白藜芦醇对氟致人神经母细胞瘤SH-SY5Y细胞线粒体生物发生障碍的影响

The effects of resveratrol on mitochondrial biogenesis dysfunction induced by fluoride in human neuroblastoma SH-SY5Y cells

目的探讨线粒体生物发生在氟神经毒性中的作用,以及白藜芦醇干预氟神经毒性的作用机制。方法不同浓度氟化钠(NaF)处理对数生长期人神经母细胞瘤SH-SY5Y细胞24h,并用20μmol/L白藜芦醇对60mg/L NaF组进行干预。NaF剂量染毒实验分组:对照组,20、40、60mg/L NaF组;白藜芦醇干预NaF染毒实验分组:对照组,60mg/LNaF组,20μmol/L白藜芦醇+60mg/LNaF组,20μmol/L白藜芦醇组。采用蛋白质免疫印迹(Western blotting)法检测线粒体生物发生关键调控因子过氧化物酶体增殖物激活受体辅激活因子1α(PGC-1α)、核呼吸因子1(NRF1)及线粒体转录因子A(TFAM)的蛋白表达水平;采用实时荧光定量PCR技术检测线粒体生物发生关键调控因子及其下游呼吸链复合物亚基细胞色素C氧化酶亚基1(CO1)和ATP合成酶亚基(ATP8)的mRNA表达水平与线粒体DNA(mtDNA)相对含量;采用流式细胞术检测线粒体膜电位。结果与对照组比较,60mg/L NaF组SH-SY5Y细胞线粒体生物发生关键调控因子PGC-1α、NRF1、TFAM mRNA与蛋白表达水平、线粒体呼吸链复合物亚基CO1和ATP8 mRNA表达水平以及mtDNA相对含量均明显降低,差异均有统计学意义(P<0.05);60mg/L NaF组线粒体膜电位与对照组比较也明显降低(P<0.05)。经白藜芦醇干预后,与60mg/L NaF组比较,20μmol/L白藜芦醇+60mg/LNaF组SH-SY5Y细胞的线粒体生物发生关键调控因子PGC-1α、NRF1、TFAM蛋白与mRNA表达水平均明显增加,线粒体呼吸链复合物亚基CO1和ATP8的mRNA表达水平、mtDNA相对含量和线粒体膜电位均明显增加,差异均有统计学意义(P<0.05)。结论白藜芦醇干预可缓解氟导致的SH-SY5Y细胞线粒体生物发生障碍。

Objective To explore the role of mitochondrial biogenesis and the neuroprotective mechanism of resveratrol in fluoride neurotoxicity. Methods SH-SY5Y cells in exponential phase were treated with different concentrations(20, 40, 60 mg/L) of sodium fluoride(NaF) for 24 h. Co-treatment with 60 mg/L NaF, 20 μmol/L resveratrol (RSV) was administrated in the resveratrol intervene trial. Western blotting was used to determine the expression levels of mitochondrial biogenesis key regulating factor of peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α), nuclear respiratory factor 1 (NRF1) and mitochondrial transcription factor A (TFAM) in SH-SY5Y cells. The mRNA levels of PGC-1α, NRF1 and TFAM were determined by Quantitative Real-time PCR in SH-SY5Y cells, as well as the relative mitochondrial DNA (mtDNA) contents and mRNA expression of mitochondrial respiratory chain complexes subunit CO1 and ATP8. Flow cytometry was used to determine mitochondrial membrane potential in SH-SY5Y cells. Results Both the protein and mRNA levels of PGC-1α, NRF1 and TFAM were decresed after 60 mg/L NaF treatment in SH-SY5Y cells (P<0.05). The relative mtDNA contents and mRNA expression of complexes subunit CO1 and ATP8 were also significantly decreased compared with control (P<0.05). Mitochondrial membrane potential were also significantly decreased after 60 mg/L NaF treatment in SH-SY5Y cells (P<0.05). Compared with 60 mg/L NaF group, the protein and mRNA levels of PGC-1α, NRF1 and TFAM in 20 μmol/L RSV+60 mg/L NaF group were significantly increased (P<0.05). The relative mtDNA contents, mitochondrial membrane potential and mRNA levels of complexes subunit CO1 and ATP8 in 20 μmol/L RSV+60 mg/L NaF group were also significantly higher than that in 60 mg/L NaF group (P<0.05). Conclusion Resveratrol may alleviate the fluoride-induced mitochondrial biogenesis dysfunction in SH-SY5Y cells.