雌激素对实验性变态反应性脑脊髓炎保护作用的研究
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摘要
目的:1.观察雌激素对实验性变态反应性脑脊髓炎(experimental allergic encephalomyelitis,EAE)的保护作用2.探讨雌激素对EAE保护作用的免疫学机制。方法:将40只雄性Wistar大鼠随机分成4组:正常对照组、EAE对照组、大剂量雌激素组和小剂量雌激素组,每组各10只。采用粗制髓鞘碱性蛋白(myelin basic protein,MBP)抗原注入EAE对照组及大、小剂量雌激素组大鼠后足掌皮下(0.2ml/100g)制作EAE模型,正常对照组注射等量生理盐水。自造模之日开始,小剂量雌激素组给予皮下注射苯甲酸雌二醇250μg/kg.d,大剂量雌激素组每日皮下注射苯甲酸雌二醇1mg/kg.d,正常对照组及EAE对照组每日皮下注射1mg/kg.d橄榄油,直至整个实验结束。EAE对照组及大、小剂量雌激素组连续3天症状评分无加重或四肢瘫痪、死亡时作为EAE发病高峰期。记录发病大鼠的发病率、发病潜伏期、进展期和高峰期神经功能障碍评分。于发病高峰期处死大鼠,正常对照组4周后处死,留取大鼠眶静脉血、脑脊髓组织。脑脊髓组织经处理切片行HE染色,光学显微镜下观察病理变化;免疫组化技术和平均光密度测定法检测各组大鼠脑和脊髓白质组织内CD44的表达情况;用酶联免疫吸附(enzyme-linked immunosorbent assay,ELISA)法检测外周血单个核细胞(peripheral blood mononuclear cell,PBMC)分泌IFN-γ、IL-4水平和高峰期脑组织细胞因子IL-10含量。采用放射免疫法测定正常对照组、EAE组和各治疗组高峰期脑组织细胞因子IL-1β、IL-2、IL-6、TNF-α含量。结果:(1)大鼠发病情况:正常对照组大鼠未发病。EAE对照组均有不同程度发病,发病率100%;大、小剂量雌激素组部分动物发病,小剂量组发病率为80%,与EAE对照组比较差异无统计学意义(P >0.05),大剂量组发病率为50%,较EAE对照组增高(P<0.05),但与小剂量组比较差异无统计学意义(P >0.05);EAE对照组发病潜伏期为11.6±2.5天,小剂量雌激素组潜伏期(17.5±1.6天)较EAE对照组延长,差异有统计学意义(P<0.01),大剂量雌激素组潜伏期(24.2±3.0天)较EAE对照组及小剂量雌激素组均明显延长(P<0.01);EAE对照组发病进展期为7.4±1.7天,小剂量雌激素组进展期(5.1±1.4天)较EAE对照组明显缩短(P<0.01),大剂量雌激素组进展期(3.2±0.8天)较EAE对照组及小剂量雌激素组明显缩短(P <0.01,P<0.05);EAE对照组发病高峰期神经功能障碍评分为3.1±1.1分,小剂量雌激素组神经功能障碍评分1.5±1.1分,较EAE对照组明显降低(P<0.01),大剂量雌激素组神经功能障碍评分(0.6±0.7分)较EAE对照组明显降低(P <0.01),大剂量雌激素组神经功能障碍评分比小剂量雌激素组低,差异有统计学意义(P<0.05)。(2)EAE对照组、大小剂量雌激素组大鼠脑及脊髓病理学改变:正常对照组大鼠脑和脊髓无异常。EAE对照组大鼠高峰期可见脑及脊髓实质内小血管充血,血管周围主要是小静脉周围有大量炎性细胞浸润,主要为单个核细胞浸润,典型者形成“袖套”状改变,血管周围白质脱髓鞘改变。而小剂量雌激素组病理改变较轻,大剂量组最轻。(3)EAE对照组、大小剂量雌激素组大鼠CNS内CD44表达情况: CD44表达部位主要在大脑、脑干、脊髓白质及灰白质交界的小血管周围。其主要分布在神经细胞胞浆,CD44高表达部位的炎症表现较重。正常对照组大鼠CNS内未发现CD44阳性细胞;EAE
对照组大鼠CNS白质及灰白质交界处可见大量阳性细胞,呈棕黄色浓染颗粒,着色较深;大小剂量雌激素组大鼠CNS内仅有少量散在淡染的阳性细胞。图像分析结果显示,与大剂量雌激素组及小剂量雌激素组比较,EAE对照组CNS白质CD44表达水平均明显升高(P <0.01);与小剂量雌激素组比较,大剂量雌激素组CNS白质的CD44表达水平显著降低(P <0.01)。(4)EAE对照组、大小剂量雌激素组大鼠PBMC分泌IFN-γ、IL-4能力:正常对照组PBMC分泌的IFN-γ水平为0.32±0.08ng/ml,EAE对照组分泌的IFN-γ水平为0.69±0.07ng/ml,EAE对照组较正常对照组明显升高(P <0.01),大、小剂量雌激素组分泌的IFN-γ水平分别为0.35±0.04ng/ml、0.42±0.04ng/ml,均较EAE对照组显著下降(P <0.01),大剂量雌激素组较小剂量雌激素组下降更明显(P <0.05);正常对照组PBMC分泌的IL-4水平为18.98±1.40 pg/ml,EAE对照组分泌IL-4的水平为11.34±1.23 pg/ml,EAE对照组分泌较IL-4的能力较正常对照组减低(P <0.01),大、小剂量雌激素组分泌的IL-4水平分别为26.19±1.74pg/ml、24.40±1.89pg/ml,均较EAE对照组显著升高(P <0.01),大剂量雌激素组较小剂量雌激素组升高明显(P <0.05);EAE对照组IFN-γ/IL-4比值(62.18±13.16)较正常对照组(16.95±5.45)明显升高(P <0.01),大、小剂量雌激素组IFN-γ/IL-4比值分别是13.39±2.07、17.36±2.82,均较EAE对照组显著下降(P <0.01),大剂量雌激素组IFN-γ/IL-4比值与小剂量雌激素组差异无统计学意义(P >0.05)。(5)细胞因子含量:EAE对照组脑组织IL-1β、IL-2、IL-6、TNF-α含量较正常对照组高(P<0.01或P<0.05),雌激素大、小剂量治疗组脑组织IL-1β、IL-2、IL-6、TNF-α含量较EAE对照组低(P<0.01或
P<0.05),雌激素大剂量治疗组脑组织IL-1β、IL-2、IL-6、TNF-α含量较雌激素小剂量治疗组明显减低(P<0.01或P<0.05)。EAE对照组脑组织IL-10含量较正常对照组明显降低(P<0.01),雌激素大、小剂量治疗组脑组织IL-10含量较EAE对照组明显增高(P<0.05,P<0.01),雌激素大剂量治疗组脑组织IL-10含量较雌激素小剂量治疗组增高(P<0.05)。(6)相关性分析:EAE对照组及大小剂量雌激素组发病高峰期CD44表达与发病潜伏期呈负相关(P<0.01或P<0.05),与发病进展期、高峰期神经功能障碍评分呈正相关(P<0.05或P<0.01)。EAE对照组及大小剂量雌激素组发病高峰期外周血IFN-γ/ IL-4比值与发病潜伏期呈负相关(P<0.01或P<0.05),与发病进展期、神经功能障碍评分呈正相关(P<0.01或P<0.05)。脑组织炎性细胞因子(IL-1β、IL-2、IL-6、TNF-α)含量与EAE对照组及雌激素各治疗组发病的潜伏期呈负相关(P<0.01或P<0.05),与进展期和高峰期神经功能障碍评分呈正相关(P<0.01或P<0.05)。脑组织IL-10含量与EAE对照组及雌激素各治疗组发病的潜伏期、进展期和高峰期神经功能障碍评分无明显相关性(P>0.05)。结论:1.本实验以豚鼠脊髓粗制MBP为免疫原诱导大鼠EAE模型,EAE大鼠神经功能障碍明显,脑和脊髓血管周围炎性细胞浸润、白质脱髓鞘,说明本法造模成功、可靠、稳定。2. EAE大鼠存在免疫失衡,发病高峰期体内Th1型细胞因子IFN-γ增多,Th2型细胞因子IL-4减少,Th1/Th2细胞比例失衡,脑组织炎性细胞因子(IL-1β、IL-2、IL-6、TNF-α)产生增加,抑制性细胞因子IL-10生成减少。免疫格局向Th1型偏移。其免疫功能异常与EAE大鼠发病的潜伏期、进展期和高峰期神经功能障碍评分密切相关。3. EAE大鼠发病期CD44表达上调,CD44表达与EAE潜伏期呈负相关,与进展期、
神经功能障碍评分呈正相关,经雌激素治疗表达降低,提示CD44与EAE发生发展有关。4.雌激素对EAE有保护作用,可有效改善EAE的临床症状,降低发病率,延缓发病时间,抑制中枢神经系统的炎症细胞浸润、髓鞘脱失和轴突损伤,其保护作用大小与剂量呈正相关。5.雌激素可能通过抑制Th1细胞活性、提高Th2细胞分泌能力、逆转Th1/Th2失衡、抑制炎性细胞因子(IL-1β、IL-2、IL-6、TNF-α)产生、促进抑制性细胞因子IL-10生成,抑制黏附分子CD44的表达而发挥对EAE大鼠的治疗作用。
