Excessive fluoride exposure decreased the expression of tight junction protein zonula occludens-1, in human vascular endothelial cells.

Abstract

Objective To observe the effect of excessive fluoride exposure on expression of tight junction protein zonula occludens-1 (ZO-1) in human vascular endothelial cells and to explore the mechanism. Methods Human umbilical vein endothelial cells (HUVECs) were cultured in vitro. Group design was adopted. Groups of 0.0 (control group) and 0.4, 0.8, 1.2 mg/L fluoride treatment groups (low, medium and high dose groups) and insulin-like growth factor-1 (IGF-1) group[before adding 1.2 mg/L fluoride ion (F-), 100 nmol/L of IGF-1 was added in advance for 2 hours] were set up respectively. Each group of cells was cultured for 24 h to extract cellular proteins. The expression of ZO-1, phosphatidylinositol kinase (PI3K), protein kinase B (AKT) and phosphorylated AKT (p-AKT) were detected and analyzed by Western blotting. Results The expression of intracellular ZO-1 (0.063 ± 0.002, 0.043 ± 0.007, 0.039 ± 0.004, 0.028 ± 0.007) decreased obviously with gradual increase of F- concentration in cultural medium (F= 21.36, P < 0.01). And the expressions of ZO-1 in all three fluorine groups were clearly lower than that in the control group (P < 0.05). At the same time, the expression of ZO-1 in high fluorine group was significantly lower than those in other fluorine groups(P < 0.05). There were significant differences in the expression of PI3K(0.032 ± 0.004, 0.031 ± 0.002, 0.017 ± 0.001, 0.017 ± 0.005) and p-AKT/AKT (0.745 ± 0.046, 0.806 ± 0.007, 0.666 ± 0.058, 0.641 ± 0.040) among all groups (F= 20.38, 9.57, P < 0.01 or < 0.05). The expressions of PI3K and p-AKT/AKT in high fluorine group were obviously lower than those of control and mild fluorine groups, and p-AKT/AKT in moderate fluorine group was clearly lower than that of mild fluorine group (P < 0.05). After pretreatment with IGF-1, the expression of ZO-1 (0.055 ± 0.015) increased significantly compared to that of high fluorine group (P < 0.05). Conclusion Excessive fluoride exposure has decreased the expression of ZO-1 in HUVECs through the PI3K/AKT signaling pathway. Key words: Fluorine; Endothelial cells; Tight junction