title: [Effects of lipopolysaccharide pretreatment on endotoxin tolerance of human umbilical cord mesenchymal stem cells].
abstract: OBJECTIVE
  To explore the effects of lipopolysaccharide (LPS) pretreatment on endotoxin tolerance of human umbilical cord mesenchymal stem cells (hUCMSCs) and its possible mechanism.
  
  
  METHODS
  hUCMSCs (1×10(4) cells/well) were exposed to 0, 0.1, 1.0, 10.0, 20.0, 30.0, 40.0, 50.0 µg/ml LPS for 24 h respectively. And the cell viability of hUCMSCs was detected by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT). 1 µg/ml and 50.0 µg/ml LPS were used as pretreatment and apoptosis induction concentrations respectively. Pyrrolidine dithiocarbamate (PDTC) (20 µmol/L, pretreatment for 20 min) was used as a specific inhibitor of nuclear transcription factor NF-κB. hUCMSCs were randomly divided by Stata software into 7 groups: control (A), LPS induction (B), pretreatment + LPS induction (C), PDTC (D), PDTC+ pretreatment + LPS induction (E), pretreatment (F) and PDTC + pretreatment (G). The apoptosis of hUCMSCs was measured by Hoechst 33258 staining and flow cytometry (FCM). The expressions of NF-κB p65 and cellular FLICE-inhibitory protein (c-FLIP) were measured by Western blot.
  
  
  RESULTS
  The cell viability of 0, 0.1, 1.0, 10.0, 20.0, 30.0, 40.0, 50.0 µg/ml LPS groups were 100%, (117.0 ± 8.8)%, (134.7 ± 6.9)%, (105.3 ± 8.3)%, (99.2 ± 8.3)%, (84.2 ± 9.3)%, (66.4 ± 6.6)% and (59.2 ± 8.0)% respectively. In comparison with 0 µg/ml LPS group, the cell viability of 1.0 µg/ml LPS group increased significantly (P = 0.004) while decreased in 40 and 50 µg/ml LPS groups (P = 0.005, 0.002). Hoechst 33258 staining indicated that chromatin of hUCMSCs was distributed evenly in group A; the apoptotic cell in group B dramatically increased; and the apoptotic cell in group C significantly decreased in comparison with that in group B. Apoptotic rates of groups A, B, C, D and E were (2.8 ± 0.8)%, (29.7 ± 3.4)%, (17.8 ± 3.0)%, (2.9 ± 0.4)% and (23.2 ± 2.6)% respectively. Compared with group A, apoptosis rate significantly increased in group B (P < 0.001). The apoptotic rate in group C significantly decreased than that in group B (P < 0.001) while group E was higher than group C (P = 0.015). The levels of NF-κB p65 and c-FLIP in group F (0.851 ± 0.031, 0.534 ± 0.053) was higher than that in group A (0.220 ± 0.021, 0.049 ± 0.009) (both P < 0.001), G (0.418 ± 0.007, 0.299 ± 0.061) (P < 0.001, P = 0.007).
  
  
  CONCLUSIONS
  LPS pretreatment can resist LPS-induced hUCMSCs apoptosis and enhance the ability of endotoxin tolerance. And the mechanism may be related with activating the NF-κB signaling pathway and up-regulating the expression of c-FLIP.
authors.affiliation: Department of Burn & Plastic Surgery, First Affiliated Hospital, PLA General Hospital, Beijing 100048, China.
authors.forename: Yusen
authors.initials: Y
authors.lastname: Hou
authors.affiliation: Department of Burn & Plastic Surgery, First Affiliated Hospital, PLA General Hospital, Beijing 100048, China. Email: cjk304@126.com.
authors.forename: Jiake
authors.initials: J
authors.lastname: Chai
authors.affiliation: Department of Burn & Plastic Surgery, First Affiliated Hospital, PLA General Hospital, Beijing 100048, China.
authors.forename: Lingying
authors.initials: L
authors.lastname: Liu
authors.affiliation: Department of Burn & Plastic Surgery, First Affiliated Hospital, PLA General Hospital, Beijing 100048, China.
authors.forename: Yonghui
authors.initials: Y
authors.lastname: Yu
authors.affiliation: Department of Burn & Plastic Surgery, First Affiliated Hospital, PLA General Hospital, Beijing 100048, China.
authors.forename: Hongjie
authors.initials: H
authors.lastname: Duan
authors.affiliation: Department of Burn & Plastic Surgery, First Affiliated Hospital, PLA General Hospital, Beijing 100048, China.
authors.forename: Quan
authors.initials: Q
authors.lastname: Hu
authors.affiliation: Department of Burn & Plastic Surgery, First Affiliated Hospital, PLA General Hospital, Beijing 100048, China.
authors.forename: Wanli
authors.initials: W
authors.lastname: Chu
authors.affiliation: Department of Burn & Plastic Surgery, First Affiliated Hospital, PLA General Hospital, Beijing 100048, China.
authors.forename: Li
authors.initials: L
authors.lastname: Ma
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004731:Endotoxins / Q000009:adverse effects*; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology*; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000187:drug effects*; D016328:NF-kappa B / Q000378:metabolism; D015398:Signal Transduction; D014470:Umbilical Cord / Q000166:cytology
chemical_list: D004731:Endotoxins; D008070:Lipopolysaccharides; D016328:NF-kappa B