The impact of para-hydroxycinnamic acid on myofibroblast trans-differentiation in MRC-5 and A549 cell lines: in silico and in vitro evaluation.

Fibrosis-related disorders like pulmonary fibrosis are chronically progressive disorders characterized by tissue scarring with a gradual decline in function. Transforming growth factor-beta1 (TGF-β1), myofibroblast trans-differentiation, and epithelial to mesenchymal transition (EMT) play a central role in fibrosis progression. Para-hydroxycinnamic acid is a naturally occurring phenolic acid metabolite having different biological activities. This study aimed to investigate the potential of para-hydroxycinnamic acid (p-HCA) in inhibiting the epithelial-mesenchymal transition in vitro and to understand the mechanism of action using in silico analysis. Molecular docking analysis was executed using Schrodinger Glide software with four selected protein targets (caspase-1, lysophosphatidic acid receptor 1, peroxisome proliferator-activated receptor-gamma, and NLRP3). In vitro studies used human normal fetal lung fibroblasts (MRC-5) and cancerous alveolar epithelial cells (A549). The MTT assay for cytotoxicity analysis was applied to p-HCA (the test agent) and pirfenidone (PFD/the standard comparator) at serial concentrations ranging from 100 to 6.25 µg/ml. Both cell lines were treated with 100 µg/ml of p-HCA and PFD following a previous induction with 10 ng/ml TGF-β1 and then incubated for 48 h. Gene expression analysis was performed using qRT-PCR for EMT genes, including α-smooth muscle actin, fibronectin, vimentin, and Smad2/3. Results revealed that p-HCA had favorable binding affinities for lysophosphatidic acid receptor 1 and PPAR-γ, with a good receptor pose, which is complemented by the in vitro results, in which p-HCA exhibited low cytotoxicity potential towards MRC-5 with IC50 191.8 µg/ml and mild cytotoxicity for A549 with IC50 34.34 µg/ml. Results of the gene expression evaluation showed a statistically significant reduction (P < 0.001) in the gene expression of all myofibroblast trans-differentiation markers by over twofold compared with the TGF-β1-induced cells and no significant difference (P > 0.05) compared with the PFD. p-HCA can halt myofibroblast formation and EMT in both MRC-5 and A549 through indirect influence on the TGF-β1/Smad2/3 pathway by interaction with LPAR1 and PPAR-γ, making it a promising agent to investigate further in pharmaceutical and clinical settings.