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目的 观察高迁移率族蛋白B1(HMGB1)对人脐静脉内皮细胞(HUVEC)屏障通透性的损伤及普通肝素(UFH)的保护作用,探讨UFH对HMGB1介导的血管内皮钙黏蛋白(VE-cadherin)表达下调的保护机制.方法 体外培养HUVEC并传代,待细胞融合生长至80%时随机分为4组:磷酸盐缓冲液(PBS)对照组(200 μL PBS)、重组人高迁移率族蛋白B1(rhHMGB1)处理组(100 μg/L rhHMGB1)、UFH对照组(10 kU/L UFH)、UFH预处理组(10 kU/L UFH+100 μg/L rhHMGB1).各组细胞经不同处理24 h后,采用四甲基偶氮唑盐(MTT)比色法测定内皮细胞活性,采用Transwell法测定内皮细胞通透性,采用免疫荧光法观察VE-cadherin的表达及分布,采用蛋白质免疫印迹试验(Western Blot)测定VE-cadherin及磷酸化p38丝裂素活化蛋白激酶(p-p38MAPK)的蛋白表达.结果 经100 μg/L的rhHMGB1处理24 h后,内皮细胞活性与PBS对照组差异无统计学意义(A值:0.230±0.004比0.255±0.006,P＞0.05),但内皮细胞通透性显著升高(葡聚糖FD40荧光强度:11.05±0.12比6.34±0.39,P＜0.05);与PBS对照组比较,荧光显微镜下显示rhHMGB1处理组VE-cadherin膜定位减少,分布松散,细胞间出现明显裂隙,定量分析显示VE-cadherin蛋白表达明显下降(VE-cadherin/β-actin:0.16±0.04比0.31±0.03,P＜0.05),p-p38MAPK蛋白表达明显升高(p-p38MAPK/β-actin:0.79±0.03比0.26±0.05,P＜0.05).UFH预处理可对HMGB1介导的内皮细胞损伤起保护作用,表现为细胞通透性明显降低(葡聚糖FD40荧光强度:9.11±0.23比11.05±0.12),VE-cadherin荧光表达明显增强,膜定位明显增加,定量分析显示VE-cadherin的蛋白表达明显上调(VE-cadherin/β-actin :0.24±0.02比0.16±0.04),且p38MAPK的磷酸化水平明显降低(p-p38MAPK/β-actin :0.54±0.05比0.79±0.03),与rhHMGB1处理组比较差异均有统计学意义(均P＜0.05).PBS对照组与UFH对照组各项指标比较差异均无统计学意义.结论 UFH可保护HMGB1所致的VE-cadherin表达减少和分布异常,进而保护内皮细胞屏障通透性,其作用机制可能与UFH抑制p38MAPK的磷酸化相关.“,”Objective To observe the damage of high mobility group box 1 (HMGB1) on human umbilical vein endothelial cell (HUVEC) barrier permeability and the protective effect of unfractionated heparin (UFH), and to explore the down-regulated protection effect mechanism of UFH on HMGB1-mediated vascular endothelial cadherin (VE-cadherin) expression. Methods The trypsin-digested HUVEC were subcultured in culture flasks. When the cells were grown to 80%, they were randomly divided into four groups: phosphate buffer (PBS) control group (200 μL PBS), recombinant human high mobility group box 1 (rhHMGB1) treatment group (100 μg/L rhHMGB1), UFH control group (10 kU/L UFH), and UFH pretreatment group (10 kU/L UFH+100 μg/L rhHMGB1). The cells in each group were challenged with different reagent for 24 hours, and the activity of endothelial cells was determined by methyl thiazolyl tetrazolium (MTT) colorimetric assay. The permeability of endothelial cells was measured by Transwell method, and the expression and distribution of VE-cadherin was observed by immunofluorescence. The protein expressions of VE-cadherin and phosphorylated p38 mitogen-activated protein kinase (p-p38MAPK) were determined by Western Blot. Results After treatment with 100 μg/L rhHMGB1 for 24 hours, the activity of endothelial cells was not significantly different from that of the PBS control group (A value: 0.230±0.004 vs. 0.255±0.006, P > 0.05), but the permeability was significantly increased (glucan FD40 fluorescence intensity: 11.05±0.12 vs. 6.34±0.39, P < 0.05). Compared with PBS control group, the fluorescence microscopy showed that the VE-cadherin membrane localization was reduced, the distribution was loose, and there were obvious fissures between cells in rhHMGB1 treatment group, and quantitative analysis showed the protein expression of VE-cadherin was decreased significantly (VE-cadherin/β-actin: 0.16±0.04 vs. 0.31±0.03, P < 0.05), and the expression of p-p38MAPK protein was significantly increased (p-p38MAPK/β-actin: 0.79±0.03 vs. 0.26±0.05, P < 0.05). UFH pretreatment could protect HMGB1-mediated endothelial cell injury, cell permeability was significantly reduced (glucan FD40 fluorescence intensity: 9.11±0.23 vs. 11.05±0.12), fluorescence expression of VE-cadherin was enhanced, membrane localization was significantly increased, quantitative analysis showed that VE-cadherin protein expression was significantly up-regulated (VE-cadherin/β-actin: 0.24±0.02 vs. 0.16±0.04), and p38MAPK phosphorylation level was significantly decreased (p-p38MAPK/β-actin: 0.54±0.05 vs. 0.79±0.03), the difference was statistically significant as compared with rhHMGB1 treatment group (all P < 0.05). There was no significant difference in all parameters between PBS control group and UFH control group. Conclusions UFH can protect the endothelial cell barrier from the HMGB1 by regulating the expression and distribution of VE-cadherin. The mechanism may be related to the inhibition of p38MAPK phosphorylation by UFH.