Regulation Role Of ERK-MAPK Pathway In The Synaptic Plasticity Damage In Hippocampal Neurons Induced By Fluoride

Objectives The study was designed to explore the regulatory role of MAPK/ERK signaling pathway in synapse plasticity injury induced by fluoride in primary hippocampal neurons after the best concentration and action time of inhibitor PD98059 of MAPK-ERK pathway were found.The purpose of the study is to elucidate the molecular mechanism of hippocampal neurons synaptic plasticity damage induced by fluoride exposure,and to provide a theoretical basis for revealing the molecular mechanism of learning and memory impairment induced by fluoride.Methods The best concentration and action time of inhibitor PD98059: The hippocampal coming from SPF-grade SD rats within 24 h after birth were cultured in vitro.The mature hippocampal neurons were exposed to inhibitors with final concentrations of 0.0(blank control),5,10,25,50,100 μmol/L for 24 h.The optimal concentration of inhibitor in the medium was 0 h,3 h,6 h,12 h,24 h,48 h.The hippocampal neuron survival rate was detected using CCK-8,LDH activity in culture supernatant of hippocampal neurons was detected using lactate dehydrogenase(LDH)kit.The mRNA expression level of ERK,CREB in cells was detected by RT-qPCR.The protein expression levels of ERK,pERK1/2,CREB,p-CREB in the cells were measured by Western-blot.Through statistical analysis,the best inhibitor concentration and action time are obtained.ERK-MAPK pathway regulation: According to previous experiments,the optimal fluoride concentration and action time were 0.8 μg/ml and 24 hours respectively.The cells were divided into four groups: blank control group,0.8 μg/ml fluoride treatment group,25 μmol/L inhibitor treatment group,25 μmol/L inhibitor + 0.8 μg/ml fluoride treatment group,cultured for 24 hours.The number of neurite protuberances in the hippocampus was observed by optical inversion microscope.The cell viability of hippocampal neurons was detected by CCK-8 method,calcium levels in cells were detected using laser confocal microscopy.The mRNA expression level of ERK1/2,CREB,BDNF,synaptophysin synaptophysin(SYN),growthassociated protein 43(GAP-43),and postsynaptic density protein 95(PSD-95)in cells was detected by RT-qPCR.The protein expression levels of ERK,p-ERK1/2,CREB,p-CREB,pCaMKII,BDNF,SYN and GAP-43 in the cells were measured by Western-blot.Results 1 There was a significant difference in cell survival rate between different inhibitor treatment groups(P<0.05),the survival rate of 50 and 100 μmol/L group cells was lower than that of the control group,5,10,and 25 μmol/L group(P<0.05).The cell survival rate was lower in the 24 and 48 hours groups than in the 0,3,6,and 12 hours.group(P<0.05).2 The LDH activity in the culture supernatant of hippocampal neurons was significantly different between different inhibitor groups(P<0.05),LDH activity of hippocampal neurons in 25,50,and 100 μmol/L groups was higher than that in the control group and 5 and 10 μmol/L groups(P<0.05),and the LDH activity of hippocampal neurons in 12,24,and 48 h groups was higher than that of 0h,3h,and 6h groups(P <0.05).3 The mRNA expression levels of ERK1/2 and CREB in different inhibitor groups were statistically significant(P<0.05),Among them,the mRNA expression levels of ERK1/2 and CREB were lower in the 25,50,and 100 μmol/L group than in the control group,5,and 10 μmol/L group(P<0.05);The mRNA expression levels of ERK1/2 and CREB were lower in the 12 h,24h and 48 h groups than in the 0h,3h and 6h groups(P<0.05).4 The protein expression levels of p-ERK1/2 and p-CREB were significantly different in different inhibitor groups(P<0.05).Among them,the expression levels of p-ERK1/2 and p-CREB in 25,50,and 100 μmol/L groups were lower than those in the control group,5,and 10 μmol/L groups(P<0.05),and p-ERK1/2,p-CREB protein levels in 12,24,and 48 h groups than in the 0,3 and 6 hours groups(P<0.05).Based on the above results,the optimal concentration and action time of the inhibitors were 25 μmol/L and 24 h.5 The difference in cell viability between different treatment groups was statistically significant(P<0.05),and the survival rate of 0.8 mg/L fluoride treatment group,inhibitor + fluoride treatment group was lower than that of the control group(P<0.05).The survival rate of the fluorine-treated group was higher than that of the 0.8 mg/L fluoride treatment group(P<0.05).6 There were statistically significant differences in calcium fluorescence intensity and pCaMKII protein expression in hippocampal neurons between different treatment groups.(P<0.05),among which 0.8 mg/L fluoride treatment group,inhibitor + fluoride treatment group The calcium fluorescence intensity and pCaMKII protein expression were higher in the control group than in the control group(P<0.05),the calcium ion fluorescence intensity and pCaMKII protein expression in the inhibitor plus fluoride treatment group were lower than those in the 0.8 mg/L fluoride treatment group(P<0.05).7 The expression levels of ERK1/2,CREB,BDNF,SYN,GAP-43 and PSD-95 mRNA in different treatment groups were statistically significant(P<0.05),the expression levels of ERK1/2 and CREB mRNA in the 0.8 mg/L fluoride treatment group,25 μmol/L inhibitor treatment group,and the inhibitor + fluoride treatment group were lower than those in the control group(P<0.05);Inhibitor + fluoride treatment group ERK1 The expression level of CREB mRNA was lower than that of fluoride treatment group(P<0.05).The expression levels of SYN,GAP-43,PSD-95 and BDNF mRNA in the 0.8 mg/L fluoride treated group and the inhibitor + fluoride treatment group were lower than those in the control group(P<0.05).The inhibitors + fluoride treated group SYN,GAP-43,The expression of PSD-95 and BDNF mRNA was higher than that of 0.8 mg/L fluoride treatment group(P<0.05).8 The expression levels of p-ERK1/2,p-CREB,BDNF,SYN,GAP-43 were significantly different among different treatment groups(P<0.05).Among them,the expression levels of p-ERK1/2 and p-CREB protein in the 0.8 mg/L fluoride treatment group,25 μmol/L inhibitor treatment group,and the inhibitor + fluoride treatment group were lower than those in the control group(P<0.05).The expression of p-ERK1/2 and p-CREB protein in the fluoride treatment group was lower than that in the 0.8 mg/L fluoride treatment group(P<0.05).In addition,the expression levels of SYN,GAP-43 and BDNF protein in the 0.8 mg/L fluoride treatment group and the inhibitor + fluoride treatment group were lower than those in the control group(P<0.05),25 μmol/L inhibitor treatment group,and the inhibitor + fluoride treatment group.The expression levels of SYN,GAP-43,BDNF protein were higher than those in fluoride treatment group(P<0.05).Conclusions 1 The MEK inhibitor PD98059 can reduce the effect of fluoride on synaptic plasticity damage in cultured hippocampal neurons in vitro.2 MAPK-ERK pathway positively regulates synaptic plasticity damage of cultured hippocampal neurons in vitro induced by fluoride.