Current Eye Research
ISSN: 0271-3683 (Print) 1460-2202 (Online) Journal homepage: http://www.tandfonline.com/loi/icey20
Effects of Eye Drops Containing a Mixture of Omega-3 Essential Fatty Acids and Hyaluronic Acid on the Ocular Surface in Desiccating Stressinduced Murine Dry Eye Zhengri Li, Jung-Han Choi, Han-Jin Oh, Soo-Hyun Park, Jee-Bum Lee & Kyung Chul Yoon To cite this article: Zhengri Li, Jung-Han Choi, Han-Jin Oh, Soo-Hyun Park, Jee-Bum Lee & Kyung Chul Yoon (2014) Effects of Eye Drops Containing a Mixture of Omega-3 Essential Fatty Acids and Hyaluronic Acid on the Ocular Surface in Desiccating Stress-induced Murine Dry Eye, Current Eye Research, 39:9, 871-878, DOI: 10.3109/02713683.2014.884595 To link to this article: https://doi.org/10.3109/02713683.2014.884595
Published online: 21 Feb 2014.
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Current Eye Research, 2014; 39(9): 871–878 ! Informa Healthcare USA, Inc. ISSN: 0271-3683 print / 1460-2202 online DOI: 10.3109/02713683.2014.884595
ORIGINAL ARTICLE
Effects of Eye Drops Containing a Mixture of Omega-3 Essential Fatty Acids and Hyaluronic Acid on the Ocular Surface in Desiccating Stress-induced Murine Dry Eye Zhengri Li1, Jung-Han Choi1, Han-Jin Oh1, Soo-Hyun Park2, Jee-Bum Lee3 and Kyung Chul Yoon1 1
Department of Ophthalmology, Chonnam National University Medical School and Hospital, Center for Creative Biomedical Scientists at Chonnam National University, Gwangju, Korea, 2Bio-therapy Human Resources Center, College of Veterinary Medicine, Chonnam National University, Gwangju, Korea, and 3Department of Dermatology, Chonnam National University Medical School and Hospital, Gwangju, Korea
ABSTRACT Purpose: 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). Methods: 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)-1b, -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. Results: 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-1b, -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. Conclusions: 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. Keywords: Dry eye, eye drops, hyaluronic acid, mixtures, omega-3 essential fatty acids
INTRODUCTION
burning, and visual disturbances, and ocular pathological changes including corneal epithelial injury and conjunctival squamous metaplasia. One of the most important mechanisms in the pathogenesis of dry eye disease is inflammation in the lacrimal functional
Dry eye is a highly prevalent ocular disease which affects 15–35% of the population worldwide.1–4 It can induce ocular symptoms, such as dryness, redness,
Received 25 June 2013; revised 5 December 2013; accepted 11 January 2014; published online 21 February 2014 Correspondence: Kyung Chul Yoon, MD, Department of Ophthalmology, Chonnam National University Medical School and Hospital, 8 Hak-Dong, Dong-Gu, Gwangju 501757, South Korea. Tel: +82 622206741. Fax: +82 622271642. E-mail: [email protected]
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872 Z. Li et al. unit composed of the ocular surface and lacrimal gland.5–10 According to recent studies, oxidative stress may also plays a pathophysiological role in dry eye disease.11–13 Oxidative stress generates reactive oxygen species (ROS), which can cause DNA alterations and damage in corneal and conjunctival epithelial cells.11 The damaged epithelial cells release cytokines and cause ocular surface inflammation, resulting in dry eye disease.11 Lipid peroxide and myeloperoxidase activity increases in tears of dry eye patients.14 Omega-3 essential fatty acids (EFAs) have a wide range of biological effects, including benefits on endothelial function and vascular reactivity.15,16 Moreover, Omega-3 EFAs modulate anti-oxidant enzyme activity and down-regulate gene expression associated with production of ROS.17,18 Omega-3 EFAs also block the expression of genes encoding inflammatory cytokines interleukin (IL)-1a, -6, interferon gamma and tumor necrosis factor -alpha (TNF-a).19,20 On the basis of these properties, topical omega-3 agents have been tested in animal models of dry eye and have been shown to improve clinical signs and inflammation on the ocular surface.19,21,22 Artificial tears is the common treatment for dry eye disease. Hyaluronic acids (HA), a linear polymer composed of long chains of repeating disaccharide units of N-acetylglucosamine and glucuronic acid, is one of the component of lubricant eye drops.23 These agents can not only relieve dry eye symptoms, but also improve corneal surface integrity. However, application of HA eye drops alone may not resolve the causative pathologic factors of dry eye. Although topical omega-3 EFAs agents have been found to be effective for experimental dry eye, the combined use of the omega-3 EFAs and HA has not been explored. In this study, we evaluated the efficacy of topical application of a mixture of omega-3 EFAs and HA for the treatment for dry eye disease, using a desiccating stress-induced mouse model, by evaluating the changes of ocular surface irregularities, fluorescein staining, inflammatory cytokines, and lipid peroxidation markers.
MATERIALS AND METHODS Mouse Model of Dry Eye and Experimental Procedure This research protocol was approved by the Chonnam National University Medical School Research Institutional Animal Care and Use Committee. All studies adhered to the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research.
Female C57BL/6 mice aged 6 to 8 weeks were used in these experiments. The mouse model of experimental dry eye (EDE) was induced by a previously described method24–26 modified by subcutaneous injection of 0.5 mg/0.2 mL scopolamine hydrobromide (Sigma-Aldrich, St. Louis, MO) four times a day (8 am, 12 am, 16 pm and 20 pm) with exposure to an air draft and 30% ambient humidity. The mice were randomly assigned to seven groups according to topical treatment administered as follows: (1) untreated (UT) control mice that were not exposed to desiccating stress or treated topically, (2) EDE control mice that received no eye drops, (3) EDE mice treated with 0.1% hyaluronic acid (HA; Alcon Korea, Seoul, Korea), (4) EDE mice treated with 0.02% omega-3 essential fatty acids (EFAs) alone (Chong Kun Dang, Seoul, Korea), (5) EDE mice treated with 0.2% omega-3 EFAs alone, (6) EDE mice treated with 0.02% omega-3 EFAs and 0.1% HA mixture, and (7) EDE mice treated with 0.2% omega3 EFAs and 0.1% HA mixture. All treatment groups received 3 mL of eye drops four times a day. Corneal irregularity scores and corneal fluorescien staining scores were measured at 5 and 10 days after treatment. Mice were euthanized at 10 days after desiccating stress, and multiplex immunobead assay and enzyme-linked immunosorbent assays (ELISA) were performed.