Objective: To observe the protective effect of estrogen on Experimental Allergic Encephalomyelitis in rats,and explore the possible immunologic mechanism of it.Method: 40 male rats were randomly divided into four groups: normal control group, EAE control group, high-dose estrogen treatment group and the low-dose estrogen treatment group, n = 10. Myelin basic protein(myelin basic protein, MBP) by crude antigen were injected (0.2ml /100g) into the EAE control group, estrogen high and low dose treatment groups, normal control group was injected with equivalent saline. Since the date of modeling, low-dose estrogen treatment group was injected subcutaneously Estradiol Benzoate 250μg/kg daily, high-dose estrogen treatment group injected subcutaneously Estradiol Benzoate 1000μg/kg daily, normal control group and the EAE control group injected equivalent saline daily until the end of the experiment. EAE control group, all dose of estrogen treatment groups had the symptom for 3 consecutive days without aggravating or quadriplegia,or the rats being dead ,would be considered as the peak of EAE disease. Record onset latency, advanced and peak disease score; the rats would be killed at he peak incidence , normal control group were killed 4 weeks later. Then we would keep the brain and spinal cord tissue,and they would be used for HE staining, to observe the pathological changes Immunohistochemical method and the mean optical density measurement to detect the rat brain and spinal cord white matter tissue CD44 expression ,the capacity of peripheral blood mononuclear cells (peripheral blood mononuclear cell, PBMC) secretion of IFN -γ, IL-4 and the levels of cytokine IL-10 in brain tissue would be teasted by enzyme-linked immunosorbent assay (ELISA).The levels of cytokine (IL-1β, IL-2, IL-6, TNF-α) in brain tissue at fastigium were analyzed by use of radio immunoassay. Result: The incidence of rats: normal control rats did not have the disease. All rats of EAE control group had varying degrees of disease, the incidence was100%; some rats of estrogen high, low-dose treatment group caught disease,the incidence were 50% and 80% respectively.EAE control group, the delitescence was 11.6±2.5 days, low-dose estrogen treatment group( 17.5±1.6 days) was longer than the EAE control group,this was statistically significant (P <0.01), high-dose estrogen treatment group was 24.2±3.0days, compared with EAE control group and low-dose group were significantly longer (P <0.01); the progression of EAE control group was 7.4±1.7days, low-dose estrogen treatment group was 5.1±1.4days,this was shorter than the EAE control group (P <0.01);high-dose estrogen treatment group was 3.2±0.8 days, and it was significantly shorter than the EAE control groupand low- dose estrogen treatment group ( P <0.01,P <0.05); EAE control group, the peak incidence of disease score was 3.1±1.1 , low-dose estrogen group disease scored 1.5±1.1, compared with EAE ,this was significantly decreased ( P <0.01), high-dose estrogen group disease scored 0.6±0.7,this was lower than the EAE control group and low-dose estrogen group (P <0.01). (2)
Pathological changes in brain and spinal cord: By light microscope, we found the slice of cerebrum, cerebellum, brain stem and spinal cord in the normal group were normal.The pathological changes of EAE in the process of maximal disease showed inflammatory cuff around small blood vessels, perivascular inflammatory cell infiltration and demyelination in white matter, especially in spinal cord. The pathological changes of estrogen treatment group lessened. (3) CD44 immunohistochemistry and image analysis: CD44 was mainly expressed in the brain, brain stem, spinal cord ,white matter and gray matter around the junction of the small blood vessels. CD44 mainly expressed in the cytoplasm of nerve cells. Expression of this part had heavier inflammation. Normal control group had no CD44 positive cells. White matter and gray matter could be seen at the junction of a large number of positive cells which was dark brown in EAE control group; high-dose estrogen and low-doses estrogen groups had only sparsely stained positive cells, low-dose estrogen group was higher than high-dose estrogen group. Image analysis showed that with high-dose estrogen group and low-dose estrogen group, EAE group of CNS white matter of CD44 expression level was significantly increased (P <0.01);the expression of CD44 in low-dose and high-dose estrogen group ware significantly different with EAE group(P <0.01). The CD44 expression in high-dose estrogen group was significantly lower than low-dose estrogen group(P <0.01). (4) The levels of IFN-γand IL-4 and the ratio of IFN-γ/IL-4: IFN-γof normal control group was 0.32±0.08ng/ml, IFN-γof EAE control group 0.69±0.07ng/ml,whith were
