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Xia Hua, Ruzhi Deng, Zongduan Zhang, Zhitao Su, Li De-Quan, Stephen C Pflugfelder; L-Carnitine Suppresses the Production of Pro-inflammatory Cytoiknes by Preventing the Hyperosmolarity-Induced Oxidative Stress in Human Corneal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3058.
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© ARVO (1962-2015); The Authors (2016-present)
To explore the anti-oxidative effects of L-carnitine that suppresses the production of pro-inflammatory cytokines by reducing oxidative stress in human corneal epithelial cells (HCECs) exposed to hyperosmotic condition.
Primary HCECs were established from fresh donor limbal tissue explants. The cultures in iso-osmolar medium (312 mOsM) were switched to hyperosmotic media (400, 450 and 500 mOsM), with or without prior incubation of 2-20mM of L-carnitine. Production of reactive oxygen species (ROS) was analyzed using the 2’, 7’-dichlorodihydrofluorescein diacetate (DCF-DA) kit. Heme oxygenase-1 (HMOX1) and cyclooxygenase 2 (COX2) enzymes were evaluated by reverse transcription and quantitative real time PCR, immunefluorescent staining and western blot. The production of cytokines (TNF-α, IL-1β and IL-6) in conditioned media was evaluated by ELISA.
Compared to iso-osmolar media, ROS production significantly increased (P<0.05) by HCECs exposed to hyperosmotic media in a hyperosmolarity-dependent manner (400-500mOsm). The mRNA expression of ROS associated enzymes, HMOX1 and COX2 was highly stimulated by hyperosmotic media at 400 mOsm (10.7 fold, P<0.01) and at 450 mOsm (30.1 fold, P<0.001). The stimulated production of these enzymes was confirmed by immunefluorescent staining and western blot at protein level. The production of pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) was also increased dramatically by hyperosmotic stress (from 312 to 450 mOsm, TNF-α from 18.9 to 81.3 pg/ml, P<0.001; IL-1β from 25.5 to 57.9 pg/ml, P<0.01; IL-6 from 3.2 to 12.8 ng/ml, P<0.01). Interestingly, prior incubation of L-carnitine significantly suppressed expression of COX2 and ROS production, which is accompanied by suppressing production of pro-inflammatory cytokines (TNF-α from 81.3 to 17.4 pg/ml, P<0.01; IL-1β from 57.9 to 29.2 pg/ml, P<0.05; IL-6 from 12.8 to 4.6 ng/ml, P<0.01) in HCECs exposed to hyperosmotic stress.
Our findings demonstrate that the hyperosmotic stress increases the production of ROS and its inducing oxidative enzymes COX2, which leads an increased production of pro-inflammatory cytokines, TNF-α, IL-1β and IL-6. L-carnitine shows suppressive effects on oxidative stress and inflammatory responses in HCECs exposed to hyperosmotic stress.
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