补阳还五汤对实验性自身免疫性脑脊髓炎单核巨噬细胞的免疫调控作用

目的:探讨补阳还五汤(BYHWD)治疗实验性自身免疫性脑脊髓炎(EAE)的有效性及对单核巨噬细胞免疫调控的作用及机制。方法:雌性C57BL/6小鼠用小鼠髓鞘少突胶质细胞糖蛋白35-55肽段(MOG_(35-55))免疫制作慢性EAE模型,随机分为生理盐水处理组和BYHWD组。在免疫后第3天开始分别予以生理盐水和BYHWD灌胃,500μL/d,持续观察临床症状和体质量变化。免疫后17 d各组统一处死部分动物,HE染色观察炎性细胞浸润情况,髓鞘染色观察脊髓髓鞘脱失比例,流式细胞术检测脾细胞M1型和M2型巨噬细胞表型;免疫荧光组织化学染色和Western blotting检测脊髓巨噬细胞iNOS、TNF-α、arginase及IL-10的表达。结果:BYHWD推迟EAE起病,减轻EAE症状,抑制中枢神经系统脊髓的炎性浸润和髓鞘脱失,促进脊髓及脾组织中M1型巨噬细胞转化为M2型。结论:BYHWD干预可缓解EAE行为学和病理学的改变,其作用机制可能与其诱导巨噬细胞极性转化相关。     

口服烟酰胺核糖抑制实验性自身免疫性脑脊髓炎的初步研究

目的：探讨口服烟酰胺核糖(nicotinamide riboside, NR)对实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis, EAE)的治疗效果和作用机制。方法：采用髓鞘少突胶质细胞糖蛋白35-55(myelin oligodendrocyte glycoprotein 35-55, MOG35-55)免疫C57BL/6雌性小鼠制备EAE模型,随机分为EAE组和NR组。从免疫后第3天开始,EAE组小鼠灌胃给予生理盐水,NR组小鼠灌胃给予NR,每天1次至免疫后第27天。免疫当天至免疫后第28天,观察并记录各组小鼠临床评分和体重。免疫后第28天处死小鼠,制备脾细胞悬液和脊髓组织冰冻切片。HE染色和髓鞘染色分别检测脊髓炎性细胞浸润和髓鞘脱失,Western blot和免疫荧光染色分别检测脊髓组织中ROCK-II、TLR4、p-NF-κB、iNOS表达及相应的阳性细胞数量,ELISA检测脾细胞培养液中TNF-α、IL-1β和IL-6的含量。结果：口服NR可推迟EAE发病时间,减轻EAE临床症状,缓解小鼠体重丢失,减少外周炎性细胞...     

黄芪甲苷对实验性自身免疫性脑脊髓炎小鼠T细胞免疫调节的影响

背景：多发性硬化初始阶段，中枢免疫细胞激活并释放大量炎症因子，引起白质脱髓鞘甚至累及灰质神经元。CD4⁺T细胞不同亚群之间的分化平衡在实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis,EAE)的病程进展中发挥着重要作用。课题组前期研究结果表明黄芪内有效成分黄芪甲苷能够调节EAE小鼠体内免疫反应，其是否对T细胞亚群分化具有调节作用尚未明确。目的：探究黄芪甲苷对EAE小鼠治疗效果及其对T细胞的免疫调控机制。方法：将C57BL/6雌性小鼠分为正常对照组、EAE疾病模型组和黄芪甲苷治疗组，每组8只，后2组使用髓鞘少突胶质细胞糖蛋白35-55(MOG35-55)制备EAE模型，免疫后第10-28天，黄芪甲苷治疗组以40 mg/(kg·d)灌胃给药。免疫当天至第28天，记录各组小鼠的体质量及临床评分；免疫后第28天取小鼠脊髓制成冰冻切片行苏木精-伊红染色、固蓝染色观察脊髓病理改变，流式细胞术检测脾脏T细胞亚群百分比，Western blot法检测脊髓组织中γ干扰素、白细胞介素17、白细胞介素6的蛋白表达，ELISA检...     