Evaluation of Corneal Surface Irregularity Corneal surface irregularities were analyzed as previously reported27–29 with an essential modification. Distortion was ranked on a 6-point scale: it was briefly as follows: 0, no distortion; 1, distortion in one quarter of the ring; 2, distortion in two quarters; 3, distortion in three quarters; 4, distortion in all four quadrants and 5, severe distortion, no ring recognizable.
Evaluation of Corneal Fluorescein Staining After instillation of 1% fluorescein (1 mL) into the inferior conjunctival sac, punctuate staining on the corneal surface was evaluated in a masked fashion using slit lamp biomicroscopy under cobalt blue light. Each cornea was divided into four quadrants that were, scored individually. The four scores were added to a final grade (possible total of, 16 points) The corneal fluorescein staining severity score was calculated using a 4-point scale: 0, absent; 1, slightly punctuate staining 530 spots; 2, punctate staining 430 spots, but not diffuse; 3, severe diffuse staining but no positive plaque; and 4, positive fluorescein plaque.30 Current Eye Research
Effectiveness of Omega-3 EFAs Mixture in Dry Eye 873 Multiplex Immunobead and Enzyme-linked Immunosorbent Assay A multiplex immunobead assay (Luminex 200; Luminex Corp, Austin, TX) measured the concentrations of interleukin (IL)-1b, -17, and interferon gamma-induced protein (IP)-10 in the conjunctiva, as previously described.28,29,31 The tissues were collected and pooled in lysis buffer containing protease inhibitors for 30 minutes. Cell extracts were centrifuged at 10,000 g for 10 minutes at 4 C, and the supernatants were stored at 70 C until required. Aliquots of the supernatants were added to wells containing the appropriate cytokine bead mixture that included mouse monoclonal antibodies specific for IL-1b, -17 and IP-10 (Panomics, Santa Clara, CA). Reactions were detected after addition of streptavidinphycoerythrin with an analysis system (xPONENT, Austin, TX). The concentrations of IL-1b, -17 and IP-10 in tissues were calculated from standard curves of known concentrations of recombinant mouse cytokines. Total protein levels of lipid peroxidation markers, hexanoyl-lys (HEL) and 4-hydroxynonenal (4-HNE), were detected using ELISA. The supernatants were collected and assayed for HEL (JaICA, Haruoka, Japan) and 4-HNE (Cell Biolabs, San Diego, CA) using ELISA kit. The samples were analyzed according to the manufacturer’s instructions.32–34
Statistical Analyses Statistical differences in the corneal irregularity and fluorecsein staining scores were evaluated by two-way ANOVA, with post hoc analysis. The Kruskal-Wallis and Mann-Whitney tests were used to compare cytokine and lipid peroxidation marker levels between different groups. A p value 50.05 was considered statistically significant.
RESULTS Corneal Surface Irregularities Scores Five days after desiccating stress, the mean corneal irregularity severity scores were significantly higher in the EDE group (3.86 ± 0.54) than in the UT control group (0.42 ± 0.49; p50.05). The mean scores after 5 days of treatment were 3.14 ± 0.57 in the HA-treated group (p = 0.39 versus EDE control), 3.13 ± 0.69 in the 0.02% omega-3 EFAs alone-treated group (p = 0.28 versus EDE control; p = 0.37 versus HA), 2.15 ± 0.59 for the 0.2% omega-3 EFAs alone-treated group (p50.05 versus EDE control; p50.05 versus HA; p50.05 versus 0.02% omega-3 EFAs alone), 3.12 ± 0.72 in the 0.02% omega-3 EFAs mixture-treated group (p = 0.40 !
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versus EDE control; p = 0.51 versus HA; p = 0.57 versus 0.02% omega-3 EFAs alone; p50.05 versus 0.2% omega-3 EFAs alone), and 1.20 ± 0.50 in the 0.2% omega-3 EFAs mixture-treated group (p50.05 versus EDE control, HA, 0.02% or 0.2% omega-3 EFAs alone, and 0.02% omega-3 EFAs mixture). The findings for the mean scores in all groups at 10 days after treatment were similar to those at 5 days (Figure 1A and B).
Corneal Fluorescein Staining Scores Five days after desiccating stress, the mean corneal fluorescein staining scores were significantly higher in the EDE group (14.25 ± 2.03) than in the UT control group (1.30 ± 0.97; p50.05). The mean scores after 5 days oftreatment were 8.12 ± 1.63 in the HA-treated group (p50.05 versus EDE control), 14.02 ± 2.29 in the 0.02% omega-3 EFAs alone-treated group (p = 0.69 versus EDE control; p50.05 versus HA), 8.07 ± 2.19 in the 0.2% omega-3 EFAs alone-treated group (p50.05 versus EDE control; p = 0.74 versus HA; p50.05 versus 0.02% omega-3 EFAs alone), 8.12 ± 1.08 in the 0.02% omega-3 EFAs mixture-treated group (p50.05 versus EDE control; p = 0.52 versus HA-treated group; p50.05 versus 0.02% omega-3 EFAs alone-treated group; p = 0.56 versus 0.2% omega-3 EFAs alonetreated group), and 4.35 ± 1.40 for the 0.2% omega-3 EFA mixture-treated group (p50.05 versus EDE control, HA, 0.02% or 0.2% omega-3 EFAs alone, and 0.02% omega-3 EFAs mixture). The findings for the mean scores in all groups at 10 days after treatment were similar to those at 5 days (Figure 2A and B).
Inflammatory Cytokine Levels in Conjunctival Tissues The concentrations of IL-1b, -17 and IP-10 in the conjunctiva of the EDE group significantly increased compared with the UT group (p50.05). In the mice treated with the mixture containing 0.2% omega-3 EFAs, the concentrations of all measured cytokines and chemokine were significantly lower compared with the EDE control, HA, 0.02% or 0.2% omega-3 EFAs alone, and 0.02% omega-3 EFAs mixture-treated groups (p50.05). In the HA, 0.2% omega-3 EFAs alone, and 0.02% omega-3 EFAs mixture-treated groups, the levels of conjunctiva IL-b was significantly lower compared with the EDE and 0.02% omega-3 EFAs alone-treated groups (p50.05). However, there was no significant differences in the levels among the HA, 0.2% omega-3 EFAs alone and 0.02% omega-3 EFAs mixture-treated groups (Figure 3A, B and C).
