Artificial tear based on an extract of Artemia Salina increases corneal epitelial permeability

Abstract

Purpose: To evaluate the effect on corneal epithelial permeability of a new developed artificial tear based on an extract of 4% Artemia Salina (AS) containing dinucleotides. Methods: AS extract composition was characterized by High Performance Liquid Chromatography (HPLC).Experiments were performed in an immortalized human corneal epithelial cell line. Cells were treated with AS extract for 15 minutes. Protein extraction was carried out 2 hours after AS treatment. Western blot assay was performed to evaluate corneal epithelial permeability by analyzing changes in the tight junction levels, specifically zonula occludens-1 (ZO-1) and Claudin-7 (CLDN-7). To decipher the intracellular pathways underlying the effect of AS, cells were treated with dinucleotides contained in AS (Gp4G and Ap4A at 10μM) and P2Y receptor antagonist (antagonists were pre-incubated 30 min before nucleotides and AS were added, and they were present during dinucleotides incubation). Results: Nucleotides (AMP, ADP), and dinucleotides (Gp4G, Ap4A and Gp3G) were identified and quantified in4% artemia salina extract, with Gp4G showing the highest concentration (10 mM). ZO-1 protein levels were significantly reduced in 30% compared to control (p<0.005) after AS extract treatment, being partially blockedby P2Y receptor antagonist. Nevertheless, no significant differences were detected in CLDN-7 protein levels after AS extract treatment. On other hand, Gp4G treatment decreased one third of CLDN-7 protein levels compared to control (p<0.005). However, this effect was not mediated by P2Y2 receptors. While Ap4A treatment significantly reduced both ZO-1 and CLDN-7 levels (p=0.017 and p<0.005, respectively), this effect was abolished by blocking P2Y2 receptor. Conclusions: Artemia Salina based artificial tears modify corneal epithelial barrier function by decreasing ZO-1 levels which increase corneal epithelial permeability. This effect is partially mediated by P2Y2 receptors.Therefore, its application could improve ocular drug delivery and consequently its efficiency.