title: Tanshinone IIA pretreatment protects free flaps against hypoxic injury by upregulating stem cell-related biomarkers in epithelial skin cells.
abstract: BACKGROUND
  Partial or total flap necrosis after flap transplantation is sometimes encountered in reconstructive surgery, often as a result of a period of hypoxia that exceeds the tolerance of the flap tissue. The purpose of this study was to determine whether Tanshinone IIA (TSA) pretreatment can protect flap tissue against hypoxic injury and improve its viability.
  
  
  METHODS
  Primary epithelial cells isolated from the dorsal skin of mice were pretreated with TSA for 2 weeks. Cell Counting Kit-8 and Trypan Blue assays were carried out to examine the proliferation of TSA-pretreated cells after exposure to cobalt chloride. Polymerase chain reaction and western blot analysis were used to assess the expression of β-catenin, vascular endothelial growth factor (VEGF), sex determining region Y-box 2 (SOX2), OCT4 (also known as POU domain class 5 transcription factor 1), Nanog, and glycogen synthase kinase-3 beta (GSK-3β) in TSA-treated cells. In other experiments, after mice were pretreated with TSA for 2 weeks, a reproducible ischemic flap model was implemented, and the area of surviving tissue in the transplanted flaps was measured. Immunohistochemistry was conducted to examine Wnt signaling as well as stem cell- and angiogenesis-related biomarkers in epithelial tissue in vivo.
  
  
  RESULTS
  Epidermal cells, pretreated with TSA, showed enhanced resistance to hypoxia. Activation of the Wnt signaling pathway in TSA-pretreated cells was characterized by the upregulation of β-catenin and the downregulation of GSK-3β. The expression of SOX2, Nanog, and OCT4 were also higher in TSA-pretreated epithelial cells than in control cells. In the reproducible ischemic flap model, pretreatment with TSA enhanced resistance to hypoxia and increased the area of surviving tissue in transplanted flaps. The expression of Wnt signaling pathway components, stem-cell related biomarkers, and VEGF and CD34, which are involved in the regeneration of blood vessels, was also upregulated in TSA-pretreated flap tissue.
  
  
  CONCLUSIONS
  TSA pretreatment protects free flaps against hypoxic injury and increases the area of surviving tissue by activating Wnt signaling and upregulating stem cell-related biomarkers.
authors.forename: Zihan
authors.initials: Z
authors.lastname: Xu
authors.forename: Lijun
authors.initials: L
authors.lastname: Wu
authors.forename: Yaowen
authors.initials: Y
authors.lastname: Sun
authors.forename: Yadong
authors.initials: Y
authors.lastname: Guo
authors.forename: Gaoping
authors.initials: G
authors.lastname: Qin
authors.forename: Shengzhi
authors.initials: S
authors.lastname: Mu
authors.forename: Ronghui
authors.initials: R
authors.lastname: Fan
authors.forename: Benfeng
authors.initials: B
authors.lastname: Wang
authors.forename: Wenjie
authors.initials: W
authors.lastname: Gao
authors.affiliation: Department of Burns and Plastic Surgery, Shaanxi Provincial People's Hospital, 256 West Youyi Road, Xi'an 710068, China. zzsxa2006@126.com.
authors.forename: Zhenxin
authors.initials: Z
authors.lastname: Zhang
mesh_terms: D045784:Abietanes / Q000494:pharmacology*; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D045744:Cell Line, Tumor; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism; D058752:Free Tissue Flaps / Q000009:adverse effects*; D038362:Glycogen Synthase Kinase 3 / Q000235:genetics / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D000860:Hypoxia / Q000188:drug therapy / Q000209:etiology / Q000378:metabolism / Q000517:prevention & control*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D010766:Phosphorylation; D015398:Signal Transduction / Q000187:drug effects; D012867:Skin / Q000187:drug effects* / Q000378:metabolism; D016038:Skin Transplantation; D013234:Stem Cells / Q000187:drug effects / Q000378:metabolism*; D015854:Up-Regulation; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism; D051176:beta Catenin / Q000235:genetics / Q000378:metabolism
chemical_list: D045784:Abietanes; D015415:Biomarkers; D042461:Vascular Endothelial Growth Factor A; D051176:beta Catenin; C021751:tanshinone; D000071679:Glycogen Synthase Kinase 3 beta; C000605507:Gsk3b protein, mouse; D038362:Glycogen Synthase Kinase 3