title: Isoflurane preconditioning increases survival of rat skin random-pattern flaps by induction of HIF-1α expression.
abstract: BACKGROUND
  Survival of random-pattern skin flaps is important for the success of plastic and reconstructive surgeries. This study investigates isoflurane-induced protection against ischemia of skin flap and the underlying molecular mechanism in this process.
  
  
  METHODS
  Human umbilical vein endothelial cells (HUVECs) and human skin fibroblast cells were exposed to isoflurane for 4 h. Expression of hypoxia inducible factor-1α (HIF-1α), heme oxygenase-1 (HO-1) and vascular endothelial growth factor (VEGF) were analyzed up to 24 h post isoflurane exposure using qRT-PCR and western blot, or ELISA analyses. PI3K inhibitors--LY 294002 and wortmannin, mTOR inhibitor--rapamycin, and GSK3β inhibitor--SB 216763 were used respectively to assess the effects of isoflurane treatment and HIF-1α expression. Furthermore, 40 rats were randomly divided into 5 groups (control, isoflurane, scrambled siRNA plus isoflurane, HIF-1α siRNA plus isoflurane, and DMOG) and subjected to random-pattern skin flaps operation. Rats were prepared for evaluation of flap survival and full-feld laser perfusion imager (FLPI) (at 7 day) and microvessel density evaluation (at 10 day).
  
  
  RESULTS
  Isoflurane exposure induced expression of HIF-1α protein, HO-1 and VEGF mRNA and proteins in a time-dependent manner. Both LY 294002 and wortmannin inhibited phospho-Akt, phospho-mTOR, phospho-GSK 3β and HIF-1α expression after isoflurane exposure. Both wortmannin and rapamycin inhibited isoflurane-induced phospho-4E-BP1 (Ser 65) and phospho-P70(s6k) (Thr 389) and HIF-1α expression. SB 216763 pre-treatment could further enhance isoflurane-induced expression of phospho-GSK 3β (Ser 9) and HIF-1α protein compared to the isoflurane-alone cells. In animal experiments, isoflurane alone, scrambled siRNA plus isoflurane, or DMOG groups had significantly upregulated vascularity and increased survival of the skin flaps compared to the controls. However, HIF-1α knockdown abrogated the protective effect of isoflurane preconditioning in rats.
  
  
  CONCLUSIONS
  Isoflurane preconditioning improves survival of skin flaps by up the regulation of HIF-1α expression via Akt-mTOR and Akt-GSK 3β signaling pathways.
authors: Department of Anesthesiology, Shanghai Ninth People's Hospital Affiliated to Shanghai Jiao Tong University, School of Medicine, Shanghai.
authors: Yu
authors: Y
authors: Sun
authors: Qi-Fang
authors: QF
authors: Li
authors: Ying
authors: Y
authors: Zhang
authors: Rong
authors: R
authors: Hu
authors: Hong
authors: H
authors: Jiang
mesh_terms: D000818:Animals; D002460:Cell Line; D005786:Gene Expression Regulation / Q000187:drug effects; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D051547:Heme Oxygenase-1 / Q000235:genetics / Q000378:metabolism; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D019194:Ischemic Preconditioning; D007530:Isoflurane / Q000494:pharmacology*; D008297:Male; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D055550:Protein Stability / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000187:drug effects; D012867:Skin / Q000473:pathology; D013524:Surgical Flaps / Q000473:pathology; D058570:TOR Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D014022:Tissue Survival / Q000187:drug effects*; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism
chemical_list: D038362:Glycogen Synthase Kinase 3; D000071679:Glycogen Synthase Kinase 3 beta; D051547:Heme Oxygenase-1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007530:Isoflurane; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D051057:Proto-Oncogene Proteins c-akt; D034741:RNA, Small Interfering; D058570:TOR Serine-Threonine Kinases; D042461:Vascular Endothelial Growth Factor A; C000605506:GSK3B protein, human; C000605508:Gsk3b protein, rat