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Xia Hua, Zhitao Su, Ruzhi Deng, Jing Lin, De-Quan Li, Stephen Pflugfelder; Effects of L-carnitine, Erythritol and Betaine on Inflammatory Markers in Primary Human Corneal Epithelial Cells Exposed to Hyperosmotic Stress. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2670.
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Hyperosmolarity has been recognized to be a pro-inflammatory stress in the pathogenesis of dry eye disease. This study was to explore the suppressive effects of osmoprotectants on production of pro-inflammatory mediators in primary human corneal epithelial cells (HCECs) exposed to hyperosmotic stress.
Primary HCECs were established from donor limbal tissue. The cultures in iso-osmolar medium (312 mOsM) were switched to hyperosmotic media (400-500 mOsM) by adding 50-90 mM NaCl, with or without prior incubation of different concentrations (2, 10 or 20mM) of L-carnitine, erythritol or betaine. The mRNA expression by HCECs treated for 4 hours was determined by RT-qPCR. The protein production in the conditioned media from cultures treated for 24 hours was evaluated by ELISA.
Hyperosmotic stress (400, 450 or 500 mOsM) significantly stimulated the mRNA expression of pro-inflammatory cytokines, TNF-α (3.2 to 16.2 fold), IL-1β (2.2 to 3.5 fold) and IL-6 (3.1 to 7.3 fold), and chemokines, IL-8 (3.1 to 4.9 fold), CCL2 (6.1 to 15.3 fold) and CCL20 (2.4 to 4.1 fold) in HCECs, mostly in an osmolarity dependent fashion. Interestingly, the stimulated these pro-inflammatory markers was significantly but differentially suppressed by L-carnitine, erythritol or betaine. L-carnitine appeared to have the greatest inhibitory effects, and down-regulated 54-77%, respectively, 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) by HCECs exposed to 450 mOsM. The stimulated production of these markers by hyperosmotic stress and the suppressive effects of L-carnitine, erythritol and betaine were further confirmed at protein levels by ELISA. L-carnitine suppressed 49-79%, respectively, of the stimulated protein levels of TNF-α (down from 81.3 to 17.4 pg/ml), IL-1β (56.9 to 29.2 pg/ml), IL-6 (12.8 to 4.6 ng/ml), and IL-8 (21.2 to 10.9 ng/ml) by HCECs exposed to 450 mOsM.
Our findings demonstrate that hyperosmotic stress stimulates the expression and production of proinflammatory mediators in HCECs. L-carnitine, erythritol and betaine serve as osmoprotectants that suppress the inflammatory responses in HCECs exposed to hyperosmotic stress. L-carnitine has the best osmoprotectant effect among the three.
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