significantly higher than the normal control group (P <0.01), IFN-γof the high-dose and low-dose estrogen groups were 0.35±0.04ng/ml, 0.42±0.04ng/ml, compared with EAE control group,these were significantly decreased (P <0.01), high-dose estrogen group was decreased significantly than the low-dose(P <0.05); IL-4 of normal control group was 18.98±1.4 pg /ml, IL-4 of EAE control group was 11.34±1.23pg/ml, compared with the nomal group whith was decreased significantly (P <0.01), IL-4 of high-dose and low-doses estrogen groups were 26.19±1.74pg/ml, 24.40±1.89pg/ml, compared with EAE control group whith were significantly increased (P <0.01), low-dose estrogen group compared with high dose estrogen group, was obvious (P <0.05); EAE control group IFN-γ/IL-4ratio was significantly higher than the normal control group (P <0.01), high-dose and low-dose estrogen groups of IFN-γ/ IL-4 ratio compared with EAE control group were significantly decreased (P <0.01), high-dose and low-dose group had no significant difference (P>0.05). (5)Cytokine levels: The levels of IL-1β, IL-2, IL-6, TNF-αin brain tissue of EAE control group was higher than normal levels (P<0.05 or P<0.01), the levels of IL-1β、IL-2、IL-6、TNF-αin brain tissue of high and low dose treating group is lower than the EAE control group (P<0.01 or P<0.05), the levels of IL-1β, IL-2, IL-6, TNF-αin brain tissue of high dose treating group was significantly lower than low dose treating group (P<0.01 or P<0.05). The brain tissue IL-10 level of EAE control group was significantly lower than the normal control group(P<0.01), the brain
tissue IL-10 levels of hight dose and low dose treating EAE groups were increased significantly than the EAE control group (P<0.01or P<0.05), and the brain tissue IL-10 level of hight dose treating EAE group was increased than the low dose treating EAE group,but there was no significant difference(P>0.05).(5)Correlative analysis: the expression of CD44 in EAE control group, the low-dose estrogen group and the high-dose estrogen group were significantly negatively correlated with the delitescence and positive with the progression and miximal disease scores(P<0.01or P<0.05);the ratio of IFN-γ/IL-4 were significantly positively correlated with and the maximal disease score and the progression and negetive with delitescence; The levels of cytokine(IL-1β, IL-2, IL-6, TNF-α) in brain at fastigium were significantly negatively correlated with the delitescence period, while they were significantly positively correlated with the progression and neuro-function scores(P<0.01or P<0.05). The level of IL-10 in brain for EAE control group and estrogen groups have no obvious correlation with the delitescence period and progression and neuro-function scores. Conclusion: 1.In this experiment, crude spinal cord of guinea pigs were used to induce EAE model.EAE rats had obvious nerve dysfunction, brain and spinal cord perivascular inflammatory cell had infiltration and demyelination. The EAE model in this study was successful, reliable and stable. 2.EAE rats existed immune imbalance.EAE rats had increased Th1 type cytokines IFN-γ, and decreased Th2 type cytokines IL-4.Th1/Th2 cell had imbalance. Inflammatory
