Abstract
Pulmonary fibrosis (PF) is chronic lung disease with only two FDA approved clinically available drugs, with limited safety profile. Inadequate therapy motivated us to explore the effect of vimentin inhibitor Withaferin A, as an anti-fibrotic agent against TGF-β1-induced in vitro fibrotic events and Bleomycin induced in vivo fibrosis with an emphasis on epithelial to mesenchymal transition (EMT), extracellular matrix deposition (ECM), inflammation, and angiogenesis. In vitro EMT and fibrotic events were induced by TGF-β1 in alveolar epithelial cells and human fetal lung fibroblasts followed by treatment with Withaferin A (0.25, 0.5, and 1 μM concentrations) to explore its anti-fibrotic effects. In vivo potential of Withaferin A (2 and 4 mg/kg) was assessed in murine model of Bleomycin induced PF. All the parameters and molecular studies related to PF were performed at the end of treatment period. Withaferin A treatment reduced the progression of PF by modulating the EMT related cell markers both in vivo and in vitro. Withaferin A ameliorated the expression of inflammatory cytokines including NF-κB p65, IL-1β and TNF-α, as well as attenuated the expression of pro-fibrotic proteins including CTGF, collagen 1A2, collagen 3A1, and fibronectin. Expression of angiogenic factors like VEGF, FAK, p38 MAPK, and PLC-γ1 were also inhibited by Withaferin A. Phosphorylation of Smad 2/3 induced by TGF-β1 and Bleomycin were significantly inhibited. Withaferin A suppressed expression of pro-inflammatory, pro-fibrotic, and pro-angiogenic mediators and also reduced the ECM deposition. In a nutshell, Withaferin A could probably prove as an efficient and potential therapeutic against PF.
Highlights
Pulmonary fibrosis (PF) is a chronic, progressive and irreversible lethal medical problem hallmarked by alveolar disruption, excess scarring and edema of lungs that may significantly contribute to critical respiratory failure (American Thoracic Society and European Respiratory Society, 2002; King et al, 2011)
Withaferin A was procured from Aptus laboratories (Hyderabad, India) and TGF-β1 was obtained from Bio-legend (United States); Bleomycin sulfate was procured from Cipla labs (India); Masson’s trichrome staining kit, Sirius red, Chloramine-T, Hydroxyproline, and Ehrlich reagent were procured from Sigma-Aldrich, Anti-ZO-1, anti-E-cadherin, anti-Smad 2/3, anti-p Smad 2/3, anti-vimentin, anti-NF-κB p65, anti-p VEGF, anti-p p38 MAPK, anti-p FAK, and anti-p PLCγ1 were procured from Cell Signaling Technology, while anti-Col 1A2, anti-Col 3A1, anti-smooth muscle actin, anti-Connective tissue growth factor (CTGF), anti-fibronectin, and anti-TGF-β1 were obtained from Santa Cruz Biotechnology (United States)
It was evident that there was a significant decrease in migration rate of HFL1 cells by Withaferin A (WFA) at 0.5 and 1 μM, compared to control fibroblast cells suggesting the inhibitory effect of WFA on constitutive cell proliferation where in WFA attenuated the basal cell migration rate of fibroblasts
Summary
Pulmonary fibrosis (PF) is a chronic, progressive and irreversible lethal medical problem hallmarked by alveolar disruption, excess scarring and edema of lungs that may significantly contribute to critical respiratory failure (American Thoracic Society and European Respiratory Society, 2002; King et al, 2011). Persisting respiratory disorders like asthma, chronic obstructive pulmonary disease, severe lung injury, environmental factors, and uncontrolled wound healing culminate in the progression of PF (Chilosi et al, 2012). Esbriet (Perfenidone), a potential TGF-β inhibitor and Ofev (Nintedanib), a tyrosine kinase inhibitor are the only two USFDA approved drugs available to treat PF till date (Ley et al, 2011). These drugs could not satisfactorily overcome the progression of disease and they exhibit gastrointestinal, skin, and liver-related adverse effects (Jiang et al, 2012; Richeldi et al, 2015). Despite the advances in the arena of PF, the urge to develop efficient therapeutics prevails
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