全反式视黄酸诱导人视网膜色素上皮细胞凋亡的实验研究

目的探讨梯度浓度全反式视黄酸(all-trans retinoic acid,ATRA)诱导人视网膜色素上皮细胞凋亡的作用。方法采用流式细胞术检测视网膜色素上皮细胞凋亡、活性氧(ROS)活性。采用实时定量聚合酶链反应和Western blot,从蛋白水平检测梯度浓度ATRA对ARPE-19细胞内质网应激(endoplasmic reticulum stress,ERS)标记蛋白表达的影响。观察抗氧化剂NAC及ERS抑制剂Salubrinal抑制ARPE-19细胞诱导ROS和ERS的作用。结果 CCK-8检测结果显示:ATRA处理ARPE-19细胞24 h和48 h后的半抑制浓度(IC₅₀)分别为13.88μmol·L^-1和11.99μmol·L^-1。流式细胞术检测结果显示,10.0μmol·L^-1、15.0μmol·L^-1、20.0μmol·L^-1ATRA处理后细胞凋亡水平均较对照组显著上升,差异均有统计学意义(均为P<0.001);2.5μmol·...

All-trans retinoic acid induces apoptosis of cultured human retinal pigment epithelial cells in vitro

Objective　To investigate the apoptosis of ARPE-19 cells induced by different doses of all-trans-retinoic acid (ATRA) from the molecular level.Methods　Flow cytometry was used to evaluate the apoptosis and reactive oxygen species（ROS）in ARPE-19 cells. The effects of different doses of ATRA on the expression of endoplasmic reticulum stress (ERS) marker protein in ARPE-19 cells were examined at the protein levels using RT-PCR and Western blot, respectively. The effect of antioxidant NAC and ERS inhibitor Salubrinal on inhibiting ARPE-19 cells inducing ROS and ERS was observed. Results　The results of CCK-8 detection showed that the semi-inhibitory concentrations (IC₅₀) of ATRA treated ARPE-19 cells for 24 h and 48 h were 13.88 μmol?L^-1 and 11.99 μmol?L^-1, respectively. The results of flow cytometry showed that the apoptotic levels of the 10.0 μmol?L^-1 ATRA group, 15.0 μmol?L^-1 ATRA group, and 20.0 μmol?L^-1 ATRA group were significantly higher than those in the control group, and the differences were statistically significant (all P<0.001); ROS levels in 2.5 μmol?L^-1 ATRA group, 5.0 μmol?L^-1 ATRA group, 10.0 μmol?L^-1 ATRA group, and 20.0 μmol?L^-1 ATRA group were significantly increased compared with the control group, and the difference was statistically significant (all P<0.001). The results of Western blot showed that the expression levels of ERS-labeled protein C/EB-Phomologous protein (CHOP) and binding immunoglobulin protein (BIP) had statistically significant differences with the control group (all P<0.05). Compared with the control group, the vascular endothelial growth factor-A (VEGF-A) and CHOP protein expression levels in the ATRA-treated model group increased significantly, and the expression levels of VEGF-A and CHOP protein in the NAC-ARTA treatment group, Salubrinal-AR-TA treatment group and NAC-Salubrinal-ARTA combined treatment group were significantly decreased, and the differences were statistically significant when compared with the model group (all P<0.05).Conclusion　ATRA induces apoptosis of ARPE-19 cells via activating ROS and ERS signaling pathways.