Combined hyperosmolarity and inflammatory conditions in stressed human corneal epithelial cells and macrophages to evaluate osmoprotective agents as potential DED treatments

Exp Eye Res. 2021 Oct:211:108723. doi: 10.1016/j.exer.2021.108723. Epub 2021 Aug 9.

Abstract

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).

Keywords: DED; Hyperosmolarity; Inflammation; Osmoprotectants; Osmoprotection model; Screening.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Apoptosis / drug effects
  • Betaine / pharmacology
  • Carnitine / pharmacology
  • Cell Survival
  • Cells, Cultured
  • Dry Eye Syndromes / drug therapy*
  • Dry Eye Syndromes / physiopathology
  • Epithelium, Corneal / drug effects*
  • Epithelium, Corneal / metabolism
  • Flow Cytometry
  • Humans
  • Hyaluronic Acid / pharmacology
  • Hypertonic Solutions / pharmacology*
  • Hypromellose Derivatives / pharmacology
  • Inflammation / physiopathology*
  • Macrophages / drug effects*
  • Macrophages / metabolism
  • Mice
  • Mice, Inbred BALB C
  • Osmolar Concentration
  • Stress, Physiological / drug effects*
  • Taurine / pharmacology
  • Tumor Necrosis Factor-alpha / metabolism

Substances

  • Hypertonic Solutions
  • Tumor Necrosis Factor-alpha
  • Taurine
  • Hypromellose Derivatives
  • Betaine
  • Hyaluronic Acid
  • Carnitine