肝素通过核转录因子-κB信号通路减少脂多糖刺激人内皮细胞趋化因子的表达  

Unfractionated heparin inhibits lipopolysaccharide-induced expression of chemokines in hμman endothelial cells through nuclear factor-κB signaling pathway

作　　者：李旭[1] 马延全 陈恬璐 唐洁[1] 马晓春[1] 

机构地区：[1]中国医科大学附属第一医院重症医学科,辽宁沈阳110001 [2]辽河油田总医院重症医学科,辽宁盘锦124010

出　　处：《中华危重病急救医学》2016年第2期117-121,共5页Chinese Critical Care Medicine

基　　金：国家自然科学基金（81101411）;辽宁省自然科学基金（2013021061）

摘　　要：目的观察肝素对脂多糖（LPS）刺激人内皮细胞分泌趋化因子水平的影响，探讨肝素对核转录因子--κB（NF-κB）信号通路的作用机制。方法体外培养人肺微血管内皮细胞株（HPMEC），取传代培养至第3—5代的细胞用于实验。将细胞分成对照组、LPS刺激组、1kU／L或10kU／L肝素＋LPS组及NF-κB抑制剂甲苯磺酰赖氨酸氯甲酮（TLCK）组（TLCK＋LPS组）。LPS刺激组加LPS10mg／L；不同剂量肝素预处理组于LPS刺激前15rain分别加入1ku／L或10kU／L的普通肝素；TLCK＋LPS组于LPS刺激前30min加用TLCK10μmol／L；对照组加入与LPS等量的磷酸盐缓冲液（PBS）。各组于LPS刺激后1h收集细胞，采用免疫荧光法观察NF-κB核移位情况，以明确肝素对NF-κB的活化作用；LPS刺激后3h和6h收集细胞上清液，采用酶联免疫吸附试验（ELISA）测定白细胞介素-8（IL-8）及单核细胞趋化蛋白-1（MCP-1）的水平，以明确肝素对LPS刺激HPMEC产生趋化因子的影响以及对NF-κB信号通路的作用机制。结果①荧光显微镜下显示，对照组NF-κB大部分位于胞质中；LPS刺激组NF-κB由胞质向胞核转移明显增多；1kU／L和10ku／L肝素预处理均可明显抑制LPS诱导的NF-κB活化，以10kU／L肝素效果较好。②与对照组比较，LPS刺激后3h和6h细胞上清液中IL-8及MCP-1水平均明显增高[IL-8（ng／L）：3h为387．1±26．4比23．8±8．1，6h为645．5±69．6比125．7±18．7；MCP-1（ng／L）：3h为3654．9±467．9比721．6±61．3，6h为8178．5±792．6比1324．7±148．7，均P〈0．05]；与LPS刺激组比较，1kU／L和10ku，IJ肝素预处理均可明显降低IL-8及MCP-1水平[IL-8（ng／L）：3h为315．3±24．8、275．8±31．1比387．1±26．4，6h为557．8±43-3、496．9±38．7比645．5±69．6：MCP-1（ng／L）：3h为2924．1±267．9、2668．3±522．6比3654．9±467．9，6h为7121．7±557．2、6563．9±576．4比8178．5±792．6。均P〈O�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 Hμman pulmonary microvascular endothelial ceils （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 ehloromethyl-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 10μmol/L of TLCK 30 minutes before the addition of LPS, and HPMECs in control group were treated with an equal volμme of phosphate-buffered saline （PBS） instead. The cells were harvested 1 hour after LPS challenge, and the nuclear translocation of NF-KB was determined by immunofluorescence assay to detect the effect of UFH on NF-KB 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-KB 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 supematants 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.

关 键 词：内皮细胞 普通肝素 核转录因子-ΚB 白细胞介素-8 单核细胞趋化蛋白-1 

分 类 号：R459.7[医药卫生—急诊医学]