梓醇促進藍(lán)斑核分泌去甲腎上腺素減輕EAE動物的病理損傷
發(fā)布時間：2018-08-06 21:29
【摘要】：背景:多發(fā)性硬化(multiple sclerosis,MS)是一種以廣泛性脫髓鞘、少突膠質(zhì)細(xì)胞缺失、軸突變性為特點的中樞神經(jīng)系統(tǒng)慢性炎癥性疾病。其病因?qū)W和發(fā)病機制尚未明確。當(dāng)前,人們普遍認(rèn)為多發(fā)性硬化是由多種因素包括遺傳和環(huán)境因素引起的復(fù)雜的自身免疫性疾病。然而,越來越多的研究發(fā)現(xiàn)藍(lán)斑—去甲腎上腺素能神經(jīng)系統(tǒng)(locus coeruleus noradrenaline system,LC-NA)功能失調(diào)也許是多發(fā)性硬化的病因之一。中樞神經(jīng)系統(tǒng)內(nèi)的去甲腎上腺素(neurotransmitter noradrenaline,NA)具有抗炎和神經(jīng)保護作用,其水平下降會導(dǎo)致炎癥加劇和神經(jīng)元細(xì)胞的損傷。中樞神經(jīng)系統(tǒng)內(nèi)NA的主要來源為位于第四腦室底的藍(lán)斑核中的酪氨酸羥化酶(tyrosine hydroxylase,TH)陽性神經(jīng)元細(xì)胞。TH是NA合成的限速酶,因此,提高藍(lán)斑神經(jīng)元細(xì)胞內(nèi)TH的表達(dá)有益于增加NA的水平。梓醇是中藥地黃的提取物,對由氧化應(yīng)激和缺血造成的神經(jīng)損傷具有保護作用,而且能夠促進神經(jīng)軸突的生長。方法:使用髓鞘少突膠質(zhì)細(xì)胞糖蛋白35-55(MOG35-55)多肽免疫C57BL/6小鼠建立實驗性自身免疫性腦脊髓炎(experimental autoimmune encephalomyelitis,EAE)小鼠模型,并在使用N-(2-氯乙基)-N-乙基-2-溴芐胺鹽酸鹽[N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine,DSP-4]化學(xué)性損毀藍(lán)斑的基礎(chǔ)上建立了DSP4-EAE小鼠模型,觀察兩組動物的臨床癥狀和神經(jīng)功能評分;梓醇預(yù)處理后,觀察比較梓醇組和EAE組動物的發(fā)病情況,Wesern blot方法檢測藍(lán)斑組織的酪氨酸羥化酶(TH)蛋白含量,ELISA方法檢測腦和脊髓內(nèi)的去甲腎上腺素(NA)水平,針對NA能神經(jīng)元,使用免疫熒光進行特異性染色,對TH陽性神經(jīng)元的數(shù)量進行定量分析。此外,我們?nèi)∨咛ゴ笫蟮乃{(lán)斑組織進行原代細(xì)胞培養(yǎng),檢測其TH蛋白含量和NA釋放水平。結(jié)果:首先,本研究發(fā)現(xiàn)小鼠EAE模型中,LC的NA能神經(jīng)元數(shù)量與健康小鼠相比無明顯改變。DSP4預(yù)處理后的小鼠EAE模型,與EAE模型相比,神經(jīng)功能評分明顯加重,且發(fā)病時間提前。其次,在證實了LC的損毀對EAE發(fā)病具有促進作用以后,我們發(fā)現(xiàn)EAE小鼠腦和脊髓的NA水平下降,藍(lán)斑內(nèi)TH的含量減少。梓醇可以提高EAE小鼠腦和脊髓內(nèi)的NA水平,增加藍(lán)斑內(nèi)的TH含量。第三,在藍(lán)斑神經(jīng)元原代培養(yǎng)的體外實驗中,我們進一步證明了NA缺失導(dǎo)致神經(jīng)元細(xì)胞氧化應(yīng)激損傷加重,活性下降。梓醇可以促進藍(lán)斑神經(jīng)元分泌NA,減輕氧化應(yīng)激損傷,提高細(xì)胞活性。梓醇對原代培養(yǎng)的藍(lán)斑神經(jīng)元內(nèi)的TH含量無明顯影響。結(jié)論:研究結(jié)果表明,藍(lán)斑退變造成的中樞神經(jīng)系統(tǒng)內(nèi)NA的缺失可以導(dǎo)致EAE模型小鼠的神經(jīng)功能評分加重和發(fā)病時間提前;梓醇可以顯著減輕EAE小鼠的發(fā)病,改善藍(lán)斑TH陽性神經(jīng)元細(xì)胞的功能,提高藍(lán)斑組織內(nèi)TH的含量和中樞神經(jīng)系統(tǒng)內(nèi)NA的水平。因此,藍(lán)斑—去甲腎上腺素能神經(jīng)系統(tǒng)(LC-NA)功能失調(diào)是多發(fā)性硬化的病因之一,促進了MS的發(fā)病過程。
[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.
【學(xué)位授予單位】：首都醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R744.51
[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.
【學(xué)位授予單位】：首都醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R744.51
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