Purpose

To develop an easy-to-perform combined model in human corneal epithelial cells (HCECs) and Balb/c mice macrophages J774.A1 (MP) for preliminary screening of potential ophthalmic therapeutic substances.

Methods

HCECs were exposed to different osmolarities (350–500 mOsm/L) and MTT assay was employed for cell survival and flow cytometry to assess apoptosis-necrosis and relative cell size (RCS) distribution. Effectiveness of Betaine, L-Carnitine, Taurine at different concentrations (ranging from 20 mM to 200 mM) was studied. Also, mucoadhesive polymers such as Hyaluronic acid (HA) and Hydroxypropylmethylcellulose (HPMC) (0.4 and 0.8%) were evaluated. Cells were pre-incubated with the compounds (8h) and then exposed to hyperosmotic stress (470 mOsm/L) for 16h. Moreover, anti-inflammatory activity was performed in LPS-stimulated MP.

Results

Exposure to hyperosmotic solutions between 450 and 500 mOsm/L promoted the highest cell death after 16h exposures (p < 0.0001) with a drop in viability to 34.96% ± 11.77 for 470 mOsm/L. Pre-incubation with Betaine at 150 mM and 200 mM provided the highest cell survival against hyperosmolarity (66.01% ± 3.65 and 65.90% ± 0.78 respectively) while HA 0.4% was the most effective polymer in preventing cell death (42.2% ± 3.60). Flow cytometry showed that Betaine and Taurine at concentrations between 150-200 mM and 20–80 mM respectively presented the highest anti-apoptotic activity. Also, HA and HPMC polymers reduced apoptotic-induced cell death. All osmoprotectants modified RCS, and polymers increased their value over 100%. L-Carnitine 50 mM, Taurine 40 mM and HA 0.4% presented the highest TNF-α inhibition activity (60%) albeit all of them showed anti-inflammatory inhibition percentages higher than 20%

Conclusions

HCECs hyperosmolar model combined with inflammatory conditions in macrophages allows the screening of osmoprotectants by simulating chronic hyperosmolarity (16h) and inflammation (24h).

中文翻译：

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在受压的人角膜上皮细胞和巨噬细胞中结合高渗透压和炎症条件来评估渗透保护剂作为潜在的 DED 治疗

目的

在人角膜上皮细胞 (HCECs) 和 Balb/c 小鼠巨噬细胞 J774.A1 (MP) 中开发易于执行的组合模型，用于初步筛选潜在的眼科治疗物质。

方法

HCEC 暴露于不同的渗透压（350-500 mOsm/L），并采用 MTT 测定进行细胞存活和流式细胞术以评估细胞凋亡 - 坏死和相对细胞大小（RCS）分布。研究了不同浓度（从 20 mM 到 200 mM）的甜菜碱、L-肉碱、牛磺酸的有效性。此外，还评估了粘膜粘附聚合物，例如透明质酸 (HA) 和羟丙基甲基纤维素 (HPMC)（0.4% 和 0.8%）。细胞与化合物一起预孵育（8 小时），然后暴露于高渗应激（470 mOsm/L）16 小时。此外，在 LPS 刺激的 MP 中进行了抗炎活性。

结果

暴露于 450 和 500 mOsm/L 之间的高渗溶液促进了 16 小时暴露后最高的细胞死亡 (p < 0.0001)，470 mOsm/L 的存活率下降至 34.96% ± 11.77。用 150 mM 和 200 mM 甜菜碱预孵育可提供最高的细胞存活率（分别为 66.01% ± 3.65 和 65.90% ± 0.78），而 0.4% 的 HA 是预防细胞死亡的最有效聚合物（42.2% ± 3.60）。流式细胞术显示甜菜碱和牛磺酸分别在 150-200 mM 和 20-80 mM 之间具有最高的抗凋亡活性。此外，HA 和 HPMC 聚合物减少了凋亡诱导的细胞死亡。所有渗透保护剂都改进了 RCS，聚合物的价值增加了​​ 100% 以上。L-肉碱 50 mM、牛磺酸 40 mM 和 HA 0。

结论

HCEC 高渗模型与巨噬细胞中的炎症条件相结合，可以通过模拟慢性高渗 (16 小时) 和炎症 (24 小时) 来筛选渗透保护剂。