4-hydroxy-2-nonenal and Dry-Eye-Syndromes

Oxidative stress is widely known to be a major contributor in the pathogenesis of dry eye disease (DED). 4-Hydroxynonenal (4-HNE), a well-known byproduct frequently measured as an indicator of oxidative stress-induced lipid peroxidation, has been shown to be elevated in both human and murine corneal DED samples. This study aims to investigate if 4-HNE is responsible for the oxidative stress in human corneal epithelial cells (HCECs) and explores the underlying mechanism by which it confers its effects.. SV40-immortalized HCECs were cultured in minimum essential media (MEM) with 1% penicillin/streptomycin and 10% fetal bovine serum. HCECs were exposed to media with or without 4-HNE and cell culture supernatants were collected at 4 and 24 h. Cellular reactive oxygen species (ROS) measurement was performed using a 2',7'-dichlorofluorescein diacetate (DCFDA) assay kit according to the manufacturer's instructions. Protein levels of antioxidant enzymes copper/zinc superoxide dismutase 1 (SOD1) and NAD(P)H quinone dehydrogenase 1 (NQO1) were analyzed by Western blot. NF-κB activation and expression of IL-6 and IL-8 were measured using an NF-κB p65 Total SimpleStep ELISA Kit and Proteome Profiler Human Cytokine Array Kit. Cell viability was evaluated by LDH cytotoxicity assay.. Treatment with 4-HNE decreased cell viability of HCECs. Band intensities corresponding to levels of ROS production showed a significant increase in ROS generation after treatment with 4-HNE. 4-HNE decreased SOD1 levels and upregulated NQO1 expression in HCECs. A significant increase in activation of NF-κB and production of pro-inflammatory cytokines IL-6 and IL-8 was observed after treatment with 4-HNE. Exposure to N-acetylcysteine (NAC), an antioxidant and ROS scavenger, antagonized the oxidative effects of 4-HNE on HCECs.. 4-HNE induces oxidative stress in corneal epithelial cells by increasing levels of ROS generation and modifying the expression of antioxidant enzyme levels, decreasing cell viability of HCECs in vitro. This study demonstrates a potential pathway by which 4-HNE functions to confer its detrimental effects and provides a new therapeutic target for the treatment of DED.

Topics: Aldehydes; Blotting, Western; Cells, Cultured; Dry Eye Syndromes; Epithelium, Corneal; Humans; Inflammation; Lipid Peroxidation; Oxidative Stress; Reactive Oxygen Species; Signal Transduction

To investigate the expression of lipid peroxidation markers in the tear film and ocular surface and their correlation with disease severity in patients with dry eye disease.. The concentrations of hexanoyl-lysine (HEL), 4-hydroxy-2-nonenal (HNE), and malondialdehyde (MDA) were measured with enzyme-linked immunosorbent assays in tears obtained from 44 patients with non-Sjogren syndrome dry eye and 33 control subjects. The correlations between the marker levels and the tear film and ocular surface parameters, including tear film break-up time (BUT), Schirmer tear value, tear clearance rate, keratoepitheliopathy scores, corneal sensitivity, conjunctival goblet cell density, and symptom score, were analyzed. The expression of the lipid peroxidation markers HEL, 4-HNE, and MDA in the conjunctiva was evaluated using immunohistochemistry.. The concentrations of HEL, 4-HNE, and MDA were 279.84 ± 69.98 nmol/L, 0.02 ± 0.01 μg/mL, and 3.80 ± 1.05 pmol/mg in control subjects and 283.21 ± 89.67 nmol/L (p = 0.97), 0.20 ± 0.03 μg/mL (p < 0.01), and 13.32 ± 4.03 pmol/mg (p < 0.01) in dry eye patients. 4-HNE and MDA levels significantly correlated with BUT, Schirmer tear value, tear clearance rate, keratoepitheliopathy scores, conjunctival goblet cell density, and symptom score (p < 0.05), whereas HEL levels did not correlate with these parameters. Staining intensities for 4-HNE and MDA increased in dry eye patients.. The expression of late lipid peroxidation markers, 4-HNE and MDA, increases in the tear film and ocular surface of patients with dry eye. The levels correlate with various tear film and ocular surface parameters and may reflect the severity of dry eye disease.

Topics: Aldehydes; Biomarkers; Conjunctiva; Dry Eye Syndromes; Enzyme-Linked Immunosorbent Assay; Female; Humans; Immunohistochemistry; Lipid Peroxidation; Male; Malondialdehyde; Middle Aged; Oxidative Stress; Prospective Studies; Sjogren's Syndrome; Tears

To investigate the efficacy of topical 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) in a mouse model of experimental dry eye (EDE).. Eye drops consisting of 0.001% or 0.01% AICAR, 0.05% cyclosporine A (CsA), or balanced salt solution (BSS) were applied for the treatment of EDE. Tear volume, tear film break-up time (BUT), and corneal fluorescein staining scores were measured 10 days after treatment. Levels of interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, interferon (IFN)-γ, interferon gamma-induced protein 10 (IP-10), and monokine induced by interferon-γ (MIG) were measured in the conjunctiva. In addition, Western blot, periodic acid-Schiff staining for evaluating goblet cell density, flow cytometry for counting the number of CD4+CXCR3+ T cells, and immunohistochemistry for detection of 4-hydroxy-2-nonenal (4HNE) were performed.. Mice treated with 0.01% AICAR showed a significant improvement in all clinical parameters compared with the EDE control, vehicle control, and 0.001% AICAR groups (P < 0.001). A significant decrease in the levels of IL-1β, IL-6, TNF-α, IFN-γ, IP-10, and MIG, the number of CD4+CXCR3+ T cells, and the number of 4HNE-positive cells were also observed in the 0.01% AICAR group (P < 0.001). Although 0.05% CsA also led to an improvement in clinical parameters and inflammatory molecule levels, its therapeutic effects were comparable or inferior to those of 0.01% AICAR.. Topical application of 0.01% AICAR can markedly improve clinical signs and decrease inflammation in the ocular surface of EDE, suggesting that AICAR eye drops may be used as a therapeutic agent for dry eye disease.

