The Potential of Gelam Honey in Promoting the Proliferative Phase of Corneal Reepithelialization

Original Research

The Potential of Gelam Honey in Promoting the Proliferative Phase of Corneal Reepithelialization

Keywords

corneal epithelial cells

Gelam honey

phenotype

proliferation

November 2017

ISSN 1044-7946

Index Wounds 2017;29(11):327–332. Epub 2017 June 28

Abstract

Objective. The aim of this study is to investigate the potential benefits of Gelam honey (GH) in promoting proliferation of ex vivo corneal epithelial cells (CECs) and its effects on the phenotypical features. Materials and Methods. Corneal epithelial cells were isolated from the corneas of rabbits (n = 6). The optimal dose of GH for CEC proliferation in both basal medium (BM) and cornea medium (CM) was determined via MTT (3-[4, 5-dimethyl thiazolyl-2]-2, 5-diphenyl tetrazolium bromide) assay. Morphology, gene and protein expressions, and cell cycle analysis of CECs were evaluated via phase contrast microscopy, real-time polymerase chain reaction, immunocytochemistry, and flow cytometry, respectively. Results. Corneal epithelial cells cultured in 0.0015% GH-supplemented media (BM + 0.0015% GH; CM + 0.0015% GH) demonstrated optimal proliferative capacity with normal polygonal-shaped morphology. Gelam honey potentiates cytokeratin 3 (CK3) gene expression in accordance with the cytoplasmic CK3 protein expression while retaining normal cell cycle of CECs. Conclusion. Culture media treated with 0.0015% GH increased CEC proliferation while preserving its phenotypical features. This study demonstrated the potential development of GH-based topical treatment for superficial corneal injury.

Introduction

A superficial corneal wound or abrasion can lead to a cascade of changes as part of the healing process, including corneal epithelial cell (CEC) migration to the injured area, proliferation, and differentiation to form the multilayered epithelium.¹ Proliferation, as an essential phase in corneal reepithelialization, requires energy and growth factors to promote mitotic activities.²

Honey, a substance produced by bees via nectar foraging, contains a high concentration of energy-providing carbohydrates (glucose and fructose). About 5% of honey comprises components such as water, amino acids, enzymes, vitamins, and trace elements.^3-5 Numerous medicinal properties such as anti-inflammatory, antineoplastic, antioxidant, and antibacterial are attributed to honey.^6-12 The remedial effect of honey on skin injuries involves several processes, including promotion of cellular proliferation.¹³

Gelam honey (GH) is among the famous Malaysian local honey, obtained from the Apis mellifera beehive of wild Gelam (Melaleuca spp) trees.¹⁴ The honey contains the highest sugar content among monofloral honeys in addition to the substantial concentration of flavonoid and vitamins B1, B3, and C.¹⁵ Its high phenolic content is accredited to a number of therapeutic benefits such as antibacterial, anti-inflammatory, and skin healing properties.^16-19

The positive effect of GH on the proliferative capacity of corneal keratocytes was reported recently.²⁰ The present study investigates the proliferative effects of GH on ex vivo rabbit corneal epithelium via cell viability assay, morphological examination, gene and protein expression, and cell cycle analyses.

Materials and Methods

Preparation of GH
The GH irradiated at 25 kGy was obtained from the National Apiary Centre, Department of Agriculture, Johor, Malaysia and stored at room temperature (25°C to 30°C).^21,22

Isolation and culture of CECs
Corneas were isolated from 6 New Zealand white strain rabbits (n = 6) and immersed in dispase (a neutral protease used to dissociate epithelial and stromal layers of the cornea) solution 2 mg/mL (Gibco Invitrogen, Thermo Fisher Scientific, Waltham, MA) for 18 to 24 hours to facilitate separation of epithelium from the underlying stroma.²³ Then, the epithelium was trypsinized (0.05% trypsin-EDTA; Gibco Invitrogen, Thermo Fisher Scientific) to form a single-layer epithelial cell suspension. The CECs were centrifuged at 1200 rpm for 10 minutes. The obtained pellet was then washed with phosphate-buffered solution (PBS pH 7.2; Gibco Invitrogen, Thermo Fisher Scientific) prior to cell counting using hemocytometer and trypan blue dye (Gibco Invitrogen, Thermo Fisher Scientific). EpiLife (Cascade Biologics, Portland, OR), a cornea medium (CM) containing human corneal growth supplement (HCGS; Cascade Biologics), and 1% of antibiotic-antimycotic (Gibco Invitrogen, Thermo Fisher Scientific) were then transferred into tubes containing CEC, followed by cell seeding at the density of 1 x 10⁵cells/cm² in each well of a 6-well plate. Samples were incubated at 37°C, 5% CO₂, and 95% humidity, and the culture medium was changed every 48 hours. Once confluency was achieved, CECs were trypsinized with 0.05% trypsin-EDTA then culture expanded to passage 1.

