Molecular Vision 2016; 22:284-293
Received 22 October 2015 | Accepted 31 March 2016 | Published 02 April 2016
The last two authors contributed equally to this study.
1Natural Products Research Center, Korea Institute of Science and Technology (KIST), Gangneung, Korea; 2Department of Ophthalmology, University of Ulsan, Gangneung Asan Hospital, Gangneung, Korea; 3Department of Biological Chemistry, Korea University of Science and Technology (UST), Daejeon, Korea
Correspondence to: Sung Jae Yang, Department of Ophthalmology, University of Ulsan, Gangneung Asan Hospital #415, Bangdong-ri, Sacheon-myeon, Gangneung-si, Gangwon-do, Korea 210-711; Phone: +82-33-610-3943; FAX: +82-33-610-4960; email: email@example.com
Purpose: In this study, the beneficial effects of the oral administration of ethanol extract of Diospyros kaki (EEDK) were tested on a mouse dry eye model induced by benzalkonium chloride (BAC).
Methods: A solution of 0.2% BAC was administered topically to mouse eyes for 14 days, twice daily, to induce dry eye. Various concentrations of EEDK were administrated daily by oral gavage for 14 days after BAC treatment. Preservative-free eye drops were instilled in the positive-control group. The tear secretion volume (Schirmer’s test), tear break-up time (BUT), and fluorescein score were measured on the ocular surface. BAC-induced corneal damage was tested with hematoxylin-eosin staining. Moreover, apoptotic cell death in the corneal epithelial layer was investigated with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining. The protein expression level of interleukin-1alpha (IL-1α), IL-1β, IL-6, tumor necrosis factor- alpha (TNF-α), and monocyte chemotactic protein-1 (MCP-1) was determined with western blot analysis. Furthermore, squamous metaplasia in the corneal epithelial layer was detected with immunofluorescent staining for cytokeratine-10. The cellular proliferation in the cornea was examined with immunohistochemical staining for Ki-67.
Results: EEDK treatment resulted in prolonged BUT, decreased fluorescein score, increased tear volume, and smoother epithelial cells compared with BAC treatment alone in the cornea. Moreover, EEDK treatment inhibited the inflammatory response and corneal epithelial cell death in a BAC-induced murine dry eye model, and changes in squamous cells were inhibited. Proliferative activity in the corneal epithelium cells was improved with EEDK.
Conclusions: EEDK could be a potential therapeutic agent in the clinical treatment of dry eye.
Dry eye is a common and multifactorial ocular surface disease that results in symptoms of ocular discomfort, visual disturbance, and tear film instability with potential damage to the ocular surface . Patients often complain of dryness, redness, burning, and a foreign body sensation. In more severe cases, patients suffer from dry eye–related ocular complications, including corneal epithelium damage, squamous metaplasia, and neovascularization [2,3]. Although the definitive mechanism of dry eye has not been entirely clarified, ocular surface inflammation may have a prominent role in the pathogenesis of dry eye [4-8]. Tear secretory gland dysfunction results in changes in tear composition, which stimulates production of inflammatory mediators on the ocular surface .
Most ophthalmic solutions contain a preservative to protect the ingredients and prevent bacterial contamination after the bottle is opened. Benzalkonium chloride (BAC), an antimicrobial agent, is the most widely used preservative agent in ophthalmic solutions today . BAC’s effect is largely due to its detergent properties, which destroy bacterial walls, membranes, and cytoplasmic layers.
However, long-term use of BAC-containing ophthalmic solution for glaucoma has been shown to cause dry eye diseases . In vitro experiments demonstrated that BAC induced apoptosis in conjunctival cells associated with inflammatory cytokines [12,13]. In vivo experiments reported that BAC causes the upregulation of inflammatory mediators and infiltration of inflammatory cells in the cornea [12-14].
