In Vitro Inhibition of NFAT5-Mediated Induction of CCL2 in Hyperosmotic Conditions by Cyclosporine and Dexamethasone on Human HeLa-Modified Conjunctiva-Derived Cells

PurposeTo investigate the pro-inflammatory intracellular mechanisms induced by an in vitro model of dry eye disease (DED) on a Hela-modified conjunctiva-derived cells in hyperosmolarity (HO) stress conditions. This study focused on CCL2 induction and explored the implications of the nuclear factor of activated T-cells 5 (NFAT5) as well as mitogen-activated protein kinases (MAPK) and nuclear factor kappa B (NFĸB). This work was completed by an analysis of the effects of cyclosporine A (CsA), dexamethasone (Dex) and doxycycline (Dox) on HO-induced CCL2 and NFAT5 induction.MethodsA human HeLa-modified conjunctiva-derived cell line was cultured in NaCl-hyperosmolar medium for various exposure times. Cellular viability, CCL2 secretion, NFAT5 and CCL2 gene expression, and intracytoplasmic NFAT5 were assessed using the Cell Titer Blue® assay, enzyme-linked immunosorbent assay (ELISA), RT-qPCR and immunostaining, respectively. In selected experiments, inhibitors of MAPKs or NFκB, therapeutic agents or NFAT5 siRNAs were added before the hyperosmolar stimulations.ResultsHO induced CCL2 secretion and expression as well as NFAT5 gene expression and translocation. Adding NFAT5-siRNA before hyperosmolar stimulation led to a complete inhibition of CCL2 induction and to a decrease in cellular viability. p38 MAPK (p38), c-Jun NH2-terminal kinase (JNK) and NFĸB inhibitors, CsA and Dex induced a partial inhibition of HO-induced CCL2, while Dox and extracellular signal-regulated kinase (ERK) inhibitor did not. Dex also induced a partial inhibition of HO-induced NFAT5 gene expression but not CsA or Dox.ConclusionsThese in vitro results suggest a potential role of CCL2 in DED and highlight the crucial role of NFAT5 in the pro-inflammatory effect of HO on HeLa-modified conjunctiva-derived cells, a rarely studied cellular type. This inflammatory pathway involving NFAT5 and CCL2 could offer a promising target for developing new therapies to treat DED, warranting further investigations to fully grasp the complete intracellular mechanisms.**研究目的** 本研究旨在探讨高渗（HO）应激条件下，干眼症（DED）体外模型作用于经HeLa修饰的结膜来源细胞所诱导的促炎细胞内机制。本研究聚焦于CCL2的表达诱导，并探讨活化T细胞核因子5（NFAT5）、丝裂原活化蛋白激酶（MAPK）以及核因子κB（NFκB）的作用。此外，本研究还分析了环孢素A（CsA）、地塞米松（Dex）与多西环素（Dox）对高渗诱导的CCL2及NFAT5表达的影响。 **研究方法** 本研究将人源HeLa修饰的结膜来源细胞系培养于氯化钠高渗培养基中，设置不同的作用时长。分别采用Cell Titer Blue®细胞活力检测试剂盒、酶联免疫吸附试验（ELISA）、实时定量聚合酶链反应（RT-qPCR）以及免疫染色技术，检测细胞活力、CCL2分泌水平、NFAT5与CCL2的基因表达水平以及胞质内NFAT5的表达情况。在部分实验中，于高渗刺激前分别加入MAPK或NFκB抑制剂、治疗性药物或NFAT5小干扰RNA（siRNA）。 **研究结果** 高渗刺激可诱导CCL2的分泌与表达，同时上调NFAT5的基因表达并促进其转位。在高渗刺激前加入NFAT5 siRNA，可完全抑制CCL2的表达诱导，并降低细胞活力。p38丝裂原活化蛋白激酶（p38）、c-Jun氨基末端激酶（JNK）以及NFκB抑制剂，联合CsA与Dex，可部分抑制高渗诱导的CCL2表达；而多西环素与细胞外调节蛋白激酶（ERK）抑制剂则无此效果。此外，Dex可部分抑制高渗诱导的NFAT5基因表达，但CsA与Dox无此作用。 **研究结论** 本体外实验结果表明，CCL2可能在干眼症的发病过程中发挥潜在作用；同时证实了NFAT5在高渗刺激对HeLa修饰的结膜来源细胞（一种鲜有研究的细胞类型）产生的促炎效应中具有关键作用。这一涉及NFAT5与CCL2的炎症通路，可为干眼症的新型治疗药物开发提供极具前景的靶点，未来仍需开展进一步研究以全面阐明其完整的细胞内作用机制。