细胞核受体RXRα在2,2′,4,4′-四溴二苯醚神经毒性中的作用及其机制

Effect of BDE-47 on nuclear receptor RXRα and toxicity in human neuroblastoma SK-N-SH cells

目的:探讨视黄醛X受体(RXRs)为核心的多个核受体在2,2′,4,4′-四溴二苯醚(BDE-47)致神经细胞毒性中的相互作用,揭示受体介导的多溴二苯醚(PBDEs)神经毒性效应的分子机制。方法:构建人神经母细胞瘤细胞SK-N-SH的RXRα基因敲除细胞株及高表达细胞株(分别命名为RXR/KO-SK-N-SH和RXR/OE-SK-N-SH细胞);CCK-8法分别检测SK-N-SH(野生型)、RXR/KO-SKN-SH和RXR/OE-SK-N-SH细胞在BDE-47浓度分别为1、5、10、20、50、100、150、200μmol/L处理12、24、48、72 h时的细胞增殖率;qPCR和Western blot法分别检测上述3种细胞中RXRα、TRα、TRβ、PPARα、PPARγ的mRNA和蛋白表达水平。结果:在染毒时间分别为12、24、48、72 h时,不同浓度BDE-47作用下3种细胞的增殖率比较,差异有统计学意义(P<0.05)。BDE-47对RXR/KO-SK-N-SH细胞的IC50是野生型和RXR/OE-SK-N-SH细胞的1/2。BDE-47处理3种细胞后,RXRα的mRNA和蛋白表达水平在野生型和RXR/OE-SK-N-SH细胞中显著升高,SK-N-SH细胞和RXR/OE-SK-N-SH细胞中TRα和PPARα的表达升高,此两种受体在RXR/KO-SK-N-SH细胞中表达降低。在本实验所采用的BDE-47处理浓度下,TRβ和PPARγ表达在3种细胞中均无明显变化。结论:RXRα在提高SK-N-SH细胞生存能力中发挥重要作用。RXRα可以介导TRα和PPARα的表达,而BDE-47并未激活或者抑制SK-N-SH细胞TRβ和PPARγ的表达。说明BDE-47对于SK-N-SH细胞的毒性主要是通过激活RXRα,进一步诱导TRα和PPARα的表达而介导的。

OBJECTIVE:To explore interactions between nucleus receptor RXRs and other nuclear receptors in BDE-47-induced cytotoxicity in human neuroblastoma SK-N-SH cells.METHODS:RXRα-knockout cell lines and RXRαhigh-expression cell lines from human neuroblastoma SK-N-SH cells(named RXR/KO-SK-N-SH and RXR/OE-SK-N-SH cells,respectively)were constructed.The three cell lines were exposed to different concentrations of BDE-47(1,5,10,20,50,100,150,200 mol/L)for 12,24,48,72 h,and their proliferation rates were determined by the CCK-8 method.mRNA and protein levels of RXR,TR,TR,PPAR and PPAR were measured by qPCR and Western blot,respectively.RESULTS:Proliferation rates of three cell lines which were exposed to BDE-47 for 12,24,48,72 h were significantly different from each other,(P<0.05).The IC50 of BDE-47 to RXR/KO-SK-N-SH was 1/2 to SK-N-SH and RXR/OE-SK-NSH.After treatment with BDE-47,the wild-type and RXR/OE-SK-N-SH cell lines showed significant increase of RXRα mRNA and protein expression levels.Their protein expression levels of TRα and PPARα showed the same changes.However,the knockout SK-N-SH cells showed lower expression level in RXRα.The expression levels of TRβ and PPARγ were not dramatically changed in all three SK-N-SH cell lines after the BDE-47 treatment.CONCLUSION:RXRαplayed a critical role in improving cell viability.After treatment with BDE-47,RXRαmediated the expression of TRαand PPARα in the three SK-N-SH cell lines,but not the expression of TRβ and PPARγ.These data demonstrate that toxicity of BDE-47 on SK-N-SH cells was medicated by activation of RXRαwhich enhanced TRαand PPARαexpression.