title: Heat shock pretreatment improves stem cell repair following ischemia-reperfusion injury via autophagy.
abstract: OBJECTIVE
  To investigate whether heat shock pretreatment (HSP) improves mesenchymal stem cell (MSC) repair via autophagy following hepatic ischemia-reperfusion injury (HIRI).
  
  
  METHODS
  Apoptosis of MSCs was induced by 250 mM hydrogen peroxide (H2O2) for 6 h. HSP was carried out using a 42 °C water bath for 1, 2 or 3 h. Apoptosis of MSCs was analyzed by flow cytometry, and Western blot was used to detect Bcl-2, Bax and cytochrome C expression. Autophagy of MSCs was analyzed by flow cytometry and transmission electron microscopy, and the expression of beclin I and LC3-II was detected by Western blot. MSCs were labeled in vivo with the fluorescent dye, CM-Dil, and subsequently transplanted into the portal veins of rats that had undergone HIRI. Liver levels of proliferating cell nuclear antigen (PCNA) were quantified by fluorescent microscopy. Serum aminotransferase activity and the extent of HIRI were also assessed at each time point.
  
  
  RESULTS
  HSP for 2 h reduced apoptosis of MSCs induced by H2O2 as seen by a decrease in apoptotic rate, a decrease in Bax and cytochrome C expression and an increase in Bcl-2 expression (P < 0.001). In addition, HSP for 2 h induced autophagy of MSCs exposed to H2O2 as shown by an increase in acidic vesicular organelle-positive cells, beclin 1 and LC3-II expression, and autophagosome formation (P < 0.05). Treatment with 3-methyladenine attenuated HSP-induced autophagy and abolished the protective effects of HSP on the apoptosis of MSCs. Rapamycin failed to have additional effects on either autophagy or apoptosis compared with HSP alone. The phosphorylation of p38MAPK was significantly elevated and the phosphorylation of mTOR was downregulated in heat shock pretreated MSCs. Treatment with the p38MAPK inhibitor, SB203580, reduced HSP-induced autophagy in MSCs. In vivo studies showed that the transplantation of HSP-MSCs resulted in lower serum aminotransferase levels, lower Suzuki scores, improved histopathology and an increase in PCNA-positive cells (P < 0.05).
  
  
  CONCLUSIONS
  HSP effectively induces autophagy following exposure to H2O2 via the p38MAPK/mTOR pathway, which leads to enhanced MSC survival and improved MSC repair following HIRI in rats.
authors.affiliation: Peng-Fei Qiao, Lei Yao, Xin-Chen Zhang, De-Quan Wu, Department of General Surgery, the Second Affiliated Hospital of Harbin Medical University, Harbin 150001, Heilongjiang Province, China.
authors.forename: Peng-Fei
authors.initials: PF
authors.lastname: Qiao
authors.affiliation: Peng-Fei Qiao, Lei Yao, Xin-Chen Zhang, De-Quan Wu, Department of General Surgery, the Second Affiliated Hospital of Harbin Medical University, Harbin 150001, Heilongjiang Province, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Yao
authors.affiliation: Peng-Fei Qiao, Lei Yao, Xin-Chen Zhang, De-Quan Wu, Department of General Surgery, the Second Affiliated Hospital of Harbin Medical University, Harbin 150001, Heilongjiang Province, China.
authors.forename: Xin-Chen
authors.initials: XC
authors.lastname: Zhang
authors.affiliation: Peng-Fei Qiao, Lei Yao, Xin-Chen Zhang, De-Quan Wu, Department of General Surgery, the Second Affiliated Hospital of Harbin Medical University, Harbin 150001, Heilongjiang Province, China.
authors.forename: Guo-Dong
authors.initials: GD
authors.lastname: Li
authors.affiliation: Peng-Fei Qiao, Lei Yao, Xin-Chen Zhang, De-Quan Wu, Department of General Surgery, the Second Affiliated Hospital of Harbin Medical University, Harbin 150001, Heilongjiang Province, China.
authors.forename: De-Quan
authors.initials: DQ
authors.lastname: Wu
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D001343:Autophagy / Q000187:drug effects; D000071186:Beclin-1; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004195:Disease Models, Animal; D005434:Flow Cytometry; D018869:Heat-Shock Response; D006358:Hot Temperature; D006861:Hydrogen Peroxide / Q000633:toxicity; D008107:Liver Diseases / Q000378:metabolism / Q000473:pathology / Q000601:surgery*; D008297:Male; D045164:Mesenchymal Stem Cell Transplantation; D059630:Mesenchymal Stem Cells / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure*; D046529:Microscopy, Electron, Transmission; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D017207:Rats, Sprague-Dawley; D015427:Reperfusion Injury / Q000378:metabolism / Q000473:pathology; D015398:Signal Transduction / Q000187:drug effects; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism; D013997:Time Factors; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
chemical_list: D051017:Apoptosis Regulatory Proteins; D000071186:Beclin-1; D006861:Hydrogen Peroxide; D008869:Microtubule-Associated Proteins; D058570:TOR Serine-Threonine Kinases; D048051:p38 Mitogen-Activated Protein Kinases; C512829:Becn1 protein, rat; C526109:Anxa3 protein, rat; C546845:mTOR protein, rat
keywords: Autophagy; Heat shock pretreatment; Hepatic ischemia-reperfusion injury; Mesenchymal stem cells; Transplantation