cytokines (IL-1β, IL-2, IL-6 and TNF-α) in brain increasing, immune suppression of cytokines IL-10 level reducing, immune style shifted to the Th1 type pattern.The immune dysfunction has close correlation with the delitescence of EAE, the progression and maximal disease scores. 3.EAE rats had upregulation of CD44. CD44 expression was negatively correlated with delitescence, and positively correlated with progression, disease score.After estrogen treatment ,CD44 expression reduced, which indicated that CD44 had a relationship with the development of EAE. 4.Estrogen on EAE had a therapeutic effect, whith could improve the clinical symptoms, delay the onset time, improve the central CNS inflammation, de- crease the demyelination and axonal injury, and even promote the regeneration of neuraxon. The protective effect of estrogen is increased with the dose in- crease. 5. The protective mechanism of estrogen to EAE might be through inhibiting the activity of Th1 cells, improving the Th2 Cell capacity, regulating the Th1/Th2 balance role and reducing the brain inflammatory cytokines(IL-1β, IL-2, IL-6 and TNF-α) levels, increasing brain immune suppression of cytokines IL-10 level,and downregulating CD44 expression.
对照组大鼠CNS白质及灰白质交界处可见大量阳性细胞,呈棕黄色浓染颗粒,着色较深;大小剂量雌激素组大鼠CNS内仅有少量散在淡染的阳性细胞。图像分析结果显示,与大剂量雌激素组及小剂量雌激素组比较,EAE对照组CNS白质CD44表达水平均明显升高(P <0.01);与小剂量雌激素组比较,大剂量雌激素组CNS白质的CD44表达水平显著降低(P <0.01)。(4)EAE对照组、大小剂量雌激素组大鼠PBMC分泌IFN-γ、IL-4能力:正常对照组PBMC分泌的IFN-γ水平为0.32±0.08ng/ml,EAE对照组分泌的IFN-γ水平为0.69±0.07ng/ml,EAE对照组较正常对照组明显升高(P <0.01),大、小剂量雌激素组分泌的IFN-γ水平分别为0.35±0.04ng/ml、0.42±0.04ng/ml,均较EAE对照组显著下降(P <0.01),大剂量雌激素组较小剂量雌激素组下降更明显(P <0.05);正常对照组PBMC分泌的IL-4水平为18.98±1.40 pg/ml,EAE对照组分泌IL-4的水平为11.34±1.23 pg/ml,EAE对照组分泌较IL-4的能力较正常对照组减低(P <0.01),大、小剂量雌激素组分泌的IL-4水平分别为26.19±1.74pg/ml、24.40±1.89pg/ml,均较EAE对照组显著升高(P <0.01),大剂量雌激素组较小剂量雌激素组升高明显(P <0.05);EAE对照组IFN-γ/IL-4比值(62.18±13.16)较正常对照组(16.95±5.45)明显升高(P <0.01),大、小剂量雌激素组IFN-γ/IL-4比值分别是13.39±2.07、17.36±2.82,均较EAE对照组显著下降(P <0.01),大剂量雌激素组IFN-γ/IL-4比值与小剂量雌激素组差异无统计学意义(P >0.05)。(5)细胞因子含量:EAE对照组脑组织IL-1β、IL-2、IL-6、TNF-α含量较正常对照组高(P<0.01或P<0.05),雌激素大、小剂量治疗组脑组织IL-1β、IL-2、IL-6、TNF-α含量较EAE对照组低(P<0.01或
P<0.05),雌激素大剂量治疗组脑组织IL-1β、IL-2、IL-6、TNF-α含量较雌激素小剂量治疗组明显减低(P<0.01或P<0.05)。EAE对照组脑组织IL-10含量较正常对照组明显降低(P<0.01),雌激素大、小剂量治疗组脑组织IL-10含量较EAE对照组明显增高(P<0.05,P<0.01),雌激素大剂量治疗组脑组织IL-10含量较雌激素小剂量治疗组增高(P<0.05)。(6)相关性分析:EAE对照组及大小剂量雌激素组发病高峰期CD44表达与发病潜伏期呈负相关(P<0.01或P<0.05),与发病进展期、高峰期神经功能障碍评分呈正相关(P<0.05或P<0.01)。EAE对照组及大小剂量雌激素组发病高峰期外周血IFN-γ/ IL-4比值与发病潜伏期呈负相关(P<0.01或P<0.05),与发病进展期、神经功能障碍评分呈正相关(P<0.01或P<0.05)。脑组织炎性细胞因子(IL-1β、IL-2、IL-6、TNF-α)含量与EAE对照组及雌激素各治疗组发病的潜伏期呈负相关(P<0.01或P<0.05),与进展期和高峰期神经功能障碍评分呈正相关(P<0.01或P<0.05)。脑组织IL-10含量与EAE对照组及雌激素各治疗组发病的潜伏期、进展期和高峰期神经功能障碍评分无明显相关性(P>0.05)。结论:1.本实验以豚鼠脊髓粗制MBP为免疫原诱导大鼠EAE模型,EAE大鼠神经功能障碍明显,脑和脊髓血管周围炎性细胞浸润、白质脱髓鞘,说明本法造模成功、可靠、稳定。2. EAE大鼠存在免疫失衡,发病高峰期体内Th1型细胞因子IFN-γ增多,Th2型细胞因子IL-4减少,Th1/Th2细胞比例失衡,脑组织炎性细胞因子(IL-1β、IL-2、IL-6、TNF-α)产生增加,抑制性细胞因子IL-10生成减少。免疫格局向Th1型偏移。其免疫功能异常与EAE大鼠发病的潜伏期、进展期和高峰期神经功能障碍评分密切相关。3. EAE大鼠发病期CD44表达上调,CD44表达与EAE潜伏期呈负相关,与进展期、
神经功能障碍评分呈正相关,经雌激素治疗表达降低,提示CD44与EAE发生发展有关。4.雌激素对EAE有保护作用,可有效改善EAE的临床症状,降低发病率,延缓发病时间,抑制中枢神经系统的炎症细胞浸润、髓鞘脱失和轴突损伤,其保护作用大小与剂量呈正相关。5.雌激素可能通过抑制Th1细胞活性、提高Th2细胞分泌能力、逆转Th1/Th2失衡、抑制炎性细胞因子(IL-1β、IL-2、IL-6、TNF-α)产生、促进抑制性细胞因子IL-10生成,抑制黏附分子CD44的表达而发挥对EAE大鼠的治疗作用。
Objective: To observe the protective effect of estrogen on Experimental Allergic Encephalomyelitis in rats,and explore the possible immunologic mechanism of it.Method: 40 male rats were randomly divided into four groups: normal control group, EAE control group, high-dose estrogen treatment group and the low-dose estrogen treatment group, n = 10. Myelin basic protein(myelin basic protein, MBP) by crude antigen were injected (0.2ml /100g) into the EAE control group, estrogen high and low dose treatment groups, normal control group was injected with equivalent saline. Since the date of modeling, low-dose estrogen treatment group was injected subcutaneously Estradiol Benzoate 250μg/kg daily, high-dose estrogen treatment group injected subcutaneously Estradiol Benzoate 1000μg/kg daily, normal control group and the EAE control group injected equivalent saline daily until the end of the experiment. EAE control group, all dose of estrogen treatment groups had the symptom for 3 consecutive days without aggravating or quadriplegia,or the rats being dead ,would be considered as the peak of EAE disease. Record onset latency, advanced and peak disease score; the rats would be