Determination of GH optimal dose via MTT assay
The GH optimal dose for CEC proliferation was determined via MTT (3-[4, 5-dimethyl thiazolyl-2]-2, 5-diphenyl tetrazolium bromide) assay (Sigma-Aldrich, St Louis, MO). After trypsinization, the CECs were seeded in CM at a density of 5 x 10³ cells/cm² in each well of a 96-well plate (Cellstar, Wiesbaden, Germany). After 24 hours, the medium was replaced with basal medium (BM) and CM with serially diluted GH from 0% to 6.25%. After 48 hours, 10 µL of MTT solution was added into each well, incubated in the dark for 4 hours, and then solubilized with 100 µL of dimethyl sulfoxide (DMSO), producing purple formazan precipitate. The plates were then analyzed by the microplate reader (Thermo Fisher Scientific) to measure absorbance at 570 nm. The concentration that produced the highest level of cell proliferation was chosen as the optimal dose of GH and used in the subsequent tests for phenotypic evaluation.

Gene expression analysis
Gene expression of all experimental groups (BM, BM + GH, CM, and CM + GH) was performed via the real-time polymerase chain reaction (qRT-PCR) protocol. Ribonucleic acid (RNA) extraction from the sample was initially carried out by adding 1 mL of TRI Reagent Solution (Molecular Research Center Inc, Cincinnati, OH), and RNA Isolation Reagent (Molecular Research Center Inc) into the culture wells on day 3. Lysed CECs kept in 1.5-mL safe-lock microcentrifuge tubes (Eppendorf North America, Hauppauge, NY) were then treated with 200 µL of chloroform (Merck, Darmstadt, Germany) and 500 µL of isopropanol (Merck) to visualize the 3-layered protein-DNA-RNA. The clear RNA liquid layer was transferred into a fresh tube followed by the addition of 5 µL of PolyAcryl Carrier (Molecular Research Center Inc) to precipitate the RNA. The RNA pellet produced was then washed with 75% sterile ethanol (Merck), air dried for 25 minutes, and dissolved in 21 µL of ribonuclease- and deoxyribonuclease-free distilled water (Invitrogen, Thermo Fisher Scientific, Carlsbad, CA). The subsequent synthesis of complimentary-DNA (cDNA) was performed using cDNA SuperScript III First-Strand Synthesis Supermix (Thermo Fisher Scientific). The qRT-PCR, using SYBR Green qPCR Supermix for the iCycler kit (Thermo Fisher Scientific) on the CEC-specific marker and cytokeratin (CK3) with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the housekeeping gene, was performed. The primers for both genes were designed from National Institutes of Health GenBank (Bethesda, MD; eTable 1). The PCR products subsequently visualized via 2% agarose gel electrophoresis using 0.5 µm/mL ethidium bromide at 115 mV.

Immunocytochemistry
Immunocytochemistry staining for cytoplasmic CK3 detection of the CECs was performed using modified Abcam Dako immunostaining kit (Abcam, Cambridge, UK) protocol. Samples from all experimental groups were fixed with 4% paraformaldehyde (Sigma-Aldrich) for 3 days. The CECs were then treated with acetone (Merck) at 4°C for 5 minutes and incubated with blocking agent (3% hydrogen peroxide; Merck) for 5 minutes. Samples were then heated at 95ºC with trypsin-EDTA for 20 minutes for antigen retrieval, followed by 1:100 diluted anti-CK3 antibody (Abcam) incubation for 30 minutes. After washing with the Tris-buffered solution 0.1 M pH 7.2 three times, samples were then incubated with the secondary antibody, streptavidin-horseradish peroxidase, for 30 minutes and followed by 3, 3’-diaminobenzidine substrate for 7 minutes. Finally, the nuclei staining using Harris hematoxylin (Sigma-Aldrich) was performed with subsequent sample dehydration by soaking in increasing concentrations of ethanol and xylene prior to mounting. Examination of stained samples was done via confocal laser scanning microscopy (Axiovert S100; Carl Zeiss, Dublin, CA).

