氟中毒大鼠脑组织中NMDA受体及内质网应激相关通路蛋白的表达

Expression of N-methyl-D-aspartic acid receptor and endoplasmic reticulum stress related pathway proteins in brain tissue of fluorosis rats

背景:前期研究发现N-甲基-D-天冬氨酸(N-methyl-D-aspartic acid receptor,NMDA)受体与氟相关,但其在氟诱导的内质网应激中的作用尚不明确。目的:观察实验性氟中毒大鼠脑组织中兴奋性神经递质NMDA受体及内质网应激IRE1α-ASK1-JNK通路蛋白表达变化,并应用NMDA受体抑制剂干预SH-SY5Y细胞,探讨氟中毒神经系统损伤的发病机制。方法:(1)动物模型:18只1月龄SD大鼠随机分为对照组(饮水含氟量<0.5 mg/L)、低氟组(饮水含氟量为10.0 mg/L)、高氟组(饮水含氟量为100.0 mg/L),每组6只,雌雄各半。饮水摄氟饲养6个月后,观察大鼠氟斑牙发生情况,测定24 h尿氟含量;大鼠麻醉处死后取脑组织观察组织病理变化,蛋白印迹法检测脑组织中NMDA受体及IRE1α、ASK1、JNK蛋白表达。(2)细胞模型:体外培养SH-SY5Y细胞,选择终浓度为0.3 mmol/L和3 mmol/L的氟化钠染氟处理,并用10μmol/L的NMDA受体拮抗剂Ifenprodil、MK-801对染氟细胞进行干预,观察相关蛋白改变。结果与结论:(1)高氟组大鼠氟斑牙发生率、尿氟水平显著高于对照组和低氟组(P<0.05);(2)与对照组相比,低氟组大鼠海马CA3区神经细胞胞质嗜碱性略增加,高氟组CA3区神经细胞排列紊乱,嗜碱性增加,部分细胞核固缩;(3)大鼠脑组织内,高氟组NR2A与低氟组NR2B的蛋白表达明显高于对照组(P<0.05),且高氟组NR2B、IRE1、ASK1、p-JNK蛋白表达明显高于对照组和低氟组(P<0.05);(4)SH-SY5Y细胞内,高氟组NR1、NR2A、NR2B蛋白表达明显高于对照组(P<0.05),且高氟+Ifenprodil组、高氟+MK-801组NR1、NR2A蛋白表达均明显低于高氟组(P<0.05),高氟+Ifenprodil组NR2B蛋白表达也明显低于高氟组(P<0.05);(5)SH-SY5Y细胞内,高氟组IRE1、ASK1、p-JNK蛋白表达明显高于对照组(P<0.05),且高氟+Ifenprodil组、高氟+MK-801组ASK1、p-JNK蛋白表达均明显低于高氟组(P<0.05),高氟+Ifenprodil组IRE1蛋白水平也明显低于高氟组(P<0.05);(6)提示:过量氟摄入可激活中枢神经系统NMDA受体,引起内质网应激IRE1α、ASK1、p-JNK蛋白表达增加,使用NMDA受体抑制剂对氟中毒所致内质网应激具有一定缓解作用。

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:181-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 10μmol/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.