title: Oxidative stress markers induced by hyperosmolarity in primary human corneal epithelial cells.
abstract: Oxidative stress has been known to be involved in pathogenesis of dry eye disease. However, few studies have comprehensively investigated the relationship between hyperosmolarity and oxidative damage in human ocular surface. This study was to explore whether and how hyperosmolarity induces oxidative stress markers in primary human corneal epithelial cells (HCECs). Primary HCECs were established from donor limbal explants. The hyperosmolarity model was made in HCECs cultured in isosmolar (312 mOsM) or hyperosmotic (350, 400, 450 mOsM) media. Production of reactive oxygen species (ROS), oxidative damage markers, oxygenases and anti-oxidative enzymes were analyzed by DCFDA kit, RT-qPCR, immunofluorescent and immunohistochemical staining and Western blotting. Compared to isosmolar medium, ROS production significantly increased at time- and osmolarity-dependent manner in HCECs exposed to media with increasing osmolarities (350-450 mOsM). Hyperosmolarity significantly induced oxidative damage markers in cell membrane with increased toxic products of lipid peroxidation, 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA), and in nuclear and mitochondria DNA with increased aconitase-2 and 8-OHdG. Hyperosmotic stress also increased the mRNA expression and protein production of heme oxygenase-1 (HMOX1) and cyclooxygenase-2 (COX2), but reduced the levels of antioxidant enzymes, superoxide dismutase-1 (SOD1), and glutathione peroxidase-1 (GPX1). In conclusion, our comprehensive findings demonstrate that hyperosmolarity induces oxidative stress in HCECs by stimulating ROS production and disrupting the balance of oxygenases and antioxidant enzymes, which in turn cause cell damage with increased oxidative markers in membrane lipid peroxidation and mitochondrial DNA damage.
authors.affiliation: School of Optometry and Ophthalmology, Wenzhou Medical University, Wenzhou, China; Ocular Surface Center, Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine, Houston, Texas, United States of America.
authors.forename: Ruzhi
authors.initials: R
authors.lastname: Deng
authors.affiliation: School of Optometry and Ophthalmology, Wenzhou Medical University, Wenzhou, China; Tianjin Eye Hospital, Tianjin Key Lab of Ophthalmology and Visual Science, Clinical College of Ophthalmology, Tianjin Medical University, Tianjin, China.
authors.forename: Xia
authors.initials: X
authors.lastname: Hua
authors.affiliation: School of Optometry and Ophthalmology, Wenzhou Medical University, Wenzhou, China; Ocular Surface Center, Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine, Houston, Texas, United States of America.
authors.forename: Jin
authors.initials: J
authors.lastname: Li
authors.affiliation: Ocular Surface Center, Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine, Houston, Texas, United States of America.
authors.forename: Wei
authors.initials: W
authors.lastname: Chi
authors.affiliation: School of Optometry and Ophthalmology, Wenzhou Medical University, Wenzhou, China; Ocular Surface Center, Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine, Houston, Texas, United States of America.
authors.forename: Zongduan
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: School of Optometry and Ophthalmology, Wenzhou Medical University, Wenzhou, China.
authors.forename: Fan
authors.initials: F
authors.lastname: Lu
authors.affiliation: Ocular Surface Center, Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine, Houston, Texas, United States of America.
authors.forename: Lili
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Ocular Surface Center, Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine, Houston, Texas, United States of America.
authors.forename: Stephen C
authors.initials: SC
authors.lastname: Pflugfelder
authors.affiliation: Ocular Surface Center, Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine, Houston, Texas, United States of America.
authors.forename: De-Quan
authors.initials: DQ
authors.lastname: Li
mesh_terms: D000975:Antioxidants / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D002478:Cells, Cultured; D003315:Cornea / Q000166:cytology / Q000378:metabolism*; D004249:DNA Damage; D004272:DNA, Mitochondrial / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism; D005128:Eye Diseases / Q000378:metabolism / Q000473:pathology; D006801:Humans; D015227:Lipid Peroxidation; D009994:Osmolar Concentration; D018384:Oxidative Stress; D010105:Oxygenases / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism*
chemical_list: D000975:Antioxidants; D015415:Biomarkers; D004272:DNA, Mitochondrial; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D010105:Oxygenases