Diospyros kaki has been cultivated throughout Eastern Asia for centuries and is traditionally used in herbal medicine for its beneficial properties, which include anti-atherosclerosis, anti-diabetes, anti-inflammatory, and anti-neurodegenerative effects [15-19]. D. kaki leaves contain an abundance of active constituents, including the most common groups of polyphenolic compounds, such as flavonoids, terpenoids, resins, polysaccharides, chlorophyll, carotene, and so on [20,21]. A previous study showed that certain flavonoids and terpenoids inhibit inflammatory mediators, which could be beneficial in inflammatory disease’s prevention and treatment . Recently, we reported that EEDK attenuates corneal neovascularization induced by alkali burn in vivo . Interestingly, it was also shown that EEDK was effective in ameliorating retinal degeneration in vitro and in vivo .
These results suggest that EEDK may be a potential candidate for the treatment of various ocular diseases. However, the beneficial effects of EEDK have not been assessed in a dry eye model. Therefore, the purpose of this study was to investigate the natural protective effects and possible mechanisms of EEDK in a BAC-induced mouse dry eye model.
Male BALB/c mice (6-weeks-old, 20–25 g; Central Lab. Animal Inc., Seoul, South Korea) were housed at 23±0.5 °C and 10% humidity in a 12 h:12 h light-dark cycle. All animals were acclimated at least 1 week, were caged, and were fed animal chow and water ad libitum.
All animal studies were performed in a pathogen-free barrier zone at the Korea Institute of Science and Technology (KIST) Gangneung Institute in accordance with the Association for Research and Vision in Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research guidelines. Procedures used in this study were approved by the Animal Care and Use Committee of KIST (No. 2014–011).
In June 2013, D. kaki leaves were collected from Gangneung in the Gangwon province of South Korea. A voucher specimen (D-521) has been deposited at the KIST Gangneung Institute. Dried D. kaki leaves were extracted in 95% ethanol twice and concentrated to dryness with rotary evaporation to obtain ethanol extract of D. kaki (EEDK) . For testing, EEDK was dissolved in distilled water.
BAC was used to induce dry eye in mice, as previously described [25-27]. Mice received 5 μl of 0.2% BAC (Sigma-Aldrich, St. Louis, MO) in sterile PBS (8 g/l, 0.2 g/l KCl, 1.44 g/l Na2HPO4, 0.24 g/l NaH2PO4, and pH 7.4) buffer, topically in the eyes, for 14 days, twice daily (10:00 AM and 10:00 PM). Control groups received an equal volume of physiologic saline.
Forty mice were randomly divided into five groups (n = 8 mice per group). 0.2% BAC was instilled alone in a dry eye (DE) groups. Various concentrations of EEDK (50 mg/kg or 10 mg/kg bodyweight) were administered daily by oral gavage for 14 days after BAC treatment. Control groups were administered an equal volume of distilled water without EEDK. Preservative-free eye drops (Refresh Plus® lubricant eye drops, Allergan, CA) were instilled in the positive-control group, 4 times daily, for 14 days.
Animals were anesthetized with an intraperitoneal injection of a mixture of 1.6 μg/g tiletamine hydrochloride/zolazepam hydrochloride (Zoletil; Virbac Laboratories, Fort Worth, TX) and 0.05 μl/g xylazine hydrochloride (Rompun, Newbury, UK).
To test the tear break-up time (BUT), 2% fluorescein was instilled on the conjunctival sac in anesthetized mice. Lids were closed manually three to four times; thereafter, with the eye held open, the BUT was recorded (in seconds). At the first appearance of dry spots, the stopwatch was stopped.
After the BUT score was recorded, corneal epithelial damage was graded using a blue filter under a slit-lamp microscope (Haag−Streit, Wedel, Germany). The cornea was divided into four quadrants, which were scored separately. Scores from these four regions were summed for a final grade (Total possible: 16 points). The stained area was graded using the 2007 Dry Eye Work Shop (DEWS)-recommended grading system, and grading was performed by two investigators in a blinded manner, as previously described  with essential modification, briefly, as follows: 0, absent; 1, slightly punctuate staining with fewer than 30 spots; 2, punctate staining with more than 30 spots but not diffuse; 3, severe, diffuse staining but no positive plaque; 4, positive for fluorescein plaque.
