title: Effect of Ambroxol and Beclomethasone on Lipopolysaccharide-Induced Nitrosative Stress in Bronchial Epithelial Cells.
abstract: BACKGROUND
  Nitrosative stress is involved in different airway diseases. Lipopolysaccharide (LPS) induces neutrophil-related cytokine release and nitrosative stress in human bronchial epithelial (BEAS-2B) cells alone or with human polymorphonuclear neutrophils (PMNs). Ambroxol protects against oxidative stress, and beclomethasone dipropionate is an anti-inflammatory drug.
  
  
  OBJECTIVE
  We evaluated the ability of ambroxol and/or beclomethasone dipropionate to inhibit LPS-induced expression/release of RANTES, IL-8, inducible NO synthase (iNOS), myeloperoxidase (MPO) and 3-nitrotyrosine (3-NT: nitrosative stress biomarker) in BEAS-2B ± PMNs stimulated with LPS (1 μg/ml).
  
  
  METHODS
  The effect of ambroxol and/or beclomethasone dipropionate on IL-8, RANTES and iNOS levels was assessed by Western blot analysis; IL-8, MPO and 3-NT levels were measured by ELISA. Cell viability was assessed by the trypan blue exclusion test.
  
  
  RESULTS
  In BEAS-2B alone, LPS (at 12 h) increased RANTES/iNOS expression and IL-8 levels (p < 0.001). Ambroxol suppressed LPS-induced RANTES expression and IL-8 release (p < 0.001), whilst inhibiting iNOS expression (p < 0.05). Beclomethasone dipropionate had no effect on RANTES but halved iNOS expression and IL-8 release. Coculture of BEAS-2B with PMNs stimulated IL-8, MPO and 3-NT production (p < 0.001), potentiated by LPS (p < 0.001). Ambroxol and beclomethasone dipropionate inhibited LPS-stimulated IL-8, MPO and 3-NT release (p < 0.05). Ambroxol/beclomethasone dipropionate combination potentiated the inhibition of IL-8 and 3-NT production in BEAS-2B with PMNs (p < 0.05 and p < 0.01, respectively). Ambroxol and/or beclomethasone dipropionate inhibited nitrosative stress and the release of neutrophilic inflammatory products in vitro.
  
  
  CONCLUSIONS
  The additive effect of ambroxol and beclomethasone dipropionate on IL-8 and 3-NT inhibition suggests new therapeutic options in the treatment of neutrophil-related respiratory diseases such as chronic obstructive pulmonary disease and respiratory infections.
authors.affiliation: Department of Clinical and Biological Sciences, University of Torino, San Luigi Hospital, Orbassano, Italy.
authors.forename: Fabio L M
authors.initials: FL
authors.lastname: Ricciardolo
authors.forename: Valentina
authors.initials: V
authors.lastname: Sorbello
authors.forename: Sabrina
authors.initials: S
authors.lastname: Benedetto
authors.forename: Davide
authors.initials: D
authors.lastname: Paleari
mesh_terms: D000551:Ambroxol / Q000494:pharmacology*; D001507:Beclomethasone / Q000494:pharmacology*; D001980:Bronchi / Q000166:cytology; D002460:Cell Line; D018946:Chemokine CCL5 / Q000187:drug effects / Q000378:metabolism; D004847:Epithelial Cells / Q000187:drug effects*; D005100:Expectorants / Q000494:pharmacology*; D005938:Glucocorticoids / Q000494:pharmacology*; D006801:Humans; D016209:Interleukin-8 / Q000187:drug effects / Q000378:metabolism; D008070:Lipopolysaccharides / Q000633:toxicity; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000187:drug effects / Q000378:metabolism; D015538:Nitrosation / Q000187:drug effects; D009195:Peroxidase / Q000187:drug effects / Q000378:metabolism; D020545:Respiratory Mucosa / Q000166:cytology; D013312:Stress, Physiological / Q000187:drug effects*; D014443:Tyrosine / Q000031:analogs & derivatives / Q000187:drug effects / Q000378:metabolism
chemical_list: C483024:CCL5 protein, human; C508606:CXCL8 protein, human; D018946:Chemokine CCL5; D005100:Expectorants; D005938:Glucocorticoids; D016209:Interleukin-8; D008070:Lipopolysaccharides; D000551:Ambroxol; C002744:3-nitrotyrosine; D014443:Tyrosine; D009195:Peroxidase; C496319:NOS2 protein, human; D052247:Nitric Oxide Synthase Type II; D001507:Beclomethasone