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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 investigate the mechanism of octapeptide cholecystokinin (CCK 8) regulating lipopolysaccharide (LPS) -induced nuclear factor kappa B (NFκB) expression in vascular endothelial cells. Methods Cultured human umbilical vein endothelial cell line ECV 304 was treated with solvent (saline), LPS, CCK 8, CCK receptor antagonist (CCK R) non-specific antagonist valproic acid, CCK A receptor (CCK AR) specific antagonist CR 1409, CR 2945, a CCK B receptor (CCK BR) specific antagonist, stimulated ECV 304 cells for 1 h, respectively. The expression of NFκB p65 protein was detected by Western blotting. The nuclear translocation of NFκB p65 protein was detected by immunocytochemistry. Results Compared with the solvent control group, LPS induced the nuclear translocation of NFκB p65 protein in ECV 304 cells and its expression was significantly up-regulated. CCK 8 inhibited LPS-induced nuclear translocation and up-regulation in a dose-dependent manner. CCK receptor antagonist The above inhibitory effect of CCK 8 can be reversed, in which the effects of CR 1409, CR 2945 and proglumide are successively enhanced. Conclusions CCK AR and CCK BR are involved in the mediation of the inhibitory effect of CCK 8 on LPS-induced NFκB expression in ECV 304 cells. CCK BR is slightly stronger than CCK AR.