促吞噬肽(Tuftsin)在实验性自身免疫性脑脊髓炎小鼠中的神经功能改善作用

目的:探索促吞噬肽(Tuftsin)在实验性自身免疫性脑脊髓炎(Experimental autoimmune encephalomyelitis,EAE)小鼠模型中的神经功能改善作用。方法:用髓鞘少突胶质细胞糖蛋白35-55(MOG35-55)免疫C57BL/6小鼠,应用微量注射泵皮下缓慢注射500μmol/L Tuftsin。每天对各组小鼠进行神经功能评分,免疫后28 d处死各组小鼠并取其脑和脊髓组织,观察并比较各组炎性细胞浸润部位及程度、有无脱髓鞘及脱失程度,以及比较各组IL-10和TNF-α的表达情况。结果:1Tuftsin干预组小鼠发病率比EAE模型组低,平均神经功能评分小于EAE模型组(P0.05)。2Tuftsin干预组较EAE模型组炎性细胞的浸润程度和髓鞘的脱失程度及范围减轻(P0.05)。3Tuftsin干预组的IL-10的表达量比EAE模型组高(P0.05),TNF-α的表达比EAE模型组低。结论:Tuftsin通过降低炎性反应、增强抗炎作用在中枢神经系统中发挥保护功能,降低EAE模型小鼠的炎性细胞浸润及脱髓鞘程度,减缓EAE小鼠的症状、促进其神经功能的恢复。     

降纤药治疗实验性自身免疫性脑脊髓炎小鼠的机制

目的:研究降纤药巴曲酶对实验性自身免疫性脑脊髓炎(EAE)小鼠的预防及治疗作用,初步探讨纤维蛋白沉积在多发性硬化(MS)中的作用机制。方法:采用MOG35-55免疫的C57雌性小鼠,诱发EAE动物模型,分别在免疫后立即(预防组)及免疫后出现症状(治疗组)隔日连续给予降纤药巴曲酶注射至实验结束,观察发病情况并进行临床症状评分。于免疫后30天、40天及60天分别将预防组、治疗组及EAE对照组小鼠脊髓与小脑组织取材后进行组织病理学与免疫组化染色分析,Western blot及实时荧光定量PCR技术观察药物干预对炎性浸润、脱髓鞘、纤维蛋白沉积及胶质细胞活化的影响。结果:降纤预防组及治疗组均可明显减轻EAE小鼠的发病症状、降低临床评分。预防组和治疗组较EAE未用药对照组小鼠炎性细胞浸润明显减少、髓鞘脱失及胶质细胞活化减轻,但轴索损害的改善作用不明显。免疫组化及免疫荧光显示,预防组和治疗组较EAE未用药对照组的髓鞘碱性蛋白(MBP)表达升高而胶质纤维酸性蛋白(GFAP)表达降低,Western blot结果示MBP蛋白表达升高而p-Akt表达降低。实时荧光定量PCR技术也证实了预防及治疗组MBP的mRNA表达升高,组织型纤溶酶原激活物(-tPA)的mRNA表达也升高。结论:在EAE的发病过程中纤维蛋白沉积发挥了重要作用,巴曲酶通过有效降低EAE小鼠体内的纤维蛋白从多个方面改善临床症状和发病过程。     

羟基法舒地尔对实验性自身免疫性脑脊髓炎小鼠氧化应激的影响

目的：探究羟基法舒地尔（HF）对实验性自身免疫性脑脊髓炎（EAE）小鼠氧化应激的影响及可能机制。方法：将BV2细胞随机分为正常对照组、LPS组和LPS+HF组。HF预处理2 h后给予LPS刺激24 h，收取细胞上清液。将16只C57BL/6小鼠随机平均分为EAE组和HF组。HF组小鼠于免疫后第3天起以HF腹腔注射干预，剂量40 mg/（kg·d）。EAE组小鼠注射等量生理盐水。从免疫当天开始，每隔1 d记录小鼠的临床评分。DPPH法和ABTS法检测不同浓度HF对自由基的清除作用。MTT法检测BV2细胞在不同浓度HF的干预下细胞活力的改变。HE和LFB染色检测脊髓炎症和髓鞘脱失。ELISA检测细胞上清液和小鼠脊髓中RNS、ROS、MDA、SOD、CAT和GSH-Px含量。免疫荧光染色和Western blot检测小鼠脊髓中Nrf2、HO-1表达。结果：HF在3.75～60μg/ml浓度范围内对DPPH和ABTS自由基几乎没有清除作用，在0～15μg/ml浓度范围内对BV2细胞活力没有影响，HF可抑制EAE小鼠的临床症状，改善脊髓炎症和髓鞘脱失，减少氧化产物ROS、RNS和MDA产生，增加...     

