title: NADPH oxidase-dependent formation of reactive oxygen species contributes to transforming growth factor β1-induced epithelial-mesenchymal transition in rat peritoneal mesothelial cells, and the role of astragalus intervention.
abstract: OBJECTIVE
  To investigate the role of nicotinamide-adenine dinucleotide phosphate (NADPH) oxidasedependent formation of reactive oxygen species (ROS) in the transforming growth factor β1 (TGF-β1)-induced epithelial-mesenchymal transition (EMT) in rat peritoneal mesothelial cells (RPMCs), and the effect of Astragalus injection (AGI) intervention.
  
  
  METHODS
  Primary RPMCs were cultured to the second generation in vitro. After synchronization for 24 h, the cells were randomly assigned to the following groups: control (Group A), AGI (2 g/mL; Group B), TGF-β1 (10 ng/mL; Group C), TGF-β1 (10 ng/mL) + AGI (2 g/mL; Group D; pretreated for 1 h with AGI before TGF-β1 stimulation). Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis were employed to evaluate the mRNA and protein expression of the NADPH oxidase subunit p67phox, α-smooth muscle actin (α-SMA) and E-cadherin. The dichlorofluorescein-sensitive cellular ROS levels were measured by a fluorometric assay and confocal microscopy.
  
  
  RESULTS
  TGF-β1 significantly induced NADPH oxidase subunit p67phox mRNA and protein expression in RPMCs, as well as inducing the production of intracellular ROS. AGI inhibited this TGF-β1-induced up-regulation by 39.3% and 47.8%, respectively (P<0.05), as well as inhibiting the TGF-β1-induced ROS generation by 56.3% (P<0.05). TGF-β1 also induced α-SMA mRNA and protein expression, and down-regulated E-cadherin mRNA and protein expression (P<0.05). This effect was suppressed by AGI (P<0.05).
  
  
  CONCLUSIONS
  NADPH oxidase-dependent formation of ROS may mediate the TGF-β1-dependent EMT in RPMCs. AGI could inhibit this process, providing a theoretical basis for AGI in the prevention of peritoneal fibrosis.
authors.affiliation: Department of Nephrology, Zhejiang Provincial Hospital of Integrated Chinese and Western Medicine, Hangzhou, 310003, China.
authors.forename: Xiao-xian
authors.initials: XX
authors.lastname: Liu
authors.forename: Hong-juan
authors.initials: HJ
authors.lastname: Zhou
authors.forename: Long
authors.initials: L
authors.lastname: Cai
authors.forename: Wen
authors.initials: W
authors.lastname: Zhang
authors.forename: Ji-lin
authors.initials: JL
authors.lastname: Ma
authors.forename: Xiao-juan
authors.initials: XJ
authors.lastname: Tao
authors.forename: Jian-ning
authors.initials: JN
authors.lastname: Yu
mesh_terms: D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D058750:Epithelial-Mesenchymal Transition / Q000502:physiology*; D004848:Epithelium; D019255:NADPH Oxidases / Q000378:metabolism*; D010529:Peritoneal Cavity / Q000166:cytology*; D016133:Polymerase Chain Reaction; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D053773:Transforming Growth Factor beta1 / Q000502:physiology*
chemical_list: D017931:DNA Primers; D017382:Reactive Oxygen Species; D053773:Transforming Growth Factor beta1; D019255:NADPH Oxidases