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FIGURE 1 Mean corneal smoothness scores (A) and representative figures (B) in the untreated (UT) control, experimental dry eye (EDE) control, 0.1% hyaluronic acid (HA)-treated group, 0.02% omega-3 essential fatty acids (EFAs) alone-treated group, 0.2% omega3 EFAs alone-treated group, 0.02% omega-3 EFAs and 0.1% HA mixtures-treated group, and 0.2% omega-3 EFAs and 0.1% HA mixtures-treated groups at days 5 and 10. *p50.05 versus EDE. yp50.05 versus 0.1% HA. zp50.05 versus 0.02% omega-3 EFAs alone. xp50.05 versus 0.2% omega-3 EFAs alone. #p50.05 versus 0.02% omega-3 EFAs mixture.
FIGURE 2 Mean corneal fluorescein staining scores (A) and representative figures (B) in the untreated (UT) control, experimental dry eye (EDE) control, 0.1% hyaluronic acid (HA)-treated group, 0.02% omega-3 essential fatty acids (EFAs) alone-treated group, 0.2% omega-3 EFAs alone-treated group, 0.02% omega-3 EFAs and 0.1% HA mixtures-treated group, and 0.2% EFAs and 0.1% HA mixturestreated groups at days 5 and 10. *p50.05 versus EDE. yp50.05 versus 0.1% HA. zp50.05 versus 0.02% omega-3 EFAs alone. xp50.05 versus 0.2% omega-3 EFAs alone. #p50.05 versus 0.02% omega-3 EFAs mixture. Current Eye Research
Effectiveness of Omega-3 EFAs Mixture in Dry Eye 875
FIGURE 3 Concentrations of IL-1b (A), IL-17 (B), and IP-10 (C) in the conjunctiva of untreated (UT) control, experimental dry eye (EDE) control, 0.1% hyaluronic acid (HA)-treated group, 0.02% omega-3 essential fatty acids (EFAs) alone-treated group, 0.2% omega3 EFAs alone-treated group, 0.02% omega-3 EFAs and 0.1% HA mixtures-treated group, and 0.2% omega-3 EFAs and 0.1% HA mixtures-treated groups, measured by multiplex immunobead assay. *p50.05 versus EDE. yp50.05 versus 0.1% HA. zp50.05 versus 0.02% omega-3 EFAs alone. xp50.05 versus 0.2% omega-3 EFAs alone. #p50.05 versus 0.02% omega-3 EFAs mixture.
FIGURE 4 Concentrations of HEL (A) and 4-HNE (B) in the conjunctiva of untreated (UT) control, experimental dry eye (EDE) control, 0.1% hyaluronic acid (HA)-treated group, 0.02% omega-3 essential fatty acids (EFAs) alone-treated group, 0.2% omega-3 EFAs alonetreated group, 0.02% omega-3 EFAs and 0.1% HA mixtures-treated group, and 0.2% omega-3 EFAs and 0.1% HA mixtures, measured with enzyme-linked immunosorbent assays. *p50.05 versus EDE. yp50.05 versus 0.1% HA. zp50.05 versus 0.02% omega-3 EFAs alone. xp50.05 versus 0.2% omega-3 EFAs alone. #p50.05 versus 0.02% omega-3 EFAs mixture.
Lipid Peroxidation Marker Level in Conjunctival Tissues Although HEL levels were higher in the EDE group (17.02 ± 4.70 nmol/L) than in the UT group (2.01 ± 0.99 nmol/L; p50.05), there were no significant differences in the levels between groups after !
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desiccating stress (Figure 4A). The levels of 4-HNE in the conjunctiva were significantly higher in the EDE (2.98 ± 0.40 mg/mL) group than the UT group (0.52 ± 0.20 mg/mL; p50.05) after desiccating stress. Conjunctival 4-HNE levels were 2.79 ± 0.58 mg/mL in the HA-treated group (p = 0.63 versus EDE control), 2.71 ± 0.49 in the 0.02% omega-3 EFAs alone-treated group (p = 0.49 versus EDE control; p = 0.65 versus
876 Z. Li et al. HA), 1.51 ± 0.30 for the 0.2% omega-3 EFAs alonetreated group (p50.05 versus EDE control; p50.05 versus HA; p50.05 versus 0.02% omega-3 EFAs alone), 2.50 ± 0.30 mg/mL in the 0.02% omega-3 EFAs mixture-treated group (p = 0.37 versus EDE control; p = 0.54 versus HA), and 0.79 ± 0.24 mg/mL in the 0.2% omega-3 EFAs mixture-treated group (p50.05 versus EDE control, HA, 0.02% or 0.2% omega-3 EFAs alone, and 0.02% omega-3 EFAs mixture) (Figure 4B).