敲除toll样受体4基因对小鼠实验性自身免疫性脑脊髓炎的影响

目的:研究toll样受体4（TLR4）在小鼠实验性自身免疫性脑脊髓炎（EAE）发病中的作用。方法:C57BL/6野生型（WT）和Tlr4敲除(Tlr4^-/-)小鼠注射髓鞘少突胶质细胞糖蛋白35-55(MOG_35-55)建立EAE模型。定期记录神经功能评分及体重变化;苏木素-伊红（HE）和劳克坚牢蓝（LFB）染色观察脊髓组织内炎症细胞浸润及髓鞘脱失情况;免疫荧光染色检测髓鞘碱性蛋白（MBP）的表达。结果:MOG_35-55免疫后,与WT组相比,Tlr4^-/-小鼠体重下降程度较轻、发病时间延迟、神经功能评分降低、炎性细胞浸润减少、髓鞘脱失减轻、MBP表达增加。结论:敲除Tlr4可减轻EAE小鼠的病理症状。     

银杏提取物对实验性自身免疫性脑脊髓炎小鼠的影响

目的:探讨银杏提取物(GBE)对实验性自身免疫性脑脊髓炎(EAE)小鼠炎症脱髓鞘病变的影响。方法:应用髓鞘少突胶质细胞糖蛋白33-55(MOG33-55)配以完全弗氏佐剂(CFA)免疫小鼠,诱发EAE模型。将小鼠分为CFA对照组、EAE模型组和GBE治疗组(每日腹腔注射GBE70mg/kg)。通过神经功能评分、行为学实验以及免疫荧光染色,观察GBE对EAE小鼠的影响。结果:GBE组小鼠各时间段神经功能评分均低于EAE组(P0.05),行为学检测显示发病高峰期falling latency时间较EAE组延长10s;GBE组较EAE组视神经髓鞘碱性蛋白(MBP)表达水平增高,可见MBP阳性髓鞘结构包绕轴突;海马伞矢状切片免疫荧光染色证实GBE组CD11b阳性小胶质细胞较EAE组明显减少,但是GFAP阳性星形胶质细胞数量与EAE组无明显差别。结论:GBE可能通过抑制小胶质细胞激活从而延缓EAE小鼠脱髓鞘进程,提示GBE对多发性硬化具有一定的治疗作用。     

墨西哥钝口螈脊髓全切后胶质细胞的变化

背景：墨西哥钝口螈脊髓切断可以再生,再生过程伴随胶质细胞数目及分布的改变,研究墨西哥钝口螈脊髓全切后胶质细胞的变化,对进一步探讨其脊髓切断再生机制有重要意义。 目的：观察墨西哥钝口螈脊髓全切后小胶质细胞、星形胶质细胞及少突胶质细胞的变化。 方法：选用成年墨西哥钝口螈,分为脊髓全切组和对照组,利用免疫组织化学法观察脊髓全切后1,3和10 d的损伤脊髓及周围区cd11b标记的小胶质细胞、胶质细胞原纤维酸性蛋白标记的星形胶质细胞及髓鞘碱性蛋白标记的少突胶质细胞的变化。 结果与结论：脊髓全切后短期内cd11b染色阴性;脊髓损伤后胶质细胞原纤维酸性蛋白及髓鞘碱性蛋白阳性细胞染色强度,1 d组阳性细胞染色强度与对照组比较无显著差异,3及10 d组阳性细胞染色强度较对照组低。墨西哥钝口螈小胶质细胞染色阴性,可能存在不同于哺乳动物的标记蛋白;脊髓全切后3及10 d在损伤脊髓及周围区的胶质细胞原纤维酸性蛋白及髓鞘碱性蛋白阳性细胞染色强度较对照组低,提示钝口螈脊髓急性损伤早期未见星形胶质细胞及少突胶质细胞增生,无胶质瘢痕形成。     

