title: [Combined effect of high mobile group box-1 protein and lipopolysaccharide on release of cytokines from human HepG2 cells].
abstract: OBJECTIVE
  To study the effects of high mobile group box-1 protein (HMGB1) and lipopolysaccharide (LPS) singly or in combination on release of cytokines from human liver carcinoma cell line (HepG2).
  
  
  METHODS
  HepG2 cells were cultured, and purified HMGB1 protein was prepared by chromatography on Ni(2+)-NTA Sepharose column under natural conditions with recombinant expression plasmid pET14b-HMGB1. Different concentrations of HMGB1 (0, 0.01, 0.1, 1, 10 mg/L) and LPS (0, 0.1, 1, 10, 100 mg/L) were added into the cultured cells for 24 hours, respectively. Then the supernatant were collected to detect the levels of granulocyte/macrophage colony stimulating factor (GM-CSF), interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), and interleukin-1 beta (IL-1 beta), IL-2, IL-4, IL-6, IL-8, IL-10, and IL-12 by using LiquiChip system. Lastly, HepG2 cells were co-stimulated with 10 mg/L LPS and 1 mg/L HMGB1 for 24 hours. The supernatant were collected to determine the levels of above ten of cytokines .
  
  
  RESULTS
  The expression and release of IL-6 and IL-8 increased from HepG2 cells after being stimulated by LPS in a dose-dependent manner, but there were no changes in other eight kinds of cytokines (P<0.05 or P<0.01). Low concentration of HMGB1 could up-regulate the expression of IL-6 and IL-8 in HepG2 cells (both P<0.01). But the extent of induction decreased with higher concentration of HMGB1. Similar to LPS, there was no effect of HMGB1 on the expression of other eight kinds of cytokines from cultured HepG2 cells. Furthermore, high concentration of HMGB1 could obviously inhibit the upregulation of IL-6 and IL-8 by high concentration of LPS when the HepG2 cells were co-stimulated with LPS and HMGB1 (both P<0.01).
  
  
  CONCLUSIONS
  HepG2 cells could only express and release a few kinds of cytokines when the cells were stimulated with pro-inflammatory agents, such as LPS or HMGB1. Two kinds of cytokines, IL-6 and IL-8 could be up-regulated by LPS and low concentration of HMGB1, and HMGB1 acted as an inhibitor of LPS to down-regulate the expression and release of IL-6 and IL-8 from HepG2 cells.
authors.affiliation: Department of Intensive Care Unit, Guangzhou General Hospital of Guangzhou Military Command, Guangdong, China.
authors.forename: Zhi-feng
authors.initials: ZF
authors.lastname: Liu
authors.forename: Juan
authors.initials: J
authors.lastname: Wang
authors.forename: Zheng
authors.initials: Z
authors.lastname: Liu
authors.forename: You-qing
authors.initials: YQ
authors.lastname: Tang
authors.forename: Fan-su
authors.initials: FS
authors.lastname: Meng
authors.forename: Jing-hua
authors.initials: JH
authors.lastname: Liu
authors.forename: Lei
authors.initials: L
authors.lastname: Su
mesh_terms: D016207:Cytokines / Q000378:metabolism*; D024243:HMGB1 Protein / Q000494:pharmacology*; D056945:Hep G2 Cells; D006801:Humans; D015850:Interleukin-6 / Q000378:metabolism*; D016209:Interleukin-8 / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology*; D018805:Sepsis / Q000378:metabolism / Q000503:physiopathology; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: C508606:CXCL8 protein, human; D016207:Cytokines; D024243:HMGB1 Protein; C508600:IL6 protein, human; D015850:Interleukin-6; D016209:Interleukin-8; D008070:Lipopolysaccharides; D014409:Tumor Necrosis Factor-alpha