DISCUSSION Omega-3 EFAs are natural modulators of inflammatory activity via their metabolism to eicosanoids, which are locally acting hormones involved in the control of inflammatory and immune responses.35 Omega-3 EFAs metabolites include alpha-linolenic acid and its elongation and desaturation products, stearidonic acid, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA).19 EPA is the precursor of novel potent anti-inflammatory molecules, which inhibits IL-1a, b and TNF-a.35 DHA, the precursor of neuroprotectin D1, can protect the lacrimal gland and ocular surface from TNF-a-induced apoptosis.19,20 Moreover, EPA and DHA work to allow conversion of dihomo-gammalinoleic acid to prostaglandin E 1(PGE 1), which has anti-inflammatory properties.36 Anti-inflammatory PGE 3 and leukotriene B5 can be also synthesis by EFAs.37 Omega-3 EFAs modulate protein levels of antioxidant enzymes, such as superoxide dismutase and catalase.17 Omega-3 EFAs can suppress production of ROS in stimulated leukocytes.38–44 In addition, omega-3 EFAs can inhibit pro-oxidant enzyme phospholipase A2 and, stimulate antioxidant enzymes in membrane lipids and lipoproteins.45,46 In the present study, the application of a mixture containing omega-3 EFAs and HA led to an improvement of clinical signs and a decrease of inflammatory cytokines on the ocular surface of desiccating stressinduced dry eye. The corneal irregularity and fluorescein staining scores were significantly lower in the 0.2% omega-3 EFAs mixture-treated group than in the EDE, HA, 0.02% or 0.2% omega-3 EFAs alone, and 0.02% omega-3 EFAs mixture-treated groups. The 0.2% omega-3 EFAs groups also had lower IL-1b, -17, and IP-10 concentrations in the conjunctiva compared with the other groups. In our study, the products of oxidative stress was evaluated by measuring the levels of HEL, an acute marker of lipid peroxidation, and 4-HNE, a chronic marker of lipid peroxidation in the conjunctiva. Similar to previous reports, the HEL and 4-HNE levels increased after dry eye stress.47,48 After treatment, the HEL levels did not show significant differences between groups, whereas the 4-HNE levels significantly decreased after treatment with the 0.2% omega-3 EFAs mixture. Although the reason for
this discrepancy between two markers is unclear, we suppose that a compensatory mechanism might work during the acute stage of oxidative stress. Results of multiplex immunobead and ELISA assays indicate that eye drops containing a mixture of 0.2% omega-3 EFAs can reduce inflammation and oxidative stress on the ocular surface. This is consistent with the findings of a number of prior studies. Rashid et al.19 reported that topical omega-3 EFAs could decrease inflammatory markers such as CD11b+, IL-1a and TNF-a at both cellular and molecular levels in murine dry eye. De Paiva et al.21 also reported that resolvin, which was derived from the omega-3 EFAs, improved the outcome measures of corneal staining and goblet cell density in murine dry eye, suggesting the potential utility in the treatment of dry eye. In addition, treatment with omega-3 EFAs reportedly down-regulates products of lipid peroxidation.49 All parameters were significantly improved using the mixture containing 0.2% EFAs compared with the EDE, HA, 0.02% or 0.2% omega-3 EFAs alone, and 0.02% omega-3 EFAs mixture-treated groups, while mice treated with 0.2% omega-3 EFAs alone-treated group showed an improvement in corneal irregularity scores, and IL-17, IP-10 and 4-HNE levels compared with EDE, HA, 0.02% omega-3 EFAs alone, and 0.02% omega-3 EFAs mixture-treated group. In addition, HA, 0.2% omega-3 EFAs alone, and 0.02% omega-3 EFAs mixture-treated group showed an improvement in corneal fluorescein staining scores and IL-1b level compared with EDE control and 0.002% omega-3 EFAs alone-treated group. However, there were no significant differences in the parameters among the HA, 0.2% omega-3 EFAs alone, and 0.02% omega-3 EFAs mixture-treated groups. These results suggest that eye drops containing a mixture of 0.2% EFAs is more effective for dry eye than the 0.2% omega-3 EFAs alone and 0.02% omega-3 EFAs mixture. The HA-treated group had significantly lower corneal staining scores and lower levels of IL-b compared with the EDE group. HA promotes corneal epithelial proliferation and migration, stabilizes the ocular surface epithelial barrier, having intrinsic wound healing properties.50,51 Moreover, HA can also play an important role in controlling localized inflammation.52,53 According to a human study of dry eye disease, HA suppressed inflammatory markers including HLA-DR and CD 40, and up-regulated protective the markers MUC5AC and CD63.54 These wound healing and inflammation-controlling properties of HA might have resulted in an improvement of corneal staining and decrease of the pro-inflammatory cytokine. In conclusion, 0.2% omega-3 EFAs and 0.1% HA mixture eyedrops could improve corneal irregularity and epithelial barrier disruption, and decrease inflammatory cytokines and oxidative stress marker on the Current Eye Research
Effectiveness of Omega-3 EFAs Mixture in Dry Eye 877 ocular surface of murine dry eye. Considering wound healing property of HA and anti-inflammatory and anti-oxidant effects of omega-3 EFAs, the mixture of both components would be synergistically helpful for improving symptoms and signs of dry eye. Our results showed that the topical omega-3 EFAs and HA mixture may have a greater therapeutic effect on clinical signs and inflammation in dry eye diseases compared with topical HA.
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DECLARATION OF INTEREST 17.
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. The study was partially supported by the CNUH Biomedical Research Institute (CRI 11076-21 and CRI 13906-22) and Forest Science & Technology Projects (Project No. S121313L50100) by Korea Forest Service.
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of desiccating stress-induced dry eye. Invest Ophthalmol Vis Sci 2013;54:155–162. Pauly A, Brignole-Baudouin F, Labbe´ A, Liang H, Warnet JM, Baudouin C. New tools for the evaluation of toxic ocular surface changes in the rat. Invest Ophthalmol Vis Sci 2007;48:5473–5483. Yoon KC, Ahn KY, Choi W, Li Z, Choi JS, Lee SH. Tear production and ocular surface changes in experimental dry eye after elimination of desiccating stress. Invest Ophthalmol Vis Sci 2011;52:7267–7273. Oh HJ, Jang JY, Li Z, Park SH, Yoon KC. Effects of umbilical cord serum eye drops in a mouse model of ocular chemical burn. Curr Eye Res 2012;37:1084–1090. Park JW, Li Z, Choi JS, Oh HJ, Park SH, Yoon KC. Expression of CXCL9, -10 and -11 in the aqueous humor of patients with herpetic endotheliitis. Cornea 2012;31: 1246–1250. Choi W, Li Z, Oh HJ, Im SK, Lee SH, Park SH, et al. Expression of CCR5 and its ligands CCL3, -4 and -5 in the tear film and ocular surface of patients with dry eye disease. Curr Eye Res 2012;37:12–17. Roncone M, Bartlett H, Eperjesi F. Essential fatty acids for dry eye: a review. Cont Lens Anterior Eye 2010;33:49–54. Aragona P, Bucolo C, Spinella R, Giuffrida S, Ferreri G. Systemic omega-6 fatty acid treatment and PGE1 tear content in Sjogren’s patients. Invest Ophthalmol Vis Sci 2005;46:4474–4479. Calder P, Zurier R. Polyunsaturated fatty acids and rheumatoid arthritis. Curr Opin Clin Nutr Metab Care 2001;4:115–121. Hiramatsu K, Arimori S. Increased superoxide production by mononuclear cells of patients with hypertriglyceridemia and diabetes. Diabetes 1988;37:832–837. Thompson PJ, Misso NL, Passarelli M, Phillips MJ. The effect of eicosapentaenoic acid consumption on human neutrophil chemiluminescence. Lipids 1991;26:1223–1226. Varming K, Schmidt EB, Svaneborg N, Møller JM, Lervang HH, Grunnet N, et al. The effect of n-3 fatty acids on neutrophil chemiluminescence. Scand J Clin Lab Invest 1995;55:47–52. Fisher M, Levine PH, Weiner BH, Johnson MH, Doyle EM, Ellis PA, et al. Dietary n-3 fatty acid supplementation reduces superoxide production and chemiluminescence in a monocyte-enriched preparation of leukocytes. Am J Clin Nutr 1990;51:804–808. Fisher M, Upchurch KS, Levine PH, Johnson MH, Vaudreuil CH, Natale A, et al. Effects of dietary fish oil
