硒对氟诱导NRK-52E细胞凋亡干预作用机制

Mechanism of interference effect of selenium on sodium fluoride-induced apoptosis in NRK-52E cells

目的探讨硒对氟诱导NRK-52E细胞凋亡的拮抗作用。方法实验设对照组、染氟组、染硒组、硒干预组,培养NRK-52E细胞,染毒72 h,采用流式细胞仪检测细胞凋亡,Western blot检测AMPK及线粒体通路相关蛋白表达水平。结果与对照组[(5.97±0.09)%]比较,5、20 mg/L Na F组细胞凋亡率[分别为(7.92±0.24)%、(11.06±0.17)%]明显升高(P<0.05);与20 mg/L Na F组比较,17.1、34.2μg/L硒干预组细胞凋亡率[分别为(8.46±0.09)%、(9.88±0.08)%]明显降低(P<0.05);与对照组比较,20 mg/L Na F组细胞AMPK磷酸化蛋白、bax、Cyt-C蛋白表达[分别为(0.73±0.16)、(0.99±0.16)、(0.73±0.08)]、活性caspase-9和caspase-3蛋白表达[分别为(1.17±0.17)、(1.51±0.42)]均升高(P<0.05);与20 mg/L Na F组比较,17.1、34.2 g/L硒干预组AM PK磷酸化蛋白表达量[分别为(0.46±0.12)、(0.48±0.15)]、bax、Cyt-C蛋白表达量[分别为(0.55±0.09)、(0.61±0.16)与(0.30±0.06)、(0.34±0.05)]及活性caspase-9、caspase-3蛋白表达量[分别为(0.76±0.11)、(0.40±0.12)与(0.35±0.12)、(0.27±0.04)]均降低(P<0.05)。结论 AMPK介导的线粒体通路参与了硒拮抗氟诱导NRK-52E细胞凋亡过程。

Objective To study antagonistic effect of selenium on sodium fluoride-induced apoptosis of NRK-52E cells. Methods NRK-52E cells were cultured and divided into following groups： a control group, two sodium fluoride （NaF）-treated （5,20 mg/L） groups,two sodium selenite （Na2SeO3）-treated （17. 1,34.2 μg/L） groups,and four NaF＋ Na2SeO3-treated （5 or 20 mg/L NaF ＋ 17.1 or 34. 2 μg/L Na2SeO3 ） groups. Cell apoptosis was detected by flow cytometry 72 hours after the treatments. Protein expressions of adenosine monophosphate-activated protein kinase （AMPK） and related mitochondria pathway proteins were determined using Western blot. Results Compared to the control group （ 5.97 ± 0. 09% ）, the cell apoptosis rate of the 5 and 20 mg/L NaF-treated groups （ 7.92 ± 0. 24% and 11.06 ± 0. 17% ） were remarkably increased （P 〈0.05） ,while the rates of 20 mg/L NaF ＋ 17.1 and 34. 2 g/L Na2SeO3-treated groups （8.46 ±0. 09% and 9. 88 ±0. 08% ） were statistically significantly decreased compared to that of the 20 mg/L NaF-treated groups （ both P 〈 0.05 ）. Compared to those of the control group, the expression levels of phosphorylated-AMPK （0. 73 ±0. 16） ,bax （0. 99 ±0. 16） ,cytochrome C （Cyt-C） （0. 73 ±0. 08） ,caspase-9 （ 1.17 ±0. 17） ,and caspase-3 （ 1.51 ± 0.42 ） were obviously up-regulated in 20 mg/L NaF-treated group （P 〈 0. 05）. Compared to those of the 20 mg/L NaF- treated group, the expression levels of phosphorylated -AMPK （ 046 ± 0. 12,0.48 ± 0. 15 ）, bax （ 0. 55 ± 0. 09,0. 61 ± 0. 16） ,Cyt-C （0. 30 ±0. 06,0. 34 ±0. 05） ,caspase-9 （0. 76 ±0. 11,0. 40 ±0. 12） ,caspase-3 （0. 35 ±0. 1,0. 27 ±0. 04） were down-regulated in 20 mg/L NaF ＋17. 1 and 34. 2 g/L Na2SeO3 -treated groups （ all P 〈 0. 05 ）. Conclusion Fluoride can induce apoptosis in NRK-52E cells and the effect can be ameliorated by selenium by regulation of AMPK mediated mitochondrial pathway.