Tualang honey improves human corneal epithelial progenitor cell migration and cellular resistance to oxidative stress in vitro

Abstract

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.

Figure 1. Representative confocal images of immunofluorescence-labelled HCEP cells.

HCEP cells did not express the cornea-specific marker cytokeratin 3 (green) (A) but did express nuclear p63 transcription factor (green) (B). HCEP cells stained with AlexaFluor488 secondary antibody without any primary antibody were served as the negative control (C). Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole.

Figure 2. HCEP cell viability after treatment with 0, 0.004, 0.04, 0.4, and 3.33% Tualang honey for 48 h.

Significant lower viability was observed in HCEP cells treated with 3.33% Tualang honey compared to the other treatments. ***p<0.001.

Figure 3. mRNA expression of Tualang honey treated HCEP cells.

(A) RTPCR showed abcg and connexion-43 mRNA expressions (normalised to β-actin expression) but not cytokeratin-12 expression in HCEP cells. (B) abcg mRNA expression was down-regulated in HCEP cells treated with 3.33% Tualang honey, but the mRNA for connexin-43 remained unchanged in all groups. *p<0.05 compared to untreated control (0%). Abbreviations: abcg, ATP-binding cassette transporter; Cx-43, connexion-43.

Figure 4. HCEP cell migration following treatment with Tualang honey.

(A) Representative images of the HCEP cell scratch migration assay at baseline and after 48 h. (B) The gap size occupied by HCEP cells after 48 h was largest in the 0.4% Tualang honey treatment (*p<0.05; **p<0.01 compared to untreated control).

Figure 5. Effects of Tualang honey on hydrogen peroxide and HCEP cell resistance to oxidative stress.

(A) In vitro inhibitory effects of Tualang honey at 0.04, 0.4, and 4% (v/v) against 5, 10, 20, 30, and 40 µM H₂O₂. (B) HCEP viability in response to H₂O₂ induction at 0, 10, 20, 50, 100 and 200 µM after 24 hours using AlamarBlue assay. (C) Flow cytometric analysis showed the total number of PI-stained dead cells in Tualang honey treated HCEP cells in response to exposure to 50 µM H₂O₂ after 24 h (**p<0.01 compared to untreated control). Abbreviation: PI, propidium iodide.

Figure 6. GC-MS analysis of Tualang honey.

(A) Region A in the chromatogram of 20% Tualang honey diluted in distilled water. (B) Region A was enlarged and analysed further to identify the phytochemical constituents in Tualang Honey based on the NIST08 Mass Spectral Library.

Figure 7. AlamarBlue cell viability assay of Tualang honey (0.4%), ascorbic acid (100 µM) and 5HMF(100 µM) treated HCEP cells after H₂O₂ induction.

The resistance of treated cells against H₂O₂ was tested at (A) 0, (B) 50 and (C) 100 µM for 24 h. Significant difference in viability was found between Tualang honey and ascorbic acid-treated group using ANOVA with Tukey multiple comparisons test (*p<0.05; ***p<0.01 compared to untreated control). Abbreviation: 5HMF, 5-hydroxymethyl-2-furancarboxaldehyde.