赵宇卿, 李亚芹, 王珊, 张淑惠, 牛侨. 铝致大鼠学习记忆损伤中程序性坏死的作用[J]. 环境与职业医学, 2019, 36(8): 718-724. DOI: 10.13213/j.cnki.jeom.2019.19167
引用本文: 赵宇卿, 李亚芹, 王珊, 张淑惠, 牛侨. 铝致大鼠学习记忆损伤中程序性坏死的作用[J]. 环境与职业医学, 2019, 36(8): 718-724. DOI: 10.13213/j.cnki.jeom.2019.19167
ZHAO Yu-qing, LI Ya-qin, WANG Shan, ZHANG Shu-hui, NIU Qiao. Role of aluminum-induced necroptosis in learning and memory impairment in rats[J]. Journal of Environmental and Occupational Medicine, 2019, 36(8): 718-724. DOI: 10.13213/j.cnki.jeom.2019.19167
Citation: ZHAO Yu-qing, LI Ya-qin, WANG Shan, ZHANG Shu-hui, NIU Qiao. Role of aluminum-induced necroptosis in learning and memory impairment in rats[J]. Journal of Environmental and Occupational Medicine, 2019, 36(8): 718-724. DOI: 10.13213/j.cnki.jeom.2019.19167

铝致大鼠学习记忆损伤中程序性坏死的作用

Role of aluminum-induced necroptosis in learning and memory impairment in rats

  • 摘要: 背景 近年来研究发现铝与多种神经系统疾病的发生发展密切相关,铝的神经毒性作用包括影响中枢神经递质的含量,引起神经细胞死亡,促进Tau蛋白的异常磷酸化和聚集等方面。目前多采用细胞模型来探讨铝致神经细胞程序性坏死的机制。

    目的 本研究采用大鼠模型,探讨肿瘤坏死因子受体1(TNFR1)-受体相互作用蛋白1(RIP1)/受体相互作用蛋白3(RIP3)信号通路是否在铝麦芽酚铝,Al(mal)3致大鼠学习记忆损伤中发挥作用。

    方法 40只健康成年雄性SD大鼠,按体重随机分为手术对照组、溶剂对照组、程序性坏死抑制剂(Nec-1)组、Nec-1+Al(mal)3组和Al(mal)3组,每组8只。采用侧脑室注射的方式对大鼠进行急性染毒,Nec-1组给予1.285 4 mmol/L Nec-1 5 μL,Al(mal)3组给予50 mmol/L Al(mal)3 5 μL,Nec-1+Al(mal)3组在给予50 mmol/L Al(mal)3前1 h注射1.285 4 mmol/L Nec-1 5 μL,溶剂对照组给予等体积的生理盐水,手术对照组采用同样侧脑室插管之外不给予任何试剂处理。每2 d定时染毒1次,连续染毒10 d。染毒结束后,通过Y迷宫测定各组大鼠的自发反应交替率,从而检测大鼠空间学习记忆能力;HE染色观察海马组织病理形态变化,电镜观察海马神经元超微结构,Western blot测定各组大鼠海马组织中TNFR1、RIP1、RIP3的蛋白表达水平。

    结果 手术对照组、溶剂对照组、Nec-1组和Nec-1+Al(mal)3组大鼠的自发反应交替率没有明显差异,分别为(35.88±2.44)%、(34.38±3.06)%、(35.65±4.48)%和(32.01±6.93)%。与其他4组相比,Al(mal)3组大鼠自发反应交替率下降至(21.96±2.05)%(均P < 0.05)。光镜下Al(mal)3组大鼠海马神经元出现不同程度的胞质嗜酸性增强,胞体缩小,胞质呈深红色,细胞排列无序紊乱,神经元数量减少,其余4组大鼠海马神经细胞结构完整、清晰,细胞核清晰可见,核浆比值大,着色均匀,核仁明显,细胞排列整齐有序。电镜下Al(mal)3组大鼠海马神经元胞膜破裂,细胞器大量减少,胞质空泡化,细胞核固缩,核周空化,细胞体积膨胀,神经元之间的突触几不可见;其余4组大鼠海马神经元结构清晰,胞膜完整,胞质饱满,细胞器清晰可见,线粒体、内质网、高尔基体等细胞器围绕在核周,细胞核大而清晰,核仁明显,神经元之间的突触完整且清晰可见。手术对照组、溶剂对照组、Nec-1组和Nec-1+Al(mal)3组间的TNFR1、RIP1和RIP3蛋白表达差异没有统计学意义。与其他4组相比,Al(mal)3组TNFR1、RIP1和RIP3蛋白的表达均明显升高(均P < 0.05)。

