Abstract: BACKGROUND: Previous studies have shown that N-methyl-D-aspartic acid receptor (NMDA) receptors are associated with fluorine, but the role in fluoride-induced endoplasmic reticulum stress remains unclear. 
OBJECTIVE: To observe the changes of excitatory neurotransmitter NMDA receptor and endoplasmic reticulum stress IRE1α-ASK1-JNK pathway protein expression in brain tissue of rats with experimental fluorosis, and to investigate the pathogenesis of neurological injury in fluorosis by giving NMDA receptor inhibitor to SH-SY5Y cells. 
METHODS: (1) Animal model: 18 1-month-old SD rats were randomly divided into control group (drinking water fluoride content < 0.5 mg/L), low fluoride group (drinking water fluoride content 10.0 mg/L) and high fluoride group (drinking water fluoride content 100.0 mg/L), with 6 rats in each group, half of each sex. After 6 months of fluoride intake, the rats were observed for the occurrence of dental fluorosis, and the 24-hour urinary fluoride content was measured. After anesthesia and euthanasia, the brain tissue of rats was taken to observe the pathological changes. Western blot assay was used to detect NMDA receptors and IRE1α, ASK1 and JNK protein expression in the brain tissue. (2) Cell model: SH-SY5Y cells were cultured in vitro and treated with sodium fluoride at final concentrations of 0.3 mmol/L and 3 mmol/L. The fluoride-stained cells were interfered with 10 μmol/L NMDA receptor antagonists Ifenprodil and MK-801 to observe the relevant protein changes. 
RESULTS AND CONCLUSION: (1) The incidence of dental fluorosis and urinary fluoride level in rats in the high fluoride group were significantly higher than that in the control and low fluoride groups (P < 0.05). (2) Compared with the control group, the cytoplasm of neuronal cells in the CA3 area of the hippocampus in the low fluoride group was slightly more basophilic, while the neuronal cells in the CA3 area of the high fluoride group were disorganized, with increased basophilicity and some of the nuclei solidified. (3) In rat brain tissue, the expressions of NR2A in the high fluoride group and NR2B in the low fluoride group were significantly higher compared with the control group (P < 0.05), and NR2B, IRE1, ASK1, and p-JNK protein expression levels were increased in the high fluoride group compared with the control and low fluoride groups (P < 0.05). (4) In SH-SY5Y cells, NR1, NR2A and NR2B protein expressions were significantly increased in the high fluoride group compared to the control group (P < 0.05). The protein levels of NR1 and NR2A were significantly reduced in the high fluorine + Ifenprodil group and high fluorine + MK-801 group compared with the high fluorine group (P < 0.05). NR2B protein expression was significantly lower in the high fluorine + Ifenprodil group than that in the high fluorine group (P < 0.05). (5) In SH-SY5Y cells, IRE1, ASK1, and p-JNK protein expression was significantly higher in the high fluoride group compared with the control group (P < 0.05), while ASK1 and p-JNK protein expressions were significantly decreased in the high fluorine + Ifenprodil group and high fluorine + MK-801 group compared with the high fluorine group (P < 0.05). IRE1 protein level was significantly lower in the high fluorine + Ifenprodil group than that in the high fluorine group (P < 0.05). (6) It is concluded that excessive fluorine intake activates NMDA receptors in the central nervous system, causing increased expression of endoplasmic reticulum stress IRE1α, ASK1, and p-JNK proteins, and the use of NMDA receptor inhibitors has a mitigating effect on endoplasmic reticulum stress caused by fluorosis.

Key words: fluorosis, IRE1α, ASK1, JNK, NMDA receptor, antagonist, Ifenprodil, MK-801