中国组织工程研究

中国组织工程研究 ›› 2024, Vol. 28 ›› Issue (7): 1070-1075.doi: 10.12307/2024.118

• 干细胞基础实验 basic experiments of stem cells • 上一篇    下一篇

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

杨  淳1，温建霞1，冯江龙1，2，官志忠1，2，3，魏  娜1，2   

  1. 1贵州医科大学病理学教研室，贵州省贵阳市   550004；2贵州医科大学附属医院病理科，贵州省贵阳市   550004；3地方病与少数民族疾病(贵州医科大学)教育部重点实验室, 贵州省贵阳市   550004
  • 收稿日期:2023-02-09 接受日期:2023-03-14 出版日期:2024-03-08 发布日期:2023-07-17
  • 通讯作者: 魏娜，博士，副教授，贵州医科大学病理学教研室，贵州省贵阳市 550004；贵州医科大学附属医院病理科，贵州省贵阳市 550004
  • 作者简介:杨淳，女，1997年生，贵州省天柱县人，侗族，贵州医科大学在读硕士，主要从事氟中毒有关研究。
  • 基金资助:
    国家自然科学基金(U1812403)，项目参与人：官志忠；国家自然科学基金(81560512)，项目负责人：魏娜；贵州省科技计划项目(黔科合基础-ZK[2022]一般439]，项目负责人：魏娜；地方病与少数民族疾病教育部重点实验室(贵州医科大学)开放项目(FZSW-2021-007)，项目负责人：魏娜

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

Yang Chun1, Wen Jianxia1, Feng Jianglong1, 2, Guan Zhizhong1, 2, 3, Wei Na1, 2   

  1. 1Department of Pathology, Guizhou Medical University, Guiyang 550004, Guizhou Province, China; 2Department of Pathology, Affiliated Hospital of Guizhou Medical University, Guiyang 550004, Guizhou Province, China; 3Key Laboratory of Endemic and Minority Diseases (Guizhou Medical University), Ministry of Education, Guiyang 550004, Guizhou Province, China
  • Received:2023-02-09 Accepted:2023-03-14 Online:2024-03-08 Published:2023-07-17
  • Contact: Wei Na, MD, Associate professor, Department of Pathology, Guizhou Medical University, Guiyang 550004, Guizhou Province, China; Department of Pathology, Affiliated Hospital of Guizhou Medical University, Guiyang 550004, Guizhou Province, China
  • About author:Yang Chun, Master candidate, Department of Pathology, Guizhou Medical University, Guiyang 550004, Guizhou Province, China
  • Supported by:
    National Natural Science Foundation of China, No. U1812403 (to GZZ), 81560512 (to WN); Science and Technology Program of Guizhou Province, No. Qiankehe Foundation-ZK[2022] General 439 (to WN); Open Project of the Key Laboratory of Endemic and Minority Diseases of the Ministry of Education (Guizhou Medical University), No. FZSW-2021-007 (to WN)

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摘要：


文题释义：

N-甲基-D-天冬氨酸受体：是神经细胞膜上主要的兴奋性神经递质——谷氨酸受体，包含NR1、NR2(A、B、C、D)和NR3(A、B)3种亚基，其中NR1是基本亚基，NR2A和NR2B为调节亚基，通过受体亚单位的相互结合成为受体复合物介导钙离子内流，参与了突触可塑性、学习记忆等重要生理活动。
内质网应激：当内质网中未折叠蛋白和错误折叠蛋白过度积累时，会激活一种机体的自我保护机制——内质网应激，引发未折叠蛋白质反应，通过控制特定转录因子的表达，增强错误折叠蛋白的降解，减少新蛋白的合成来保护细胞免受内质网应激。


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10 μmol/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)；④SH-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)；⑤SH-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。

https://orcid.org/0000-0006-8984-1130 (杨淳) 

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

关键词: 氟中毒, IRE1α, ASK1, JNK, NMDA受体, 拮抗剂, Ifenprodil, MK-801

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

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