Tualang Honey Improves Human Corneal Epithelial Progenitor Cell Migration and Cellular Resistance to Oxidative Stress In Vitro

Stem cells with enhanced resistance to oxidative stress after in vitro expansion have been shown to have improved engraftment and regenerative capacities. Such cells can be generated by preconditioning them with exposure to an antioxidant. In this study we evaluated the effects of Tualang honey (TH), an antioxidant-containing honey, on human corneal epithelial progenitor (HCEP) cells in culture. Cytotoxicity, gene expression, migration, and cellular resistance to oxidative stress were evaluated. Immunofluorescence staining revealed that HCEP cells were holoclonal and expressed epithelial stem cell marker p63 without corneal cytokeratin 3. Cell viability remained unchanged after cells were cultured with 0.004, 0.04, and 0.4% TH in the medium, but it was significantly reduced when the concentration was increased to 3.33%. Cell migration, tested using scratch migration assay, was significantly enhanced when cells were cultured with TH at 0.04% and 0.4%. We also found that TH has hydrogen peroxide (H2O2) scavenging ability, although a trace level of H2O2 was detected in the honey in its native form. Preconditioning HCEP cells with 0.4% TH for 48 h showed better survival following H2O2-induced oxidative stress at 50 µM than untreated group, with a significantly lower number of dead cells (15.3±0.4%) were observed compared to the untreated population (20.5±0.9%, p<0.01). Both TH and ascorbic acid improved HCEP viability following induction of 100 µM H2O2, but the benefit was greater with TH treatment than with ascorbic acid. However, no significant advantage was demonstrated using 5-hydroxymethyl-2-furancarboxaldehyde, a compound that was found abundant in TH using GC/MS analysis. This suggests that the cellular anti-oxidative capacity in HCEP cells was augmented by native TH and was attributed to its antioxidant properties. In conclusion, TH possesses antioxidant properties and can improve cell migration and cellular resistance to oxidative stress in HCEP cells in vitro.


