title: Apocynin inhibits the upregulation of TGF-β1 expression and ROS production induced by TGF-β in skeletal muscle cells.
abstract: BACKGROUND
  Pure apocynin, which can be traditionally isolated and purified from several plant species such as Picrorhiza kurroa Royle ex Benth (Scrophulariaceae), acts as an inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) activity inhibiting its production of reactive oxygen species (ROS). Transforming growth factor type beta 1 (TGF-β1) is a growth factor that produces inhibition of myogenesis, diminution of regeneration and induction of atrophy in skeletal muscle. The typical signalling that is activated by TGF-β involves the Smad pathway.
  
  
  OBJECTIVE
  To evaluate the effect of TGF-β and the effect of apocynin on TGF-β1 expression in skeletal muscle cells.
  
  
  METHODS
  Controlled laboratory study. In vitro assays were performed with C2C12 cells incubated with TGF-β1 in presence or absence of apocynin (NOX inhibitor), SB525334 (TGF-β-receptor I inhibitor), or chelerythrine (PKC inhibitor).
  
  
  METHODS
  TGF-β1 and atrogin-1 expression was evaluated by RT-qPCR and/or ELISA; Smad3 phosphorylation by western blot; Smad4 nuclear translocation by indirect immunofluorescence; and ROS levels by DCF probe fluorescent measurements.
  
  
  RESULTS
  We show that myoblasts respond to TGF-β1 by increasing its own gene expression in a time- and dose-dependent fashion which was abolished by SB525334 and siRNA for Smad2/3. TGF-β1 also induced ROS. Remarkably, apocynin inhibited the TGF-β1 induced ROS as well as the autoinduction of TGF-β1 gene expression. We also show that TGF-β-induced ROS production and TGF-β1 expression require PKC activity as indicated by the inhibition using chelerythrine.
  
  
  CONCLUSIONS
  These results strongly suggest that TGF-β induces its own expression through a TGF-β-receptor/Smad-dependent mechanism and apocynin is able to inhibit this process, suggesting that requires NOX-induced ROS in skeletal muscle cells.
authors: Laboratorio de Biología y Fisiopatología Molecular, Departamento de Ciencias Biológicas, Facultad de Ciencias Biológicas and Facultad de Medicina, Universidad Andres Bello, Santiago, Chile; Millennium Institute on Immunology and Immunotherapy, Santiago, Chile.
authors: Johanna
authors: J
authors: Abrigo
authors: Laboratorio de Biología y Fisiopatología Molecular, Departamento de Ciencias Biológicas, Facultad de Ciencias Biológicas and Facultad de Medicina, Universidad Andres Bello, Santiago, Chile; Millennium Institute on Immunology and Immunotherapy, Santiago, Chile.
authors: María Gabriela
authors: MG
authors: Morales
authors: Laboratorio de Fisiopatología Integrativa, Departamento de Ciencias Biológicas, Facultad de Ciencias Biológicas and Facultad de Medicina, Universidad Andres Bello, Santiago, Chile; Millennium Institute on Immunology and Immunotherapy, Santiago, Chile.
authors: Felipe
authors: F
authors: Simon
authors: Departamento de Ciencias Químicas y Biológicas, Universidad Bernardo O´Higgins, Santiago, Chile.
authors: Daniel
authors: D
authors: Cabrera
authors: Laboratorio de Biología y Fisiopatología Molecular, Departamento de Ciencias Biológicas, Facultad de Ciencias Biológicas and Facultad de Medicina, Universidad Andres Bello, Santiago, Chile; Millennium Institute on Immunology and Immunotherapy, Santiago, Chile.
authors: Gabriella
authors: G
authors: Di Capua
authors: Laboratorio de Biología y Fisiopatología Molecular, Departamento de Ciencias Biológicas, Facultad de Ciencias Biológicas and Facultad de Medicina, Universidad Andres Bello, Santiago, Chile; Millennium Institute on Immunology and Immunotherapy, Santiago, Chile. Electronic address: claudio.cabello.verrugio@gmail.com.
authors: Claudio
authors: C
authors: Cabello-Verrugio
mesh_terms: D000098:Acetophenones / Q000494:pharmacology*; D000818:Animals; D053119:Benzophenanthridines / Q000494:pharmacology; D002460:Cell Line; D007093:Imidazoles / Q000494:pharmacology; D051379:Mice; D018485:Muscle Fibers, Skeletal / Q000187:drug effects* / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D011810:Quinoxalines / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism*; D051899:Smad2 Protein / Q000378:metabolism; D051900:Smad3 Protein / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D053773:Transforming Growth Factor beta1 / Q000378:metabolism*; D015854:Up-Regulation
chemical_list: C521813:6-(2-tert-butyl-5-(6-methylpyridin-2-yl)-1H-imidazol-4-yl)quinoxaline; D000098:Acetophenones; D053119:Benzophenanthridines; D007093:Imidazoles; D011810:Quinoxalines; D017382:Reactive Oxygen Species; D051899:Smad2 Protein; C497783:Smad2 protein, mouse; D051900:Smad3 Protein; C497791:Smad3 protein, mouse; C508961:Tgfb1 protein, mouse; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; C056165:acetovanillone; C016299:chelerythrine; D011493:Protein Kinase C
keywords: Atrophy; NOX; ROS; Skeletal muscle; Smad; TGF-β1