Objective To test the effect of glutamate NMDA receptor antagonist D-2-amino-5-phosphonopentanoate (D-AP5) on the expression of synapse-associated proteins in primary cultured hippocampal neurons after fluoride and arsenic co-exposure.
Methods In vitro cultured hippocampal neurons were divided into four groups, including control group, fluoride-arsenic coexposure group (0.4 μg/mL sodium fluoride+0.1 μg/mL sodium arsenite), D-AP5 group (25 μmol/L D-AP5), and D-AP5+ fluoridearsenic co-exposure group (0.4 μg/mL sodium fluoride+0.1 μg/mL sodium arsenite+25 μmol/L D-AP5). Hippocampal neurons in each group were cultured in 5%CO2 incubator at 37℃ for 24 h. Cell viability was detected by CCK8, and the expression levels of GAP43, NR2B, and SYN proteins in hippocampal neurons were detected by Western blot.
Results Compared with the control group (100.00±0.00)%, the cell viability rates of hippocampal neurons in the fluoridearsenic co-exposure group (75.81±6.27)% and the D-AP5+ fluoride-arsenic co-exposure group (81.58±5.20)% were significantly decreased (P < 0.05). Compared with the fluoride-arsenic co-exposure group (75.81±6.27)%, the cell viability rates of hippocampal neurons in the D-AP5 group (98.78±3.53)% and the D-AP5+ fluoride-arsenic co-exposure group (81.58±5.20)% were significantly increased (P < 0.05). Compared with the control group (0.572±0.05), the protein expression levels of NR2B in the fluoride-arsenic co-exposure group (0.425±0.06), D-AP5 group (0.469±0.03), and D-AP5+ fluoride-arsenic co-exposure group (0.354±0.04) were significantly decreased (P < 0.05). The protein expression level of NR2B in the D-AP5+ fluoride-arsenic co-exposure group (0.354±0.04) were significantly lower than that of the fluoride-arsenic co-exposure group (0.425±0.06). The protein expression levels of SYN and GAP43 in the fluoride-arsenic co-exposure group and the D-AP5+ fluoride-arsenic co-exposure group were also significantly decreased (P < 0.05). The protein expression levels of SYN and GAP43 in the D-AP5 group and the D-AP5+ fluoride-arsenic co-exposure group were higher than those of the fluoride-arsenic co-exposure group (P < 0.05).
Conclusion Fluoride-arsenic co-exposure could decrease the levels of synapse-associated proteins SYN and GAP-43. Glutamate NMDA receptor antagonist D-AP5 could inhibit the effect of fluoride and arsenic co-exposure on the expression of synapse-related proteins in hippocampal neurons.
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