Xia Hua1, Ruzhi Deng2, Jin Li2, Wei Chi3, Zhitao Su4, Jing Lin4, Stephen C Pflugfelder4, De-Quan Li4. 1. 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. 2. 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. 3. 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. 4. Ocular Surface Center Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine, Houston, Texas, United States.
Abstract
PURPOSE: 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.
PURPOSE: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.
Authors: F Brignole; P J Pisella; M Goldschild; M De Saint Jean; A Goguel; C Baudouin Journal: Invest Ophthalmol Vis Sci Date: 2000-05 Impact factor: 4.799
Authors: Rodrigo G de Souza; Zhiyuan Yu; Humberto Hernandez; Claudia M Trujillo-Vargas; Andrea Lee; Kelsey E Mauk; Jiyang Cai; Milton R Alves; Cintia S de Paiva Journal: Am J Pathol Date: 2020-11-04 Impact factor: 4.307