沉默信息调节因子3对白藜芦醇诱导的人卵巢癌SKOV3细胞凋亡的影响

Effect of silencing sirtuin 3 on resveratrol-induced apoptosis of human ovarian cancer SKOV3 cells

目的:检测沉默信息调节因子3 (Sirt3)对白藜芦醇(Res)诱导的人卵巢癌SKOV3细胞凋亡的影响,探讨其促进凋亡的机制。方法:人卵巢癌SKOV3细胞加入不同浓度(0、2.5、5.0、10.0、20.0、40.0和80.0mg·L-1)Res培养24h,MTT法检测细胞存活率。将细胞随机分为对照组、Sirt3抑制剂3-(1H-1,2,3-三唑-4-基)吡啶(3-TYP)组、Res组和3-TYP+Res组,培养24h后,MTT法检各组细胞增殖抑制率;采用Hoechst 33342进行细胞核染色,激光共聚焦显微镜观察细胞核形态;采用活性氧(ROS)探针检测细胞中ROS水平;Western blotting法检测各组细胞中Sirt3、B细胞淋巴瘤/白血病-2 (Bcl-2)、Bcl-2相关X蛋白(Bax)和含半胱氨酸的天冬氨酸蛋白水解酶3 (cleaved caspase-3)蛋白表达水平。结果:MTT实验,随着Res浓度的增加,细胞存活率明显降低,Res的半数抑制浓度(IC50)为42.73mg·L^-1。与对照组比较,Res组和3-TYP+Res组细胞增殖抑制率明显升高(P<0.05);与Res组比较,3-TYP+Res组细胞增殖抑制率明显升高(P<0.05)。与对照组比较,3-TYP+Res组细胞核出现固缩、染色增强、核碎裂增多。与对照组比较,3-TYP组细胞红色荧光无明显变化,Res组和3-TYP+Res组细胞红色荧光明显减少。Western blotting法检测,与对照组比较,3-TYP组细胞中Sirt3蛋白表达水平明显降低(P<0.05),Bcl-2、Bax和cleaved caspase-3蛋白表达水平无明显变化(P>0.05),Res组细胞中Sirt3和Bcl-2蛋白表达水平明显降低(P<0.05),Bax和cleaved caspase-3蛋白表达水平明显升高(P<0.05);与Res组比较,3-TYP+Res组细胞中Bax和cleaved caspase-3蛋白表达水平明显升高(P<0.05),Bcl-2和Sirt3蛋白表达水平明显降低(P<0.05)。结论:Res可以诱导SKOV3细胞的凋亡,3-TYP通过抑制Sirt3表达可增强Res的作用。

Objective:To investigate the effect of silencing sirtuin 3(Sirt3)on the apoptosis of human ovarian cancer SKOV3 cells induced by resveratrol(Res),and to explore its mechanism of promoting apoptosis.Methods:The human ovarian cancer SKOC3 cells were cultured with different concentrations(0,2.5,5.0,10.0,20.0,40.0 and 80.0 mg·L-1)of Res for 24 h.The survival rate of cells was measured by MTT assay.The SKOV3 cells were randomly divided into control group,Sirt3 inhibitory 3-(1 H-1,2,3-triazol-4-yl)pyridine(3-TYP group,Res group and 3-TYP+Res group.After 24 hof culture,the inhibitory rates of proliferation of the cells in various groups were detected by MTT assay;the nuclei were stained with Hoechst 33342,and the morphorgy nucleus was observed by laser confocal microscope;reactive oxygen species(ROS)probe was used to detect the intracellular ROS levels;Western blotting method was used to detect the expression levels of Sirt3,Bax,Bcl-2 and cleaved caspase-3 proteins in the cells in various groups.Results:The results of MTT assay showed that the survival rates of SKOV3 cells were significantly decreased with the increase of concentration of Res,and the median inhibitory concentration(IC50)was 42.73 mg·L^-1.Compared with control group,the inhibitory rates of proliferation of cells in Res group and 3-TYP+Res group were significantly decreased(P<0.05);compared with Res group,the inhibitory rate of proliferation of the SKOV3 cells in 3-TYP+Res group was significantly inereased(P<0.01).Compared with control group,the nucleus of SKOV3 cells in 3-TYP+Res group showed pyknosis,enhanced staining and more nuclear fragmentation.Compared with control group,the red fluorescence of the SKOV3 cells in 3-TYP group had no significant change,and the red fluorescence of the SKOV3 cells in Res group and 3-TYP+Res group was significantly decreased.The results of Western blotting method showed that compared with control group,the expression level of Sirt3 protein in the cells in 3-TYP group was significantly decreased(P<0.05),and the expression levels of Bcl-2,Bax and cleaved caspase-3 in the cells did not change significantly(P>0.05);the protein expression levels of Sirt3 and Bcl-2 proteins in Res group were significantly decreased(P<0.05),and the expression levels of Bax and cleaved caspase-3 proteins were significantly increased(P<0.05).Compared with Res group,the expression levels of Bax and cleaved caspase-3 proteins in 3-TYP+Res group were significantly increased(P<0.05),and the expression levels of Bcl-2 and Sirt3 proteins in 3-TYP+Res group were significantly decreased(P<0.05).Conclusion:Res can induce the apoptosis of SKOV3 cells,and the inhibition of Sirt3 expression by 3-TYP can enhance the effect of Res.