killed at he peak incidence , normal control group were killed 4 weeks later. Then we would keep the brain and spinal cord tissue,and they would be used for HE staining, to observe the pathological changes Immunohistochemical method and the mean optical density measurement to detect the rat brain and spinal cord white matter tissue CD44 expression ,the capacity of peripheral blood mononuclear cells (peripheral blood mononuclear cell, PBMC) secretion of IFN -γ, IL-4 and the levels of cytokine IL-10 in brain tissue would be teasted by enzyme-linked immunosorbent assay (ELISA).The levels of cytokine (IL-1β, IL-2, IL-6, TNF-α) in brain tissue at fastigium were analyzed by use of radio immunoassay. Result: The incidence of rats: normal control rats did not have the disease. All rats of EAE control group had varying degrees of disease, the incidence was100%; some rats of estrogen high, low-dose treatment group caught disease,the incidence were 50% and 80% respectively.EAE control group, the delitescence was 11.6±2.5 days, low-dose estrogen treatment group( 17.5±1.6 days) was longer than the EAE control group,this was statistically significant (P <0.01), high-dose estrogen treatment group was 24.2±3.0days, compared with EAE control group and low-dose group were significantly longer (P <0.01); the progression of EAE control group was 7.4±1.7days, low-dose estrogen treatment group was 5.1±1.4days,this was shorter than the EAE control group (P <0.01);high-dose estrogen treatment group was 3.2±0.8 days, and it was significantly shorter than the EAE control groupand low- dose estrogen treatment group ( P <0.01,P <0.05); EAE control group, the peak incidence of disease score was 3.1±1.1 , low-dose estrogen group disease scored 1.5±1.1, compared with EAE ,this was significantly decreased ( P <0.01), high-dose estrogen group disease scored 0.6±0.7,this was lower than the EAE control group and low-dose estrogen group (P <0.01). (2)
Pathological changes in brain and spinal cord: By light microscope, we found the slice of cerebrum, cerebellum, brain stem and spinal cord in the normal group were normal.The pathological changes of EAE in the process of maximal disease showed inflammatory cuff around small blood vessels, perivascular inflammatory cell infiltration and demyelination in white matter, especially in spinal cord. The pathological changes of estrogen treatment group lessened. (3) CD44 immunohistochemistry and image analysis: CD44 was mainly expressed in the brain, brain stem, spinal cord ,white matter and gray matter around the junction of the small blood vessels. CD44 mainly expressed in the cytoplasm of nerve cells. Expression of this part had heavier inflammation. Normal control group had no CD44 positive cells. White matter and gray matter could be seen at the junction of a large number of positive cells which was dark brown in EAE control group; high-dose estrogen and low-doses estrogen groups had only sparsely stained positive cells, low-dose estrogen group was higher than high-dose estrogen group. Image analysis showed that with high-dose estrogen group and low-dose estrogen group, EAE group of CNS white matter of CD44 expression level was significantly increased (P <0.01);the expression of CD44 in low-dose and high-dose estrogen group ware significantly different with EAE group(P <0.01). The CD44 expression in high-dose estrogen group was significantly lower than low-dose estrogen group(P <0.01). (4) The levels of IFN-γand IL-4 and the ratio of IFN-γ/IL-4: IFN-γof normal control group was 0.32±0.08ng/ml, IFN-γof EAE control group 0.69±0.07ng/ml,whith were
