Effect Of Fluorosis On NMDA Receptor Protein Subunits And Phosphorylation In SH-SY5Y Cells

Objective: Invitro culture SH-SY5 Y cells,apply N-Cymene-D-aspartate acid(NMDA)the noncompetitive antagonist of receptor,dizocilpine(MK-801)as the instrument to protect neural,through detect cytoactive,apoptosis,intracellular Ca2+,NMDA receptor subunit protein and the expression of phosphorylated protein,discuss the mechanism that the NMDA receptor participate in the neural injure cause by fluorosis.Methods: The SH-SY5 Y cells cultured in vitro were intervened with different concentrations of Na F and MK-801,and subsequently divided into six groups:(1)normal control group;(2)low fluorine group(0.2mmol/L Na F);(3)high fluorine group(2mmol/L Na F);(4)antagonist control group(10μmol/L MK-801);(5)low fluorine antagonist group(0.2mmol/L Na F+10μmol/L MK-801);and(6)high fluorine antagonist group(2mmol/L Na F+10μmol/L MK-801).The cell viability was assayed by CCK-8 to screen the appropriate treatment concentrations of Na F and MK-801;the intracellular Ca2+ concentration was determined by Fluo-3-AM intracellular calcium ion fluorescence staining;the apoptosis rate was detected by Annexin V-FITC and PI double staining as well as flow cytometry;and the expression of NMDAR subunit proteins and their phosphorylated proteins such as NMDAR1,NMDAR2 A,NMDAR2B,phospho-NMDAR1,phospho-NMDAR2 A and phospho-NMDAR2 B were examined by Western blot.The statistical analyses were done through SPSS 22.0 software.Results: 1.The damage of Na F on SH-SY5 Y cells showed concentration-and time-dependent way.The screened concentration of Na F in the low fluorine and the high fluorine was 0.2mmol/L and 2mmol/L,respectively,and the applied concentration of MK-801 was 10μmol/L.2.Intracellular Ca2+ concentration: Compared with the control group,both average intracellular Ca2+ fluorescence value was significantly increased in the low and high fluorine group,whereas it was decreased markedly in the high fluorine group followed by intervention with MK-801,All P were smaller than 0.05.3.Apoptosis of SH-SY5 Y cells: Compared with the control group,the apoptosis rate of SH-SY5 Y cells had no significant change in the low fluoride group,so did in the cells treated with MK-801.However,the apoptosis rate of the cells was obviously higher in the high fluorine group than the control group,and somewhat decreased after intervention with MK-801(P<0.05).4.Expression of NMDA: The expression levels of NMDAR1,NMDAR2 A,phospho-NMDAR1 and phospho-NMDAR2 B were up-regulated in SH-SY5 Y cells exposed to high concentration of fluorine.The increasing expression could be reversed by MK-801(P<0.05).Conclusions: NMDA receptor is associated with the pathogenesis of nervous system injury by excessive fluorine.The possible pathway may be as follows,the phosphorylation of NMDA receptor subunits —overactivation of NMDA receptor—overload of intracellular Ca2+—apoptosis and necrosis of nerve cells.