Ocular Surface Center, Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine, Houston, TX, 77030, USA; State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Guangdong Provincial Key Laboratory of Ophthalmology and Visual Science, Sun Yat-Sen University, Guangzhou, 510060, China.
Ocular Surface Center, Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine, Houston, TX, 77030, USA; National Clinical Research Center for Ocular Diseases, Eye Hospital, Wenzhou Medical University, Wenzhou, 325027, China.
Ocul Surf. 2024 Apr;32:182-191. doi: 10.1016/j.jtos.2024.03.002. Epub 2024 Mar 13.
To explore novel role and molecular mechanism of a natural osmoprotectant ectoine in protecting corneal epithelial cell survival and barrier from hyperosmotic stress.
Primary human corneal epithelial cells (HCECs) were established from donor limbus. The confluent cultures in isosmolar medium were switched to hyperosmotic media (400-500 mOsM), with or without ectoine or rhIL-37 for different time periods. Cell viability and proliferation were evaluated by MTT or WST assay. The integrity of barrier proteins and the expression of cytokines and cathepsin S were evaluated by RT-qPCR, ELISA, and immunostaining with confocal microscopy.
HCECs survived well in 450mOsM but partially damaged in 500mOsM medium. Ectoine well protected HCEC survival and proliferation at 500mOsM. The integrity of epithelial barrier was significantly disrupted in HCECs exposed to 450mOsM, as shown by 2D and 3D confocal immunofluorescent images of tight junction proteins ZO-1 and occludin. Ectoine at 5-20 mM well protected these barrier proteins under hyperosmotic stress. The expression of TNF-α, IL-1β, IL-6 and IL-8 were dramatically stimulated by hyperosmolarity but significantly suppressed by Ectoine at 5-40 mM. Cathepsin S, which was stimulated by hyperosmolarity, directly disrupted epithelial barrier. Interestingly, anti-inflammatory cytokine IL-37 was suppressed by hyperosmolarity, but restored by ectoine at mRNA and protein levels. Furthermore, rhIL-37 suppressed cathepsin S and rescued cell survival and barrier in HCECs exposed to hyperosmolarity.
Our findings demonstrate that ectoine protects HCEC survival and barrier from hyperosmotic stress by promoting IL-37. This provides new insight into pathogenesis and therapeutic potential for dry eye disease.
探索天然渗透保护剂羟乙基呱嗪乙磺酸（ectoine）在保护角膜上皮细胞生存和屏障功能免受高渗应激方面的新作用和分子机制。
从供体角膜缘建立原代人角膜上皮细胞（HCEC）。将处于等渗培养基中的汇合培养物转换至高渗培养基（400-500mOsM），加入或不加入 ectoine 或 rhIL-37 培养不同时间。通过 MTT 或 WST 测定法评估细胞活力和增殖。通过 RT-qPCR、ELISA 和共聚焦免疫荧光显微镜评估屏障蛋白的完整性以及细胞因子和组织蛋白酶 S 的表达。
HCEC 在 450mOsM 中生长良好，但在 500mOsM 培养基中部分受损。ectoine 在 500mOsM 下可很好地保护 HCEC 的存活和增殖。暴露于 450mOsM 中的上皮屏障完整性明显受损，如紧密连接蛋白 ZO-1 和 occludin 的 2D 和 3D 共聚焦免疫荧光图像所示。在高渗应激下，5-20mM 的 ectoine 可很好地保护这些屏障蛋白。TNF-α、IL-1β、IL-6 和 IL-8 的表达在高渗时显著增加，但在 5-40mM 的 ectoine 下显著受到抑制。高渗刺激组织蛋白酶 S，直接破坏上皮屏障。有趣的是，抗炎细胞因子 IL-37 受高渗抑制，但在 mRNA 和蛋白水平上被 ectoine 恢复。此外，rhIL-37 在高渗暴露的 HCEC 中抑制组织蛋白酶 S 并挽救细胞存活和屏障。
我们的研究结果表明，ectoine 通过促进 IL-37 来保护 HCEC 免受高渗应激的影响。这为干眼症的发病机制和治疗潜力提供了新的认识。
