氟暴露下SH-SY5Y细胞ERK5信号转导通路的改变

Changes of ERK5 signal transduction pathway in SH-SY5Y cells exposed to fluoride

目的检测ERK5信号通路中磷酸化的细胞外信号调节激酶5(P-ERK5)及P-ERK5在氟暴露下SH-SY5Y细胞中的表达,探讨ERK5信号通路与氟暴露下SH-SY5Y神经损伤机制的关系。方法用不同剂量氟化钠(NaF,0.000、0.100、0.200、0.300、0.400、0.500、1.000、2.000、3.000、4.000及5.000 mmol/L)处理SH-SY5Y细胞24 h及48 h,采用CCK8实验筛选出低和高剂量氟中毒浓度;再以正常组SH-SY5Y细胞为参照,用低、高剂量氟化钠剂量分别处理SH-SY5Y细胞24 h和48 h,采用实时荧光定量PCR检测处理48 h时各组细胞的ERK5 mRNA水平,用Western blot检测P-ERK5、ERK5蛋白表达水平。结果染氟处理后,0.500、1.000 mmol/L NaF处理SH-SY5Y细胞24 h及48 h细胞的存活率均低于正常对照组(P<0.05),可作为低剂量组和高剂量组的染氟剂量;染氟48 h时,对照组、低剂量组和高剂量组SH-SY5Y细胞中ERK5的mRNA表达水平分别为1.00±0.00、0.85±0.08、0.94±0.21,组间比较均无统计学意义。染氟24 h时,P-ERK5蛋白高氟组较低氟组和正常组表达升高,差异有统计学意义(P<0.05),而染氟48 h时,染氟组P-ERK5蛋白表达升高,各组间比较均有统计学意义(P<0.05),染氟24 h和48 h正常组、低氟组、高氟组P-ERK5蛋白表达水平分别为(0.20±0.05、0.28±0.04、0.50±0.18)、(0.28±0.01、0.46±0.02、0.61±0.08);染氟24 h后,总ERK5蛋白升高,高氟组较低氟组、正常组有意义(F=14.731,P<0.05),而染氟48 h后,染氟组较正常组升高有统计学意义(F=17.877,P<0.05),染氟24 h和48 h总ERK蛋白表达水平分别为(0.34±0.04、0.36±0.05、0.67±0.12),(0.55±0.02、0.72±0.03、0.78±0.08)。结论ERK5信号通路中P-ERK5蛋白及ERK5蛋白在慢性氟中毒时被激活,随着染氟时间与染氟剂量增加其表达水平出现逐渐升高,其可能参与代偿性神经保护机制对抗氟的神经毒性作用。

Objective To investigate the expression of phosphorylated extracellular signal regulated kinase 5(P-ERK5)in ERK5 signal transduction pathway and extracellular signal regulated kinase 5 in SH-SY5Y cells exposed to fluoride,and to investigate the mechanism between ERK5 signal transduction pathway and nerve injury in SH-SY5Y cells exposed to fluoride.Methods SH-SY5Y cells were treated with different doses of sodium fluoride(NaF,0.000,0.100,0.200,0.300,0.400,0.500,1.000,2.000,3.000,4.000,and 5.000 mmol/L)for 24 and 48 hours respectively.The low and high doses of fluoride intoxication concentration were screened by CCK8 experiment;SH-SY5Y cells in normal group were used as reference,SH-SY5Y cells were treated with low and high doses of sodium fluoride for 24 h and 48 h respectively,ERK5 mRNA level was detected by real-time fluorescent quantitative PCR on each group at 48 h;P-ERK5 and ERK5 protein expression level was detected by Western blot.Results After fluoride treatment,the 24h and 48h survival rates of SH-SY5Y cells treated with 0.500 and 1.000 mmol/L sodium fluoride were lower than those of the normal control group(P<0.05),which could be used as the fluoride staining dose of low-dose and high-dose group.After 48 hours of fluoride exposure,the mRNA expression levels of ERK5 in three groups were 1.00±0.00,0.85±0.08,and 0.94±0.21,respectively,with no statistical significance between groups.After 24 hours of fluoride exposure,the expression of P-ERK5 protein in the high fluoride group increased more than that of low fluoride group and the normal group,difference was statistically significant(P<0.05).After 48 hours of fluoride exposure,the expression of P-ERK5 protein in the fluoride group increased,comparison among groups showed statistical significance(P<0.05).The expression levels of P-ERK5 protein in normal group,low fluoride group and the high fluoride group were(0.20±0.05,0.28±0.04,0.50±0.18;0.28±0.01,0.46±0.02,0.61±0.08)respectively after 24 and 48 hours of fluoride exposure.After 24 hours of fluoride exposure,the total ERK5 protein expression increased in general;high fluoride group showed more statistical significance than that of low fluoride group and normal group(F=14.731,P<0.05);while after 48 hours of fluoride exposure,the total ERK5 protein expression level in the fluoride group was significantly higher than that of normal group(F=17.877,P<0.05);total ERK protein expression levels were(0.34±0.04,0.36±0.05,0.67±0.12)and(0.55±0.02,0.72±0.03,0.78±0.08),respectively.Conclusion P-ERK5 protein and ERK5 protein in ERK5 signal pathway are activated in chronic fluorosis,and their expression levels gradually increase with the increase of fluoride exposure time and dosage.Such mechanism may be involved in compensatory neuroprotective mechanism against the neurotoxicity of fluoride.