普通肝素改善高迁移率族蛋白1介导的血管内皮细胞屏障通透性增加的实验研究

Protective Effects of Unfractionated Heparin on HMGB1-Induced Increased Permeability of Endothelial Cells

目的观察普通肝素(UFH)对高迁移率族蛋白1(HMGB 1)介导的人脐静脉内皮细胞屏障通透性损伤的保护作用,探讨UFH对HMGB 1介导的胞质紧密粘连蛋白-1(ZO-1)表达缺失的保护机制。方法将人脐静脉内皮细胞进行体外培养,人脐静脉内皮细胞株传代培养后分为4组(n=5):空白对照组(加入等量PBS)、HMGB 1处理组(100 ng/ml)、UFH对照组(UFH 10 U/ml)、HMGB 1及UFH处理组(100 ng/ml HMGB 1+UFH 10 U/ml)。MTT法测定内皮细胞存活率,用Transwell小室法测定单层内皮屏障通透性,用免疫荧光染色法测定ZO-1的表达分布,蛋白免疫印迹(Western blot)法检测ZO-1蛋白及核因子-κB(NF-κB)的表达。结果 HMGB 1(100 ng/ml)对内皮细胞活性无抑制作用(P>0.05)。UFH预处理后可减少HMGB1所致的内皮细胞通透性增加(P<0.05)。UFH预处理后可降低HMGB 1所致内皮细胞ZO-1密闭环的减少和破坏,使ZO-1荧光强度增强,ZO-1蛋白表达增加,并使NF-κB的核易位减少。结论 UFH可保护HMGB 1介导的内皮细胞ZO-1的表达缺失,进而改善内皮细胞屏障通透性,其机制与减少NF-κB核易位相关。

Objective To observe the protective effects of unfractionated heparin （UFH） on high-mobility group box-1 protein （HMGB 1） induced increased permeability of endothelial cells, and investigate the protective mechanism of UFH on HMGB1 induced defective expression of zonula occludens-1 （ZO-1）. Methods Human umbilical vascular endothelial cells （HUVECs） were cultured in vitro and divided into 4 groups （n=5）, namely a control group, a HMGB1 group （100 ng/ml）, a heparin group （UFH 10 U/ml）, a HMGB1/heparin group （100 ng/ml HMGB1 ＋ UFH 10 U/ml）. Endothelial cell viability was measured by methyl thiazolyl tetrazolium （MTT） colorimetric method. Endothelial permeability was determination by TransweU chamber method. Immunofluorescence and laser confocal microscopy were used to assess the distribution of ZO-1. The protein expressions of tight junction protein ZO-1 and nuclear factor kappa B （NF-κB） were detected by Western blot. Results HMGB1 （100 ng/ml） had no inhibitory effect on endothelial cell viability （P 〉 0.05）. UFH pretreatment could reduce the permeability increment of endothelial cells induced by HMGB1. UFH pretreatment could reduce the close loop reduction and damage of ZO-1 induced by HMGB1, enhance the fluorescence intensity and expression of ZO-1, and decrease the NF-κB translocation. Conclusions UFH can protect HMGB 1-mediated defect of ZO-1 expression and increased permeability of the endothelial cells. The mechanism may be related to the decreased nuclear translocation of NF-κB.