Objective To explore the role of chromogranin A (CGA) derived peptide CGA47-66 (Chromfungin,CHR) on septic serum induced high permeability of vascular endothelial cells.Methods Human umbilical venous endothelial cell line (EA.hy926 cells) was exposed to CHR,serum of septic shock patient,and tumor necrosis factor-α (TNF-α) respectively.Methyl thiazolyl tetrazolium (MTT) method,Transwell assay and immunofluorescence were performed to determine cell viability [absorbance (A) value],permeability of monolayer endothelial cells (A value),and the morphological characteristic and distribution of F-actin respectively.Results Compared with the blank control group,when EA.hy926 were exposed to CHR with 1,10,100 nmol/L the cell activity was not significantly affected (A value:1.219 ±0.253,1.179 ±0.065,1.179 ±0.062 vs.1.306 ±0.162,all P＞0.05),while when the cells was exposed to CHR in 1 000 nmol/L the cell activity was significantly inhibited (A value:1.049 ± 0.256 vs.1.306 ± 0.162,t=-2.390,P=0.031).Compared with blank control group,when the cells were exposed to CHR of 1,10,100 nmol/L a significant decrease in permeability in EA.hy926 cells was observed (A value:1.619 ±0.324,1.496 ±0.356,1.132 ± 0.280 vs.2.315 ± 0.440,P＜0.05 or P＜0.01).Treatment of septic shock patient's serum or TNF-α to EA.hy926 produced an obvious increase in its permeability (septic serum group A value:1.204 ± 0.248 vs.0.277 ± 0.017,P＜0.01 ; TNF-α group A value:2.485 ± 0.113 vs.1.602 ± 0.679,P＜0.05).High-permeability induced by TNF-α or septic shock patient's serum was alleviated by CHR in the concentration of 1,10,100 nmol/L in a dose-dependent manner (septic serum + C HR group A value:0.299 ± 0.065,0.224 ± 0.028,0.131 ± 0.015 vs.1.204 ± 0.248; TNF-α + CHR group A value:1.995 ± 0.394,1.920 ± 0.096,1.744 ± 0.475 vs.2.485 ± 0.113,P＜0.05 or P＜0.01).Under a laser scanning confocal microscope,it was found that the F-actin cytoskeleton of EA.hy926 cells was redistributed,and more stress fibers were found in the septic shock patient's serum group and TNF-α group,while CHR obviously alleviated the above effects induced by septic shock patient's serum or TNF-α.Conclusion In a dose-dependent manner,CHR may inhibit increased permeability of vascular endothelial cells induced by septic shock patient's serum,its underlying mechanism may be related to inhibition of the effect of TNF-α.%目的 观察嗜铬粒蛋白A衍生多肽CGAn47-66(Chromfungin,CHR)对脓毒症患者血清引起的血管内皮细胞高通透性的影响.方法 分别用CHR、脓毒性休克患者血清、肿瘤坏死因子-α(TNF-α)作用于人脐静脉内皮细胞株EA.hy926,采用四甲基偶氮唑盐(MTT)比色法、Transwell小室法测定EA.hy926的活性[吸光度(A)值]和通透性(A值),免疫荧光法镜下观察细胞纤维肌动蛋白(F-actin)的形态和分布.结果 与空白对照组比较,1、10、100 nmol/L CHR对EA.hy926细胞活性无明显影响(A值:1.219±0.253、1.179±0.065、1.179±0.062比1.306±0.162,均P＞0.05),而1 000 nmol/L CHR对EA.hy926细胞活性有显著抑制作用(A值:1.049±0.256比1.306±0.162,t=-2.390,P=0.031).与空白对照组比较,1、10、100 nmol/L CHR作用下EA.hy926细胞通透性明显降低(A值:1.619±0.324、1.496±0.356、1.132±0.280比2.315±0.440,P＜0.05或P＜0.01);脓毒性休克患者血清或TNF-α单独作用下EA.hy926细胞通透性明显升高(血清组A值:1.204±0.248比0.277±0.017,P＜0.01;TNF-α组A值:2.485±0.113比1.602±0.679,P＜0.05);1、10、100 nmol/L的CHR既可明显降低脓毒性休克患者血清引起的高通透性(A值:0.299±0.065、0.224±0.028、0.131±0.015比1.204±0.248,均P＜0.01),也可明显降低TNF-α引起的高通透性(A值:1.995±0.394、1.920±0.096、1.744±0.475比2.485±0.113,P＜0.05或P＜0.01),且在1～100 nmol/L范围内,CHR的上述作用呈现一定的剂量依赖性.与空白对照组比较,脓毒性休克患者血清或TNF-α单独作用下,F-actin细胞骨架发生明显重组和再分布,应力纤维形成增多,而CHR可以抑制上述变化的发生.结论 CHR可以抑制脓毒性休克患者血清引起的血管内皮细胞的高通透性,且呈现一定的剂量依赖性,其机制与抑制TNF-α的作用有关.
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