miR-30a对实验性自身免疫性脑脊髓炎模型小鼠发病的作用

目的:探讨在体过表达miR-30a对实验性自身免疫性脑脊髓炎(EAE)模型小鼠脱髓鞘病变的作用。方法:构建含稳定表达miR-30a的shRNA慢病毒载体,以含有空载体的慢病毒为对照(LV-ctrl)。雌性C57BL/6小鼠随机分为正常对照组(normal)、对照慢病毒(LV-ctrl)组和miR-30a慢病毒(LV-miR-30a)组,2组慢病毒经尾静脉注射,注射7 d后用MOG35-55多肽免疫小鼠。Kono 5分法检测发病情况,快蓝(LFB)染色及透射电镜检测脊髓脱髓鞘程度及髓鞘结构的变化。结果:LV-ctrl组小鼠在MOG_(35-55)多肽免疫的第10天开始发病,到第21天,其Kono评分为2.57±0.79,LV-miR-30a组小鼠的Kono评分显著低于LV-ctrl组小鼠;miR-30a处理可显著提高小鼠体质量增长率,同时LFB评分也较LV-ctrl组显著增加;髓鞘超微结构显示LV-ctrl组小鼠脊髓髓鞘板层松解且稀疏,而LV-miR-30a组小鼠的髓鞘结构相对完好,髓鞘厚度显著大于LV-ctrl组。结论:过表达miR-30a可以明显减缓EAE的发病,减轻脊髓脱髓鞘程度。     

Progesterone attenuates neurological behavioral deficits of experimental autoimmune encephalomyelitis through remyelination with nucleus-sublocalized Olig1 protein

Multiple sclerosis (MS) is the most common demyelination disease of central nervous system (CNS). The deterioration of the disease is characterized by the axonal loss with defective remyelination. Progesterone can promote the remyelination, but whether it exerts beneficial effect on treatment of MS still remains unclear. Olig1 protein is a key regulator in the remyelination, when the intracellular sublocalization plays an import role too. We observed the effect of progesterone on experimental autoimmune encephalomyelitis (EAE) in rats by injecting the progesterone after the neurological behavioral deficits were shown up. The results showed no continuous increase of the nervous function score from day 10 after injection (p < 0.05). Electron microscopy and LFB staining found prominent increase of OD value of normal myelin in the brain from day 6 after injection (p < 0.05). Olig1 protein was localized almost completely in the cytoplasm of Olig1-positive cells from normal rats’ brain. In EAE rats, the Olig1 protein has been translocated to the nucleus of 32.17% of Olig1-positive cells, which was increased to 68.52% after injection with progesterone at day 6 after injection (p < 0.01). The results indicate that the progesterone is beneficial to attenuating neurological behavioral deficits, for it can promote more successful remyelination of EAE with aid of the nucleus-sublocalized Olig1 protein.     