The tear volume was measured using a phenol red cotton thread  at a similar time of day (9:00 AM) on days 0, 7, and 14. The lower eyelid was pulled down gently, and a 1 mm portion of thread was placed at a specified point approximately one third of the distance from the lateral can thus for 15 s. The red portion of the thread was measured in millimeters. Each eye was tested three times, and the average length of the red portion was used for statistical analysis. After the test, the eyes were closed to avoid excessive exposure and irritation due to the dryness of the ocular surface.
Formalin-fixed eyes were embedded in paraffin and cut into 4 μm vertical slices. Sections were washed in distilled water for 5 min and treated with 0.1% hematoxylin buffer (Sigma-Aldrich) for 8 min at room temperature. Then, the sections were washed and dipped in 1% Eosin Y (Sigma-Aldrich) solution for 1 min. Slides were washed and mounted with mounting solution. Histological analyses of retinal tissues were evaluated under a light microscope (Olympus, Tokyo, Japan).
Corneal sections were deparaffinized and incubated in serum blocking solution to block non-specific binding for 2 h at room temperature. Sections were incubated with primary antibodies, viz., anti-cytokeratin-10, or anti-Ki-67 (Abcam, Cambridge, UK), at 4 °C overnight.
Immunofluorescent staining: Sections were washed and incubated with a secondary antibody, anti-rabbit Alexa 488 (Invitrogen, Carlsbad, CA), for 2 h at room temperature in the dark. The sections were washed, then mounted with mounting solution, and covered with a glass coverslip. The fluorescence intensity of the staining was examined using a Leica TCS SP5 confocal system (Leica, Wetzlar, Germany). Appropriate fluorescence filters (Leica) were used: 4′,6-diamidino-2-phenylindole (DAPI; ex 360/em 460) and Alexa Fluor 488 (ex 480/em 512).
Immunochemical staining: Sections were washed and incubated with a secondary antibody (Horseradish peroxidase [HRP]-conjugated goat anti-mouse immunoglobulin [IgG]; Santa Cruz Biotechnology, Santa Cruz, CA) for 2 h at room temperature in the dark by using the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA). The reaction products were developed with diaminobenzidine (DAB, Vector Laboratories, Burlingame, CA), and then mounted with mounting medium. Counterstaining with hematoxylin (Vector Laboratories) was performed with Ki-67 staining. The stained sections were examined under a light microscope (Olympus).
We used an in situ terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end-labeling (TUNEL) detection kit (Roche, Welwyn Garden, UK) to indicate apoptosis according to the manufacturer’s instructions. TUNEL-positive cells in the cornea epithelial layer were observed using a Leica TCS SP5 confocal system (Leica). The quantification of nuclei with fragmented DNA was performed by calculating the percentage of TUNEL-positive cells from ten random fields for each condition.
Corneal and conjunctival tissues were surgically removed under anesthesia and washed with cold PBS. Tissues were centrifuged at 14,000 ×g at 4 °C for 10 min and then homogenized using a modular homogenizer (CAT Scientific, Paso Robles, CA) in RIPA buffer (150 mM NaCl, 1.0% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS], 50 mM Tris, pH 8.0, 1× protease inhibitors, and 1 mM phenylmethylsulfonyl fluoride [PMSF]). Homogenates were centrifuged at 14,000 ×g for 20 min, and total protein concentrations were determined with the Bio-Rad Protein Assay kit (Bio-Rad Laboratories, Hercules, CA). Protein concentrations were evaluated based on the standard curve using bovine serum albumin (BSA).
