Glucan phosphate inhibits HMGB-1 release from rat myocardial H9C2 cells in sepsis via TLR4/NF-кB signal pathway

PURPOSE: The effect of glucan phosphate (GP) on the release of HMGB-1 from rat myocardial cells (H9C2) during lipopolysaccharide-induced sepsis, and the underlying mechanisms, were investigated. METHODS: H9C2 cells were divided into three groups: normal; lipopolysaccharide (LPS) (1 mg/ml LPS); and, LPS+GP (2 mg/ml GP). Western blot was used to determine toll-like receptor 4 (TLR4) levels, and electrophoretic mobility-shift assays (EMSA) was used to determine nuclear factor-кB (NF-кB) activity 3, 6 and 9 h after treatment. HMGB-1 mRNA levels in cultured cells were determined by real-time PCR and supernatant HMGB-1 protein levels were evaluated by ELISA at 12, 24, 36 and 48 h after treatment. Following the transfection of H9C2 cells with Ad5-IкBα, which inhibits NF-кB activity, TLR4, NF-кB and HMGB-1 levels were determined. RESULTS: Intracellular TLR4 levels and NF-кB activity in LPS and LPS+GP groups increased 3-9 h after stimulation, but the increased levels of TLR4 and elevated activity of NF-кB were significantly lower in the LPS+GP group vs. the LPS group. HMGB-1 mRNA levels in both LPS and LPS+GP groups, increased gradually from 24 h after stimulation, but the increase was more obvious in the LPS group vs. the LPS+GP group. Supernatant HMGB-1 levels in the LPS and LPS+GP groups increased gradually from 9 h after stimulation, and also increased markedly in the LPS group. After the inhibition of NF-кB activity, LPS-induced HMGB-1 release decreased significantly (p.