Abstract: Objective　To investigate the effect of the extract of Empetrum nigrum var. Japonicum on the apoptosis of human epithelial ovarian cancer cells and to explore the mechanism. Methods　The fresh tumor tissue of 10 patients with epithelial ovarian cancer who underwent gynecological surgery in Department of Obstetrics, Liaocheng People's Hospital from January 2019 to September 2020 was collected. The expressions of Beclin-1 and Bcl-2 genes in the epithelial ovarian cancer tissue were detected. The ovarian cancer cells, SKOV3, A2780, and OVCAR3, and the normal ovarian cells, IOSE80, were cultured. The cells were co-cultured with different concentrations of the extracts. The cell activity was determined by the cell counting kit-8 (CCK-8) method, the cell apoptosis was detected by the flow cytometry method, and the mRNA and protein expressions of Beclin-1 and Bcl-2 were detected by real-time fluorescent quantitative polymerase chain reaction (PCR) and Western blot. SPSS 22.0 and GraphPad Prism were used for the statistical analysis. The cell 50% inhibiting concentration (IC₅₀) was analyzed by one way ANOVA. GraphPad Prism was used to analyze the graphs. The measurement data were compared between the groups by independent-sample t test. Results　The expressions of Bcl-2 and Beclin-1 were (0.759±0.110) and (0.537±0.150) in the epithelial ovarian cancer tissue, and were (0.294±0.160) and (1.612±0.130) in the normal ovarian tissue, with statistical differences (both P < 0.01). The Ⅱ, Ⅲ, Ⅳ, and Ⅴ extracts of Emperum nrgrum var. Japorcum significantly inhibited the proliferation of the SKOV3, A2780 and OVCAR3 epithelial ovarian cancer cells, and the apoptosis rate was significantly increased (P < 0.01); it was dose-time dependent; the order of inhibition was as below: Ⅴ>Ⅳ>Ⅱ>Ⅲ; extract I had no obvious inhibitory effect on the proliferation of tumor cells (P>0.05); all the extracts from Emperum nrgrum var. Japorcum had no inhibitory effect on the proliferation of the normal ovarian cells IOSE80 (P>0.05). The relative expression levels of Beclin-1 mRNA and protein in the SKOV3, A2780 and OVCAR3 cells treated with extract Ⅱ, Ⅲ, Ⅳ, and Ⅴ of Emperum nrgrum var. Japorcum were increased (P < 0.01), while the relative expression levels of Bcl-2 mRNA and protein were decreased (P < 0.01). The expression of Beclin-1 protein was significantly increased and the expression of Bcl-2 protein was significantly decreased in the epithelial ovarian cancer cell line SKOV3 treated with extract Ⅱ, Ⅲ, Ⅳ, and Ⅴ of Emperum nrgrum var. Japorcum (P<0.01). Conclusion　The extract Ⅱ, Ⅲ, Ⅳ, and Ⅴ of Emperum nrgrum var. Japorcum have significant inhibitory effects on the proliferation of SKOV3, A2780, and OVCAR3 cells, and the apoptosis rates of SKOV3, A2780, and OVCAR3 are significantly increased. After treatment, the relative expression of Beclin-1 mRNA and protein is significantly increased, while the relative expression of Bcl-2 mRNA and protein is significantly decreased, suggesting that the extract of Emperum nrgrum var. Japorcum may induce apoptosis by regulating the expression of autophagy gene.

Key words: Emperum nrgrum var. Japorcum, Epithelial ovarian cancer, Bcl-2, Beclin-1, Apoptosis, Autophagy

摘要： 目的　探讨东北岩高兰提取物二氢查耳酮类化合物对上皮性卵巢癌细胞抗肿瘤作用，并初步探讨其作用机制。方法　选取2019年1月至2020年9月于聊城市人民医院行妇科手术治疗的上皮性卵巢癌患者10例新鲜肿瘤组织，检测上皮性卵巢癌组织中的Bcl-2、Beclin-1基因表达情况，培养上皮性卵巢癌细胞SKOV3、A2780、OVCAR3及正常卵巢细胞IOSE80，将不同浓度的东北岩高兰提取物与细胞共培养，采用CCK-8（cell counting kit-8）法测定细胞活性，流式细胞术检测细胞凋亡，采用实时荧光定量聚合酶链式反应（polymerase chain reaction，PCR）及Western blot检测药物反应后细胞中Bcl-2、Beclin-1 mRNA及蛋白表达情况。使用统计软件SPSS 22.0、GraphPad Prism进行统计学分析，单因素方差分析进行细胞IC₅₀值测定，GraphPad Prism进行作图分析，计量资料组间比较采用独立样本t检验。结果　Bcl-2在上皮性卵巢癌组织中相对表达量为（0.759±0.110），Beclin-1表达量为（0.537±0.150），正常卵巢组织中分别为（0.294±0.160）、（1.612±0.130），相比正常卵巢组织，上皮性卵巢癌组织Bcl-2表达明显升高、Beclin-1表达显著降低（均P<0.01）。东北岩高兰提取物Ⅱ、Ⅲ、Ⅳ、Ⅴ对上皮性卵巢癌细胞SKOV3、OVCAR3、A2780增殖抑制作用显著，细胞凋亡率明显升高（P<0.01），呈剂量-时间依赖性，按抑制作用排序为：提取物Ⅴ>Ⅳ>Ⅱ>Ⅲ，岩高兰提取物Ⅰ对于肿瘤细胞无增殖抑制作用。经东北岩高兰提取物Ⅱ、Ⅲ、Ⅳ、Ⅴ处理的上皮性卵巢癌细胞SKOV3、A2780、OVCAR3组Bcl-2 mRNA相对表达量降低（P<0.01），Beclin-1 mRNA相对表达量升高（P<0.01）。经东北岩高兰提取物Ⅱ、Ⅲ、Ⅳ、Ⅴ处理的上皮性卵巢癌细胞SKOV3中Beclin-1蛋白表达显著升高（P<0.01），Bcl-2蛋白表达明显减少（P<0.01）。结论　东北岩高兰提取物Ⅱ、Ⅲ、Ⅳ、Ⅴ对上皮性卵巢癌细胞SKOV3、A2780、OVCAR3增殖有显著抑制作用，细胞凋亡率明显升高，说明东北岩高兰提取物对于卵巢癌有抗肿瘤特性；经提取物处理后，肿瘤细胞Beclin-1 mRNA及蛋白相对表达量显著升高，Bcl-2 mRNA及蛋白相对表达量明显降低，提示东北岩高兰提取物可能通过调节自噬基因的表达来诱导细凋亡。

关键词: 东北岩高兰, 上皮性卵巢癌, Bcl-2, Beclin-1, 细胞凋亡, 自噬