significantly higher than the normal control group (P <0.01), IFN-γof the high-dose and low-dose estrogen groups were 0.35±0.04ng/ml, 0.42±0.04ng/ml, compared with EAE control group,these were significantly decreased (P <0.01), high-dose estrogen group was decreased significantly than the low-dose(P <0.05); IL-4 of normal control group was 18.98±1.4 pg /ml, IL-4 of EAE control group was 11.34±1.23pg/ml, compared with the nomal group whith was decreased significantly (P <0.01), IL-4 of high-dose and low-doses estrogen groups were 26.19±1.74pg/ml, 24.40±1.89pg/ml, compared with EAE control group whith were significantly increased (P <0.01), low-dose estrogen group compared with high dose estrogen group, was obvious (P <0.05); EAE control group IFN-γ/IL-4ratio was significantly higher than the normal control group (P <0.01), high-dose and low-dose estrogen groups of IFN-γ/ IL-4 ratio compared with EAE control group were significantly decreased (P <0.01), high-dose and low-dose group had no significant difference (P>0.05). (5)Cytokine levels: The levels of IL-1β, IL-2, IL-6, TNF-αin brain tissue of EAE control group was higher than normal levels (P<0.05 or P<0.01), the levels of IL-1β、IL-2、IL-6、TNF-αin brain tissue of high and low dose treating group is lower than the EAE control group (P<0.01 or P<0.05), the levels of IL-1β, IL-2, IL-6, TNF-αin brain tissue of high dose treating group was significantly lower than low dose treating group (P<0.01 or P<0.05). The brain tissue IL-10 level of EAE control group was significantly lower than the normal control group(P<0.01), the brain
tissue IL-10 levels of hight dose and low dose treating EAE groups were increased significantly than the EAE control group (P<0.01or P<0.05), and the brain tissue IL-10 level of hight dose treating EAE group was increased than the low dose treating EAE group,but there was no significant difference(P>0.05).(5)Correlative analysis: the expression of CD44 in EAE control group, the low-dose estrogen group and the high-dose estrogen group were significantly negatively correlated with the delitescence and positive with the progression and miximal disease scores(P<0.01or P<0.05);the ratio of IFN-γ/IL-4 were significantly positively correlated with and the maximal disease score and the progression and negetive with delitescence; The levels of cytokine(IL-1β, IL-2, IL-6, TNF-α) in brain at fastigium were significantly negatively correlated with the delitescence period, while they were significantly positively correlated with the progression and neuro-function scores(P<0.01or P<0.05). The level of IL-10 in brain for EAE control group and estrogen groups have no obvious correlation with the delitescence period and progression and neuro-function scores. Conclusion: 1.In this experiment, crude spinal cord of guinea pigs were used to induce EAE model.EAE rats had obvious nerve dysfunction, brain and spinal cord perivascular inflammatory cell had infiltration and demyelination. The EAE model in this study was successful, reliable and stable. 2.EAE rats existed immune imbalance.EAE rats had increased Th1 type cytokines IFN-γ, and decreased Th2 type cytokines IL-4.Th1/Th2 cell had imbalance. Inflammatory
cytokines (IL-1β, IL-2, IL-6 and TNF-α) in brain increasing, immune suppression of cytokines IL-10 level reducing, immune style shifted to the Th1 type pattern.The immune dysfunction has close correlation with the delitescence of EAE, the progression and maximal disease scores. 3.EAE rats had upregulation of CD44. CD44 expression was negatively correlated with delitescence, and positively correlated with progression, disease score.After estrogen treatment ,CD44 expression reduced, which indicated that CD44 had a relationship with the development of EAE. 4.Estrogen on EAE had a therapeutic effect, whith could improve the clinical symptoms, delay the onset time, improve the central CNS inflammation, de- crease the demyelination and axonal injury, and even promote the regeneration of neuraxon. The protective effect of estrogen is increased with the dose in- crease. 5. The protective mechanism of estrogen to EAE might be through inhibiting the activity of Th1 cells, improving the Th2 Cell capacity, regulating the Th1/Th2 balance role and reducing the brain inflammatory cytokines(IL-1β, IL-2, IL-6 and TNF-α) levels, increasing brain immune suppression of cytokines IL-10 level,and downregulating CD44 expression.
