Regulatory Role Of I Group MGluR On Hippocampal Neurons Synaptic Plasticity Damage Induced By Fluoride And Arsenic Combined Exposure

Objective The present study was designed to explore the effect of fluoride and arsenic joint exposure on growth of primary cultured hippocampal neurons, synaptic plasticity, the protein expressions ofⅠgroup metabolic glutamate receptor.The study would be useful for clarifing the molecular mechanism of learning and memory damage induced by fluoride and arsenic joint exposure and providing theoretical basis for the prevention and control for fluoride and arsenic poisoning.Methods 1 The hippocampal neurons taken from newborn SD rats were cultured in vitro. The cell viability was determined with CCK8 kit, the activity of lactate dehydrogenase(lactate dehydrtogenase, LDH) in cell culture supernatant was detected using LDH kit. The cell apoptosis was detected by Annexin V-FITC apoptosis detection kit, the protein expressions of SYN and GAP-43 were detected by the Western-blot method in hippocampal neurons after the cells were incubated with sodium fluoride at the concentrations of 0.00,0.10,0.20,0.40,0.80 μg/ml and sodium arsenite at the concentrations of 0, 0.05, 0.10, 0.20, 0.40 μg/ml for 24 h,respectively. 2 At the same time, the primary cultured hippocampal neurons from neonatal SD rats in the fluoride-arsenic exposure were given three kinds of glutamate receptor antagonist D-AP5(NMDAR inhibitor), LY367385(m Glu R1 inhibitor), MPEP(m Glu R5 inhibitor) intervention for 24 h. The cell viability was determined with CCK8 kit, the protein expressions of NR2 B, m Glu R1α, m Glu R5,SYN and GAP-43 were detected by the Western-blot method.Results 1 The survival rate of primary cultured hippocampal neurons and the pro portion of hippocampal neurons with rich neural network decreased gradually with the increase of sodium fluoride and sodium arsenite concentration in the groups exposed to fluoride and arsenic. 2 The apoptosis rate of primary cultured hippoca mpal neurons and the activities of LDH in cell culture supernatant lactate increase d with the increase of sodium fluoride and sodium arsenite concentration in the gr oups exposed to fluoride and arsenic. 3 With the concentration of sodium fluoride exposure increased, there was an appreciable decrease in GAP-43 protein expressi on, SYN protein expression decrease after the first rise. 4 There was an appreciab le decrease in SYN and GAP-43 protein expressions with the concentration of ars enic exposure increased. 5 Compared with the blank control group, the cell surviv al rate and the corresponding glutamate receptor protein expressions(NR2B, m Glu R1α, m Glu R5) in the primary cultured hippocampal neurons in the fluoride-arsenic exposure group and different concentrations of three kinds of glutamate receptor antagonist groups( D-AP5 group, LY367385 group, MPEP group) significantly decreased(P<0.05). Compared with the fluoride-arsenic exposure group, the cell s urvival rate and the expression level of the corresponding glutamate receptor prote in(NR2B, m Glu R1α, m Glu R5) increased in primary cultured hippocampal neurons in different concentration of three kinds of glutamate receptor antagonist groups(P<0.05). And with the increase of glutamate receptor inhibitor concentration, the cell survival rate and the corresponding protein expression levels were significantly higher. 6 Compared with blank control group, the SYN and GAP-43 protein expr ession levels significantly decreased in hippocampal neurons in the fluoride arsenic exposure group, 50μM D-AP5 group, 25μM LY367385 group and 20μM MPEP group(P<0.01).Compared with the fluoride-arsenic exposure group, the levels of S YN and GAP-43 protein expression were significantly higher in hippocampal neur ons in 50μM D-AP5, 25μM LY367385 and 20μM MPEP group(P<0.05).Conclusion 1 Fluorine and arsenic exposure alone may reduce the cell survival and LDH activity of primary cultured hippocampal neurons.Fluorine and arsenic exposure alone induced apoptosis,reduced the expression of SYN and GAP-43 protein and damaged synaptic plasticity in the primary cultured hippocampal neurons. 2 Fluoride and arsenic joint exposure may affect synaptic plasticity in primary cultured hippocampal neurons through the regulation the expression levels of NR2 B, m Glu R1α, m Glu R5 protein.