高氟暴露降低人血管内皮细胞紧密连接蛋白ZO-1表达及其机制的初步研究

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

目的观察高氟暴露对人血管内皮细胞紧密连接胞质带状闭合蛋白1(ZO-1)表达的影响,并初步探讨其机制。方法体外培养人脐静脉血管内皮细胞,采用成组设计,分别设置0.0(对照组)和0.4、0.8、1.2mg/L氟处理组(低、中、高剂量组),以及胰岛素样生长因子-1(IGF-1)组[加入1.2mg/L的氟离子(F-)前,预先加入100nmol/L的IGF-1处理2h],各组细胞培养24h,提取细胞蛋白,应用蛋白免疫印迹法(Western Blot)检测各组细胞中ZO-1、磷脂酰肌醇激酶(PI3K)、蛋白激酶B(AKT)和磷酸化AKT(p-AKT)的表达并进行分析。结果对照,低、中、高剂量组间内皮细胞中ZO-1的表达(0.063±0.002、0.043±0.007、0.039±0.004、0.028±0.007)比较差异有统计学意义(F=21.36,P<0.01);各加氟处理组ZO-1的表达均显著低于对照组(P均<0.05);高剂量组ZO-1的表达显著低于低、中剂量组(P均<0.05)。对照,低、中、高剂量组间内皮细胞中PI3K表达(0.032±0.004、0.031±0.002、0.017±0.001、0.017±0.005)和p-AKT与AKT表达比(p-AKT/AKT:0.745±0.046、0.806±0.007、0.666±0.058、0.641±0.040)比较差异有统计学意义(F=20.38、9.57,P<0.01或<0.05);其中高剂量组PI3K表达和p-AKT/AKT均显著低于对照组和低剂量组(P均<0.05);中剂量组p-AKT/AKT显著低于低剂量组(P<0.05)。加入PI3K/AKT通路的激动剂IGF-1后,IGF-1组内皮细胞中ZO-1的表达(0.055±0.015)高于高剂量组(P<0.05)。结论高氟暴露可通过PI3K/AKT信号途径引起血管内皮细胞ZO-1的表达降低。

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.