    结论 铝可能通过触发TNFR1-RIP1/RIP3信号通路引起程序性坏死,产生神经毒性,进而损伤学习记忆能力。

     

    Abstract: Background In recent years, studies have found that aluminum is closely related to the occurrence and development of various neurological diseases. The neurotoxic effects of aluminum include affectng the content of central neurotransmiters, causing nerve cell death, and promotng abnormal phosphorylaton and aggregaton of Tau protein. Current studies use cell models to investgate the mechanism of necroptosis induced by aluminum.

    Objective This experiment is designed to investigate the role of tumor necrosis factor receptor 1 (TNFR1)-receptor interacting protein 1 (RIP1)/receptor interacting protein 3 (RIP3) signaling pathway in aluminum Al(mal)3-induced learning and memory impairment in rats.

    Methods Forty healthy adult male SD rats were randomly divided into surgical control group, solvent control group, necrostain-1 (Nec-1) group, Nec-1+Al(mal)3 group, and Al(mal)3 group, with eight rats in each group. By intracerebroventricular injecton, the Nec-1 group was given 1.258 4 mmol/L Nec-1 at 5 μL, the Al(mal)3 group was given 50 mmol/L Al(mal)3 at 5 μL, the Nec-1+Al(mal)3 group was injected 5 μL 1.258 4 mmol/L Nec-1 1 hour before being given 5 μL 50 mmol/L Al(mal)3, the solvent control group was given an equal volume of normal saline, and the surgical control group was treated with the same lateral ventricle cannula without any reagent treatment. They were treated every two days and contnuously for 10 days. The spontaneous learning reacton rate of each group of rats was determined by Y maze to detect spatal learning and memory ability of rats; the pathological changes of hippocampus were observed afer HE staining under light microscope; the ultrastructure of hippocampus was observed under electron microscope; the expression levels of TNFR1, RIP1, and RIP3 in hippocampus were measured by Western blot.

    Results There was no significant difference in the rate of spontaneous reaction alternation among the surgical control group(35.88±2.44)%, the solvent control group(34.38±3.06)%, the Nec-1 group(35.65±4.48)%, and the Nec-1+Al(mal)3 group(32.01±6.93)%. Compared with the other four groups, the spontaneous reacton alternaton rate of rats in the Al(mal)3 group decreased to (21.96±2.05)% (Ps < 0.05). The light microscope observaton showed that the hippocampal neurons of the Al(mal)3 group had different degrees of cytoplasmic acidophilia enhancement, shrunk cell body, dark red cytoplasm, disorderly distributed cells, and reduced neurons; by contrast, the hippocampal neurons of the other four groups had intact and clear structure, clear nuclei, large nucleoplasm ratio, obvious nucleolus, even color, and neatly distributed cells. The electron microscope observaton showed that the Al(mal)3 group had ruptured hippocampal neurons, greatly reduced organelles, vacuolated cytoplasm, pyknotc nucleus, perinuclear cavitaton, expanded cell volume, and invisible synapses between neurons; by contrast, the hippocampal neurons in the other four groups had clear structure, intact membrane, full cytoplasm, clear organelles such as perinuclear mitochondria, endoplasmic retculum, and Golgi bodies, large and clear nucleus, obvious nucleolus, and complete and clearly visible synapses between neurons. There was no signifcant difference in the expression of TNFR1, RIP1, and RIP3 among the surgical control group, the solvent control group, the Nec-1 group, and the Nec-1+Al(mal)3 group. Compared with the other four groups, the expression levels of TNFR1, RIP1, and RIP3 of rats in the Al(mal)3 group decreased signifcantly (Ps < 0.05).

    Conclusion Aluminium may cause necroptosis by triggering TNFR1-RIP1/RIP3 signaling pathway, thereby producing neurotoxicity and impairing learning and memory ability.

     

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