表达质粒后,SVOG细胞再经H2O2处理,同样观察上述指标的变化。结果·250μmol/L H2O2处理SVOG细胞12 h,可诱发细胞活力明显下降(P=0.017),细胞核固缩,MDA含量增加(P=0.001),SOD活性下降(P=0.006);伴随SIRT1、BCL-2 mRNA和蛋白表达降低,P66SHC表达升高。SIRT1过表达后再经H2O2处理,与H2O2处理组相比,SVOG细胞核恢复正常形态,MDA含量下调(P=0.038),SOD活性回升(P=0.021),P66SHC表达降低(P=0.002),BCL-2表达升高(P=0.013)。结论·SIRT1可能通过下调P66SHC和上调BCL-2的表达发挥对抗人卵巢颗粒细胞氧化应激损伤的作用。

Objective·To investigate the effect and possible mechanism of silent information regulator 1(SIRT1)on H2O2-induced oxidative damage in human ovarian granulosa cells.Methods·Human ovarian granulosa cells SVOG were treated with 0,100,250,500,1000μmol/L H2O2 for 4,8,12,24 h,respectively.The cell viability was measured by CCK-8 method,and the appropriate H2O2 concentration and treatment time were used to establish the oxidative stress injury model of granulosa cells in vitro.The nuclear morphological changes after H2O2 treatment were observed with fluorescence microscope.The malondialdehyde(MDA)levels and superoxide dismutase(SOD)activities were detected by chemical chromatometry kits.Real-time quantitative PCR and Western blotting were respectively used to analyze the mRNA and protein levels of SIRT1,P66SHC(the 66 kDa Src homology 2 domain containing isoform)and B-cell lymphoma-2(BCL-2).The changes of the above indicators were also assessed after SVOG cells were transfected with SIRT1 overexpression plasmid by liposome and treated with H2O2.Results·After treatment with 250μmol/L H2O2 for 12 h,the SVOG cell viability decreased significantly(P=0.017),the cell nuclei shrank,the MDA level increased(P=0.001),the SOD activity decreased(P=0.006),and the expression of P66SHC increased with the decreased expression of SIRT1 and BCL-2 at mRNA and protein levels.After overexpression of SIRT1 and treatment with H2O2,the nuclei of SVOG cells returned to normal morphology,the MDA level decreased(P=0.038),the SOD activity increased(P=0.021),the expression of P66SHC decreased(P=0.002)and the expression of BCL-2 increased(P=0.013).Conclusion·SIRT1 can protect human ovarian granulosa cells from oxidative damage by down-regulating the expression of P66SHC and up-regulating BCL-2.