AITC inhibits fibroblast-myofibroblast transition via TRPA1-independent MAPK and NRF2/HO-1 pathways and reverses corticosteroids insensitivity in human lung fibroblasts

<jats:title>Abstract</jats:title><jats:sec> <jats:title>Background</jats:title> <jats:p>Little is known on the role of transient receptor potential ankyrin 1 (TRPA1) in fibroblast—myofibroblast transition (FMT) that can lead to airway remodeling which is a major problem for severe asthma and fibrosis. Thus, this study investigated the effect of TRPA1 modulators on transforming growth factor beta 1(TGF-β1) -treated lung fibroblasts.</jats:p> </jats:sec><jats:sec> <jats:title>Methods</jats:title> <jats:p>MRC-5 cells were preincubated with TGF-β1 for 24 h. TRPA1 agonist or antagonist were added and further incubated for 24 h. The changes in TRPA1 and alpha-smooth muscle actin (α-SMA) expressions by stimuli were evaluated using qRT-PCR, western blot and immunohistochemical analyses. Statistical significance was determined by using one- or two-way ANOVA, followed by Bonferroni’s post hoc analysis for comparison of multiple groups and paired 2-tailed Student’s t-test between 2 groups.</jats:p> </jats:sec><jats:sec> <jats:title>Results</jats:title> <jats:p>MRC-5 cells treated by TGF-β1 significantly upregulated α-SMA mRNA expressions (<jats:italic>P</jats:italic> < 0.01), but downregulated TRPA1 gene expression (<jats:italic>P</jats:italic> < 0.001). Post-treatment of TRPA1 activator, allyl isothiocyanate (AITC), after TGF-β1 significantly downregulated the α-SMA gene induction (<jats:italic>P</jats:italic> < 0.01 at 24 h), protein expression (<jats:italic>P</jats:italic> < 0.05) and immunoreactivity with stress fibers (<jats:italic>P</jats:italic> < 0.05). On the other hand, TRPA1 antagonist HC-030031 did not prevent this effect, and instead tended to facilitate the suppressive effect of AITC when co-stimulated. AITC significantly increased phosphorylated- extracellular signal-regulated kinase (ERK) 1/2 and heme oxygenase (HO)-1 protein expressions (<jats:italic>P</jats:italic> < 0.05) in TGF-β1-treated cells. Combined inhibition with ERK1/2 mitogen-activated protein kinase (MAPK) and nuclear factor erythroid 2-related factor (NRF2) almost completely reversed AITC-induced α-SMA suppression (<jats:italic>P</jats:italic> < 0.05). Dexamethasone was not able to inhibit the upregulated α-SMA induction by TGF-β1. However, AITC improved dexamethasone-insensitive myodifferentiation in the presence of the corticosteroid (<jats:italic>P</jats:italic> < 0.01).</jats:p> </jats:sec><jats:sec> <jats:title>Conclusion</jats:title> <jats:p>We found that AITC exerts protective effect on TGF-β1-induced α-SMA induction by activating ERK1/2 MAPK and NRF2/HO-1 pathways in lung fibroblasts. It also overcomes corticosteroids insensitivity in TGF-β1-induced α-SMA induction. TRPA1 antagonist modulates the suppressive effect, but not prevent it. AITC and TRPA1 antagonist may be therapeutic agents in treating chronic respiratory diseases.</jats:p> </jats:sec>