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Ruzhi Deng, Zhitao Su, Jing Lin, Xia Hua, De-Quan Li, Stephen Pflugfelder; L-carnitine, Erythritol and Betaine Suppress the Production and Activity of Matrix Metalloproteinases in Primary Human Corneal Epithelial Cells Exposed to Hyperosmotic Stress. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4315.
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Hyperosmolarity has been recognized to be a pro-inflammatory stress in the pathogenesis of dry eye disease. This study investigated the suppressive effect of osmoprotectants (L-carnitine, erythritol and betaine) on the production and activity of matrix metalloproteinases (MMPs) in primary human corneal epithelial cells (HCECs) exposed to hyperosmotic stress.
Primary HCECs were established from fresh donor limbal tissue explants. 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 with different concentrations (2, 10 or 20mM) of L-carnitine, erythritol or betaine. The mRNA expression of MMPs by HCECs treated for 4 hours was determined by reverse transcription and quantitative real time PCR. Their protein production and activity in the conditioned media from cultures treated for 24-48 hours were evaluated by zymography, immunofluorescent staining, ELISA and activity assays.
Hyperosmotic stress (400, 450 or 500 mOsM) significantly stimulated the mRNA expression of collagenase MMP13 (3.1 to 7.5 fold), gelatinase MMP9 (1.7 to 2.5 fold), stromelysin MMP3 (2.2 to 4.1 fold) and matrilysin MMP7 (2.3 to 4.5 fold), mostly in an osmolarity dependent fashion. Interestingly, the stimulated expression of these MMPs was significantly, but differentially suppressed by L-carnitine, erythritol or betaine. L-carnitine appeared to have the greatest inhibitory effects, and down-regulated 52-78%, respectively, of the stimulated mRNA levels of MMP13 (down from 7.0 to 3.1 fold), MMP9 (2.5 to 1.2 fold), MMP3 (4.1 to 0.9 fold), and MMP7 (4.5 to 1.5 fold) by HCECs exposed to 450 mOsM. The stimulated production and activity of these MMPs by hyperosmotic stress and the suppressive effects of L-carnitine, erythritol and betaine were further confirmed at protein levels by gelatin and casein gel zymography, immunofluorescent staining, ELISA and/or activity assays, respectively.
Our findings demonstrate that hyperosmotic stress stimulates the expression, production and activity of MMPs in HCECs. L-carnitine, erythritol and betaine serve as osmoprotectants that suppress the production and activity of MMPs in HCECs exposed to hyperosmotic stress. L-carnitine shows the best inhibitory effect among the three.
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