普通肝素对高迁移率族B1介导的内皮细胞紧密连接蛋白Claudin-5下调的保护作用

Protective effect of unfractionated heparin on high mobility group box-1 protein mediated down-regulation of endothelial Claudin-5

目的 研究高迁移率族蛋白B1（high mobility group box-1 protein, HMGB1） 对人脐静脉内皮细胞屏障通透性的影响，以及普通肝素对HMGB1 介导的内皮细胞紧密连接蛋白Claudin-5 下调的保护作用。方法 将经胰酶消化后的人脐静脉内皮细胞于培养瓶中传代培养，并分成4 组：空白对照组（加入等量PBS）、rhHMGB1 处理组（100 ng/mL）、普通肝素对照组（UFH10 U/mL）、rhHMGB1＋ 普通肝素处理组（100 ng/mL rhHMGB1 ＋ UFH 10 U/mL）。传代培养后的人脐静脉内皮细胞，通过MTT 法测定内皮细胞的存活率。Transwell 法测定内皮细胞通透性；免疫荧光法对Claudin-5 表达及分布进行测定；通过蛋白免疫印迹（Western blot） 法检测Claudin-5 蛋白表达。结果 用rhHMGB1（100 ng/mL） 处理内皮细胞后，与空白对照组比较细胞活力无明显变化（P〉0.05）；用rhHMGB1（100 ng/mL） 分别处理内皮细胞3 h、6 h 后，内皮细胞屏障通透性较空白对照组无明显变化（P〉0.05）；而经rhHMGB1 处理12 h、24 h 后，与空白对照组相比，内皮细胞屏障通透性显著升高，差异有统计学意义（P〈0.05）。而普通肝素和rhHMGB1 共同孵育内皮细胞12 h、24 h 后，其内皮细胞屏障通透性均低于rhHMGB1 处理组（P〈0.05）。免疫荧光和Westernblot研究表明，rhHMGB1 处理内皮细胞24 h 后，细胞紧密连接相关蛋白Claudin-5 分布和表达降低；而经普通肝素和rhHMGB1 共同孵育后，细胞紧密连接相关蛋白Claudin-5 的表达提高，Claudin-5的荧光强度增强。结论 HMGB1 可介导内皮细胞紧密连接相关蛋白Claudin-5 的分布异常和表达下降，以及增加内皮细胞屏障的通透性；普通肝素可改善HMGB 1 介导的内皮细胞紧密连接相关蛋白Claudin-5 的表达和分布及内皮细胞屏障通透性。

Objective To investigate the effect of high mobility group box-1 protein （HMGB1） on the permeability of human umbilical vein endothelial cells, and the protective effect of unfractionated heparin on HMGBl-mediated, endothelial cell tightly junction-related protein Claudin-5. Methods The human umbilical vein endothelial cells after trypsin digestion were subcultured in culture flasks and divided into 4 groups： blank control group （addition of PBS equivalent）, rhHMGB1 treatment group （100 ng/mL）, unfractionated heparin control group （UFH, 10 U/mL） and rhHMGBI＋ unfractionated heparin-treated group （100 ng/mL rhHMGB1 ＋ UFH 10 U/mL）. After human umbilical vein endothelial cells were cultured： the viability of endothelial cells was determined by MTT assay; transwell method was used to measure the permeability of endothelial cells; the expression and distribution of Claudin-5 were determined by immunofluorescence; and Claudin-5 protein expression was detected by Western blotting. Results After treated with rhHMGB 1 （100 ng/mL）, the viability of endothelial cells was not significantly different from that of the blank control group （P〉 0,05）. After treated with rhHMGB1 （100 ng/mL） for 3 and 6 h respectively, the permeability of endothelial cells was not significantly different from that of the blank control group （P〉 0.05）. After 12 h and 24 h treatment of rhHMGB1, the permeability of endothelial cells was significantly increased compared with the blank control group （P〈 0.05）. However, after endothelial ceils were incubated with unfractionated heparin and rhHMGB1 for 12 and 24 h, the permeability of endothelial cell was lower than that of rhHMGB1 treatment group （P〈 0.05）. Immunofluorescence and Western-blot showed that after treatment of rhHMGB 1 for 24 h, the distribution and expression of claudin-5, a tightly junction-associated protein, was decreased. After incubation with unfractionated heparin and rhHMGB1, the expression of tightly junction-associated protein Claudin-5 was increased, as well as the fluorescence intensity of Claudin-5. Conclusions HMGB1 can increase the permeability of endothelial cells by mediating the abnormal distribution and decreased expression of Claudin-5. Unfractionated heparin can improve the expression and distribution of Claudin-5, improving the permeability of endothelial cells.