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55.Ishizuka Y, Yokota A, Nishimura M.et al..Ligation of CD44 leads to killing activity in human peripheral mononuclear cells via MAP kinase and tyrosine kinases[J]. Hematology.2008 Aug;13(4):230-235.
56.汪贵明,赵志伟。去势大鼠骨髓细胞CD44表达及其意义[J].四川生理科学杂志2009,31(3):101-102
57.Salem ML, Hossain MS, Nomoto K.et al. Mediation of the immunomodulatory effect of beta-estradiol on inflammatory responses by inhibition of recruitment and activation of inflammatory cells and their gene expression of TNF-alpha and IFN-gamma[J]. Int Arch Allergy Immunol. 2000 Mar;121(3):235-245.
[1] Stefan M Gold and Rhonda R Voskuhl. Estrogen and Testosterone Therapies in Multiple Sclerosis[J].Prog Brain Res,2009; 175: 239–251.
[2] Subramanian S,Matejuk A,Zamora A, et a1.Oral feeding with ethinyl estradiol suppresses and treats experimental autoimmune encephalomyelitis in SJL mice and inhibits the recruitment of inflammatory cells into the central nervous system [J]. Immunology, 2003,170(3):1548—1555。
[3] Seema Tiwari-Woodruff, Laurie Beth J,Morales.et al. Differential neuroprotective and antiinflammatory effects of estrogen receptor ERαand ERβligand treatment[J]. Proc Natl Acad Sci U S A. 2007 September 11; 104(37): 14813–14818.
[4]Lang TJ. Estrogen as an immunomodulator[J]. Clin Immunol, 2004,113(3): 224-230.
[5]Drew PD, Chavis JA, Bhatt R. Sex steroid regulation of microglial cell activation: relevance to multiple sclerosis[J]. Ann N Y Acad Sci,2003, 1007: 329-334.
[6] Drew PD, Chavis JA, B hatt R. Sex steroid regulation of microglial cell activation: relevance to multiple sclerosis[J]. Ann N Y Acad Sci,2003, 1007: 329-334.
[7] Sicotte NL, Liva SM, Klutch R, et al. Treatment of multiple sclerosis with the pregnancy hormone estriol [J]. Ann Neurol, 2002, 52 (4):421-428.
[8] Matejuk A, Dwyer J, Zamora A, et al. Evaluation of the effects of17beta-estradiol (17beta-e2) on gene expression in experimental autoimmune encephalomyelitis using DNA microarray [J].Endocrinology, 2002, 143(1): 313-319.
[9] Kim S, Liva S, Dalal M, et al. Estriol ameliorates autoimmune demyelinating disease: implications for multiple sclerosis [J].Neurology, 1999, 52(6): 1230-1238.
[10] Karen MP, Liu HB, Loo KK, et al. Estriol treatment ameliorates disease in males with experimental autoimmune encephalomyelitis implications for multiple sclerosi [J]. J Neurosci, 2004, 149 (1-2):84-89.
[11] Laurie Beth J. Morales, Kyi Kyi Loo.etal.Treatment with an Estrogen ReceptorαLigand Is Neuroprotective in Experimental Autoimmune Encephalomyelitis[J]. The Journal of Neuroscience, June 21, 2006, 26(25):6823-6833.
[12] ]Xiao BG,Liu X,Link H.Antigen -specific T cell functions are suppressed over the estrogen - dendritic cell - indoleamine 2 ,3 -dioxygenase axis [ J ].Steroids.2004 ,69 ( 1 0 ) :6 5 3 - 6 5
[13]MoralesLB,LooKK,LiuHB,eta1.Treatment with an estrogen receptor alpha ligand is neuroprotective in experimental autoimmune encephalomyelitis [J].J Neurosci,2006,26(25):6823-6833.
