Effects ofl-Carnitine, Erythritol and Betaine on Pro-inflammatory Markers in Primary Human Corneal Epithelial Cells Exposed to Hyperosmotic Stress

Abstract

Purpose To explore the effects of osmoprotectants on pro-inflammatory mediator production in primary human corneal epithelial cells (HCECs) exposed to hyperosmotic stress. Methods HCECs cultured in iso-osmolar medium (312mOsM) were switched to hyperosmotic media with or without prior incubation with 2–20mM of L-carnitine, erythritol or betaine for different time periods. The mRNA expression and protein production of pro-inflammatory markers in HCECs were evaluated by RT-qPCR and ELISA. Results Hyperosmolar media significantly stimulated the mRNA and protein expression of pro-inflammatory cytokines, TNF-α, IL-1β and IL-6, and chemokines, IL-8, CCL2 and CCL20 in HCECs in an osmolarity dependent manner. The stimulated expression of these pro-inflammatory mediators was significantly but differentially suppressed by L-carnitine, erythritol or betaine. L-carnitine displayed the greatest inhibitory effects and down-regulated 54–77% of the stimulated mRNA levels of TNF-α (down from 12.3 to 5.7 fold), IL-1β (2.2 to 0.9 fold), IL-6 (7.3 to 2.9 fold), IL-8 (4.6 to 2.0 fold), CCL2 (15.3 to 3.5 fold) and CCL20 (4.1 to 1.5 fold) in HCECs exposed to 450mOsM. The stimulated protein production of TNF-α, IL-1β, IL-6 and IL-8 was also significantly suppressed by L-carnitine, erythritol and betaine. L-carnitine suppressed 49–79% of the stimulated protein levels of TNF-α (down from 81.3 to 17.4pg/ml), IL-1β (56.9 to 29.2pg/ml), IL-6 (12.8 to 4.6ng/ml), and IL-8 (21.2 to 10.9ng/ml) by HCECs exposed to 450mOsM. Interestingly, hyperosmolarity stimulated increase in mRNA and protein levels of TNF-α, IL-1β and IL-6 were significantly suppressed by a TRPV1 activation inhibitor capsazepine. Conclusions L-carnitine, erythritol and betaine function as osmoprotectants to suppress inflammatory responses via TRPV1 pathway in HCECs exposed to hyperosmotic stress. Osmoprotectants may have efficacy in reducing innate inflammation in dry eye disease.

Evaluation of an enhanced viscosity artificial tear for moderate to severe dry eye disease: A multicenter, double-masked, randomized 30-day study

“…Further studies could explore patient preference while rheological studies could evaluate viscosity under shear stress in gel-based formulations to determine which components provide optimal performance in terms of symptom improvement and tolerability. A number of osmoprotectants, including L-carnitine and erythritol, have already shown a potential anti-inflammatory effect in corneal epithelial cells under osmotic stress [36]. Additional studies that include measures of markers of cellular stress could help elucidate effects of the additional components in the CMC-GLY formulation.…”

Protective Effects of L-Carnitine Against Oxidative Injury by Hyperosmolarity in Human Corneal Epithelial Cells

“…L-carnitine as an organic osmoprotectant 19,20 has been proven to have protective roles against production of proinflammatory mediators 21 and apoptosis 16 in primary HCECs exposed to hyperosmotic media, as well as in dry-eye patients 36,37 and animal models. 15 However, the antioxidative effects of Lcarnitine are not well understood in an in vitro dry-eye model.…”

“…[18][19][20] Recently, osmoprotectants, L-carnitine, erythritol, and betaine, have been demonstrated to inhibit in HCECs and in a dry-eye mouse model hyperosmotic-induced increases in proinflammatory cytokines (TNF-a, IL-1b, IL-6, and IL-17) and chemokines (IL-8, CCL2, and CCL20). 15,21 We further showed their suppressive effects on the expression, production, and activity of matrix metalloproteinases (MMP-13, MMP-2, MMP-9, MMP-3, and MMP-7) induced by hyperosmotic media in primary HCECs. 22 We also observed that L-carnitine and erythritol protected HCECs from hyperosmotic stress via suppressive effect on activation of c-Jun N-terminal kinases (JNK) and p38MAP kinase.…”

Matrix regeneration agents improve wound healing in non-stressed human corneal epithelial cells

“…Osmoprotectants such as L-carnitine, erithritol, or betaine have been shown to significantly reduce HO-induced proinflammatory responses, 15 to promote the survival of corneal epithelial cells, and protect the integrity of the epithelium. [16][17][18] We therefore tested the effects of these molecules on wound healing under HO conditions ( Figure 5).…”