title: [Unfractionated heparin inhibits lipopolysaccharide-induced expression of chemokines in human endothelial cells through nuclear factor-ΚB signaling pathway].
abstract: OBJECTIVE
  To determine the effect of unfractionated heparin (UFH) on lipopolysaccharide (LPS)-induced expression of chemokines and nuclear factor-ΚB (NF-ΚB) signaling pathway.
  
  
  METHODS
  Human pulmonary microvascular endothelial cells (HPMECs) were cultured in vitro, and the cells between passages 3 and 5 were used in the experiments. The cells were divided into control group, LPS challenge group, 1 kU/L or 10 kU/L UFH+LPS group, and NF-ΚB inhibitor N-tosyl-L-lysyl chloromethyl-ketone (TLCK) group (TLCK+LPS group). HPMECs in LPS challenge group were treated with 10 mg/L LPS. UFH pretreatment with different dosages groups were treated with 1 kU/L or 10 kU/L UFH 15 minutes before LPS challenge. Cells in the TLCK+LPS group were treated with 10 μmol/L of TLCK 30 minutes before the addition of LPS, and HPMECs in control group were treated with an equal volume of phosphate-buffered saline (PBS) instead. The cells were harvested 1 hour after LPS challenge, and the nuclear translocation of NF-ΚB was determined by immunofluorescence assay to detect the effect of UFH on NF-ΚB activation. The levels of interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) in cell culture supernatants were determined by enzyme linked immunosorbent assay (ELISA) 3 hours and 6 hours after LPS challenge to detect the effect of UFH on LPS induced expression of chemokines and its mechanism of effect on NF-ΚB signaling pathway in HPMECs.
  
  
  RESULTS
  (1) In the control group, NF-ΚB was mostly located in the cytosol as shown by immunofluorescence. Treatment of HPMECs with LPS significantly increased the translocation of NF-ΚB from the cytosol to nucleus. UFH suppressed LPS-induced NF-ΚB activation both in 1 kU/L and 10 kU/L dosages, and 10 kU/L UFH gave even better results. (2) Compared with control group, the levels of IL-8 and MCP-1 in the supernatants in LPS challenge group were significantly increased at 3 hours and 6 hours after LPS challenge [IL-8 (ng/L): 387.1±26.4 vs. 23.8±8.1 at 3 hours, 645.5±69.6 vs. 125.7±18.7 at 6 hours; MCP-1 (ng/L): 3 654.9±467.9 vs. 721.6±61.3 at 3 hours, 8 178.5±792.6 vs. 1 324.7±148.7 at 6 hours, all P < 0.05]. Compared with that of LPS challenge group, in 1 kU/L and 10 kU/L UFH pretreatment groups, the levels of IL-8 and MCP-1 were significantly decreased [IL-8 (ng/L): 315.3±24.8, 275.8±31.1 vs. 387.1±26.4 at 3 hours, 557.8±43.3, 496.9±38.7 vs. 645.5±69.6 at 6 hours; MCP-1 (ng/L): 2 924.1±267.9, 2 668.3±522.6 vs. 3 654.9±467.9 at 3 hours, 7 121.7±557.2, 6 563.9±576.4 vs. 8 178.5±792.6 at 6 hours, all P < 0.05]. The results indicated that 10 kU/L UFH yielded better results. However, inhibition study using the known NF-ΚB inhibitor TLCK could decrease LPS-induced increase in IL-8 and MCP-1 levels [IL-8 (ng/L): 162.4±21.3 vs. 387.1±26.4 at 3 hours, 274.1±22.6 vs. 645.5±69.6 at 6 hours; MCP-1 (ng/L): 1 478.2±138.5 vs. 3 654.9±467.9 at 3 hours; 3 667.6±259.4 vs. 8 178.5±792.6 at 6 hours, all P < 0.05].
  
  
  CONCLUSIONS
  The levels of IL-8 and MCP-1 were increased obviously in LPS treated HPMECs. UFH might suppress LPS-activated NF-ΚB signaling pathway, contributing to the inhibitory effects of chemokines in HPMECs.
authors.affiliation: Department of Critical Care Medicine, the First Affiliated Hospital of China Medical University, Shenyang 110001, Liaoning, China (Li X, Chen TL, Tang J, Ma XC); Department of Critical Care Medicine, General Hospital of Panjin Oilfield, Panjin 124010, Liaoning, China (Ma YQ). Corresponding author: Ma Xiaochun, Email: xcma2972@sina.com.
authors.forename: Xu
authors.initials: X
authors.lastname: Li
authors.forename: Yanquan
authors.initials: Y
authors.lastname: Ma
authors.forename: Tianlu
authors.initials: T
authors.lastname: Chen
authors.forename: Jie
authors.initials: J
authors.lastname: Tang
authors.forename: Xiaochun
authors.initials: X
authors.lastname: Ma
mesh_terms: D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000378:metabolism*; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects*; D006493:Heparin / Q000494:pharmacology*; D006801:Humans; D016209:Interleukin-8 / Q000378:metabolism*; D008070:Lipopolysaccharides; D008168:Lung / Q000166:cytology; D016328:NF-kappa B / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: C111835:CCL2 protein, human; C508606:CXCL8 protein, human; D018932:Chemokine CCL2; D016209:Interleukin-8; D008070:Lipopolysaccharides; D016328:NF-kappa B; D006493:Heparin