Introduction
The cornea, which is a transparent and tough tissue that covers the anterior segment of the eye, is responsible for transmitting and reflecting light onto the retina. As the first layer of defence against external insults and oxidative insult from the sunlight, the corneal epithelium possesses mechanisms to maintain its cellular homeostasis. These mechanisms are orchestrated by a population of selfrenewing stem cells that reside in the basal limbal epithelium, which is a niche area situated at the peripheral edge of the cornea called the limbus [1]. These cells express keratinocyte stem cell markers (p63, EGFR, K19, ABCG2 and integrin b1) and exhibit low expression of corneal differentiation markers (K3, involucrin, and connexin-43) [2], and they are responsible for sustaining the clarity and integrity of the corneal epithelium required for normal vision. Complete depletion or dysfunction of the corneal epithelial stem cells that occurs in some severe ocular surface abnormalities as a result of caustic injuries such as burns, inflammatory conditions, or hereditary genetic disorders such as aniridia [3,4] will lead to conjunctivalisation, keratinisation, and opacification and result in impaired vision or blindness. Under these circumstances, visual acuity can only be restored by transplanting corneal epithelial stem cells onto the injured cornea. To minimise the risk of immune rejection, autologous stem cell transplantation may be the only option. Hence, culturing corneal epithelial stem cells and preserving their stemness and functions in vitro are pivotal for ensuring successful regeneration following transplantation.
Reactive oxygen species (ROS) are common metabolic byproducts of aerobic metabolism, and their level is maintained through intrinsic antioxidant mechanisms in healthy cells. When maintained at the appropriate physiological level, ROS are vital in modulating several cellular signalling pathways that affect cell growth and function, including the phosphoinositide 3-kinase (PI3K) [5] and mitogen-activated protein kinase (MAPK) pathways [6]. In addition, ROS have the capability to dictate stem cell fate at physiological levels [7][8][9]. However, abnormal redox homeostasis involving ROS overproduction can induce oxidative stress, a physiological condition that renders cells susceptible to damage. Studies have confirmed that overproduction of ROS can compromise genomic stability [10] and trigger mutations and promote cancer growth [11]. High ROS levels also contribute to poor cell engraftment and viability, which impede regeneration after transplantation [12]. Although stem cells have greater antioxidant capacity compared to differentiated cells [13,14], they can exhibit telomere shortening-induced replicative senescence and reduced self-renewal capability under oxidative stress [15]. Hence, protecting stem cells from oxidative damage may help to promote cell survival, homing, and regeneration after transplantation. This protection could be achieved by maintaining a reduced environment at the site of transplantation through adjunctive therapy with dietary antioxidant [16] or by adding an antioxidant supplement to cells during in vitro expansion prior to transplantation. The efficacy of the latter strategy is supported by studies showing the potential for supplemental antioxidant in the culture medium to enhance the intracellular antioxidant activity of stem cells [15], prevent cellular damage, and salvage cultureinduced loss of stemness [17].
