Melatonin Improves Oxidative Stress Injury in Retinopathy of Prematurity by Targeting miR-23a-3p/Nrf2

Melatonin has promising protective effects for retinopathy. However, its roles in retinopathy of prematurity (ROP) and the underlying mechanisms remain unknown. We aimed to explore its roles and mechanisms in a ROP model. Hematoxylin and eosin staining were used to observe the morphology of the retina. Immunofluorescence was used to detect positive (Nrf2+ and VEGF+) cells. Immunohistochemistry was used to detect the level of nuclear expression of PCNA in retinal tissue. Transmission electron microscope (TEM) was used to observe the morphology and structure of pigment cells. qRT-PCR was used to assay the expression of miR-23a-3p, Nrf2, and HO-1. Western blotting was used to detect the expression of Nrf2, HO-1, β-actin, and Lamin B1. Melatonin or miR-23a-3p antagomir treatment could ameliorate the Oxygen-induced pathological changes, increased the expression of Nrf2 and HO-1, SOD, and GSH-Px, and decreased the expression of VEGF, miR-23a-3p, MDA and the apoptosis in the ROP model. Further target prediction and luciferase reporter assays confirmed the targeted binding relationship between miR-23a-3p and Nrf2. Our study showed that melatonin could ameliorate H₂O₂-induced apoptosis and oxidative stress injury in RGC cells by mediating miR-23a-3p/Nrf2 signaling pathway, thereby improving retinal degeneration.

褪黑素对视网膜病变展现出良好的保护潜力，然而其在早产儿视网膜病变（retinopathy of prematurity, ROP）中的作用及潜在分子机制仍未明确。本研究旨在探究褪黑素在ROP模型中的作用及相关机制。实验采用苏木精-伊红染色（Hematoxylin and eosin staining）观察视网膜形态学变化；采用免疫荧光染色（Immunofluorescence）检测核因子E2相关因子2（Nuclear factor erythroid 2-related factor 2, Nrf2）+与血管内皮生长因子（Vascular endothelial growth factor, VEGF）+阳性细胞；采用免疫组织化学染色（Immunohistochemistry）检测视网膜组织中增殖细胞核抗原（Proliferating Cell Nuclear Antigen, PCNA）的核表达水平；采用透射电子显微镜（Transmission electron microscope, TEM）观察色素细胞的形态与结构；采用实时定量聚合酶链反应（quantitative real-time polymerase chain reaction, qRT-PCR）检测miR-23a-3p、核因子E2相关因子2（Nrf2）及血红素氧合酶1（Heme Oxygenase-1, HO-1）的表达水平；采用蛋白质免疫印迹（Western blotting）检测Nrf2、HO-1、β-肌动蛋白（β-actin）及核纤层蛋白B1（Lamin B1）的表达水平。结果显示，褪黑素或miR-23a-3p拮抗剂（antagomir）干预可改善氧诱导的ROP模型病理损伤，上调Nrf2、HO-1、超氧化物歧化酶（Superoxide Dismutase, SOD）及谷胱甘肽过氧化物酶（Glutathione Peroxidase, GSH-Px）的表达水平，同时下调VEGF、miR-23a-3p、丙二醛（Malondialdehyde, MDA）的表达并抑制细胞凋亡。进一步的靶基因预测及双荧光素酶报告基因实验（luciferase reporter assays）证实了miR-23a-3p与Nrf2之间存在靶向结合关系。本研究证实，褪黑素可通过调控miR-23a-3p/Nrf2信号通路，改善H₂O₂诱导的视网膜神经节细胞（Retinal Ganglion Cells, RGC）凋亡及氧化应激损伤，进而缓解视网膜退行性病变。