Cell cycle analysis
The CECs (5 x 10⁵ cells /cm²) in all experimental groups were stained with DNA CycleTest PLUS reagent kit (BD Biosciences, Singapore) and analyzed in flow cytometry BD FACSCalibur (BD Biosciences) according to manufacturer protocols. The software used for cell cycle analysis was Cellquest Pro Version 5.2.1 (BD Biosciences) and ModFit LT Version 3.0 (Verity Software House, Topsham, ME).

Statistical analysis
All data were analyzed using SPSS Version 21 (IBM Corp, Armonk, NY). Values were expressed as mean ± standard error of mean (SEM) using Student’s t-test. Statistical significant was defined as P ≤ .05.

Results

Determination of GH optimal dose via MTT assay
eFigure 1 demonstrates the results of cell proliferation assay for the determination of the GH optimal dose for CEC proliferation. In both BM and CM groups, the addition of GH concentrations ranging from 0.00009% to 1.65% into the culture media increased the CEC proliferation in comparison with the controls (P < .05) (eFigure 1A, 1B). In the BM group, the highest proliferation activities were observed between GH concentrations of 0.0003% and 0.0061% with no statistical significance between them (eFigure 1A). In the CM groups, CECs supplemented with 0.0015% GH showed the highest proliferation compared with CM-only media (eFigure 1B). In addition, CECs cultured in the CM groups demonstrated a higher proliferative capacity compared with the BM groups with all GH concentrations (eFigure 1C). A GH concentration of 0.0015% was chosen as the optimal GH dose and used in the subsequent tests.

Cell morphology assessment
eFigure 2 shows the morphological assessment of the effects of GH on CECs. Corneal epithelial cells cultured in the BM groups were small and arranged as solitary polygonal-shaped cells and did not reach confluence at day 3 of the culture period (eFigure 2A, 2B). Supplementation of 0.0015% GH to BM resulted in a higher density of cells in comparison with its control (eFigure 2B). Corneal epithelial cells in the CM groups were smaller in size, polygonal, and showed distinct cell borders (eFigure 2C, 2D). Cell confluency was achieved in the GH-supplemented CM group (eFigure 2D) with less cell density observed in its respective control (eFigure 2C). Proliferative capacity was higher in CECs cultured in the CM groups compared with the BM groups. No morphological abnormalities were observed in the GH-supplemented groups. These results corresponded with the MTT assay results.

Gene expression analysis
eFigure 3 shows CK3 expression of CECs cultured in 0.0015% GH-supplemented BM was significantly higher (P < .05) in comparison with its untreated BM group. A similar observation was seen in the CM group. In addition, CECs cultured in CM showed higher and significant CK3 proliferation (P < .05) in comparison with the BM group. Gel electrophoresis of CK3 for the PCR products showed specific size product of CK3 in all groups as depicted in eFigure 4.

Immunocytochemistry
The CECs showed positive cytoplasmic CK3-stained cells in all groups. The highest density of brownish precipitation was seen in CECs treated with 0.0015% GH as shown in eFigure 5.

Cell cycle analysis
eFigure 6 shows cell cycle graphs of CECs in all media groups where normal peaks with no evidence of aneuploidy are demonstrated. This signified that no chromosomal abnormalities were found on CECs treated with GH. Corneal epithelial cells cultured in BM and CM media with 0.0015% GH showed a lower percentage in the G₀ and G₁ (cell growth and preparation of DNA synthesis) phases as well as higher S phase (DNA replication) and G₂/M phase (cell growth and mitosis) compared with the controls as depicted in eTable 2. A higher percentage of CECs in S phase indicated higher mitotic activity, thus possessing the greater proliferative capacity.