大黄活性成分对实验性自身免疫性脑脊髓炎相关基因蛋白表达以及炎性因子的影响

目的:探究大黄酸对实验性自身免疫性脑脊髓炎(EAE)小鼠模型中炎症因子以及Foxp3 转录因子(Foxp3) 表达量的影响。方法:通过髓鞘少突胶质细胞糖蛋白35鄄55(MOG35鄄55 )抗原乳剂免疫C57BL/6 小鼠制备EAE 模型,实验小鼠随 机分为对照组、模型组、给药组1、给药组2、给药组3;免疫当天腹腔注射大黄酸5、10、20 mg/ kg,对照组和模型组给予等量的 生理盐水,连续给药30 d;观察小鼠的发病率、死亡率,并进行临床评分。分别在免疫后第14、20、30 天取各组小鼠脑和脊髓组 织,酶联免疫吸附法(ELISA)检测白细胞介素2(IL鄄2)的水平,蛋白质印迹法(Western blot)测定Foxp3 蛋白的含量。结果:给 予EAE 模型小鼠不同剂量的大黄酸给药组较模型组发病率显著降低(P<0.05);模型组小鼠在第20 天、第30 天脑和脊髓组 织中分泌的IL鄄2 较对照组显著增加(P<0.05),脑组织中Foxp3 表达量显著降低(P<0.05);给予不同剂量的大黄酸给药组较 模型组可显著降低EAE 小鼠脑、脊髓组织中的IL鄄2 水平(P<0.05),上调Foxp3 表达量(P<0.05),其中5 mg/ kg 剂量的作用效 果相对显著(P<0.05)。结论:小剂量大黄酸可能通过抑制IL-2 水平和促进Foxp3 表达量,调控炎症反应和免疫功能,从而在 EAE 中发挥治疗作用。     

Distinct pathological patterns in relapsing-remitting and chronic models of experimental autoimmune enchephalomyelitis and the neuroprotective effect of glatiramer acetate

The respective roles of inflammatory and neurodegenerative processes in the pathology of multiple sclerosis (MS) and in its animal model experimental autoimmune encephalomyelitis (EAE) are controversial. Novel treatment strategies aim to operate within the CNS to induce neuroprotection and repair processes in addition to their anti-inflammatory properties. In this study we analyzed and compared the in situ pathological manifestations of EAE utilizing two different models, namely the relapsing-remitting PLP-induced and the chronic MOG-induced diseases. To characterize pathological changes, both transmission electron microscopy (TEM) and immunohistochemistry were employed. The effect of the approved MS drug glatiramer acetate (GA, Copaxone) on myelin damage/repair and on motor neuron loss/preservation was studied in both EAE models. Ultrastructural spinal cord analysis revealed multiple white matter damage foci, with different patterns in the two EAE models. Thus, the relapsing-remitting model was characterized mainly by widespread myelin damage and by remyelinating fibers, whereas in the chronic model axonal degeneration was more prevalent. Loss of lower motor neurons was manifested only in mice with chronic MOG-induced disease. In the GA-treated mice, smaller lesions, increased axonal density and higher prevalence of normal appearing axons were observed, as well as decreased demyelination and degeneration. Furthermore, quantitative analysis of the relative remyelination versus demyelination, provides for the first time evidence of significant augmentation of remyelination after GA treatment. The loss of motor neurons in GA-treated mice was also reduced in comparison to that of EAE untreated mice. These effects were obtained even when GA treatment was applied in a therapeutic schedule, namely after the appearance of clinical symptoms. Hence, the remyelination and neuronal preservation induced by GA are in support of the neuroprotective consequences of this treatment.     

Mimic peptides bonding specifically with the first and second extracellular loops of the CC chemokine receptor 5 derived from a phage display peptide library are potent inhibitors of experimental autoimmune encephalomyelitis

Purpose

To obtain mimic peptides that specifically bind with the first and second extracellular loops (ECL1, ECL2) of the CC chemokine receptor 5 (CCR5) and to study their treatment effects on experimental autoimmune encephalomyelitis (EAE) mice.

Methods

A phage display peptide library was applied to screen peptides that bond with ECL1 and ECL2. ELISA and DNA sequence analysis were used to identify positive clones. EAE mice were treated with synthesized peptides by intraperitoneal injection.

Results

Eighteen positive clones were obtained and four peptides with sequences STFTTTL, TPIPQLL, SLPLPKP and QTSSAAL were identified. These peptides could significantly protect against and reduce the severity of EAE. The infiltration of monocytes and lymphocytes into the spinal cord decreased significantly in treated mice, while abundant inflammatory cells and demyelination were observed in spinal cords of EAE mice.

Conclusion

CCR5 mimic peptides provided a significant protective effect to EAE mice. These potent inhibitory mimic peptides could be useful in the clinical treatment of multiple sclerosis.     

