Department of Ophthalmology, Shanghai East Hospital, School of Medicine, Tongji University, Shanghai, People's Republic of China.
Invest Ophthalmol Vis Sci. 2024 Nov 4;65(13):55. doi: 10.1167/iovs.65.13.55.
The purpose of this study was to evaluate the cytoprotective effect and the mechanism of cRGD-conjugated bilirubin nanoparticles (cNPs@BR) in dry eye disease (DED).
The binding capacity and cellular uptake of cNPs@BR in human corneal epithelial cells (HCECs) were assessed by immunofluorescence. The anti-inflammation and anti-oxidative stress effects of cNPs@BR were determined by flow cytometry, immunofluorescence, Western blot, chromatin immunoprecipitation (ChIP), and ELISA assay in LPS-stimulated RAW264.7 cells and hypertonic HCECs. The function of ocular surface barrier, tear production, and the number of goblet cells after cNPs@BR treatment were further assessed by fluorescein sodium staining, phenol red cotton threads, quantitative PCR (qPCR), hematoxylin and eosin (H&E) staining, and Periodic Acid-Schiff (PAS) staining in a 0.2% BAC-induced DED mouse model. Furthermore, the mechanism of cNPs@BR in treating DED was explored by RNA sequencing and RNA interference.
The cRGD peptide prolonged the retention time of nanoparticles on HCECs and enhanced the cellular uptake efficiency. In both cell models, 20 µM cNPs@BR pretreatment ameliorated oxidative stress by decreasing the intracellular reactive oxygen species (ROS) levels and the expression of NOX4 and 4-HNE, while promoting HO-1 and nuclear Nrf2 levels. Moreover, cNPs@BR alleviated the inflammatory response by inhibiting NF-κB p65 nuclear translocation and decreasing the expression of iNOS and the secretion of IL-1β, IL-6, and TNF-α. In addition, cNPs@BR protected ocular surface epithelium against oxidative stress and inflammation and restored conjunctival goblet cells in the mouse model of DED by activating PINK1-mediated mitophagy.
The cNPs@BR suppressed oxidative stress and inflammatory response in the ocular surface epithelium and restored goblet cells by activating PINK1-mediated mitophagy.
本研究旨在评估 cRGD 修饰胆红素纳米粒（cNPs@BR）在干燥性眼病（DED）中的细胞保护作用及其机制。
通过免疫荧光法评估 cNPs@BR 在人角膜上皮细胞（HCEC）中的结合能力和细胞摄取。通过流式细胞术、免疫荧光、Western blot、染色质免疫沉淀（ChIP）和酶联免疫吸附试验（ELISA）检测 cNPs@BR 在 LPS 刺激的 RAW264.7 细胞和高渗 HCEC 中的抗炎和抗氧化应激作用。通过荧光素钠染色、酚红棉线、定量 PCR（qPCR）、苏木精和伊红（H&E）染色和过碘酸希夫（PAS）染色进一步评估 cNPs@BR 处理后眼表屏障功能、泪液产生和杯状细胞数量，在 0.2% BAC 诱导的 DED 小鼠模型中。此外，通过 RNA 测序和 RNA 干扰探索了 cNPs@BR 治疗 DED 的机制。
cRGD 肽延长了纳米粒在 HCEC 上的保留时间并提高了细胞摄取效率。在两种细胞模型中，20µM cNPs@BR 预处理通过降低细胞内活性氧（ROS）水平和 NOX4 和 4-HNE 的表达，同时促进 HO-1 和核 Nrf2 水平，改善氧化应激。此外，cNPs@BR 通过抑制 NF-κB p65 核转位和降低 iNOS 表达和 IL-1β、IL-6 和 TNF-α 的分泌来减轻炎症反应。此外，cNPs@BR 通过激活 PINK1 介导的线粒体自噬来保护眼表上皮免受氧化应激和炎症，并在 DED 小鼠模型中恢复结膜杯状细胞。
cNPs@BR 通过激活 PINK1 介导的线粒体自噬抑制眼表上皮中的氧化应激和炎症反应并恢复杯状细胞。
