title: Inhibition of activin receptor-like kinase 5 induces matrix metallopeptidase 9 expression and aggravates lipopolysaccharide-induced pulmonary injury in mice.
abstract: BACKGROUND
  TGF-β (Transforming Growth Factor-β) mediates its biological effects through members of activin receptor-like kinase (ALK) family and TGF-β/Smad3 signaling link inflammation to pulmonary fibrosis.
  
  
  OBJECTIVE
  The aim of this study was to evaluate the role of SB431542 as a specific inhibitor of Activin receptor-Like Kinase 5 (ALK5) in pneumonic injury.
  
  
  METHODS
  Anesthetized and endo-tracheally intubated C57BL/6 mice were randomized to three groups: the control group with intra-tracheal instillation of 1.5 mg/kg normal saline (NS); LPS stimulation group with intra-tracheal instillation of 3 mg/kg LPS (lipopolysaccharide); and LPS+SB431542 group with intra-peritoneal (i.p.) injection of 4.2 mg/kg SB431542 1 h before intra-tracheal instillation of 3 mg/kg LPS. The lung tissue was obtained 6 h after injury, and the degree of pulmonary injury was evaluated by pathologic scoring. The lung wet/dry weight ratio was measured. TNF-α, IL-1β, and MMP-9 (matrix metallopeptidase-9) mRNA expression levels were assayed by real time PCR (polymerase chain reaction). The content of MMP-9 total protein was measured by Western blotting. The content of active MMP-9 was detected by gelatin zymography. Location of MMP-9 in mouse lung tissue was monitored by immunohistochemistry.
  
  
  RESULTS
  The results showed that (1) pathologic changes including interstitial pulmonary edema, neutrophil infiltration, alveolar edema and hemorrhage were observed 6 h after LPS instillation. The lung wet/dry weight rate and pathologic scores confirmed that SB431542 administration aggravated LPS injury to the mouse lung; (2) the amount of TNF-α and IL-1β mRNA expression in LPS groups was significantly higher than that in the control group, and the highest in LPS+SB431542 group; (3) the amount of MMP-9 mRNA and MMP-9 protein expression and active MMP-9 in the lung tissue of LPS groups was significantly higher than that in the control group 6 h after injury, and the highest in LPS+SB431542 group; and (4) MMP-9 expression was mainly observed in the airway epithelial cells, vascular smooth muscle cells and cytoplasm of inflammatory cells as shown by immunohistochemistry, and brownish yellow uniformed stained areas were also seen in the exudate from part of the alveoli.
  
  
  CONCLUSIONS
  These results indicate that blocking the activity of TGF-β/Smad pathway by specific inhibitor SB431542 of ALK5 promoted the releaser of large amounts of TNF-α, IL-1β and other pro-inflammatory cytokines from the lung tissue of mice sustaining acute lung injury (ALI). At the same time, the amount and activity of MMP-9 expression in the lung were increased, and MMP-9 expression was mainly located in the airway epithelial cells, vascular smooth muscle cells and inflammatory cells, causing increased permeability of the pulmonary blood vessels, degradation of the extracellular matrix and destruction of the normal lung tissue structures, which directly or indirectly promotes the progression of pulmonary inflammatory responses and aggravates ALI.
authors: Burn Institute of Chinese People's Liberation Army and Department of Burn Surgery, Changhai Hospital, Second Military Medical University, Shanghai, China.
authors: B
authors: B
authors: Ma
authors: P-Y
authors: PY
authors: Zhou
authors: W
authors: W
authors: Ni
authors: W
authors: W
authors: Wei
authors: D-F
authors: DF
authors: Ben
authors: W
authors: W
authors: Lu
authors: Z-F
authors: ZF
authors: Xia
mesh_terms: D000818:Animals; D001549:Benzamides / Q000627:therapeutic use; D004149:Dioxoles / Q000627:therapeutic use; D053583:Interleukin-1beta / Q000235:genetics; D008070:Lipopolysaccharides / Q000633:toxicity*; D055370:Lung Injury / Q000209:etiology*; D008297:Male; D020780:Matrix Metalloproteinase 9 / Q000235:genetics*; D051379:Mice; D008810:Mice, Inbred C57BL; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*; D000077293:Receptor, Transforming Growth Factor-beta Type I; D018125:Receptors, Transforming Growth Factor beta / Q000037:antagonists & inhibitors / Q000502:physiology*; D016212:Transforming Growth Factor beta / Q000037:antagonists & inhibitors; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics
chemical_list: C459179:4-(5-benzo(1,3)dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)benzamide; D001549:Benzamides; D004149:Dioxoles; D053583:Interleukin-1beta; D008070:Lipopolysaccharides; D018125:Receptors, Transforming Growth Factor beta; D016212:Transforming Growth Factor beta; D014409:Tumor Necrosis Factor-alpha; D017346:Protein Serine-Threonine Kinases; D000077293:Receptor, Transforming Growth Factor-beta Type I; C000625842:Tgfbr1 protein, mouse; D020780:Matrix Metalloproteinase 9; C501830:Mmp9 protein, mouse