title: Paeoniflorin suppresses TGF-β mediated epithelial-mesenchymal transition in pulmonary fibrosis through a Smad-dependent pathway.
abstract: OBJECTIVE
  Paeoniflorin has shown to attenuate bleomycin-induced pulmonary fibrosis (PF) in mice. Because the epithelial-mesenchymal transition (EMT) in type 2 lung endothelial cells contributes to excessive fibroblasts and myofibroblasts during multiple fibrosis of tissues, we investigated the effects of paeoniflorin on TGF-β mediated pulmonary EMT in bleomycin-induced PF mice.
  
  
  METHODS
  PF was induced in mice by intratracheal instillation of bleomycin (5 mg/kg). The mice were orally treated with paeoniflorin or prednisone for 21 d. After the mice were sacrificed, lung tissues were collected for analysis. An in vitro EMT model was established in alveolar epithelial cells (A549 cells) incubated with TGF-β1 (2 ng/mL). EMT identification and the expression of related proteins were performed using immunohistochemistry, transwell assay, ELISA, Western blot and RT-qPCR.
  
  
  RESULTS
  In PF mice, paeoniflorin (50, 100 mg·kg(-1)·d(-1)) or prednisone (6 mg·kg(-1)·d(-1)) significantly decreased the expression of FSP-1 and α-SMA, and increased the expression of E-cadherin in lung tissues. In A549 cells, TGF-β1 stimulation induced EMT, as shown by the changes in cell morphology, the increased cell migration, and the increased vimentin and α-SMA expression as well as type I and type III collagen levels, and by the decreased E-cadherin expression. In contrast, effects of paeoniflorin on EMT disappeared when the A549 cells were pretreated with TGF-β1 for 24 h. TGF-β1 stimulation markedly increased the expression of Snail and activated Smad2/3, Akt, ERK, JNK and p38 MAPK in A549 cells. Co-incubation with paeoniflorin (1-30 μmol/L) dose-dependently attenuated TGF-β1-induced expression of Snail and activation of Smad2/3, but slightly affected TGF-β1-induced activation of Akt, ERK, JNK and p38 MAPK. Moreover, paeoniflorin markedly increased Smad7 level, and decreased ALK5 level in A549 cells.
  
  
  CONCLUSIONS
  Paeoniflorin suppresses the early stages of TGF-β mediated EMT in alveolar epithelial cells, likely by decreasing the expression of the transcription factors Snail via a Smad-dependent pathway involving the up-regulation of Smad7.
authors.affiliation: Jiangsu Key Laboratory of Drug Discovery for Metabolic Diseases, Department of Pharmacology of Chinese Materia Medica, China Pharmaceutical University, Nanjing 210009, China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Ji
authors.affiliation: Jiangsu Key Laboratory of Drug Discovery for Metabolic Diseases, Department of Pharmacology of Chinese Materia Medica, China Pharmaceutical University, Nanjing 210009, China.
authors.forename: Yan-Nong
authors.initials: YN
authors.lastname: Dou
authors.affiliation: Jiangsu Key Laboratory of Drug Discovery for Metabolic Diseases, Department of Pharmacology of Chinese Materia Medica, China Pharmaceutical University, Nanjing 210009, China.
authors.forename: Qian-Wen
authors.initials: QW
authors.lastname: Zhao
authors.affiliation: Jiangsu Key Laboratory of Drug Discovery for Metabolic Diseases, Department of Pharmacology of Chinese Materia Medica, China Pharmaceutical University, Nanjing 210009, China.
authors.forename: Ji-Zhou
authors.initials: JZ
authors.lastname: Zhang
authors.affiliation: Jiangsu Key Laboratory of Drug Discovery for Metabolic Diseases, Department of Pharmacology of Chinese Materia Medica, China Pharmaceutical University, Nanjing 210009, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Yang
authors.affiliation: Jiangsu Key Laboratory of Drug Discovery for Metabolic Diseases, Department of Pharmacology of Chinese Materia Medica, China Pharmaceutical University, Nanjing 210009, China.
authors.forename: Ting
authors.initials: T
authors.lastname: Wang
authors.affiliation: Department of Chinese Materia Medica Analysis, China Pharmaceutical University, Nanjing 210009, China.
authors.forename: Yu-Feng
authors.initials: YF
authors.lastname: Xia
authors.affiliation: Jiangsu Key Laboratory of Drug Discovery for Metabolic Diseases, Department of Pharmacology of Chinese Materia Medica, China Pharmaceutical University, Nanjing 210009, China.
authors.forename: Yue
authors.initials: Y
authors.lastname: Dai
authors.affiliation: Jiangsu Key Laboratory of Drug Discovery for Metabolic Diseases, Department of Pharmacology of Chinese Materia Medica, China Pharmaceutical University, Nanjing 210009, China.
authors.forename: Zhi-Feng
authors.initials: ZF
authors.lastname: Wei
mesh_terms: D000072283:A549 Cells; D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000737:chemistry / Q000627:therapeutic use*; D001761:Bleomycin; D002470:Cell Survival / Q000187:drug effects; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D058750:Epithelial-Mesenchymal Transition / Q000187:drug effects*; D005960:Glucosides / Q000737:chemistry / Q000627:therapeutic use*; D006801:Humans; D008168:Lung / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D039821:Monoterpenes / Q000737:chemistry / Q000627:therapeutic use*; D029596:Paeonia / Q000737:chemistry; D011658:Pulmonary Fibrosis / Q000139:chemically induced / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D015398:Signal Transduction / Q000187:drug effects; D051785:Smad Proteins / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000378:metabolism*; D006027:Glycosides
chemical_list: D000894:Anti-Inflammatory Agents, Non-Steroidal; D001761:Bleomycin; D005960:Glucosides; D039821:Monoterpenes; D051785:Smad Proteins; D016212:Transforming Growth Factor beta; C015423:peoniflorin; D006027:Glycosides