Proteins were electrophoresed on SDS–polyacrylamide gel electrophoresis (PAGE), electroblotted on a polyvinylidene difluoride (PVDF) membrane (Bio-Rad), and blocked for 1 h at room temperature. The following primary antibodies were incubated at 4 °C overnight: anti-IL-1α (Biorbyt), anti-IL-1β (Abcam), anti-TNF-α (Abcam), anti-MCP-1 (Novus Biologicals), anti-IL-6 (Abcam), and anti-β-actin (loading control; Santa Cruz Biotechnology). The membrane was washed and then incubated with HRP-conjugated goat anti-rabbit or anti-mouse IgG (Santa Cruz Biotechnology) for 2 h at room temperature. After washing, membranes were developed with enhanced chemiluminescent (ECL) solution (Chemiluminescent Substrate; Thermo Scientific, Rockford, IL) band densities were evaluated using Multi Gauge 3.1 software (Fuji Photo Film, Tokyo, Japan).
All data are presented as the mean ± standard error of the mean (SEM). Statistical significance was assessed with a one-way ANOVA (ANOVA) followed by Dunnett’s test for multiple group comparisons. A p value of less than 0.5 was considered statistically significant.
We assessed the fluorescein staining score, BUT, and tear volume in BAC-induced corneal damage in mice (Figure 1). The corneal fluorescein staining scores in the control, positive control, and 50 mg/kg EEDK-treated groups were also significantly lower than for the DE group (Figure 1A,B). Similar results were obtained using the BUT. The mean BUT was lower in the DE group and 10 mg/kg EEDK-treated group at 14 days. However, the control, positive control, and 50 mg/kg EEDK-treated groups showed a higher BUT score than the DE group (Figure 1C). There was statistical significance in the BUT results, and the 50 mg/kg EEDK-treated group showed similar effects compared with the positive control.
Tear volume was measured on days 0, 7, and 14 with Schirmer’s test (Figure 1D). On day 7, the mean tear volume was lower in the mice in the DE group. The mean tear volumes in the 50 mg/kg EEDK-treated group and the positive-control group were similar but lower than in the control group on day 7. On day 14, the mean tear volumes of the control, positive control, and 50 mg/kg EEDK-treated groups were greater than those of the DE and 10 mg/kg EEDK-treated groups.
Corneal damage was also assessed with H&E staining. There were irregular corneal epithelial cells in the basal layer in the DE and 10 mg/kg EEDK-treated groups (Figure 1E, red circle and arrows). Thinning of the epithelial layer and epithelial surface irregularity were also observed in the DE (Figure 1E-b) and 10 mg/kg EEDK-treated groups (Figure 1E-d). However, the 50 mg/kg EEDK-treated group and the positive-control group were protected against irregularity of the ocular surface and thinning of the epithelial layer. Taken together, these data demonstrated that EEDK ameliorated corneal damage in the BAC-induced dry eye model.
We used western blot to examine the expression of inflammatory cytokines in corneal and conjunctival lysates of mice. Notably, compared with the controls, the expressions of IL-1α, IL-1β, TNF-α, MCP-1, and IL-6 were increased in the DE group (Figure 2). However, inflammatory cytokine expressions were significantly reduced in the 50 mg/kg EEDK-treated and positive-control groups (Figure 2). These results suggest that EEDK decreased the inflammatory reaction in the BAC-induced dry eye model, to the level of the positive-control group.
Inflammatory mediators lead to squamous metaplasia in which corneal epithelial cells are converted into keratinized cells. To evaluate squamous metaplasia, immunofluorescence staining with cytokeratin-10 (A squamous metaplasia marker) was performed. Expression of cytokeratin-10 was increased in the DE (Figure 3B) and 10 mg/kg EEDK-treated groups (Figure 3D), compared with the control group (Figure 3A). Expression of cytokeratin-10 in the 50 mg/kg EEDK-treated (Figure 3C) and positive-control groups (Figure 3E) was lower than that in the DE group.