Tualang honey is a medicinal honey that is collected from the honeycomb of Apis dorsata, the bees that built their hives on the Tualang tree (Koompassia excels) that is common in tropical forests in Malaysia. This honey has been extensively studied and has been found to have therapeutic benefits in treating various medical conditions, including protecting post-menopausal bone structure [18], promoting burn wound contraction with antibacterial effects [19,20], enhancing post-tonsillectomy healing [21], and inhibiting the growth of various cancer cells [22][23][24]. Generally, honey has been traditionally used to treat eye-related diseases [25,26]. Uwaydat et al. (2011) recently showed that raw honey accelerated healing of corneal abrasions and attenuated the inflammatory response and angiogenesis in endotoxin-induced keratitis [27]. Similar to other types of honey, previous studies of Tualang honey have also described its anti-inflammatory and antioxidant properties in treating alkali-induced eye injury in vivo [28] and its ability to save keratinocytes from inflammation and DNA damage as a result of ultraviolet radiation in vitro [29].
Although many in vivo studies of the effects of Tualang honey have been conducted, the potential for using Tualang honey in the cultivation of stem cells has not been investigated. To date, only one study described the use of Tualang honey to supplement the culture medium when cultivating a human osteoblast cell line (CRL1543) [30]. Although many studies have shown the therapeutic benefits of Tualang honey in treating cornea injury [28,29], its effects on corneal epithelial stem cells have yet to be evaluated. Herein we characterised the effects of Tualang honey on cytotoxicity, gene expression, and migration of human corneal epithelial progenitor (HCEP) cells and assessed its potential for improving cell resistance to oxidative stress.

HCEP cell culture and expansion
HCEP cells were purchased from Gibco (Invitrogen Life Technologies Co., Carlsbad, CA, US) and ATCC (Manassas, VA, US). Cells were expanded in standard keratinocyte serum-free medium (KSFM, Gibco) that was supplemented with 5 ng/ml recombinant epidermal growth factor (rEGF) and 50 mg/ml bovine pituitary tissue extracts (Invitrogen Life Technologies Co., Carlsbad, CA, US). Passage 2-5 HCEP cells were used in all of the experiments.

Preparation of Tualang honey
Tualang honey used in this experiment was from Federal Agriculture Marketing Authorities of Malaysia (FAMA) and was a gift from Professor Siti Amrah Sulaiman, Universiti Sains Malaysia. Tualang honey was diluted to 20% in serum-free DMEM/F12 (Gibco, Invitrogen Life Technologies Co., Carlsbad, CA, US) and filtered through a 0.2 mm syringe filter (Pall Co., Port Washington, NY, US) prior to use in cell culture. Filtered Tualang honey was further diluted in KSFM according to the dilution factor described in Table 1. Immunofluorescence labelling HCEP cells were seeded onto Lab-Tek8 well-chamber slides (ThermoSci., Logan, UT, USA) using honey supplemented medium. Cells were fixed with 4% paraformaldehyde at 4uC for 10 min and then permeabilised with phosphate buffered saline containing 0.1% Triton-X and 0.1% Tween-20 (all from Sigma, St Louis, MO, USA). Cell samples were washed with PBS and blocked with 10% goat serum (Cedarlane Lab, Ontario, Canada) for 1 h. The cells then were incubated with rabbit anti-p63 (Santa Cruz Biotechnology, Heidelberg, Germany) and mouse antikeratin 3 (Clone AE5, Millipore, Billerica, MA, USA) primary antibodies overnight at 4uC. Samples were washed thrice with PBS and incubated with AlexaFluor488 goat anti-rabbit secondary antibody at 37uC for 2 h. Samples were then counterstained with DAPI and imaged using a confocal microscope (Olympus, Japan).

