Wang Haizhu, Cui Zhifei, Sun Fei, Ding Huayong
Department of Cardiology, Zhoukou Central Hospital, Zhoukou 466100, China.
Clin Invest Med. 2017 Apr 26;40(2):E66-E72. doi: 10.25011/cim.v40i2.28197.
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.
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.
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.
研究磷酸葡聚糖（GP）对脂多糖诱导的脓毒症大鼠心肌细胞（H9C2）中高迁移率族蛋白B1（HMGB-1）释放的影响及其潜在机制。
将H9C2细胞分为三组：正常组；脂多糖（LPS）组（1mg/ml LPS）；LPS+GP组（2mg/ml GP）。采用蛋白质免疫印迹法检测Toll样受体4（TLR4）水平，采用电泳迁移率变动分析（EMSA）检测处理后3、6和9小时的核因子-κB（NF-κB）活性。通过实时聚合酶链反应（PCR）测定培养细胞中HMGB-1 mRNA水平，并在处理后12、24、36和48小时通过酶联免疫吸附测定（ELISA）评估上清液中HMGB-1蛋白水平。在用抑制NF-κB活性的腺病毒5-IκBα转染H9C2细胞后，测定TLR4、NF-κB和HMGB-1水平。
LPS组和LPS+GP组细胞内TLR4水平和NF-κB活性在刺激后3-9小时升高，但LPS+GP组TLR4升高水平和NF-κB活性升高程度明显低于LPS组。LPS组和LPS+GP组HMGB-1 mRNA水平均在刺激后24小时开始逐渐升高，但LPS组升高更明显。LPS组和LPS+GP组上清液中HMGB-1水平从刺激后9小时开始逐渐升高，LPS组升高更显著。抑制NF-κB活性后，LPS诱导的HMGB-1释放显著降低（p.