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supplementation on polymorphonuclear leukocyte inflammatory potential. Inflammation 1986;10:387–392. Sirtori CR, Gatti E, Tremoli E, Galli C, Gianfranceschi G, Franceschini G, et al. Olive oil, corn oil, and n-3 fatty acids differently affect lipids, lipoproteins, platelets, and superoxide formation in Type 2 hypercholesterolemia. Am J Clin Nutr 1992;56:113–122. Carbonell T, Rodenas J, Miret S, Mitjavila MT. Fish oil and oxidative stress by inflammatory leukocytes. Free Radic Res 1997;27:591–597. Applegate KR, Glomset JA. Computer-based modeling of the conformation and packing properties of docosahexaenoic acid. J Lipid Res 1986;27:658–680. von Schacky C, Siess W, Fischer S, Weber PC. A comparative study of eicosapentaenoic acid metabolism by human platelets in vivo and in vitro. J Lipid Res 1985;26:457– 464. Wakamatsu TH, Dogru M, Ayako I, Takano Y, Matsumoto Y, Ibrahim OM, et al. Evaluation of lipid oxidative stress status and inflammation in atopic ocular surface disease. Mol Vis 2010;16:2465-2475. Wakamatsu TH, Dogru M, Matsumoto Y, Kojima T, Kaido M, Ibrahim OM, et al. Evaluation of lipid oxidative stress status in Sjo¨gren syndrome patients. Invest Ophthalmol Vis Sci 2013;54:201–210. Cai F, Dupertuis YM, Pichard C. Role of polyunsaturated fatty acids and lipid peroxidation on colorectal cancer risk and treatments. Curr Opin Clin Nutr Metab Care 2012;15: 99–106. Entwistle J, Hall CL, Turley EA. HA receptors: regulators of signaling to the cytoskeleton. J Cell Biochem 1996;61: 569–577. Nishida T, Nakamura M, Mishima H, Otori T. Hyaluronan stimulates corneal epithelial migration. Exp Eye Res 1991; 53:753–758. Aragona P, Di Stefano G, Ferreri F, Spinella R, Stilo A. Sodium hyaluronate eye drops of different osmolarity for treatment of dry eye in Sjogren syndrome patients. Br J Ophthalmol 2002;86:879–884. Stern ME, Beuerman RW, Fox RI, Goa J, Mircheff AK, Pflugfelder SC. The pathology of dry eye: the interaction between the ocular surface and lacrimal gland. Cornea 1998;17:584–589. Brignole F, Pisella PJ, Dupas B, Baeyens V, Baudouin C. Efficacy and safety of 0.18% sodium hyaluronate in patients with moderate dry eye syndrome and superficial keratitis. Graefes Arch Clin Exp Ophthalmol 2005;243: 531–538.
Current Eye Research
ISSN: 0271-3683 (Print) 1460-2202 (Online) Journal homepage: http://www.tandfonline.com/loi/icey20
Effects of Eye Drops Containing a Mixture of Omega-3 Essential Fatty Acids and Hyaluronic Acid on the Ocular Surface in Desiccating Stressinduced Murine Dry Eye Zhengri Li, Jung-Han Choi, Han-Jin Oh, Soo-Hyun Park, Jee-Bum Lee & Kyung Chul Yoon To cite this article: Zhengri Li, Jung-Han Choi, Han-Jin Oh, Soo-Hyun Park, Jee-Bum Lee & Kyung Chul Yoon (2014) Effects of Eye Drops Containing a Mixture of Omega-3 Essential Fatty Acids and Hyaluronic Acid on the Ocular Surface in Desiccating Stress-induced Murine Dry Eye, Current Eye Research, 39:9, 871-878, DOI: 10.3109/02713683.2014.884595 To link to this article: https://doi.org/10.3109/02713683.2014.884595
Published online: 21 Feb 2014.
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Current Eye Research, 2014; 39(9): 871–878 ! Informa Healthcare USA, Inc. ISSN: 0271-3683 print / 1460-2202 online DOI: 10.3109/02713683.2014.884595
ORIGINAL ARTICLE
Effects of Eye Drops Containing a Mixture of Omega-3 Essential Fatty Acids and Hyaluronic Acid on the Ocular Surface in Desiccating Stress-induced Murine Dry Eye Zhengri Li1, Jung-Han Choi1, Han-Jin Oh1, Soo-Hyun Park2, Jee-Bum Lee3 and Kyung Chul Yoon1 1
Department of Ophthalmology, Chonnam National University Medical School and Hospital, Center for Creative Biomedical Scientists at Chonnam National University, Gwangju, Korea, 2Bio-therapy Human Resources Center, College of Veterinary Medicine, Chonnam National University, Gwangju, Korea, and 3Department of Dermatology, Chonnam National University Medical School and Hospital, Gwangju, Korea
ABSTRACT Purpose: 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). Methods: 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)-1b, -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. Results: 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-1b, -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. Conclusions: 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. Keywords: Dry eye, eye drops, hyaluronic acid, mixtures, omega-3 essential fatty acids
INTRODUCTION
burning, and visual disturbances, and ocular pathological changes including corneal epithelial injury and conjunctival squamous metaplasia. One of the most important mechanisms in the pathogenesis of dry eye disease is inflammation in the lacrimal functional
Dry eye is a highly prevalent ocular disease which affects 15–35% of the population worldwide.1–4 It can induce ocular symptoms, such as dryness, redness,
Received 25 June 2013; revised 5 December 2013; accepted 11 January 2014; published online 21 February 2014 Correspondence: Kyung Chul Yoon, MD, Department of Ophthalmology, Chonnam National University Medical School and Hospital, 8 Hak-Dong, Dong-Gu, Gwangju 501757, South Korea. Tel: +82 622206741. Fax: +82 622271642. E-mail: [email protected]
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872 Z. Li et al. unit composed of the ocular surface and lacrimal gland.5–10 According to recent studies, oxidative stress may also plays a pathophysiological role in dry eye disease.11–13 Oxidative stress generates reactive oxygen species (ROS), which can cause DNA alterations and damage in corneal and conjunctival epithelial cells.11 The damaged epithelial cells release cytokines and cause ocular surface inflammation, resulting in dry eye disease.11 Lipid peroxide and myeloperoxidase activity increases in tears of dry eye patients.14 Omega-3 essential fatty acids (EFAs) have a wide range of biological effects, including benefits on endothelial function and vascular reactivity.15,16 Moreover, Omega-3 EFAs modulate anti-oxidant enzyme activity and down-regulate gene expression associated with production of ROS.17,18 Omega-3 EFAs also block the expression of genes encoding inflammatory cytokines interleukin (IL)-1a, -6, interferon gamma and tumor necrosis factor -alpha (TNF-a).19,20 On the basis of these properties, topical omega-3 agents have been tested in animal models of dry eye and have been shown to improve clinical signs and inflammation on the ocular surface.19,21,22 Artificial tears is the common treatment for dry eye disease. Hyaluronic acids (HA), a linear polymer composed of long chains of repeating disaccharide units of N-acetylglucosamine and glucuronic acid, is one of the component of lubricant eye drops.23 These agents can not only relieve dry eye symptoms, but also improve corneal surface integrity. However, application of HA eye drops alone may not resolve the causative pathologic factors of dry eye. Although topical omega-3 EFAs agents have been found to be effective for experimental dry eye, the combined use of the omega-3 EFAs and HA has not been explored. In this study, we evaluated the efficacy of topical application of a mixture of omega-3 EFAs and HA for the treatment for dry eye disease, using a desiccating stress-induced mouse model, by evaluating the changes of ocular surface irregularities, fluorescein staining, inflammatory cytokines, and lipid peroxidation markers.
