重楼皂苷Ⅰ对人视网膜色素上皮细胞系ARPE-19增殖的影响

Effects of Polyphylin Ⅰ on proliferation of human retinal epithelial cells line ARPE-19

目的探讨重楼皂苷I对人视网膜色素上皮细胞系(ARPE-19)的增殖和凋亡的影响。方法不同浓度(1.5 mg·L^(-1)、3.0 mg·L^(-1)、6.0 mg·L^(-1))重楼皂苷I作用下的ARPE细胞,CCK-8法检测重楼皂苷I对ARPE-19细胞增殖的影响;流式细胞仪检测ARPE-19细胞周期变化及细胞凋亡水平;Western blot检测ARPE-19细胞内Bax、Bcl-2、cleaved caspase-3、Cyclin D1、ERK1/2、p-ERK1/2蛋白表达。结果 1.5 mg·L^(-1)、3.0 mg·L^(-1)、6.0 mg·L^(-1)重楼皂苷I处理后细胞存活率分别为(68.945±5.647)%、(58.637±5.266)%、(47.338±6.493)%,均低于空白对照组的(98.600±7.803)%;凋亡率分别为(9.622±0.315)%、(26.123±0.331)%、(34.345±0.621)%,均高于空白对照组的(4.512±0.213)%;G0/G1期比例分别为(68.520±2.630)%、(75.222±2.627)%、(81.792±3.931)%,均高于空白对照组的(54.632±2.506)%。重楼皂苷I处理对ARPE-19细胞ERK1/2蛋白表达无明显影响,但下调Cyclin D1、Bcl-2和p-ERK1/2蛋白的表达,上调Bax、cleaved caspase-3蛋白的表达。结论重楼皂苷I通过下调Cyclin D1的表达将ARPE-19细胞阻滞于G1期,抑制细胞增殖。通过抑制ERK信号通路,活化线粒体凋亡通路,诱导细胞凋亡。本研究为中药重楼治疗增生性玻璃体视网膜病变提供了依据。

Objective To study the effects of Polyphylin I on the proliferation and apoptosis of human retinal pigment epithelial cell ARPE-19.Methods CCK-8 assay was performed to investigate the effect of Polyphyllin I on the proliferation of ARPE-19 cells;The effects of Polyphylin I on the cell cycle and apoptosis were detected by flow cytometry with annexin V-FITC /PI;Western blot was used to detect Bax,Bcl-2 pleaved-easpase-3,ERK1/2,p-ERK1/2 and Cyclin D1 expressions.Results Polyphylin I inhibited proliferation of ARPE-19 cells in a dose dependent manner,the survival rates of the cells were（68.945 ±5.647）%,（58.637 ±5.266）%,（47.338 ±6.493）%,（98.600 ±7.803）%in 1.5 mg·L^-1,3.0 mg ·L^-1,6.0 mg ·L^-1 Polyphylin I groups and normal control group,respectively;Polyphylin I treatment resulted in an arrest of cell cycle at G1 phase and an elevated apoptotic percentage in ARPE49 cells,the total apoptotic rates were（9.622 ±0.315）%,（26.123 ±0.331）%,（34.345 ±0.621）%,（4.512 ±0.213）%in 1.5 mg ·L^-1,3.0mg·L^-1）,6.0 mg ·L^-1Polyphylin I groups and normal control group,respectively;The cell rates in G0/G1 phase were（68.520 ± 2.630）%,（75.222 ± 2.627）%,（81.792 ± 3.931）%,（54.632 ± 2.506）%in 1.5 mg ·L^-1,3.0 mg ·L^-1,6.0 mg ·L^-1 Polyphylin Ⅰ groups and normal control group,respectively.Polyphylin Ⅰ treatment had no effect on the ERK1/2 protein expression in ARPE-19 cells,but Cyclin D1,Bcl-2 and p-ERK1/2 protein expressions were markedly decreased Polyphylin I treatment up-regulated the expressions of Bax,cleaved-caspase-3 in ARPE49 cells.Conclusion Polyphylin I treatment can effectively inhibit the ARPE49 cells proliferation and induces cell cycle arrest at Gl phase via down-regulation of Cyclin D1 expression.Meanwhile Polyphylin Ⅰ treatment induce apoptosis of ARPE-19 cells via down-regulation of ERK activity and activation of the mitochondrial pathway.This study provides some evidences for the efficacy of paradis in the treatment of proliferative vitreoretinopathy.