FTY720, sphingosine 1-phosphate receptor modulator, ameliorates experimental autoimmune encephalomyelitis by inhibition of T cell infiltration

FTY720, a sphingosine 1-phosphate receptor modulator, induces a marked decrease in the number of peripheral blood lymphocytes and exerts immunomodulating activity in various experimental allograft and autoimmune disease models. In this study, we evaluated the effect of FTY720 and its active metabolite, （S）-enantiomer of FTY720-phosphate [（S）-FTY720-P] on experimental autoimmune encephalomyelitis （EAE） in rats and mice. Prophylactic administration of FTY720 at 0.1 to 1 mg/kg almost completely prevented the development of EAE, and therapeutic treatment with FTY720 significantly inhibited the progression of EAE and EAE-associated histological change in the spinal cords of LEW rats induced by immunization with myelin basic protein. Consistent with rat EAE, the development of proteolipid protein-induced EAE in SJL/J mice was almost completely prevented and infiltration of CD4^＋ T cells into spinal cord was decreased by prophylactic treatment with FTY720 and （S）-FTY720-P. When FTY720 or （S）-FTY720-P was given after establishment of EAE in SJL/J mice, the relapse of EAE was markedly inhibited as compared with interferon-β, and the area of demyelination and the infiltration of CD4^＋ T cells were decreased in spinal cords of EAE mice. Similar therapeutic effect by FTY720 was obtained in myelin oligodendrocyte glycoprotein-induced EAE in C57BL/6 mice. These results indicate that FTY720 exhibits not only a prophylactic but also a therapeutic effect on EAE in rats and mice, and that the effect of FTY720 on EAE appears to be due to a reduction of the infiltration of myelin antigen-specific CD4^＋ T cells into the inflammation site. Cellular ＆ Molecular Immunology. 2005;2（6）：439-448.     

神经小胶质细胞在EAE 脱髓鞘和髓鞘再生中的作用机制进展

炎性细胞浸润和脱髓鞘是中枢神经系统(CNS)的自身免疫性疾病---多发性硬化(MS)的主要病理特征,相关的病理研究多在其动物模型实验性自身免疫性脑脊髓膜炎(EAE)中开展。神经小胶质细胞(MG)是CNS 的主要免疫效应细胞,EAE 时它的激活在脱髓鞘和髓鞘再生中表现出复杂的作用。M1 型MG 是导致脱髓鞘的重要原因,抑制髓鞘再生;而M2型MG 可以促进髓鞘再生,抵抗脱髓鞘。本文综述MG 在EAE 脱髓鞘和髓鞘再生中的直接作用机制,及其通过星形胶质细胞产生的间接作用机制及进展。     

新型Rho激酶抑制剂WAR5治疗EAE的初步研究

 目的：探讨新型Rho激酶抑制剂WAR5对实验性自身免疫性脑脊髓炎（experimental autoimmune encephalomyelitis, EAE）的治疗效果及其可能的作用机制。方法：雌性C57BL/6小鼠,随机分为EAE组和WAR5治疗组。EAE模型采用MOG35-55多肽诱导,免疫后第3天起隔天腹腔注射WAR5或生理盐水至第27天,观察并比较临床症状和体重变化。免疫后第28天处死动物,取脊髓进行HE和髓鞘染色,流式细胞术检测脾细胞M1和M2型巨噬细胞表型。提取脑和脊髓组织蛋白,采用Western blotting检测脑和脊髓诱导型一氧化氮合酶（iNOS）的表达。结果：WAR5治疗能有效推迟临床发病时间,非常显著减轻EAE临床症状。病理检查发现WAR5治疗减少EAE髓鞘脱失和中枢神经系统炎症浸润。同时,WAR5抑制M1型巨噬细胞CD16/32表达,还可增加M2型巨噬细胞CD206表达,并且能够抑制脑和脊髓中iNOS的表达水平。结论：新型Rho激酶抑制剂对EAE有明显的治疗效果,可能与其诱导炎症性M1型巨噬细胞向抗炎性的M2表型转化、从而抑制中枢神经系统的炎症反应有关。