Abstract
The present study was designed to analyze the metabolites of all-frans-retinal (atRal) and compare the cytotoxicity of atRal versus its derivative all-frans-retinoic acid (atRA) in human retinal pigment epithelial (RPE) cells. We confirmed that atRA was produced in normal pig neural retina and RPE. The amount of all-trans-retinol (atROL) converted from atRal was about 2.7 times that of atRal-derived atRA after incubating RPE cells with 10 µmol/L atRal for 24 h, whereas atRA in medium supernatant is more plentiful (91 vs. 29 pmol/mL), suggesting that atRA conversion facilitates elimination of excess atRal in the retina. Moreover, we found that mRNA expression of retinoic acid-specific hydroxylase CYP26b1 was dose-dependently up-regulated by atRal exposure in RPE cells, indicating that atRA inactivation may be also initiated in atRal-accumulated RPE cells. Our data show that atRA-caused viability inhibition was evidently reduced compared with the equal concentration of its precursor atRal. Excess accumulation of atRal provoked intracellular reactive oxygen species (ROS) overproduction, heme oxygenase-1 (HO-1) expression, and increased cleaved poly(ADP-ribose) polymerase 1 (PARP1) expression in RPE cells. In contrast, comparable dosage of atRA-induced oxidative stress was much weaker, and it could not activate apoptosis in RPE cells. These results suggest that atRA generation is an antidotal metabolism pathway for atRal in the retina. Moreover, we found that in the eyes of ABCA4−/−RDH8−/− mice, a mouse model with atRal accumulation in the retina, the atRA content was almost the same as that in the wild type. It is possible that atRal accumulation simultaneously and equally promotes atRA synthesis and clearance in eyes of ABCA4−/−RDH8−/− mice, thus inhibiting the further increase of atRA in the retina. Our present study provides further insights into atRal clearance in the retina.
概要
目 的
探讨视网膜中全反式视黄醛(atRal)能否代谢生 成全反式维甲酸(atRA), 并比较两者对视网膜 色素上皮细胞(RPE)的细胞毒性作用, 以阐明 atRA 生成的意义。
创新点
建立atRA 的超高效液相串联质谱(UPLCMS/MS)检测方法, 并证明atRA 的生成是视网 膜中atRal 的重要解毒代谢通路。
方法
利用UPLC-MS/MS 分别检测猪眼神经视网膜及 RPE 层中atRA 的含量; 利用ARPE-19 细胞系模 拟atRal 在RPE 中累积, 用UPLC-MS/MS 检测细 胞内及培养基中atRA 的含量, 并用定量聚合酶 链反应(qPCR)检测CYP26b1 的表达; 利用 CCK8、DCFH-DA 染色、qPCR、western blot 等 方法对比等浓度atRA 和atRal 在RPE细胞中所诱 导的细胞毒性、氧化应激、凋亡相关蛋白表达水 平; 用UPLC-MS/MS 检测视网膜atRal 清除障碍 的ABCA4−/−RDH8−/−小鼠眼球中atRA及全反式视 黄醇。
结论
明确atRA 在正常视网膜中能够代谢产生; 证明 其形成有利于RPE 细胞中累积的atRal 迅速代谢 消除; 其自身诱导细胞氧化应激的能力显著低于 atRal, 因而能显著减弱后者的细胞毒性。
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Contributions
Qing-qing XIA performed the UPLC-MS/MS detection, analyzed the data, and wrote the manuscript. Ling-min ZHANG and Ying-ying ZHOU performed the cell culture, cell staining, qPCR, and western blot. Ya-lin WU and Jie LI designed the study, analyzed the data, and revised the manuscript. All authors have read and approved the final manuscript and, therefore, had full access to all the data in the study and take responsibility for the integrity and security of the data.
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Qing-qing XIA, Ling-min ZHANG, Ying-ying ZHOU, Ya-lin WU, and Jie LI declare that they have no conflict of interest.
All institutional and national guidelines for the care and use of laboratory animals were followed.
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Project supported by the Zhejiang Provincial Natural Science Foundation of China (No. LQ17H120001), the Medical Science and Technology Program of Zhejiang Province (Nos. 2016KYA195 and 2017KY714), the National Natural Science Foundation of China (No. 81801424), and the 211 Talents Training Program of Taizhou, China
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Xia, Qq., Zhang, Lm., Zhou, Yy. et al. All-trans-retinoic acid generation is an antidotal clearance pathway for all-trans-retinal in the retina. J. Zhejiang Univ. Sci. B 20, 960–971 (2019). https://doi.org/10.1631/jzus.B1900271
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DOI: https://doi.org/10.1631/jzus.B1900271
Key words
- All-trans-retinal
- All-trans-retinoic acid
- Antidotal pathway
- Human retinal pigment epithelial cell
- Oxidative stress