Next, we asked whether EEDK might halt apoptosis in the cornea in the BAC-induced dry eye model. Supporting this hypothesis, we detected apoptotic cells with TUNEL assay, which stains the 3′-OH ends of fragmented DNA (Figure 4). The TUNEL assays revealed that the number of apoptotic cells was increased in the DE group compared with the control group (Figure 4A,B), whereas the 50 mg/kg EEDK-treated and positive-control groups had a reduced number of apoptotic cells in the corneal epithelium (Figure 4A,B).
Ki-67 is known to be present during active phases of the cell cycle (G1, S, G2, and mitosis) but absent in resting cells (G0) . We investigated the level of Ki-67 to determine the cellular proliferation in the corneal epithelium. Immunohistochemistry for Ki-67 revealed that few Ki-67-positive cells were detected in the control group (Figure 5A) and in the DE group (Figure 5B). However, the EEDK-treated group showed a higher number of Ki-67-positive cells (Figure 5C) in a dose-dependent manner. Ki-67-positive cells were found similarly in the positive-control group (Figure 5E) compared with the 50 mg/kg EEDK-treated group.
Dry eye is a common ocular surface condition, which has various causes. Many experimental and clinical studies have shown that exposure to topical drugs, including preservatives, may cause ocular surface changes [26,29-31].
BAC is a well-known preservative. Although BAC has beneficial properties for ophthalmic solutions, long-term use of BAC decreases the density of the tear film and corneal cells, because the preservative has toxic and proinflammatory effects in humans [32,33]. Corneal cells are particularly sensitive to toxic and inflammatory stress, as they participate in regulating tear film stability . We used a BAC-induced murine dry eye model that shares common pathologic features with human dry eye, such as epithelial apoptosis, tear film defects, and inflammation, which were documented in previous studies [10,27]. We investigated whether the ethanol extract of D. kaki (EEDK) was able to protect the cornea in the BAC-induced murine dry eye model. In this study, we showed that the application of EEDK led to an improvement in clinical signs of dry eye and a decreased inflammatory response on the ocular surface of the BAC-induced dry eye model.
The fluorescein staining scores were significantly lower in the 50 mg/kg EEDK-treated group than in the 10 mg/kg EEDK-treated and DE groups (Figure 1A,B). Our results showed that the 50 mg/kg EEDK-treated group had a similar effect on dry eye compared to the positive control, which significantly inhibited corneal damage, with signs of increased BUT (Figure 1C), improved tear volume (Figure 1D), and protected epithelium (Figure 1E) in the cornea. These results support that EEDK contributed to corneal healing in the BAC-induced dry eye model.
Inflammatory cytokines are expressed at higher levels under dry eye conditions, and their overexpression is correlated with a higher rate of cell death . Several proinflammatory cytokines can trigger downstream inflammatory pathways and result in recruitment of inflammatory cells . Thus, inhibiting inflammation has been considered one of the key factors in the treatment of dry eye .
In a previous study, we showed that EEDK suppressed corneal neovascularization (CoNV) by inhibiting cytokines, such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), IL-6, and matrix metallopeptidase-2 (MMP-2) . Our data revealed that treatment with 50 mg/kg EEDK inhibited upregulation of inflammatory cytokines, such as IL-1α, IL-1β, TNF-α, MCP-1, and IL-6, in the cornea and conjunctiva compared with the DE group (Figure 2A).
Squamous metaplasia is a hallmark of various ocular surface disorders, including chronic dry eye . The severity of dry eye and the degree of squamous metaplasia have been established in patients with dry eye conditions . Proinflammatory cytokines are critical in the formation of squamous metaplasia . Cytokeratin-10 expression in the corneal epithelial layer indicates that squamous metaplasia is present, in which epithelial cells are converted into keratinized cells in the cornea. In the DE group, cytokeratin-10 expression was upregulated compared to the control group (Figure 3B). However, less cytokeratin-10 expression was observed in the corneal epithelial layer in the 50 mg/kg EEDK-treated and positive-control groups (Figure 3C,E).
