Approaches in Hydroxytyrosol Supplementation on Epithelial-Mesenchymal Transition in TGFβ1-Induced Human Respiratory Epithelial Cells

Abstract

Hydroxytyrosol (HT) is an olive polyphenol with anti-inflammatory and antioxidant properties. This study aimed to investigate the effect of HT treatment on epithelial-mesenchymal transition (EMT) in primary human respiratory epithelial cells (RECs) isolated from human nasal turbinate. HT dose-response study and growth kinetic study on RECs was performed. Several approaches on HT treatment and TGFβ1 induction with varying durations and methods was studied. RECs morphology and migration ability were evaluated. Vimentin and E-cadherin immunofluorescence staining and Western blotting [E-cadherin, vimentin, SNAIL/SLUG, AKT, phosphorylated (p)AKT, SMAD2/3 and pSMAD2/3] were performed after 72-h treatment. In silico analysis (molecular docking) of HT was performed to evaluate the potential of HT to bind with the TGFβ receptor. The viability of the HT-treated RECs was concentration-dependent, where the median effective concentration (EC₅₀) was 19.04 μg/mL. Testing of the effects of 1 and 10 µg/mL HT revealed that HT suppressed expression of the protein markers vimentin and SNAIL/SLUG while preserving E-cadherin protein expression. Supplementation with HT protected against SMAD and AKT pathway activation in the TGFβ1-induced RECs. Furthermore, HT demonstrated the potential to bind with ALK5 (a TGFβ receptor component) in comparison to oleuropein. TGFβ1-induced EMT in RECs and HT exerted a positive effect in modulating the effects of EMT.

Keywords: EMT; fibrosis; inflammation; olive; rhinosinusitis.

Figure 1

Dose-dependent effect of HT on RECs. RECs were treated with HT for 24 h, and cell viability was analyzed via MTT assay. (A) MTT assay of HT cytotoxicity. * p < 0.05: significantly different from control (0 μg/mL) (Supplementary Material Table S1). (B) Morphology of RECs treated with HT for 24 h. Scale bar represent 100 µm.

Figure 2

Long-term effect of HT supplementation on RECs. (A) Total number of cells attached vs. HT concentration. (B). Proliferation rate. * p < 0.05: significantly different from control (Supplementary Material Table S2).

Figure 3

Protein expression levels of TGFβ1- and HT-treated RECs. (A) E-cadherin and vimentin co-expression levels (%) vs. control. (B) E-cadherin expression (%) vs. control. (C) Vimentin expression (%) vs. control. * p < 0.05: significantly different from control. # p < 0.05: significantly different from Group T (Supplementary Material Table S3). (D) Immunostaining of vimentin and E-cadherin expression levels at 72 h. Green cytoplasm indicates E-cadherin expression level, red indicates vimentin, and blue is DAPI-stained nuclei. Scale bar represent 100 µm.

Figure 4

(A) REC morphology following culture in the six conditions. (B) Cell circularity. (C) Cell elongation. (D) Cell surface area. (E) Cell perimeter. * p < 0.05: significantly different from control. # p < 0.05: significantly different from Group T (Supplementary Material Table S4). Scale bar represent 100 µm.

Figure 5

(A) Migration of RECs that had been cultured in six conditions. (B) Cell migration rate. (C) Scratch closure percentage. * p < 0.05: significantly different from control. # p < 0.05: significantly different from Group T (Supplementary Material Table S5). Scale bar represent 100 µm.

Figure 6

Densitometry analysis of tested protein markers to observe the effects of HT and TGFβ1 on RECs. All Western blots were performed using a conventional Western blot system except for AKT and SMAD2/3, which were analyzed with the Jess system (A–H). E-cadherin (B), Vimentin (C), Snail-Slug (D), pAKT (F), pSMAD2 (G) and pSMAD3 (H). Solid line: Untreated REC. Dashed line: TGFβ1-induced REC. * p < 0.05: significantly different from control. # p < 0.05: significantly different from Group T (Supplementary Material Table S6).

Figure 7

Molecular docking studies show ligand binding in the active pockets of ALK5 receptors. (A) HT acetate. (B) HT. and (C) Tyrosol. The dark blue line indicates the hydrogen bond, the dashed line indicates the hydrophobic interaction. Molecular docking studies show ligand binding in the active pockets of ALK5 receptors. (D) Oleuropein. The dark blue line indicates the hydrogen bond, the dashed line indicates the hydrophobic interaction.

Figure 7

Molecular docking studies show ligand binding in the active pockets of ALK5 receptors. (A) HT acetate. (B) HT. and (C) Tyrosol. The dark blue line indicates the hydrogen bond, the dashed line indicates the hydrophobic interaction. Molecular docking studies show ligand binding in the active pockets of ALK5 receptors. (D) Oleuropein. The dark blue line indicates the hydrogen bond, the dashed line indicates the hydrophobic interaction.

Figure 8

Illustration of experimental design of HT treatment of RECs. Six groups of HT and TGFβ1 treatment conditions are shown. C = Control (untreated RECs), T = TGFβ1, and H = hydroxytyrosol.