MATERIALS AND METHODS Mouse Model of Dry Eye and Experimental Procedure This research protocol was approved by the Chonnam National University Medical School Research Institutional Animal Care and Use Committee. All studies adhered to the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research.
Female C57BL/6 mice aged 6 to 8 weeks were used in these experiments. The mouse model of experimental dry eye (EDE) was induced by a previously described method24–26 modified by subcutaneous injection of 0.5 mg/0.2 mL scopolamine hydrobromide (Sigma-Aldrich, St. Louis, MO) four times a day (8 am, 12 am, 16 pm and 20 pm) with exposure to an air draft and 30% ambient humidity. The mice were randomly assigned to seven groups according to topical treatment administered as follows: (1) untreated (UT) control mice that were not exposed to desiccating stress or treated topically, (2) EDE control mice that received no eye drops, (3) EDE mice treated with 0.1% hyaluronic acid (HA; Alcon Korea, Seoul, Korea), (4) EDE mice treated with 0.02% omega-3 essential fatty acids (EFAs) alone (Chong Kun Dang, Seoul, Korea), (5) EDE mice treated with 0.2% omega-3 EFAs alone, (6) EDE mice treated with 0.02% omega-3 EFAs and 0.1% HA mixture, and (7) EDE mice treated with 0.2% omega3 EFAs and 0.1% HA mixture. All treatment groups received 3 mL of eye drops four times a day. Corneal irregularity scores and corneal fluorescien staining scores were measured at 5 and 10 days after treatment. Mice were euthanized at 10 days after desiccating stress, and multiplex immunobead assay and enzyme-linked immunosorbent assays (ELISA) were performed.
Evaluation of Corneal Surface Irregularity Corneal surface irregularities were analyzed as previously reported27–29 with an essential modification. Distortion was ranked on a 6-point scale: it was briefly as follows: 0, no distortion; 1, distortion in one quarter of the ring; 2, distortion in two quarters; 3, distortion in three quarters; 4, distortion in all four quadrants and 5, severe distortion, no ring recognizable.
Evaluation of Corneal Fluorescein Staining After instillation of 1% fluorescein (1 mL) into the inferior conjunctival sac, punctuate staining on the corneal surface was evaluated in a masked fashion using slit lamp biomicroscopy under cobalt blue light. Each cornea was divided into four quadrants that were, scored individually. The four scores were added to a final grade (possible total of, 16 points) The corneal fluorescein staining severity score was calculated using a 4-point scale: 0, absent; 1, slightly punctuate staining 530 spots; 2, punctate staining 430 spots, but not diffuse; 3, severe diffuse staining but no positive plaque; and 4, positive fluorescein plaque.30 Current Eye Research
Effectiveness of Omega-3 EFAs Mixture in Dry Eye 873 Multiplex Immunobead and Enzyme-linked Immunosorbent Assay A multiplex immunobead assay (Luminex 200; Luminex Corp, Austin, TX) measured the concentrations of interleukin (IL)-1b, -17, and interferon gamma-induced protein (IP)-10 in the conjunctiva, as previously described.28,29,31 The tissues were collected and pooled in lysis buffer containing protease inhibitors for 30 minutes. Cell extracts were centrifuged at 10,000 g for 10 minutes at 4 C, and the supernatants were stored at 70 C until required. Aliquots of the supernatants were added to wells containing the appropriate cytokine bead mixture that included mouse monoclonal antibodies specific for IL-1b, -17 and IP-10 (Panomics, Santa Clara, CA). Reactions were detected after addition of streptavidinphycoerythrin with an analysis system (xPONENT, Austin, TX). The concentrations of IL-1b, -17 and IP-10 in tissues were calculated from standard curves of known concentrations of recombinant mouse cytokines. Total protein levels of lipid peroxidation markers, hexanoyl-lys (HEL) and 4-hydroxynonenal (4-HNE), were detected using ELISA. The supernatants were collected and assayed for HEL (JaICA, Haruoka, Japan) and 4-HNE (Cell Biolabs, San Diego, CA) using ELISA kit. The samples were analyzed according to the manufacturer’s instructions.32–34
Statistical Analyses Statistical differences in the corneal irregularity and fluorecsein staining scores were evaluated by two-way ANOVA, with post hoc analysis. The Kruskal-Wallis and Mann-Whitney tests were used to compare cytokine and lipid peroxidation marker levels between different groups. A p value 50.05 was considered statistically significant.
RESULTS Corneal Surface Irregularities Scores Five days after desiccating stress, the mean corneal irregularity severity scores were significantly higher in the EDE group (3.86 ± 0.54) than in the UT control group (0.42 ± 0.49; p50.05). The mean scores after 5 days of treatment were 3.14 ± 0.57 in the HA-treated group (p = 0.39 versus EDE control), 3.13 ± 0.69 in the 0.02% omega-3 EFAs alone-treated group (p = 0.28 versus EDE control; p = 0.37 versus HA), 2.15 ± 0.59 for the 0.2% omega-3 EFAs alone-treated group (p50.05 versus EDE control; p50.05 versus HA; p50.05 versus 0.02% omega-3 EFAs alone), 3.12 ± 0.72 in the 0.02% omega-3 EFAs mixture-treated group (p = 0.40 !
