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.

中文翻译：

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Ectoine 通过促进抗炎细胞因子 IL-37 保护角膜上皮存活和屏障免受高渗应激


探索天然渗透保护剂四氢嘧啶在保护角膜上皮细胞存活和高渗应激屏障中的新作用和分子机制。原代人角膜上皮细胞（HCEC）是从供体角膜缘建立的。将等渗培养基中的汇合培养物切换至高渗培养基（400-500 mOsM），在不同时间段内添加或不添加四氢嘧啶或 rhIL-37。通过MTT或WST测定评估细胞活力和增殖。通过 RT-qPCR、ELISA 和共聚焦显微镜免疫染色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 培养基中存活良好，但在 500mOsM 培养基中部分受损。四氢嘧啶在 500mOsM 时可以很好地保护 HCEC 的存活和增殖。紧密连接蛋白 ZO-1 和 occludin 的 2D 和 3D 共聚焦免疫荧光图像显示，暴露于 450mOsM 的 HCEC 中上皮屏障的完整性显着破坏。 5-20 mM 的四氢嘧啶在高渗应激下可以很好地保护这些屏障蛋白。高渗透压会显着刺激 TNF-α、IL-1β、IL-6 和 IL-8 的表达，但 5-40 mM 的 Ectoine 会显着抑制其表达。高渗透压刺激的组织蛋白酶S直接破坏上皮屏障。有趣的是，抗炎细胞因子 IL-37 被高渗透压抑制，但在 mRNA 和蛋白质水平被四氢嘧啶恢复。此外，rhIL-37 抑制组织蛋白酶 S 并挽救暴露于高渗透压的 HCEC 中的细胞存活和屏障。我们的研究结果表明，四氢嘧啶通过促进 IL-37 来保护 HCEC 存活并抵御高渗应激。这为干眼病的发病机制和治疗潜力提供了新的见解。