氟暴露对HT22海马神经元细胞miR-204和BDNF-TrkB通路的影响

Effects of fluorine exposure on miR-204 and BDNF-TrkB pathways in HT22 hippocampal neurons

目的探讨氟暴露对小鼠海马神经元细胞(HT22细胞)微小核糖核酸(microRNA,miRNA,miR)-204、脑源性神经营养因子(BDNF)、酪氨酸激酶B(TrkB)表达的影响。方法将HT22细胞暴露于0(对照)、2、4、6、8、10 mg/L氟化钠(NaF)。培养24 h后,CCK-8法检测细胞活性,根据细胞存活率结果,选取0.0(对照)、2.5、5.0、10.0 mg/L NaF,作用于未转染和转染(加入miR-204激动剂)的HT22细胞24 h,实时荧光定量PCR(qRT-PCR)和蛋白免疫印迹(Western blot)法分别检测细胞中miR-204表达,BDNF、TrkB mRNA和蛋白表达。结果与对照组比较,各染氟组细胞存活率降低(P均<0.01)。未转染细胞中,与对照组比较,miR-204表达升高(P<0.05或<0.01);5.0、10.0 mg/L染氟组BDNF mRNA表达降低(P均<0.01),各染毒组BDNF蛋白表达降低(P<0.05或<0.01);各染毒组TrkB mRNA表达降低(P<0.05或<0.01),5.0、10.0 mg/L染氟组TrkB蛋白表达降低(P均<0.01)。转染细胞中,与对照组比较,各染毒组miR-204表达升高(P均<0.01),BDNF、TrkB mRNA和蛋白表达下降(P均<0.01)。细胞存活率与染氟浓度呈负相关(r=-0.989,P<0.01);染氟浓度与BDNF、TrkB mRNA、蛋白表达呈负相关(r=-0.746、-0.853、-0.889、-0.827,P均<0.01);miR-204表达与染氟浓度呈正相关(r=0.889,P<0.01),与BDNF、TrkB mRNA和蛋白表达呈负相关(r=-0.766、-0.770,-0.594、-0.523,P均<0.01);TrkB mRNA和蛋白表达与BDNF mRNA和蛋白表达呈正相关(r=0.657、0.869,P均<0.01)。结论氟降低HT22细胞活性,作用机制可能与上调miR-204表达后抑制了BDNF-TrkB通路有关。

Objective To explore the changes of microRNA(miRNA,miR)-204,brain-derived neurotrophic factor(BDNF)and tyrosine kinase receptor B(TrkB)expression levels in HT22 hippocampal neurons exposed to fluorine.Methods The HT22 cells were exposed to NaF at 0,2,4,6,8,10 mg/L.After 24 h,the cell viability was detected by CCK-8.According to the cell survival rate,the NaF concentrations[0.0(control),2.5,5.0 and 10.0 mg/L]were selected for subsequent experiments.The infected(without transfection)and transfected(with the addition of miR-204 agonist)HT22 cells were both exposed to NaF for 24 h.The miR-204,BDNF and TrkB mRNA expression levels in cells were detected by Real time fluorescence quantitative PCR(qRT-PCR);the BDNF and TrkB protein expression levels in cells were detected by Western blotting.Results Compared with the control group,the cell viabilities in fluorine exposure groups were decreased(P<0.01).In the infected groups,compared with the control group,and the miR-204 expression levels were increased(P<0.05 or<0.01).The expressions of BDNF mRNA were decreased in fluorine exposure groups at 5.0 and 10.0 mg/L(P<0.01)and the BDNF protein expressions were decreased in all fluorine exposure groups(P<0.05 or<0.01).In the exposure groups,TrkB mRNA expressions were decreased(P<0.05 or<0.01).The TrkB protein expressions were decreased in fluorine exposure groups at 5.0 and 10.0 mg/L(P<0.01).In the transfected groups,compared with the control group,the expressions of miR-204 were increased(P<0.01)and the mRNA and protein expressions of BDNF and TrkB were decreased(P<0.01).The negative correlation was found between NaF concentration and cell survival rate(r=-0.989,P<0.01).Moreover,the mRNA and protein expressions of BDNF and TrkB were negative correlated with NaF concentration(r=-0.746,-0.853,-0.889,-0.827,P<0.01).A positive correlation was found between NaF concentration and miR-204 expression(r=0.889,P<0.01).However,the mRNA and protein expressions of BDNF and TrkB were negative correlated with miR-204 expression(r=-0.766,-0.770,-0.594,-0.523,P<0.01).The positive correlations were found between BDNF mRNA and protein expressions and those of TrkB(r=0.657,0.869,P<0.01).Conclusion Fluorine has inhibited the cell activity of HT22,and the mechanism of action may be related to the inhibition of BDNF-TrkB pathway after up-regulation of miR-204 expression.