Protective Effects of L-Carnitine Against Oxidative Injury by Hyperosmolarity in Human Corneal Epithelial Cells.
OBJECTIVE
L-carnitine suppresses inflammatory responses in human corneal epithelial cells (HCECs) exposed to hyperosmotic stress. In this study, we determined if L-carnitine induces this protective effect through suppression of reactive oxygen species (ROS)-induced oxidative damage in HCECs.


METHODS
Primary HCECs were established from donor limbal explants. A hyperosmolarity dry-eye model was used in which HCECs are cultured in 450 mOsM medium with or without L-carnitine for up to 48 hours. Production of reactive oxygen species (ROS), oxidative damage markers, oxygenases and antioxidative enzymes were analyzed by 2',7'-dichlorofluorescein diacetate (DCFDA) kit, semiquantitative PCR, immunofluorescence, and/or Western blotting.


RESULTS
Reactive oxygen species production increased in HCECs upon substitution of the isotonic medium with the hypertonic medium. L-carnitine supplementation partially suppressed this response. Hyperosmolarity increased cytotoxic membrane lipid peroxidation levels; namely, malondialdehyde (MDA) and hydroxynonenal (HNE), as well as mitochondria DNA release along with an increase in 8-OHdG and aconitase-2. Interestingly, these oxidative markers were significantly decreased by coculture with L-carnitine. Hyperosmotic stress also increased the mRNA expression and/or protein production of heme oxygenase-1 (HMOX1) and cyclooxygenase-2 (COX2), but inhibited the levels of antioxidant enzymes, superoxide dismutase-1 (SOD1), glutathione peroxidase-1 (GPX1), and peroxiredoxin-4 (PRDX4). However, L-carnitine partially reversed this altered imbalance between oxygenases and antioxidant enzymes induced by hyperosmolarity.


CONCLUSIONS
Our findings demonstrate for the first time that L-carnitine protects HCECs from oxidative stress by lessening the declines in antioxidant enzymes and suppressing ROS production. Such suppression reduces membrane lipid oxidative damage markers and mitochondrial DNA damage.
Ocular Surface Center Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine, Houston, Texas, United States 2Tianjin Eye Hospital, Tianjin Key Lab of Ophthalmology and Visual Science, Clinical College of Ophthalmology, Tianjin Medic.
Xia
X
Hua
Ocular Surface Center Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine, Houston, Texas, United States 3School of Optometry and Ophthalmology, Wenzhou Medical University, Wenzhou, China.
Ruzhi
R
Deng
Ocular Surface Center Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine, Houston, Texas, United States 3School of Optometry and Ophthalmology, Wenzhou Medical University, Wenzhou, China.
Jin
J
Li
Ocular Surface Center Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine, Houston, Texas, United States 4Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China.
Wei
W
Chi
Ocular Surface Center Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine, Houston, Texas, United States.
Zhitao
Z
Su
Ocular Surface Center Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine, Houston, Texas, United States.
Jing
J
Lin
Ocular Surface Center Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine, Houston, Texas, United States.
Stephen C
SC
Pflugfelder
Ocular Surface Center Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine, Houston, Texas, United States.
De-Quan
DQ
Li
D002331:Carnitine / Q000494:pharmacology*; D002478:Cells, Cultured; D019573:Epithelium, Corneal / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D009994:Osmolar Concentration; D018384:Oxidative Stress / Q000187:drug effects*; D012313:RNA / Q000235:genetics*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014803:Vitamin B Complex / Q000494:pharmacology
D017382:Reactive Oxygen Species; D014803:Vitamin B Complex; D012313:RNA; D002331:Carnitine