title: NO-releasing xanthine KMUP-1 bonded by simvastatin attenuates bleomycin-induced lung inflammation and delayed fibrosis.
abstract: OBJECTIVE
  Pulmonary fibrosis (PF) is a progressing lung injury initiated by pulmonary inflammation (PI). Bleomycin (BLM) is the most common pathogenesis of PF through early PI and extensive extracellular matrix deposition. This study is aimed to determine whether NO-releasing KMUP-1 inhibits PI and PF, and if so, the benefits of KMUP-1S resulted from simvastatin (SIM)-bonding to KMUP-1.
  
  
  METHODS
  C57BL/6 male mice were intra-tracheally administered BLM (4 U/kg) at day 0. KMUP-1 (1-5 mg/kg), KMUP-1S (2.5 mg/kg), SIM (5 mg/kg), Plus (KMUP-1 2.5 mg/kg + SIM 2.5 mg/kg), and clarithromycin (CAM, 10 mg/kg) were orally and daily administered for 7 and 28 days, respectively, to mice, sacrificed at day-7 and day-28 to isolate the lung tissues, for examining the inflammatory and fibrotic signaling and measuring the cell population and MMP-2/MMP-9 activity in broncholaveolar lavage fluid (BAL).
  
  
  RESULTS
  KMUP-1 and KUP-1S significantly decreased neutrophil counts in BAL fluid. Fibroblastic foci were histologically assessed by H&E and Masson's trichrome stain and treated with KMUP-1 and references. Lung tissues were determined the contents of collagen and the expressions of TGF-β, α-SMA, HMGB1, CTGF, eNOS, p-eNOS, RhoA, Smad3, p-Smad3, MMP-2 and MMP-9 by Western blotting analyses, respectively. These changes areregulated by NO/cGMP and inhibited by various treatments. KMUP-1 and KMUP-1S predominantly prevented HMGB1/MMP-2 expression at day-7 and reduced TGF-β/phosphorylated Smad3 and CTGF at day-28.
  
  
  CONCLUSIONS
  KMUP-1 and KMUP-S restore eNOS, inhibit iNOS/ROCKII/MMP-2/MMP-9, attenuate histologic collagen disposition and reduce BALF inflammatory cells, potentially useful for the treatment of BLM-lung PF.
authors.affiliation: Department of Cardiology, Yuan's General Hospital, Kaohsiung, Taiwan.
authors.forename: C P
authors.initials: CP
authors.lastname: Liu
authors.affiliation: Department of Pharmacology, Kaohsiung Medical University, Kaohsiung, Taiwan.
authors.forename: M S
authors.initials: MS
authors.lastname: Kuo
authors.affiliation: Department of Pharmacology, Kaohsiung Medical University, Kaohsiung, Taiwan.
authors.forename: B N
authors.initials: BN
authors.lastname: Wu
authors.affiliation: Department of Pathology, Kaohsiung Medical University, Kaohsiung, Taiwan.
authors.forename: C Y
authors.initials: CY
authors.lastname: Chai
authors.affiliation: Department of Anatomy, Kaohsiung Medical University, Kaohsiung, Taiwan.
authors.forename: H T
authors.initials: HT
authors.lastname: Huang
authors.affiliation: Department of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.
authors.forename: P W
authors.initials: PW
authors.lastname: Chung
authors.affiliation: Department of Pharmacology, Kaohsiung Medical University, Kaohsiung, Taiwan. Electronic address: ingjun@kmu.edu.tw.
authors.forename: I J
authors.initials: IJ
authors.lastname: Chen
mesh_terms: D000818:Animals; D001761:Bleomycin / Q000633:toxicity; D015153:Blotting, Western; D001992:Bronchoalveolar Lavage Fluid; D017291:Clarithromycin / Q000494:pharmacology; D004195:Disease Models, Animal; D005786:Gene Expression Regulation / Q000187:drug effects; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D010880:Piperidines / Q000008:administration & dosage / Q000737:chemistry / Q000494:pharmacology*; D011014:Pneumonia / Q000188:drug therapy* / Q000473:pathology; D011658:Pulmonary Fibrosis / Q000188:drug therapy* / Q000473:pathology; D015398:Signal Transduction / Q000187:drug effects; D019821:Simvastatin / Q000008:administration & dosage / Q000737:chemistry / Q000494:pharmacology*; D013997:Time Factors; D014970:Xanthines / Q000008:administration & dosage / Q000737:chemistry / Q000494:pharmacology*
chemical_list: D010880:Piperidines; D014970:Xanthines; D001761:Bleomycin; C439304:KMUP 1; D019821:Simvastatin; D017291:Clarithromycin
keywords: ALT; AST; BAL; Fibrosis; HMGB1; IHC; Inflammation; MMP-2/-9; MMPs; NO; NO/eNOS; PDE-5A; PF; PI; PVDF; Pulmonary; ROCKII; ROS; Rho kinase II; SDS; Smad3; TGF-β1; TGFβ; alanine aminotransferase; aspartate aminotransferase; broncholaveolar lavage fluid; cGMP; eNOS; endothelial nitric oxide synthase; guanosine 3′,5′-cyclic monophosphate; high mobility group box 1; iNOS; immunohistochemistry; inducible NOS; matrix metalloproteinases; matrix metalloproteinases-2 and -9; mouse mothers against decapentaplegic homolog 3; nitric oxide; p-Smad3; p-eNOS; phosphodiesterase 5A; phosphorylated Smad3; phosphorylated eNOS; polyvinylidene; pulmonary fibrosis; pulmonary inflammation; reactive oxygen species; sodium dodecyl sulfate-polyacrylamide gel electrophoresis; transforming growth factor-β1