梓醇促进蓝斑核分泌去甲肾上腺素减轻EAE动物的病理损伤

发布时间：2018-08-06 21:29

【摘要】：背景:多发性硬化(multiple sclerosis,MS)是一种以广泛性脱髓鞘、少突胶质细胞缺失、轴突变性为特点的中枢神经系统慢性炎症性疾病。其病因学和发病机制尚未明确。当前,人们普遍认为多发性硬化是由多种因素包括遗传和环境因素引起的复杂的自身免疫性疾病。然而,越来越多的研究发现蓝斑—去甲肾上腺素能神经系统(locus coeruleus noradrenaline system,LC-NA)功能失调也许是多发性硬化的病因之一。中枢神经系统内的去甲肾上腺素(neurotransmitter noradrenaline,NA)具有抗炎和神经保护作用,其水平下降会导致炎症加剧和神经元细胞的损伤。中枢神经系统内NA的主要来源为位于第四脑室底的蓝斑核中的酪氨酸羟化酶(tyrosine hydroxylase,TH)阳性神经元细胞。TH是NA合成的限速酶,因此,提高蓝斑神经元细胞内TH的表达有益于增加NA的水平。梓醇是中药地黄的提取物,对由氧化应激和缺血造成的神经损伤具有保护作用,而且能够促进神经轴突的生长。方法:使用髓鞘少突胶质细胞糖蛋白35-55(MOG35-55)多肽免疫C57BL/6小鼠建立实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis,EAE)小鼠模型,并在使用N-(2-氯乙基)-N-乙基-2-溴苄胺盐酸盐[N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine,DSP-4]化学性损毁蓝斑的基础上建立了DSP4-EAE小鼠模型,观察两组动物的临床症状和神经功能评分;梓醇预处理后,观察比较梓醇组和EAE组动物的发病情况,Wesern blot方法检测蓝斑组织的酪氨酸羟化酶(TH)蛋白含量,ELISA方法检测脑和脊髓内的去甲肾上腺素(NA)水平,针对NA能神经元,使用免疫荧光进行特异性染色,对TH阳性神经元的数量进行定量分析。此外,我们取胚胎大鼠的蓝斑组织进行原代细胞培养,检测其TH蛋白含量和NA释放水平。结果:首先,本研究发现小鼠EAE模型中,LC的NA能神经元数量与健康小鼠相比无明显改变。DSP4预处理后的小鼠EAE模型,与EAE模型相比,神经功能评分明显加重,且发病时间提前。其次,在证实了LC的损毁对EAE发病具有促进作用以后,我们发现EAE小鼠脑和脊髓的NA水平下降,蓝斑内TH的含量减少。梓醇可以提高EAE小鼠脑和脊髓内的NA水平,增加蓝斑内的TH含量。第三,在蓝斑神经元原代培养的体外实验中,我们进一步证明了NA缺失导致神经元细胞氧化应激损伤加重,活性下降。梓醇可以促进蓝斑神经元分泌NA,减轻氧化应激损伤,提高细胞活性。梓醇对原代培养的蓝斑神经元内的TH含量无明显影响。结论:研究结果表明,蓝斑退变造成的中枢神经系统内NA的缺失可以导致EAE模型小鼠的神经功能评分加重和发病时间提前;梓醇可以显著减轻EAE小鼠的发病,改善蓝斑TH阳性神经元细胞的功能,提高蓝斑组织内TH的含量和中枢神经系统内NA的水平。因此,蓝斑—去甲肾上腺素能神经系统(LC-NA)功能失调是多发性硬化的病因之一,促进了MS的发病过程。
[Abstract]:Background: multiple sclerosis (MS) is a chronic inflammatory disease of central nervous system characterized by extensive demyelination, oligodendrocyte deletion and axonal degeneration. Its etiology and pathogenesis are not clear. At present, multiple sclerosis is a complex autoimmune disease caused by many factors, including genetic and environmental factors. However, a growing number of studies have found that locus coeruleus-noradrenergic nervous system (locus coeruleus noradrenaline-na dysfunction may be one of the causes of multiple sclerosis. Neurotransmitter noradrenaline (na) in the central nervous system (CNS) has anti-inflammatory and neuroprotective effects, and a decrease in its level can lead to the exacerbation of inflammation and the injury of neuronal cells. The main source of na in the central nervous system is the tyrosine hydroxylase (th) -positive neurons located in the locus coeruleus of the fourth ventricle. Th is the rate-limiting enzyme of na synthesis. Increasing th expression in neurons of locus coeruleus is beneficial to increase na level. Catalpol is the extract of Rehmannia glutinosa, which has protective effect on nerve injury caused by oxidative stress and ischemia, and can promote the growth of nerve axon. Methods: C57BL/6 mice were immunized with myelin oligodendrocyte glycoprotein 35-55 (MOG35-55) peptide to establish an experimental autoimmune encephalomyelitis (experimental autoimmune encephalomyelitis) model. On the basis of using N- (2-chloroethyl) -N-ethyl-2-bromobenzylamine DSP-4 (N- (2-chloroethyl) -N-ethyl-2-bromobenzylamine DSP-4) chemical damage to locus coeruleus, the DSP4-EAE mouse model was established. To observe and compare the incidence of catalpol group and EAE group, to detect tyrosine hydroxylase (TH) protein content in locus coeruleus tissue by Wesern blot method and to detect noradrenaline (NA) level in brain and spinal cord by Elisa. The number of th positive neurons was quantitatively analyzed by immunofluorescence staining. In addition, we took the locus coeruleus tissue from embryonic rats for primary cell culture, and detected the th protein content and na release level. Results: first of all, we found that the number of na neurons of LC in mouse EAE model had no significant change compared with that of healthy mice. Compared with EAE model, the neural function score was significantly aggravated and the onset time was earlier than that in EAE model pretreated with DSP4. Secondly, after confirming that LC lesion can promote the pathogenesis of EAE, we found that na level in brain and spinal cord of EAE mice decreased and th content in locus coeruleus decreased. Catalpol increased na levels in brain and spinal cord of EAE mice and increased th content in locus coeruleus. Thirdly, in the primary culture of locus coeruleus neurons in vitro, we further demonstrated that na deficiency resulted in increased oxidative stress damage and decreased activity of neurons. Catalpol can promote NAsecretion of locus coeruleus neurons, reduce oxidative stress injury and increase cell activity. Catalpol had no effect on th content in primary cultured locus coeruleus neurons. Conclusion: the results showed that the absence of na in the central nervous system caused by locus coeruleus degeneration could lead to the aggravation of neurological function score and the advance of onset time in EAE model mice, and catalpol could significantly reduce the onset of EAE mice. To improve the function of th positive neurons in locus coeruleus, increase the content of th in locus coeruleus tissue and na level in central nervous system. Therefore, locus coeruleus-noradrenergic nervous system (LC-NA) dysfunction is one of the etiology of multiple sclerosis, which promotes the pathogenesis of MS.
【学位授予单位】：首都医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R744.51

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