RNA extraction and cDNA synthesis
The total RNA was isolated using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The isolated RNA was treated with DNase I (Sigma, St. Louis, MO, USA) to ensure the absence of genomic DNA contamination. Total RNA samples were quantitatively and qualitatively assessed using UV-spectrophotometric measurement and agarose gel electrophoresis, respectively. The cDNA was synthesised using the QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany) according to manufacturer's protocol.

Primer design
Full-length exon spanning gene sequences were obtained from the NCBI GenBank Database (http://www.ncbi.nlm.nih.gov/ genbank/). All primers were designed from the gene sequences using Primer-BLAST online software (NCBI, NIH, USA). All primers are listed in Table 2. The sequences of the primers were compared to the GenBank Database using BLAST in order to  determine their specificity. Primers that could have resulted in non-specific signals were excluded. All primers were purchased from BioBasic Inc. (Ontario, Canada).

Polymerase chain reactions (PCR)
PCR amplification was performed using the MyCycler Thermal Cycler (Bio-Rad, Hercules, CA, USA). All PCR reagents used in this study were purchased from Biotools B&M Labs (S.A., Madrid, Spain). Total cDNA used in each reaction was 1 mL unless specified otherwise. Reaction buffer (1 X), 1.5 mM MgCl 2 , 1.75 mM of each dNTP, 0.5 mM of each primer, 100 ng of cDNA, and 1 U of DNA Taq Polymerase were added to all samples to reach a final volume of 20 ml in each reaction. A negative control (sample without template) was also included. PCR was performed at 95uC for 2 min as initial denaturation followed by 35 cycles of 30 sec at 95uC, 30 sec at 60uC, and 30 sec at 72uC, and then at 72uC for another 2 min. The PCR products were loaded with gel loading dye (Promega, Madison, WI, USA) onto 4% agarose gel in 1X TAE buffer and electrophoresed for 60 min at 80 V. A 25 base pair molecular ladder (Promega, Madison, WI, USA) was used. Subsequently, the gel was stained with ethidium bromide (Bio-Rad, Hercules, CA, USA) to visualize the presence of PCR amplicons.

Cytotoxicity assay
HCEP cells were seeded at 5000 cells/well onto a 96-well plate and left in an incubator with 5% CO 2 at 37uC overnight. The culture medium then was changed to test medium supplemented with Tualang honey at various concentrations (Table 1), and the cells were incubated for 48 h. Cell viability was tested using AlamarBlue (Molecular Probes, Invitrogen Life Technologies Co., Carlsbad, CA, USA) according to the manufacturer's protocol. After 3 h of incubation with AlamarBlue, the fluorescence intensity of each sample was read using a FLUOstar Omega multi-mode microplate reader (BMG Labtech, Germany) with an excitation wavelength of 570 nm and an emission wavelength of 585 nm. Controls were HCEP cells in regular medium without Tualang honey. Cell viability was expressed as a percentage using the following formula: Where FI treated = Fluorescence intensity of test samples (Tualang honey treated HCEP cells) FI non-treated = Fluorescence intensity of control samples (nontreated HCEP cells) Scratch migration assay HCEP cells were seeded onto a 6-well plate and allowed to expand and reach 90% confluence. A scratch was made onto each HCEP cell-containing well using a 200 ml pipette tip. The culture medium then was changed to standard KSFM supplemented with 0.004, 0.04, 0.4, or 3.33% (v/v) Tualang honey, and the cells were incubated at 37uC for 2 days. Ten representative images were taken using a charge-coupled device camera under an inverted light microscope pre-and post-treatment. Images obtained were analysed for wound closure using ImageJ software (NIH). All experimental samples were compared to a control (i.e., HCEP cells without Tualang honey treatment). The percentage of closure of the gap area was calculated using the following formula:

