Anti-fibrotic effects of pirfenidone and rapamycin in primary IPF fibroblasts and human alveolar epithelial cells

M Molina-Molina,A Montes-Worboys,E Sala-Llinas,P Luburich-Hernaiz,J Dorca,I Escobar,R Llatjós,C Machahua-Huamani,V Vicens-Zygmunt

doi:10.1186/s12890-018-0626-4

Abstract

BackgroundPirfenidone, a pleiotropic anti-fibrotic treatment, has been shown to slow down disease progression of idiopathic pulmonary fibrosis (IPF), a fatal and devastating lung disease. Rapamycin, an inhibitor of fibroblast proliferation could be a potential anti-fibrotic drug to improve the effects of pirfenidone.MethodsPrimary lung fibroblasts from IPF patients and human alveolar epithelial cells (A549) were treated in vitro with pirfenidone and rapamycin in the presence or absence of transforming growth factor β1 (TGF−β). Extracellular matrix protein and gene expression of markers involved in lung fibrosis (tenascin-c, fibronectin, collagen I [COL1A1], collagen III [COL3A1] and α-smooth muscle actin [α-SMA]) were analyzed. A cell migration assay in pirfenidone, rapamycin and TGF−β-containing media was performed.ResultsGene and protein expression of tenascin-c and fibronectin of fibrotic fibroblasts were reduced by pirfenidone or rapamycin treatment. Pirfenidone-rapamycin treatment did not revert the epithelial to mesenchymal transition pathway activated by TGF−β. However, the drug combination significantly abrogated fibroblast to myofibroblast transition. The inhibitory effect of pirfenidone on fibroblast migration in the scratch-wound assay was potentiated by rapamycin combination.ConclusionsThese findings indicate that the combination of pirfenidone and rapamycin widen the inhibition range of fibrogenic markers and prevents fibroblast migration. These results would open a new line of research for an anti-fibrotic combination therapeutic approach.

Highlights

Pirfenidone, a pleiotropic anti-fibrotic treatment, has been shown to slow down disease progression of idiopathic pulmonary fibrosis (IPF), a fatal and devastating lung disease
Pirfenidone and rapamycin are well tolerated by human lung fibroblasts and alveolar epithelial cells Cell viability was assayed with the WST reagent in fibroblasts and A549 cells cultured in 96-well plates and treated with transforming growth factor (TGF)−β (5 ng/ml), pirfenidone (1 mg/ml) and rapamycin (1 μg/ml) for 72 h
The cell toxicity assay demonstrated that pirfenidone and rapamycin, in addition to the combination of the 2 agents with transforming growth factor β1 (TGF−β) (5 ng/ml), did not cause cellular death when compared to untreated cells and were well tolerated for the treatment period of 72 h (Fig. 1)

Summary

Introduction

Pirfenidone, a pleiotropic anti-fibrotic treatment, has been shown to slow down disease progression of idiopathic pulmonary fibrosis (IPF), a fatal and devastating lung disease. An inhibitor of fibroblast proliferation could be a potential anti-fibrotic drug to improve the effects of pirfenidone. The current clinical objective in IPF treatment is to inhibit disease progression with anti-fibrotic therapy [14], and the current research objective is to find an anti-fibrotic capable of completely halting or abrogate fibrosis. In this context, the in vitro antifibrotic effect of drug combinations, together with the safety profile of this therapeutic approach, should be explored in future clinical trials

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