八肽缩胆囊素调节脂多糖诱导ECV-304细胞核转录因子-κB表达的受体机制研究

Receptor mechanisms underlying the modulation of lipopolysaccharide-induced nuclear factor-κB expression in vascular endothelial cells by cholecystokinin octapeptide

目的探讨八肽缩胆囊素(CCK 8)调节脂多糖(LPS)诱导血管内皮细胞核转录因子κB(NFκB)表达的受体机制。方法培养人脐静脉内皮细胞株ECV 304;用溶剂(生理盐水)、LPS、CCK 8、CCK受体(CCK R)非特异性拮抗剂丙谷胺、CCK A受体(CCK AR)特异性拮抗剂CR 1409、CCK B受体(CCK BR)特异性拮抗剂CR 2945分别或联合刺激ECV 304细胞1 h。用蛋白质免疫印迹法(W esternb lot)检测NFκB p65蛋白表达;用免疫细胞化学技术检测NFκB p65蛋白核移位。结果与溶剂对照组比较,LPS可诱导ECV 304细胞NFκB p65蛋白核移位,且其表达明显上调;CCK 8可呈剂量依赖性地抑制LPS诱导的核移位及表达上调;CCK受体拮抗剂可翻转CCK 8的上述抑制效应,其中CR 1409、CR 2945、丙谷胺作用依次增强。结论CCK AR和CCK BR参与介导了CCK 8对LPS诱导ECV 304细胞NFκB表达的抑制作用,其中CCK BR的作用比CCK AR稍强。

Objective To elucidate the receptor mechanisms underlying the modulation of lipopolysaccharide （LPS）-induced nuclear factor -κB （NF -κB） expression in human umbilical vein endothelial cell line ECV - 304 cells by cholecystokinin octapeptide （CCK - 8）. Methods Human umbilical vein endothelial cell line ECV -304 cells were stimulated with vehicle, LPS, CCK - 8 （10^-9- 10^-7mol/L）, CCK receptor non - specific antagonist proglumide, CCK -A receptor （CCK -AR） specific antagonist CR -1409 or CCK- B receptor （CCK - BR） specific antagonist CR - 2945 singularly or in combination. The NF -κB p65 protein level was determined by Western blot and immunocytochemistry technique. Results LPS resulted in an increase in the up - regulatory expression and nuclear translocation of NF -κB p65 protein in ECV 304 compared with vehicle stimulation. CCK - 8 obviously inhibited LPS - induced the changes in NF-κB p65 protein in a dose - dependent manner. The inhibitory effects of CCK - 8 on NF -κB p65 protein expression were attenuated by proglumide〉CR - 2945〉CR - 1409. Conclusion CCK - AR and CCK - BR are involved in the mediation of CCK - 8 inhibitive regulation for LPS - induced NF-κB protein expression in ECV - 304 cells, whereas the effect of CCK -BR are more than that of CCK AR.