Gas Chromatography-Mass Spectrometry (GC-MS) analysis
GC-MS analysis of the honey was conducted using a Shimadzu system (Japan) consisting of a GC-MS-QP2010 gas chromatograph and a quadrupole mass spectrometer. The interface and source temperatures were 280uC and 250uC, respectively. Electron impact mass spectra were recorded in the 20-650 amu range at 70 eV ionization energy. Separation was performed on a fused-silica bonded-phase capillary column BP X5 (30 mx0.25 mm ID and 0.25 mm film thickness). The injector temperature was set at 250uC; and the samples were run in split mode with the ratio being adjusted to 25:1 and an injection volume of 1 mL. The temperature program was isothermal at 35uC for 1 min, then it was raised to 280uC at 25uC/min and to 310uC at 10uC/min. This temperature was held for 2 min. Samples in the chromatograms were identified by comparing their mass spectra with NIST08 library data as well as the retention times against known standards.

Statistical analysis
All experiments were repeated three times and data were expressed as mean 6 standard error (SEM). All statistical analyses were performed using Graphpad Prism (La Jolla, CA, USA). The differences between groups were analysed using one-way ANOVA with Dunnett post-hoc test. Differences were considered significant at p,0.05.

Cultured HCEP cells possessed stem cell characteristics
The cultured HCEP cells were highly proliferative and capable of forming holoclones. Immunofluorescence staining confirmed that the cultured HCEP cells used in this study expressed nuclear p63 but not cytokeratin 3/12 (Figure 1), suggesting a keratinocyte stem cell phenotype. However, the cultured HCEP cells exhibited signs of replicative senescence as soon as they reached passage 5 or 6.

Low levels of Tualang honey favoured HCEP cell viability, gene expression, and migration
Viability of HCEP cells was assessed after 24 h and no sign of cytotoxicity was observed in groups treated with 0.004, 0.04, or 0.4%Tualang honey ( Figure 2). However, cell viability was significantly reduced to 63.162.1% at the concentration of 3.33% compared to the untreated control (p,0.001). We also sought to investigate whether Tualang honey could affect HCEP cell stemness or trigger corneal epithelial differentiation. To do this, the mRNA level of ATP-binding cassette transporter 2 (abcg2), a marker which was found to express in human corneal epithelial stem cells, and corneal epithelial differentiation marker connexin-43 and cytokeratin-12, were examined. RTPCR revealed that the mRNA expression for connexin-43 and abcg2 were detected in all groups, but expression of cytokeratin-12 was not ( Figure 3A). Semi-quantitative analysis of the expressed mRNA revealed that connexin-43 mRNA remained unaffected by Tualang honey in all groups, which also indicates that Tualang honey had minimal or no potential in triggering corneal epithelial differentiation. However, a significant down-regulation of abcg2 mRNA was found in HCEP cells treated with 3.33% Tualang honey; this represents a decrease of 47% when compared with the expression in untreated cells (p,0.05) ( Figure 3B). These results suggest that Tualang honey did not affect the HCEP stemness and differentiation at non-cytotoxic level.
The in vitro scratch migration assay showed that the percentage of gap area covered by migrated HCEP cells after 48 h of treatment increased in a dose-dependent manner (Figure 4). HCEP cells treated with 0.04 and 0.4% Tualang honey occupied 17.360.9% (p,0.05) and 2061.5% (p,0.01) of the gap area, respectively, compared to 1266% in the untreated group. In the 3.33% Tualang honey treatment, only 13.360.3% of the gap area was occupied after 48 h.
Tualang honey scavenged H 2 O 2 in a dose-dependent manner and enhanced HCEP cell resistance to oxidative stress When Tualang honey was tested for its H 2 O 2 scavenging ability, the level of inhibition was found to increase with increasing Tualang honey concentration ( Figure 5A). This result suggests that Tualang honey is a source of anti-H 2 O 2 antioxidant. Notably, only negligible scavenging effects were observed at 30 and 40 mM H 2 O 2 when the Tualang honey was diluted to 0.04%. This indicates that over-dilution of Tualang honey may compromise its H 2 O 2 scavenging ability. To identify the cytotoxicity of H 2 O 2 , HCEPs were exposed to 10, 20, 50, 100 and 200 H 2 O 2 and the viability was assessed after 24 h using AlamarBlue assay. As shown in Figure 5B, HCEP viability was reduced by H 2 O 2 in a dosedependent manner, and the difference became apparent and statistically significant when H 2 O 2 level was increased to $50 mM (p,0.01).
This study was extended to investigate the potential for Tualang honey to improve HCEP cell resistance to H 2 O 2 -induced oxidative stress. Interestingly, HCEP cells treated with 0.4% honey had a higher survival rate than the untreated HCEP cells, with a significantly lower number of dead cells (15.360.4%) compared to the control (20.560.9%, p,0.01) ( Figure 5C).
The improvement in HCEP resistance to H 2 O 2-induced oxidative stress was attributed to the antioxidant properties of Tualang honey at its native form To test whether the observed improvement in HCEP resistance to H 2 O 2 -induced oxidative stress following the treatment with Tualang honey was attributed to 5-hydroxymethyl-2-furancarboxaldehyde (5HMF), the viability of HCEPs treated with 100 mM of 5HMF for 48 h were examined with AlamarBlue after exposed to 50 and 100 mM H 2 O 2 for 24 h. As shown in Figure 7, HCEP viability was significantly decreased after treated with 5HMF for 48 h compared to the untreated group (p,0.05). However, the viability became comparable to that of untreated control after 24 h induction of H 2 O 2 at 50 and 100 mM. This indicates that although 5HMF had little effects in improving HCEP resistance to H 2 O 2 , the benefit was nullified by its mild cytotoxicity.   To assess whether the antioxidant property of Tualang honey was responsible for the elevated resistance to H 2 O 2 -induced oxidative stress, HCEPs treated with 100 mM ascorbic acid, a known powerful antioxidant, was included as the positive control. AlamarBlue assay showed better HCEP survival following H 2 O 2 insults at 50 mM and 100 mM after treated with ascorbic acid or Tualang honey as compared to the untreated group. More importantly, the resistance to 100 mM H 2 O 2 was found significantly greater in Tualang honey-treated HCEP cells than that of ascorbic acid-treated ( Figure 7C). Taken together, these results confirm that the enhanced HCEP resistance to oxidative stress is conferred by the antioxidant property of Tualang honey in its native form.

