紫檀芪诱导人视网膜母细胞瘤WERI-Rb-1细胞的细胞凋亡及其机制研究

Pterostilbene induces retinoblastoma WERI-Rb-1 cell apoptosis via autophagy induction

背景与目的:紫檀芪是一种天然抗氧化剂,其抗视网膜母细胞瘤的效果仍不明确。拟探讨紫檀芪对人视网膜母细胞瘤WERI-Rb-1细胞的细胞增殖、细胞凋亡及细胞自噬的作用,并初步阐明其作用机制。方法:采用细胞计数试剂盒(cell counting kit-8,CCK-8)法测定细胞的增殖活力,Annexin V/PI流式细胞术检测细胞凋亡,吖啶橙染色观察细胞内自噬囊泡的变化,蛋白[质]印迹法(Western blot)检测LC3B及P62蛋白的表达。结果:紫檀芪显著抑制WERI-Rb-1细胞的增殖活力(P<0.01),以25、50和100μmol/L的药物浓度作用细胞24 h时,细胞增殖活力分别为(93.02±0.47)%、(55.10±2.04)%和(30.33±1.45)%;50μmol/L紫檀芪处理细胞12、24和48 h时,细胞增殖活力分别为(88.38±3.70)%、(53.37±1.17)%和(29.60±1.05)%。紫檀芪显著诱导细胞凋亡(P<0.01),对照组、24和48 h细胞的凋亡率分别为(4.08±0.79)%、(13.44±2.12)%和(23.49±2.01)%。紫檀芪能够激活WERI-Rb-1细胞发生细胞自噬,上调LC3蛋白的表达,下调P62蛋白的表达,增加细胞内自噬囊泡的数量(P<0.01)。3-MA及Beclin1 si RNA抑制细胞自噬后能够部分逆转紫檀芪的抗肿瘤作用(P<0.01)。3-MA抑制细胞自噬后,紫檀芪处理组的细胞凋亡率为(12.97±2.09)%,3-MA+紫檀芪组为(8.35±1.11)%。siRNA敲低Beclin1后,紫檀芪处理组和siRNA+紫檀芪组的细胞凋亡率分别为(13.80±2.19)%和(9.62±0.52)%。结论:紫檀芪可以显著抑制WERI-Rb-1细胞的细胞增殖并激活细胞自噬促进细胞凋亡。

Background and purpose： Pterostilbene is a natural antioxidant, whose role in retinoblastoma remains unclear. The aim of this study is to probe the effects of pterostilbene on the proliferation, apoptosis and autophagy in retinoblastoma WERI-Rb-1 cell lines. Methods： Cell counting kit-8 （CCK-8） assays were used to analyze the effects of pterostilbene on the proliferation of WERI-Rb-1 cells. Apoptosis rate was determined by Annexin V/PI. Autophagic vacuoles were observed by acridine orange staining. LC3 and P62 protein expressions were determined using Western blot. Results： Pterostilbene significantly inhibited the proliferation of WERI-Rb-1 cells （P〈0.01）. The cell viability were （93.02±0.47）%, （55.10±2.04）% and （30.33±1.45）% after WERI-Rb-1 cells were treated with 25, 50 and 100 μmol/L pterostilbene for 24 h, and the cell viability were （88.38±3.70）%, （53.37±1.17）%, （29.60±1.05）% after WERI-Rb-1 cells were treated with 50 μmol/L pterostilbene for 12, 24 and 48 h. Pterostilbene induced cell apoptosis （P〈0.01）, the apoptosis rates of control group, 24 h treated group and 48 h treated group were （4.08±0.79）%, （13.44±2.12）% and （23.49±2.01）%. Pterostilbene induced autophagy of WERI-Rb- 1 cells, increased LC3 expression, downregulated P62 expression and increased the number of autophagic vacuoles in WERI-Rb-1 cells （P〈0.01）. 3-MA and Beclinl were able to rescue pterostilbene-induced cell death （P〈0.01）. After 3-MA was used to blunt autophagosome formation, the apoptosis rate markedly decreased in 3-MA＋pterostilbene-treated cells compared with cells treated with pterostilbene alone [（12.97±2.09）% vs （8.35±1.11）%], and after siRNA was used to knockdown Beclinl, the apoptosis rate had the same change [（13.80±2.19）% vs （9.62±0.52）%]. Conelusiou： Pterostilbene can inhibit the proliferation of WERI-Rb-1 cells and induce cell apoptosis via autophagy activation.