A study on the effect of kelulut honey on the epithelial to mesenchymal transition of primary human epidermal keratinocyte in wound healing

Abstract

The use of honey in wound healing has been well-established. Despite the extensive clinical data collected, little is known regarding the cellular and molecular mechanism of honey effect in wound healing. Human dermal fibroblasts (HDF) and human dermal keratinocytes (HEK) is the two major cells in the human skin. HDF and HEK interact with each other to ensure successful skin regeneration process. Reepithelialisation process by HEK during wound healing resembles the epithelial to mesenchymal transition (EMT) process. Objective of this study can be divided into five phases. First, to elucidate the effect of Kelulut honey (KH) on the HDF viability and proliferation. Second, to establish in vitro model of transforming growth factor β1 (TGFβ1)-induced EMT in HEK. Third, to elucidate the effect of KH on the HEK viability and proliferation. Fourth, to investigate the effect of KH on TGFβ1-induced EMT in HEK. Finally, to investigate molecular interaction underlying KH effect on TGFβ1-induced EMT in HEK. Skin samples were collected from consented donors undergoing abdominoplasty or circumcision. HDF and HEK were isolated from the skin samples and cultured. MTT assay was used to investigate viability and proliferation of HDF and HEK. HDF cell cycle was evaluated using flowcytometry. Cell circularity, E-cadherin expression and vimentin expression were evaluated to indicate phenotypic changes in HEK. HEK were recorded under time-lapse imaging to evaluate its rate of healing, rate of individual migration, and directionality of migration. Molecular interaction was measured using phosphor-specific antibody against Smad 2/3 complex as well as antibody against Snail/Slug and E-cadherin. KH improved the viability of HDF at 0.024 µg/ml dose. The 0.024 µg/ml dose also maintains HDF proliferation, morphology, and cell cycle. TGFβ1-induced EMT model in HEK was achieved with 5 ng/ml TGFβ where decrease in cell circularity, upregulation of E-cadherin, downregulation of vimentin, increase in wound closure and increase in individual migration rate were observed. No changes in terms of directionality of migration was observed compared to the control. In HEK, KH maintained viability at low dose of 15 to 240 µg/ml. Cytotoxicity was detected at the KH dose of 15 600 µg/ml. Next, treatment of 15 µg/ml KH reverted the 5 ng/ml TGFβ1 induction in HEK when increase in cell circularity, downregulation of E-cadherin, decrease in wound closure and decrease in individual migration rate were observed. No changes in terms of vimentin expression was observed compared to the TGFβ1-induced HEK. Treatment of 15 µg/ml KH improved the directionality of migration compared to the control. KH or TGFβ1, alone or in combination, inhibit Smad 2/3 complex phosphorylation, upregulated Snail/Slug, and downregulated E-cadherin. These findings suggest that KH have positive effect on HDF viability and the EMT process in HEK during the reepithelialisation process of wound healing.