Discussion
Tualang honey has been used to treat bacterial infections [33], promote burn wound healing [19,34], and protect bone in postmenopausal women [18]. However, its potential as a supplement in cell culture medium for stem cell expansion has not been studied, and little is known about the effects of honey on cells at the cellular level. This is the first study to evaluate the use of Tualang honey in culturing HCEP cells and its effects in enhancing cell function in vitro. Thus, it is a pivotal study for exploring the potential of this honey and its application in corneal regeneration.
The ATP binding cassette transport 2 (abcg2) is known for its capability to efflux Hoechst 33342 and has been used as a marker to identify side population cells [35]. This marker was also found to present in clonogenic human limbal-derived epithelial stem cells [36]. A study by Kubota et al. (2010) suggested that abcg2 has a vital role in maintaining endogenous anti-oxidative capacity in HCEPs [37]. Here, the cultured human corneal epithelial cells used in this study expressed mRNA for abcg2 and nuclear p63 protein, the putative corneal epithelial stem cell markers [38,39]. However, significant downregulation of abcg2 mRNA was detected when Tualang honey was introduced to the cells at cytotoxic level, 3.33%. This may also suggest that the cytotoxicity of Tualang honey in HCEP cells is via the downregulation of abcg2 expression, reduces the cell anti-oxidative capacity which renders the cells susceptible to oxidative damage. Although no terminal corneal epithelial differentiation was detected (based on the lack of cytokeratin 3 protein and cytokeratin 12 mRNA expression), we found that the cells constantly expressed gap junction protein connexin-43 mRNA, a negative marker for corneal epithelial stem cells [40]. This suggests that the cells had partial commitment to the corneal epithelial lineage. Hence, in order to accurately describe the cell population, we called them human corneal epithelial progenitor cells (i.e., HCEPs).
Previous studies showed that the bactericidal effects of honey were partly, if not entirely, due to its acidity, high osmolality, and H 2 O 2 content [41,42]. These characteristics could also be cytotoxic to cells. We addressed the cytotoxicity of Tualang honey to cultured HCEP cells by lowering the concentration to a level that favoured cell growth (#0.4%). Our data show that the pH of the culture medium was not altered by Tualang honey at low concentration (0.004-0.4%), but the medium became acidic at high concentration (3.33%) (Table 4). Furthermore, a high level of honey supplementation could produce a hyperosmotic culture medium, which could induce ROS generation and apoptosis in corneal epithelial cells [43]. In contrast to our finding, Ghashm et al. (2010) reported that the pH of the culture medium remained suitable for cell culture after adding Tualang honey at high concentrations (3.5-20%) compared to the control medium without Tualang honey [23]. This discrepancy may be due to differences in preparation and storage conditions of the Tualang honey used in the experiments, as storage conditions can alter the content and properties of the honey [44].
We also found that Tualang honey diluted to 0.04% did not affect gene expression and was favourable to HCEP cells, as shown by the enhanced cell migration in vitro in this treatment. This result suggests that dilution of Tualang honey does not hamper its beneficial effects on HCEP cells. These data coincide with a past study that showed improvement in proliferation of human osteoblast cells (CRL 1543) when the culture medium was supplemented with 0.0195% Tualang honey [30]. The underlying mechanism for the observed improvements is unknown, but it could be related to the active compounds present in Tualang honey or to the presence of H 2 O 2 , a ROS that is essential for modulating stem cell behaviours at the physiological level [45][46][47]. Pan et al. (2011) recently suggested that low levels of H 2 O 2 (10-50 mM) could promote rabbit corneal epithelial cell attachment, mobility, and wound repair [48]. This contradicts our findings using human cells that H 2 O 2 showed significant cytotoxicity at 50 mM and no noticeable changes in cell number at 10 and 20 mM when compared with the untreated control. Nonetheless, this discrepancy may due to the difference in the species of origin of corneal epithelial cells used in the experiments.
Furthermore, we also found that H 2 O 2 was present in Tualang honey, but only at a negligible level of ,2 mM after dilution to # 0.4%. This finding suggests that the H 2 O 2 level may not be sufficient to exert significant changes in cell behaviours. Although H 2 O 2 is present in naturally occurring Tualang honey, it also contains several phytochemical compounds with antioxidant properties that are capable of mitigating H 2 O 2 . The predominant phytocompound identified in Tualang honey was 5-hydroxymethyl-2-furancarboxaldehyde, which is a five carbon ring aromatic aldehyde antioxidant that is commonly found in fruits [49] and marine products [50]. Compared to other types of honey, Tualang honey had been identified as a new source of antioxidant with superior activity that is attributed to its high phenolic content [51].
Oxidative stress has been shown to cause chromosomal instability, shortened telomeres, and cellular replicative senescence in stem cells in vitro [52,53], all of which hamper cell function and limit the therapeutic outcome of cell transplantation. Accumulating evidence supports the premise that cellular antioxidant levels in stem cells play a vital role in dictating their fate, regenerative capability, and therapeutic outcome after transplantation [54,55]. Overexpression of cellular redox regulators such as Cu/Zn superoxide dismutase enzymes and nuclear factor erythroid 2- related factor-2 (Nrf-2) has been shown to improve stem cell functions [56]. The oxidative stress tolerance of stem cells can also be augmented by the addition of exogenous catalase [57] or antioxidants from natural products [16,58,59]. Herein, Tualang honey at 0.4% was found to improve HCEP cell resistance to oxidative stress, as shown by the significantly lower number of dead cells after treatment with 50 mM H 2 O 2 in the treatment versus control group. This indicates that Tualang honey can be used as a source of antioxidant for preconditioning HCEP cells prior to transplantation. However, more evidence is needed to confirm the changes in HCEP cell antioxidant enzyme levels that likely account for the Tualang honey-induced enhancement of the oxidative stress tolerance of HCEP cells.
In summary, this study describes a novel approach to integrating the application of natural products to the process of corneal regeneration. Our data indicate that Tualang honey is an antioxidant and contains active phytocompounds that enhance HCEP cell migration and resistance to oxidative stress in vitro in a dose-dependent manner. The beneficial effects, however, are offset by the cytotoxicity of Tualang honey at high concentration. Indepth studies to isolate and identify the active components in Tualang honey and to identify the mechanism responsible for the observed benefits in vitro and in vivo are needed to enable use of this natural product for the treatment of corneal diseases.