摘要
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·L-1、5.0μmol·L-1、10.0μmol·L-1、20.0μmol·L-1ATRA处理后细胞的ROS水平均较对照组显著增加,差异均有统计学意义(均为P<0.001)。Western blot检测结果显示,ERS标记蛋白C/EBP同源蛋白(C/EBPhomologous protein,CHOP)、结合免疫球蛋白(binding immunoglobulin protein,BIP)的蛋白表达水平与对照组差异均有统计学意义(均为P<0.05);经ATRA处理的模型组较对照组的血管内皮生长因子A(VEGF-A)、CHOP蛋白表达水平均显著上升,NAC-ARTA处理组、Salubrinal-ARTA处理组及NAC-Salubrinal-ARTA联合处理组较模型组的VEGF-A、CHOP蛋白表达水平均显著下降,差异均有统计学意义(均为P<0.05)。结论ATRA通过激活ROS和ERS信号通路诱导ARPE-19细胞凋亡。
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 13.88μmol·L-1 and 11.99μmol·L-1,respectively.The results of flow cytometry showed that the apoptotic levels of the 10.0μmol·L-1 ATRA group,15.0μmol·L-1 ATRA group,and 20.0μmol·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 2.5μmol·L-1 ATRA group,5.0μmol·L-1 ATRA group,10.0μmol·L-1 ATRA group,and 20.0μmol·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-Phomolo-gous 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.
作者
吴娟
崔冬梅
曾骏文
WU Juan;CUI Dongmei;ZENG Junwen(State Key Laboratory of Ophthalmology,Zhongshan Ophthalmic Center,Sun Yat-Sen University,Guangzhou 510060,Guangdong Province,China)
出处
《眼科新进展》
CAS
北大核心
2020年第12期1101-1106,共6页
Recent Advances in Ophthalmology
基金
国家自然科学基金资助(编号81100872)。
关键词
全反式视黄酸
视网膜色素上皮
凋亡
活性氧
内质网应激
all-trans-retinoic acid
retinal pigment epithelium
apoptosis
reactive oxygen species
endoplasmic reticulum stress
作者简介
吴娟(ORCID:0000-0002-9644-855X),女,1983年4月出生,广东广州人,博士,主治医师,研究方向:视网膜疾病,E-mail:wujuan25@mail2.sysu.edu.cn;通信作者:曾骏文(ORCID:0000-0001-7883-7677),男,1961年12月出生,广东广州人,教授,研究方向:低视力,E-mail:zeng_zoc@163.com。