Topics: Administration, Topical; Aldehydes; Aminoimidazole Carboxamide; Animals; Blotting, Western; Chemokine CXCL9; Disease Models, Animal; Dry Eye Syndromes; Female; Flow Cytometry; Hypoglycemic Agents; Immunohistochemistry; Interferon-gamma; Interleukin-1beta; Mice; Mice, Inbred C57BL; Ophthalmic Solutions; Ribonucleotides; Tears; Tumor Necrosis Factor-alpha

To investigate the efficacy of the topical application of omega-3 essential fatty acids (EFAs) and hyaluronic acid (HA) mixtures in a mouse model of experimental dry eye (EDE).. Eye drops consisting of 0.1% HA, 0.02%, or 0.2% omega-3 EFAs alone and mixture of 0.02%, or 0.2% omega-3 EFAs and 0.1% HA were applied in desiccating stress-induced murine dry eye. Corneal irregularity scores and fluorescein staining scores were measured 5 and 10 days after treatment. Levels of interleukin (IL)-1β, -17, and interferon gamma-induced protein (IP)-10 were measured in the conjunctiva at 10 days using a multiplex immunobead assay. The concentrations of hexanoyl-lys (HEL) and 4-hydroxynonenal (4-HNE) in conjunctiva tissue were measured with enzyme-linked immunosorbent assays.. Mice treated with the mixture containing 0.2% omega-3 EFAs showed a significant improvement in corneal irregularity scores and corneal fluorescein staining scores compared with EDE, HA, 0.02% or 0.2% omega-3 EFAs alone, and 0.02% omega-3 EFAs mixture-treated mice. A significant decrease in the levels of IL-1β, -17, and IP-10 were observed in the 0.2% EFAs mixture-treated group, compared with the other groups. In the mice treated with the mixture containing 0.2% omega-3 EFAs, the concentration of 4-HNE was also lower than the other groups. Although 0.2% omega-3 EFAs alone group also had a significant improvement in corneal irregularity scores and IL-17, IL-10, and 4 HNE levels compared with the other groups, the efficacy was lower than 0.2% omega-3 mixture group.. Topically applied eye drops containing a mixture of omega-3 EFAs and HA could improve corneal irregularity and corneal epithelial barrier disruption, and decrease inflammatory cytokines and oxidative stress markers on the ocular surface. Topical omega-3 EFAs and HA mixture may have a greater therapeutic effect on clinical signs and inflammation of dry eye compared with HA artificial tears.

Topics: Aldehydes; Animals; Chemokine CXCL10; Conjunctiva; Cornea; Desiccation; Disease Models, Animal; Drug Combinations; Dry Eye Syndromes; Enzyme-Linked Immunosorbent Assay; Fatty Acids, Omega-3; Female; Hyaluronic Acid; Interleukin-17; Interleukin-1beta; Lipid Peroxidation; Mice; Mice, Inbred C57BL; Ophthalmic Solutions; Oxidative Stress

To investigate whether oxidative stress is involved in the etiology of the corneal disorder in blink-suppressed dry eye in a clinically relevant in vivo rat model.. A series of treatments were performed under continuous exposure to low-humidity airflow. Rats were placed on a jogging board (JB) made of a plastic pipe for 7.5 h/d, and for 16.5 hours, they were placed in individual cages without a JB. This protocol was repeated for up to 30 days. Corneal surface alteration was evaluated by the score of punctate fluorescein staining. To assess oxidative stress status, the levels of damaged DNA, and the protein modification by reactive aldehydes in corneal epithelia were detected by immunohistochemistry, using 8-hydroxy-2-deoxyguanosine, 4-hydroxynonenal- and malondialdehyde-specific antibodies.. Significant increases in the fluorescein staining score were observed from days 1 to 30 compared with the initial value. The average score for the dry eye group was significantly increased compared with that for the nontreatment group at all time points throughout the experiment. Immunoreactivity of all oxidative stress markers increased in the dry eye treatment. Quantitative analysis of the positive-stained cells showed a significant increase in the number of positive cells after 10 and 30 days in the dry eye treatment group compared with the nontreatment group.. These results suggest a relationship between the accumulation of oxidative stress and the etiology of corneal epithelial alterations in blink-suppressed dry eye.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Aldehydes; Animals; Apoptosis; Biomarkers; Blinking; Cell Differentiation; Deoxyguanosine; Disease Models, Animal; Dry Eye Syndromes; Epithelium, Corneal; Female; Immunoenzyme Techniques; In Situ Nick-End Labeling; Malondialdehyde; Microscopy, Fluorescence; Oxidative Stress; Oxidoreductases; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Reverse Transcriptase Polymerase Chain Reaction