Inflammation also results in increased apoptosis, which has also been implicated in the pathogenesis of dry eye . Furthermore, apoptotic cells have been detected in the cornea and conjunctiva of dry eye animal models [40,41]. We found that treatment with BAC alone resulted in apoptotic cell death in the cornea and stromal layers. However, few apoptotic cells were detected in the positive-control and 50 mg/kg EEDK-treated groups (Figure 3).
Topical instillation of BAC caused ocular surface changes (Figure 1E); it resulted in irregularity, instability, and cracks in the ocular surface (Figure 1E). For these reasons, the negative effects of BAC may include direct damage, such as apoptosis in the endothelium and posterior stroma. A previous study also showed toxicity to the whole cornea in a dry eye murine model . Together, these results suggest that upregulated inflammatory cytokines, upregulated squamous metaplasia, and increased numbers of apoptotic cells were inhibited by treatment with EEDK (Figure 2, Figure 3, Figure 4).
Based on the Ki-67 cell proliferative index, abnormal proliferation in the conjunctiva has been reported in patients with aqueous-deficient dry eye and Sjögren’s syndrome . However, these results did not correlate with previous studies, in which epithelial cells showed decreased proliferative activity in dry eye conditions [12,28]. In the DE group, few Ki-67 -positive cells were detected (Figure 5B) compared to the control group (Figure 5A), whereas several Ki-67 -positive cells were detected in the 50 mg/kg EEDK-treated (Figure 5C) and positive-control groups (Figure 5E).
Ki-67 and DNA content can be used to determine cell cycle status. Ki-67 protein is expressed during the G1, S, G2, and M phases of the cell cycle but not in the quiescent G0 phase. Wu et al. showed that BAC induces DNA damage in human corneal epithelial cells , which ultimately leads to a decrease in DNA content and Ki-67 protein synthesis in corneal cells. However, flavonoids isolated from the leaves of D. kaki have been reported to alleviate oxidative stress-induced DNA damage and apoptosis . Overall, these results support the conclusion that treatment with EEDK contributed to proliferation of corneal epithelial cells in a BAC-induced dry eye model.
Based on these results, the presumed mechanisms of the underlying effects of D. kaki in the treatment of dry eye in this study include epithelium maintenance by promoting epithelium proliferation and apoptosis inhibition through anti-inflammatory and antioxidant effects.
The leaves of D. kaki have been used for various treatments, without reports of toxic effects, over the past hundred years . In the present study, daily oral administration of EEDK in mice did not result in any noticeable toxicity throughout the experiment. The leaves of D. kaki have also been approved by the Korea Food and Drug Administration (KFDA) for manufacturing of functional foods and medicines. However, our previous study showed that a high dose may elicit retinotoxicity . Although D. kaki might be considered a reliable material in common use, further studies are needed to elucidate the potential effects of D. kaki, such as drug−drug interactions, as well as its pharmacokinetics, and its pharmacodynamics.
We performed standardization of EEDK and isolated nine compounds, such as quercetin, kaempferol, and their glycoside-, galactoside-, or galloylated-derivatives in our previous study (Appendix 1) . Quercetin isolated from EEDK attenuates negative influences to the retina following N-methyl-N-nitrosourea (MNU)-induced retinal damage . However, it is difficult to identify which of these ingredients acted as the bioactive compound in the positive effects of EEDK on dry eye. In the future, studies should be conducted to determine the bioactive compounds of EEDK that are useful for dry eye diseases.
In summary, EEDK can benefit tear film maintenance under dry eye conditions. EEDK showed clinical improvements in BAC-induced mouse dry eye by stabilizing the tear film and maintaining the integrity of the epithelium, and was supported by histologic assessments. Our study indicated that EEDK has great potential to be a beneficial agent in the clinical treatment of dry eye.
Sang Hoon Jung (firstname.lastname@example.org) and Sung Jae Yang (email@example.com) are co-corresponding authors for this study. This work was funded by Gangneung Asan Hospital (Biomedical Research Center Promotion Fund, 2014-007).