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).
Conclusion: 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.