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versus EDE control; p = 0.51 versus HA; p = 0.57 versus 0.02% omega-3 EFAs alone; p50.05 versus 0.2% omega-3 EFAs alone), and 1.20 ± 0.50 in the 0.2% omega-3 EFAs mixture-treated group (p50.05 versus EDE control, HA, 0.02% or 0.2% omega-3 EFAs alone, and 0.02% omega-3 EFAs mixture). The findings for the mean scores in all groups at 10 days after treatment were similar to those at 5 days (Figure 1A and B).
Corneal Fluorescein Staining Scores Five days after desiccating stress, the mean corneal fluorescein staining scores were significantly higher in the EDE group (14.25 ± 2.03) than in the UT control group (1.30 ± 0.97; p50.05). The mean scores after 5 days oftreatment were 8.12 ± 1.63 in the HA-treated group (p50.05 versus EDE control), 14.02 ± 2.29 in the 0.02% omega-3 EFAs alone-treated group (p = 0.69 versus EDE control; p50.05 versus HA), 8.07 ± 2.19 in the 0.2% omega-3 EFAs alone-treated group (p50.05 versus EDE control; p = 0.74 versus HA; p50.05 versus 0.02% omega-3 EFAs alone), 8.12 ± 1.08 in the 0.02% omega-3 EFAs mixture-treated group (p50.05 versus EDE control; p = 0.52 versus HA-treated group; p50.05 versus 0.02% omega-3 EFAs alone-treated group; p = 0.56 versus 0.2% omega-3 EFAs alonetreated group), and 4.35 ± 1.40 for the 0.2% omega-3 EFA mixture-treated group (p50.05 versus EDE control, HA, 0.02% or 0.2% omega-3 EFAs alone, and 0.02% omega-3 EFAs mixture). The findings for the mean scores in all groups at 10 days after treatment were similar to those at 5 days (Figure 2A and B).
Inflammatory Cytokine Levels in Conjunctival Tissues The concentrations of IL-1b, -17 and IP-10 in the conjunctiva of the EDE group significantly increased compared with the UT group (p50.05). In the mice treated with the mixture containing 0.2% omega-3 EFAs, the concentrations of all measured cytokines and chemokine were significantly lower compared with the EDE control, HA, 0.02% or 0.2% omega-3 EFAs alone, and 0.02% omega-3 EFAs mixture-treated groups (p50.05). In the HA, 0.2% omega-3 EFAs alone, and 0.02% omega-3 EFAs mixture-treated groups, the levels of conjunctiva IL-b was significantly lower compared with the EDE and 0.02% omega-3 EFAs alone-treated groups (p50.05). However, there was no significant differences in the levels among the HA, 0.2% omega-3 EFAs alone and 0.02% omega-3 EFAs mixture-treated groups (Figure 3A, B and C).
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FIGURE 1 Mean corneal smoothness scores (A) and representative figures (B) in the untreated (UT) control, experimental dry eye (EDE) control, 0.1% hyaluronic acid (HA)-treated group, 0.02% omega-3 essential fatty acids (EFAs) alone-treated group, 0.2% omega3 EFAs alone-treated group, 0.02% omega-3 EFAs and 0.1% HA mixtures-treated group, and 0.2% omega-3 EFAs and 0.1% HA mixtures-treated groups at days 5 and 10. *p50.05 versus EDE. yp50.05 versus 0.1% HA. zp50.05 versus 0.02% omega-3 EFAs alone. xp50.05 versus 0.2% omega-3 EFAs alone. #p50.05 versus 0.02% omega-3 EFAs mixture.
FIGURE 2 Mean corneal fluorescein staining scores (A) and representative figures (B) in the untreated (UT) control, experimental dry eye (EDE) control, 0.1% hyaluronic acid (HA)-treated group, 0.02% omega-3 essential fatty acids (EFAs) alone-treated group, 0.2% omega-3 EFAs alone-treated group, 0.02% omega-3 EFAs and 0.1% HA mixtures-treated group, and 0.2% EFAs and 0.1% HA mixturestreated groups at days 5 and 10. *p50.05 versus EDE. yp50.05 versus 0.1% HA. zp50.05 versus 0.02% omega-3 EFAs alone. xp50.05 versus 0.2% omega-3 EFAs alone. #p50.05 versus 0.02% omega-3 EFAs mixture. Current Eye Research
Effectiveness of Omega-3 EFAs Mixture in Dry Eye 875
FIGURE 3 Concentrations of IL-1b (A), IL-17 (B), and IP-10 (C) in the conjunctiva of untreated (UT) control, experimental dry eye (EDE) control, 0.1% hyaluronic acid (HA)-treated group, 0.02% omega-3 essential fatty acids (EFAs) alone-treated group, 0.2% omega3 EFAs alone-treated group, 0.02% omega-3 EFAs and 0.1% HA mixtures-treated group, and 0.2% omega-3 EFAs and 0.1% HA mixtures-treated groups, measured by multiplex immunobead assay. *p50.05 versus EDE. yp50.05 versus 0.1% HA. zp50.05 versus 0.02% omega-3 EFAs alone. xp50.05 versus 0.2% omega-3 EFAs alone. #p50.05 versus 0.02% omega-3 EFAs mixture.
FIGURE 4 Concentrations of HEL (A) and 4-HNE (B) in the conjunctiva of untreated (UT) control, experimental dry eye (EDE) control, 0.1% hyaluronic acid (HA)-treated group, 0.02% omega-3 essential fatty acids (EFAs) alone-treated group, 0.2% omega-3 EFAs alonetreated group, 0.02% omega-3 EFAs and 0.1% HA mixtures-treated group, and 0.2% omega-3 EFAs and 0.1% HA mixtures, measured with enzyme-linked immunosorbent assays. *p50.05 versus EDE. yp50.05 versus 0.1% HA. zp50.05 versus 0.02% omega-3 EFAs alone. xp50.05 versus 0.2% omega-3 EFAs alone. #p50.05 versus 0.02% omega-3 EFAs mixture.
Lipid Peroxidation Marker Level in Conjunctival Tissues Although HEL levels were higher in the EDE group (17.02 ± 4.70 nmol/L) than in the UT group (2.01 ± 0.99 nmol/L; p50.05), there were no significant differences in the levels between groups after !
