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

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 HMGB1-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 rhHMGB1+ 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 rhHMGB1 (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 cells 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 rhHMGB1 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. Key words: Unfractionated heparin; High mobility group box1; Tight junction; Claudin-5; Permeability of the endothelial