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

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80.0 mg·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（cleavedcaspase-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42.73 mg·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。

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

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-(1H-1,2,3-triazol-4-yl)pyridine(3-TYP group, Res group and 3-TYP+Res group. After 24 h of 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.