Discussion

Superficial corneal wound healing is an essential physiological process to retain the protective function of epithelium and maintain normal vision.^24-27 Acceleration of cellular proliferation, which is the key initial phase of reepithelialization, potentially improves the rate of healing and prevents other complications such as superimposed infection and progression to deeper injury.^1,28 Prophylactic antibiotics are commonly prescribed; however, the development of resistance was implicated in cases requiring prolonged treatment.^28-30In addition, benzalkonium chloride, a substance used as the preservative in the eye drop preparations, evidently destabilized the corneal epithelial barrier with a series of other ophthalmological complications.^31-34

Facilitating high-energy delivery in the form of adenosine triphosphate to the actively proliferating cells subsequently improves reepithelialization.^35,36 In this ex vivo study, the investigators demonstrated that supplementation of GH to the culture media profoundly stimulates CEC proliferation. The GH comprised 50.45% glucose, 44.91% fructose, and 4.64% sucrose and possesses the ability to significantly supply energy to the target cells.³⁷ The obtained optimal GH dose of 0.0015% was in agreement with previous findings,³⁸ where a mild-to-moderate concentration of sugar-induced proliferation was observed. Reduction or inhibition of proliferation was seen in cells treated with overly concentrated sugar solutions.³⁹

The function of glucose oxidase in GH is to transform glucose into hydrogen peroxide, which is attributed to its antibacterial effect.^6,12 In addition, the little amount of water in honey was found to have an osmotic inhibitory effect towards bacterial activities.³⁹ Methylglyoxal and bee defensin-1 peptide found in GH were reported to promote its antibacterial potency.^{40, 41} In the present study, the investigators observed infrequent contaminations that were possibly due to this antibacterial property of the honey, thus providing a favorable environment for CECs to proliferate. Further ex vivo study on this aspect should be investigated.

Another important condition for upregulated cell proliferation is the presence of an increased concentration of growth factors in the healing tissue. This was evident in the study herein where CECs cultured in CM showed a significantly higher proliferative capacity in comparison with the BM groups with or without the addition of GH. Human corneal growth supplements used in this experiment contained bovine pituitary extract (BPE), which possesses similar growth effects such as transforming growth factor-β, platelet-derived growth factor, and epidermal growth factor. Its growth effects were reported to be 70 times higher than BPE-deficient media.^42-48 The potential of GH in promoting CEC proliferation in the absence of growth factors was seen in GH-supplemented BM culture, which showed higher proliferative capacity compared with the BM only. Deductively, the evidence of the synergistic effects of GH and HCGS in promoting CEC proliferation was apparent. This also raised the question of the possibility of growth-like substances present in GH.

Cytokeratin 3 paired with CK12 is a specific marker for CEC differentiation and important in maintaining CEC integrity.^49,50 It is highly expressed by CECs in central epithelium and suprabasal cells in the limbal area.^51,52 The increase in CK3 gene expression with GH supplementation corresponding to the increase of CEC proliferation showed preservation of CEC phenotypic characterization; this is visually supported by the photomicrograph assessment of cytoplasmic CK3 protein expression. In addition, the absence of morphological abnormalities on phase contrast microscopic examination and the preservation of the normal cell cycle analysis provide promising facts that no promutagenic effects were seen in GH-supplemented culture media.

Limitations

There are a couple limitations in this study. The action of GH on the corneal epithelial stem cell markers (namely CK 19, ABCG2, and Notch-1) were not measured. Under such condition, it is difficult to comment on whether the proliferative capacity of GH was due to the effects of growth factors in the honey preparation or due to its action in preserving the colony of corneal epithelial stem cell in vitro. Another limitation is that conjunctival epithelium was not excluded in the cultures.

Conclusions

This study demonstrated that GH has the potential to facilitate corneal reepithelialization by stimulating the cell proliferation process while maintaining its phenotype. Further ex vivo research with subsequent animal and clinical studies is cardinal in the development and manufacturing of the new therapeutic topical agent for corneal abrasion.

Acknowledgments

The authors would like to thank the staff of the Anatomy, Physiology, and Biochemistry Departments, Faculty of Medicine, Universiti Kebangsaan Malaysia Medical Centre for their technical assistance.

Affiliations: Department of Anatomy, Universiti Kebangsaan Malaysia Medical Centre, Bandar Tun Razak, Kuala Lumpur, Malaysia; Discipline of Anatomy, Faculty of Medicine, Universiti Teknologi MARA, Jalan Hospital, Selangor, Malaysia; Department of Ophthalmology, Universiti Kebangsaan Malaysia Medical Centre; and Department of Physiology, Universiti Kebangsaan Malaysia Medical Centre

Correspondence:
Norzana Abd Ghafar, PhD, MBChB
Department of Anatomy
Pre-Clinical Block,
Universiti Kebangsaan Malaysia Medical Centre
Jalan Yaacob Latif
Cheras, Kuala Lumpur,
56000 Malaysia
norzana@Ukm.Edu.My

Disclosure: This study was funded by Universiti Kebangsaan Malaysia research grant FF-2014-367.

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