BackgroundPirfenidone is a novel anti-fibrotic and anti-inflammatory agent that inhibits the progression of fibrosis in animal models and in patients with idiopathic pulmonary fibrosis (IPF). We previously showed that pirfenidone inhibits the over-expression of collagen type I and of heat shock protein (HSP) 47, a collagen-specific molecular chaperone, in human lung fibroblasts stimulated with transforming growth factor (TGF)-β1 in vitro. The increased numbers of HSP47-positive type II pneumocytes as well as fibroblasts were also diminished by pirfenidone in an animal model of pulmonary fibrosis induced by bleomycin. The present study evaluates the effects of pirfenidone on collagen type I and HSP47 expression in the human alveolar epithelial cell line, A549 cells in vitro.MethodsThe expression of collagen type I, HSP47 and E-cadherin mRNAs in A549 cells stimulated with TGF-β1 was evaluated by Northern blotting or real-time PCR. The expression of collagen type I, HSP47 and fibronectin proteins was assessed by immunocytochemical staining.ResultsTGF-β1 stimulated collagen type I and HSP47 mRNA and protein expression in A549 cells, and pirfenidone significantly inhibited this process. Pirfenidone also inhibited over-expression of the fibroblast phenotypic marker fibronectin in A549 cells induced by TGF-β1.ConclusionWe concluded that the anti-fibrotic effects of pirfenidone might be mediated not only through the direct inhibition of collagen type I expression but also through the inhibition of HSP47 expression in alveolar epithelial cells, which results in reduced collagen synthesis in lung fibrosis. Furthermore, pirfenidone might partially inhibit the epithelial-mesenchymal transition.
Objectives: Mucus hypersecretion is a prominent feature in patients with chronic respiratory tract infections such as cystic fibrosis and diffuse panbronchiolitis, and the clinical effectiveness of macrolide antibiotics has been reported in these patients. Because human neutrophil peptide-1 (HNP-1), an antimicrobial peptide in neutrophils, exists in high concentrations in the airway fluid of these patients, we examined the direct effect of HNP-1 on MUC5AC mucin production using NCI-H292 cells. The effects of macrolide antibiotics on the response were also examined.Methods: MUC5AC synthesis was assayed using RT-PCR and ELISA. Phosphorylation of ERK1/2 was determined by western blotting.Results: Stimulation with HNP-1 or lipopolysaccharide (LPS) derived from Pseudomonas aeruginosa increases the production of MUC5AC mRNA and protein, and an additive effect was found upon co-stimulation with both HNP-1 and LPS. Azithromycin and clarithromycin had inhibitory effects on overproduction of MUC5AC induced by HNP-1 or LPS stimulation. Telithromycin also had an inhibitory effect on MUC5AC production induced by LPS, but not on production by HNP-1. Phosphorylation of ERK1/2 was induced by HNP-1 or LPS stimulation, and azithromycin, clarithromycin and telithromycin had inhibitory effects on ERK1/2 phosphorylation induced by LPS, but not by HNP-1.Conclusions: These findings suggest that neutrophil-derived defensins as bacterial components contribute to excessive mucus production in patients with respiratory tract infections, and that macrolide and ketolide antibiotics directly inhibit these actions by interfering with intracellular signal transduction. However, the mechanism of telithromycin inhibition of MUC5AC synthesis may differ from the response induced by azithromycin and clarithromycin.
Animals and plants express endogenous peptide antibiotics called defensins. Defensins show broad-spectrum antimicrobial activity, even against bacteria that have resistance to conventional antibiotics, which has made them viable candidates for new antibiotics.However, human defensins have failed to reach the market because of their cytotoxic effects and non-antimicrobial bioactivities. Plectasin is a defensin that has shown promise but has not had its potentially negative effects clarified. To address this issue, we examined plectasin's cytotoxicity in human cells using an AlamarBlue reduction assay, its interleukin (IL)-8-inducing capacity using real-time PCR and ELISA, and measured its MIC against bacteria. We confirmed that plectasin has specific antibacterial activity against S. pneumoniae. Plectasin showed no cytotoxicity to A549 cells, normal human bronchial epithelial cells, or lung fibroblasts, and it did not induce IL-8 transcription or production in A549 cells. Our results suggest that plectasin could be an inoffensive alternative antibiotic for clinical application.
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