脂肪细胞通过TNF-α-p38 MAPK信号通路促进内皮细胞ICAM-1表达

Adipocytes promote the expression of ICAM-CAM-1 in endothelial cells through TNF-α-p38 MAPK signal pathpathway

目的血管内皮细胞功能紊乱是腹膜透析患者心血管疾病(cardiovascular disease,CVD)发生的重要原因。本研究模拟腹膜透析液的高糖环境,观察高糖作用下脂肪细胞对微血管内皮细胞细胞间粘附分子-1(intercellular adhesion molecule,ICAM-1)表达的影响,探讨腹膜透析患者内皮功能紊乱导致CVD发生的可能机制。方法体外诱导3T3-L1分化成为脂肪细胞。将实验所用内皮细胞分成6组,分别是:对照组,高糖组,高渗组,对照脂肪细胞组(普通培养基培养脂肪细胞所得上清干预内皮细胞),高糖脂肪细胞组(高糖培养脂肪细胞所得上清干预内皮细胞)及高渗脂肪细胞组(高渗培养脂肪细胞所得上清作干预内皮细胞)。细胞增殖检测试剂盒检测b End.3增殖能力。酶联免疫吸附试验检测脂肪细胞高糖、高渗作用后上清中肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)表达量的改变。Western blot法检测脂肪细胞条件培养基对内皮细胞ICAM-1表达量的影响,及加用丝裂原激活的蛋白激酶(mitogen-activated protein kinase,MAPK)信号通路阻断剂SB203580作用于内皮细胞后ICAM-1表达量的变化。结果与对照组相比,高糖培养液直接干预内皮细胞或培养脂肪细胞后干预内皮细胞均可以促进内皮细胞增殖(高糖组/对照组:1.600±0.104比1.000±0.000,t=9.954,P=0.010;高糖脂肪细胞组/对照脂肪细胞组:1.563±0.181比1.213±0.097,t=2.945,P=0.042)。高糖可显著促进脂肪细胞TNF-α的表达(2515.313±277.434比788.683±167.267,t=9.232,P=0.001)。高糖培养脂肪细胞的上清显著促进内皮细胞ICAM-1表达(1.533±0.058比1.133±0.153,t=4.243,P=0.013),该作用可被p38 MAPK信号通路阻断剂SB203580部分抑制(0.850±0.111比1.124±0.108,t=-3.053,P=0.038)。结论高糖培养脂肪细胞后,可通过TNF-α-p38 MAPK信号通路促进内皮细胞ICAM-1表达,可能和腹膜透析患者内皮细胞功能紊乱及CVD发生有关。

Objectives Endothelial dysfunction is the main cause of cardiovascular disease（CVD） in peritoneal dialysis（PD） patients. This study mimicked the high glucose condition of dialysate, and observed the role of adipocytes on the expression of ICAM-1 in endothelial cells, aiming to explore the mechanism of endothelial dysfunction. Methods 3T3-L1 cells were differentiated into mature adipocytes. Endothelial cells（ECs） were divided into six groups according to the medium used： blank control group（ECs cultured in DMEM）, high glucose group（ECs treated with 139 mmol/L glucose）, high mannitol group（ECs treated with139 mmol/L mannitol）, control-adipocyte group（ECs treated with the supernatant of adipocytes in DMEM）,glucose-adipocyte group（ECs treated with the supernatant of adipocytes in higher glucose concentration）, and mannitol-adipocyte group（ECs treated with the supernatant of adipocytes in higher mannitol concentration）.Proliferation of ECs in 24 h was measured by cell counting kit-8（CCK-8） assay. Expression of tumor necrosis factor-α（TNF-α） by adipocytes was measured by enzyme-linked immunosorbent assay（ELISA）. ICAM-1 level with or without the addition of SB203580（a blocker of p38 mitogen- activated protein kinase, MAPK） in ECs was detected by western blot. Results More than 90% of the 3T3- L1 cells differentiated into adipocytes. The proliferation of ECs was promoted by the treatment of ECs with high glucose, high mannitol, super-natant of adipocytes in high glucose or high mannitol（high glucose group/control group, 1.600 ± 0.104 vs.1.000±0.000, t=9.954, P=0.010; high mannitol group/control group, 1.240±0.100 vs. 1.000±0.000, t=4.157, P=0.014; glucose- adipocyte group/control- adipocyte group, 1.563 ± 0.181 vs. 1.213 ± 0.097, t=2.945, P=0.042;mannitol- adipocyte group/control- adipocyte group, 1.520 ± 1.473 vs. 1.213 ± 0.097, t=3.010, P=0.040）. High glucose significantly promoted the production of TNF- α in adipocytes（2515.313 ± 277.434 vs. 788.683 ±167.267, t=9.232, P=0.001）. The supernatant of high glucose treated adipocytes significantly promoted the expression of ICAM-1 in endothelial cells（1.533±0.058 vs. 1.133±0.153, t=4.243, P=0.013）, and this promotion was partially inhibited by the p38 MAPK blocker SB203580（0.850 ± 0.111 vs. 1.124 ± 0.108, t=- 3.053, P=0.038）. Conclusion Adipocytes exposed to high glucose promoted the expression of ICAM-1 in endothelial cells through TNF-α- p38 MAPK signal pathway. This process may relate to endothelial dysfunction and cardiovascular events in PD patients.