氟化钠对人成骨肉瘤Saos-2细胞PI3K/Akt信号表达及凋亡的影响

Effect of sodium fluoride on expression of PI3K/Akt signal molecules and apoptosis in human osteosarcoma Saos-2 cells

目的观察氟化钠(NaF)对人成骨肉瘤Saos-2细胞磷脂酰肌醇-3-激酶/蛋白激酶B(PI3K/Akt)信号表达及凋亡的影响。方法采用成组设计,按NaF剂量将Saos-2细胞分为0(对照)、5、10、20、40 mg/L染氟组(n=3)。体外培养24、48 h后收集细胞,采用蛋白免疫印迹(Western blot)法检测PI3K、Akt和线粒体凋亡途径相关分子Forkhead转录因子(FoxO)1的蛋白表达,采用流式细胞仪检测Saos-2细胞的凋亡水平。结果体外培养24、48 h时,各组Saos-2细胞PI3K、Akt蛋白表达比较,差异均无统计学意义(P均>0.05)。24 h时,5、10、20、40 mg/L染氟组磷酸化PI3K(p-PI3K)蛋白表达(0.40±0.06、0.45±0.02、0.37±0.06、0.32±0.06)均高于对照组(0.28±0.04,P均<0.05);48 h时,5、10 mg/L染氟组p-PI3K蛋白表达(0.46±0.06、0.58±0.03)均高于对照组(0.29±0.04,P均<0.05),而40 mg/L染氟组(0.21±0.03)低于对照组(P<0.05)。24 h时,5、10、20 mg/L染氟组磷酸化Akt(p-Akt)蛋白表达(0.27±0.01、0.30±0.03、0.27±0.03)均高于对照组(0.20±0.02,P均<0.05);48 h时,5、10 mg/L染氟组p-Akt蛋白表达(0.42±0.04、0.60±0.02)均高于对照组(0.27±0.01,P均<0.05),而40 mg/L组(0.18±0.02)低于对照组(P<0.05)。24 h时,10、20、40 mg/L染氟组FoxO1蛋白表达(0.38±0.07、0.41±0.06、0.47±0.08)均高于对照组(0.34±0.04,P均<0.05);48 h时,5、10、20、40 mg/L染氟组FoxO1蛋白表达(0.36±0.08、0.37±0.10、0.54±0.04、0.73±0.04)均高于对照组(0.28±0.04,P均<0.05)。24、48 h时,对照组和5、10、20、40 mg/L染氟组细胞凋亡率分别为(2.867±0.583)%、(3.647±0.035)%、(3.773±0.095)%、(5.440±0.325)%、(7.203±0.476)%,(3.707±0.286)%、(4.023±0.241)%、(4.970±0.368)%、(12.473±0.949)%、(19.387±1.892)%。24 h时,40 mg/L染氟组凋亡水平高于对照组(P<0.05);48 h时,20、40 mg/L染氟组凋亡水平均高于对照组(P均<0.05)。结论氟可以直接激活成骨细胞内的PI3K/Akt信号通路,进而产生抗凋亡作用。

Objective To observe the effect of sodium fluoride(NaF)on the expression of phosphatidylinositol-3-kinase/protein kinase B(PI3K/Akt)signal and apoptosis in human osteosarcoma Saos-2 cells.Methods Saos-2 cells were divided into 0(control),5,10,20 and 40 mg/L fluoride groups(n=3)according to the dose of NaF.The cells were collected after 24 and 48 h of culture in vitro.Western blotting was used to detect the expressions of PI3K/Akt signal and Forkhead transcription factor(FoxO)1,and the apoptosis level of Saos-2 cells was detected by flow cytometry.Results There were no significants differences in the expressions of PI3K and Akt in Saos-2 cells at 24 and 48 h(P>0.05).At 24 h,the expressions of phosphorylated PI3K(p-PI3K)in 5,10,20 and 40 mg/L fluoride groups(0.40±0.06,0.45±0.02,0.37±0.06,0.32±0.06)were higher than that in the control group(0.28±0.04,P<0.05);at 48 h,the expressions of p-PI3K in 5 and 10 mg/L fluoride groups(0.46±0.06,0.58±0.03)were higher than that in the control group(0.29±0.04,P<0.05),and that in the 40 mg/L fluoride group(0.21±0.03)was lower than that in the control group(P<0.05).At 24 h,the expressions of phosphorylated Akt(p-Akt)in 5,10 and 20 mg/L fluoride groups(0.27±0.01,0.30±0.03,0.27±0.03)were higher than that in the control group(0.20±0.02,P<0.05);at 48 h,the expressions of p-Akt in 5 and 10 mg/L fluoride groups(0.42±0.04,0.60±0.02)were higher than that in the control group(0.27±0.01,P<0.05),and that in the 40 mg/L fluoride group(0.18±0.02)was lower than that in the control group(P<0.05).The expressions of FoxO1 in 10,20 and 40 mg/L fluoride groups at 24 h(0.38±0.07,0.41±0.06,0.47±0.08)were higher than that in the control group(0.34±0.04,P<0.05).At 48 h,the expressions of FoxO1 in 5,10,20 and 40 mg/L fluoride groups(0.36±0.08,0.37±0.10,0.54±0.04,0.73±0.04)were higher than that in the control group(0.28±0.04,P<0.05).At 24 and 48 h,the apoptosis rates of control group and 5,10,20 and 40 mg/L fluoride groups were(2.867±0.583)%,(3.647±0.035)%,(3.773±0.095)%,(5.440±0.325)%,(7.203±0.476)%;(3.707±0.286)%,(4.023±0.241)%,(4.970±0.368)%,(12.473±0.949)%,(19.387±1.892)%,respectively.The apoptosis level of 40 mg/L fluoride group was higher than that of control group at 24 h(P<0.05),and that of 20 and 40 mg/L fluoride groups were higher than that of control group at 48 h(P<0.05).Conclusion Fluoride can directly activate the PI3K/Akt signaling pathway in osteoblasts,and then has anti-apoptotic effect.