Objective: This study aims to explore the relationship between PBEF and VEGF and p-MLC and the mechanism of PBEF increasing permeability of endothelial cells in hypoxia/re-oxygenation.
Methods: Hypoxia/re-oxygenation model was established and PBEF siRNA was synthesized. According to the different HUVEC treatment, it can be divided into normal control group, PBEF siRNA group; hypoxia (20 hours) and re-oxygenation (3 h) group, hypoxia (20 h) and re-oxygenation (6 h) group, hypoxia (20 h) and re-oxygenation (9 hours) group, hypoxia (20 h)/re-oxygenation (12 h). The expressions of PBEF, VEGF and p-MLC were tested by RT-PCR and Western blot.
Results: The mRNA and protein expression of PBEF in PBEF siRNA group were significantly lower compared to liposome group and the negative controls (P < 0.05). The expression of PBEF protein in hypoxia/re-oxygenation group was significantly higher than the normal control group. It increased in the 3 h of re-oxygenation group, peaked at 9 h, until 12 h started to decline (P < 0.05). When the PBEF gene was knockdown, the expression of VEGF and p-MLC in hypoxia and re-oxygenation are significantly lower.
Conclusions: PBEF siRNA can effectively inhibit the expression of PBEF in endothelial cells. The expression of PBEF, VEGF and p-MLC were significantly higher in endothelial cell after Hypoxia/re-oxygenation. PBEF may change the permeability of endothelial cells by regulating the expression of VEGF and the phosphorylation of MLC.
Keywords: Pre-B-cell colony enhancing factor (PBEF); RNA interference; VEGF; hypoxia/reoxygenation; p-MLC.