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desiccating stress (Figure 4A). The levels of 4-HNE in the conjunctiva were significantly higher in the EDE (2.98 ± 0.40 mg/mL) group than the UT group (0.52 ± 0.20 mg/mL; p50.05) after desiccating stress. Conjunctival 4-HNE levels were 2.79 ± 0.58 mg/mL in the HA-treated group (p = 0.63 versus EDE control), 2.71 ± 0.49 in the 0.02% omega-3 EFAs alone-treated group (p = 0.49 versus EDE control; p = 0.65 versus
876 Z. Li et al. HA), 1.51 ± 0.30 for the 0.2% omega-3 EFAs alonetreated group (p50.05 versus EDE control; p50.05 versus HA; p50.05 versus 0.02% omega-3 EFAs alone), 2.50 ± 0.30 mg/mL in the 0.02% omega-3 EFAs mixture-treated group (p = 0.37 versus EDE control; p = 0.54 versus HA), and 0.79 ± 0.24 mg/mL in the 0.2% omega-3 EFAs mixture-treated group (p50.05 versus EDE control, HA, 0.02% or 0.2% omega-3 EFAs alone, and 0.02% omega-3 EFAs mixture) (Figure 4B).
DISCUSSION Omega-3 EFAs are natural modulators of inflammatory activity via their metabolism to eicosanoids, which are locally acting hormones involved in the control of inflammatory and immune responses.35 Omega-3 EFAs metabolites include alpha-linolenic acid and its elongation and desaturation products, stearidonic acid, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA).19 EPA is the precursor of novel potent anti-inflammatory molecules, which inhibits IL-1a, b and TNF-a.35 DHA, the precursor of neuroprotectin D1, can protect the lacrimal gland and ocular surface from TNF-a-induced apoptosis.19,20 Moreover, EPA and DHA work to allow conversion of dihomo-gammalinoleic acid to prostaglandin E 1(PGE 1), which has anti-inflammatory properties.36 Anti-inflammatory PGE 3 and leukotriene B5 can be also synthesis by EFAs.37 Omega-3 EFAs modulate protein levels of antioxidant enzymes, such as superoxide dismutase and catalase.17 Omega-3 EFAs can suppress production of ROS in stimulated leukocytes.38–44 In addition, omega-3 EFAs can inhibit pro-oxidant enzyme phospholipase A2 and, stimulate antioxidant enzymes in membrane lipids and lipoproteins.45,46 In the present study, the application of a mixture containing omega-3 EFAs and HA led to an improvement of clinical signs and a decrease of inflammatory cytokines on the ocular surface of desiccating stressinduced dry eye. The corneal irregularity and fluorescein staining scores were significantly lower in the 0.2% omega-3 EFAs mixture-treated group than in the EDE, HA, 0.02% or 0.2% omega-3 EFAs alone, and 0.02% omega-3 EFAs mixture-treated groups. The 0.2% omega-3 EFAs groups also had lower IL-1b, -17, and IP-10 concentrations in the conjunctiva compared with the other groups. In our study, the products of oxidative stress was evaluated by measuring the levels of HEL, an acute marker of lipid peroxidation, and 4-HNE, a chronic marker of lipid peroxidation in the conjunctiva. Similar to previous reports, the HEL and 4-HNE levels increased after dry eye stress.47,48 After treatment, the HEL levels did not show significant differences between groups, whereas the 4-HNE levels significantly decreased after treatment with the 0.2% omega-3 EFAs mixture. Although the reason for
this discrepancy between two markers is unclear, we suppose that a compensatory mechanism might work during the acute stage of oxidative stress. Results of multiplex immunobead and ELISA assays indicate that eye drops containing a mixture of 0.2% omega-3 EFAs can reduce inflammation and oxidative stress on the ocular surface. This is consistent with the findings of a number of prior studies. Rashid et al.19 reported that topical omega-3 EFAs could decrease inflammatory markers such as CD11b+, IL-1a and TNF-a at both cellular and molecular levels in murine dry eye. De Paiva et al.21 also reported that resolvin, which was derived from the omega-3 EFAs, improved the outcome measures of corneal staining and goblet cell density in murine dry eye, suggesting the potential utility in the treatment of dry eye. In addition, treatment with omega-3 EFAs reportedly down-regulates products of lipid peroxidation.49 All parameters were significantly improved using the mixture containing 0.2% EFAs compared with the EDE, HA, 0.02% or 0.2% omega-3 EFAs alone, and 0.02% omega-3 EFAs mixture-treated groups, while mice treated with 0.2% omega-3 EFAs alone-treated group showed an improvement in corneal irregularity scores, and IL-17, IP-10 and 4-HNE levels compared with EDE, HA, 0.02% omega-3 EFAs alone, and 0.02% omega-3 EFAs mixture-treated group. In addition, HA, 0.2% omega-3 EFAs alone, and 0.02% omega-3 EFAs mixture-treated group showed an improvement in corneal fluorescein staining scores and IL-1b level compared with EDE control and 0.002% omega-3 EFAs alone-treated group. However, there were no significant differences in the parameters among the HA, 0.2% omega-3 EFAs alone, and 0.02% omega-3 EFAs mixture-treated groups. These results suggest that eye drops containing a mixture of 0.2% EFAs is more effective for dry eye than the 0.2% omega-3 EFAs alone and 0.02% omega-3 EFAs mixture. The HA-treated group had significantly lower corneal staining scores and lower levels of IL-b compared with the EDE group. HA promotes corneal epithelial proliferation and migration, stabilizes the ocular surface epithelial barrier, having intrinsic wound healing properties.50,51 Moreover, HA can also play an important role in controlling localized inflammation.52,53 According to a human study of dry eye disease, HA suppressed inflammatory markers including HLA-DR and CD 40, and up-regulated protective the markers MUC5AC and CD63.54 These wound healing and inflammation-controlling properties of HA might have resulted in an improvement of corneal staining and decrease of the pro-inflammatory cytokine. In conclusion, 0.2% omega-3 EFAs and 0.1% HA mixture eyedrops could improve corneal irregularity and epithelial barrier disruption, and decrease inflammatory cytokines and oxidative stress marker on the Current Eye Research
Effectiveness of Omega-3 EFAs Mixture in Dry Eye 877 ocular surface of murine dry eye. Considering wound healing property of HA and anti-inflammatory and anti-oxidant effects of omega-3 EFAs, the mixture of both components would be synergistically helpful for improving symptoms and signs of dry eye. Our results showed that the topical omega-3 EFAs and HA mixture may have a greater therapeutic effect on clinical signs and inflammation in dry eye diseases compared with topical HA.
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DECLARATION OF INTEREST 17.
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. The study was partially supported by the CNUH Biomedical Research Institute (CRI 11076-21 and CRI 13906-22) and Forest Science & Technology Projects (Project No. S121313L50100) by Korea Forest Service.
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