[14] McClain MA,Gat son NN,Powell ND ,et al . Preg2 nancy suppresses experimental auto-Immune encephalomyelitis through immunoregulatory cytokine production [J ] .J Immunol , 2007,179 ( 12):8146—8152.
[15] Hong J ,ZangY C,Ni e H et al. C D 4+ regulatory T cell responses induced by T cell vacci nation i n patients with multiple sclerosis [ J ]. Pro Natl acad Sci US A,2006:1 0 3 ( 1 3 ):502 4 - 5 029.
[16] Offner H,Polanezyk M.A potential role for estrogen in experimental autoimmune enceph- alomyelitis and multiple cslerosis[J].Ann N Y Acad Sci,2006;l089:343-372.
[17] Okasha SA,Ryu S,Do Y e t al.Evidenceforestradiol -induced apoptosi and dysregulated T cell matttration in the thymus [ J ].Toxicology, 2 0 0 1 163:49-62
[18] Hoffman-Goetz L, Cherie F, McCutcheon D, et al. Effects o17bate-estradiol and voluntary exercise or lymphocyte apoptosis in mice[J]. Physiol Behave, 2001, 74(4-5): 653-658.
[19]宋向凤,孙翔,王辉.雌孕激素对T淋巴细胞生长的调节作用[J].细胞与分子免疫学杂志, 2005, 21(2): 249-253.
[20] Urich E,Guteher I,Prim M et a1.Autoantibndy-mediated demyelination depends on complement activation but not activatroy Fc-receptors[J].Proc Natl Acad Sci USA,2006;103(49):18697-18702.
[21] Magdalena J,Polanczyk,Richard E,et a1.T lymphocytes do not directly mediate the protective effect of estrogen on experimental autoimmune encephalomyelitis[J].Am J Pathol,2004,165(6):2069—2077.
[22] Pettersson A,Ciumas C,Chirsky V,et a1.Dendritic cells exposed to estrogen in vitro exhibit therapeutic effects in ongoing experimental allergicencephalomyeliris[J].J Neuroimmunol,2004,156(1-2):58—65。
[23] Xiao B G,Liu X,Link H.Antigen-spcific Tcell functions are suppressed over the estrogen-dendritic cell-indoleamine 2,3- dioxygenaseaxis [J].Steroids,2004;69(10):653-659
[24] Janis K,Hoeltke J,Nazareth M et al.Estrogen decreasese xpression of chemokine receptors,and suppresses chemoke bioactivity in murine monocytes[J].Am J Reprod Immunol,2004;51(1):22—31.
[25] Vlotides G,Gaertner R,Angstwurm M W.Modulation of monocytic lipopolysaccharide– induced tissue factor expression and tumor necrosis factor alpha release by estrogen and calcitrol [J].Hormones (Athens),2007;6(1):52-61
[26] Bruce-Keller AJ,KeelingJL,KellerJN,et a1.Anti-inflammatory effects of estrogen on microglial activation[J].Endocrinology,2000,141(10):3646-3656
[27]Baker A E,Brautigam V M,Watters JJ.Estrongen modulatesmicroglial inflammatory mediator production via interaction with estrogen receptorbeta[J].Endocrinology,2004;145(11):5021-5032
[28]Nilsson N,Carlsten H.Estrogen inducessuppression ofNatural Killer cell cytotoxicitiy and augmentation of Bcell activation[J].Cell Immunol,1994,158(1):131-139
[29] Takao T,Flint N,Lee L etal.17beta-estradiol protects oligodendrocytes from cytotoxicity induced cell death[J].J Neurochem,2004;89(3):660-673.
[30] Comabella M,Pericot L,Goertsches R,et al. Plasma osteopontin levels in multiple sclerosis[J]. Neuroimmunol,2005;158(1/2):231-239.
[31] Oksala O,Salo T,Tammi R,et al.Expression of proteoglycans and hyaluronan during wound healing [J] .Histochen Cytochem,1995;43:125-135
[32] Garin T, Rubinstein A, Grigoriadis N et al. CD44 variant DNA vaccination with virtual lymph node ameliorates experimental autoimmune encephalomyelitis through the induction of apoptosis[J] Neurol Sci. 2007 Jul 15;258(1-2):17-26.
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54.Monteiro de Castro G, Eduarda Zanin M, et al.Th1 and Th2 cytokine immunomodulation by gangliosides in experimental autoimmune encephalomyelitis [J]. Cytokine, 2004; 26(4):155-163
55.Ishizuka Y, Yokota A, Nishimura M.et al..Ligation of CD44 leads to killing activity in human peripheral mononuclear cells via MAP kinase and tyrosine kinases[J]. Hematology.2008 Aug;13(4):230-235.
56.汪贵明,赵志伟。去势大鼠骨髓细胞CD44表达及其意义[J].四川生理科学杂志2009,31(3):101-102
57.Salem ML, Hossain MS, Nomoto K.et al. Mediation of the immunomodulatory effect of beta-estradiol on inflammatory responses by inhibition of recruitment and activation of inflammatory cells and their gene expression of TNF-alpha and IFN-gamma[J]. Int Arch Allergy Immunol. 2000 Mar;121(3):235-245.
[1] Stefan M Gold and Rhonda R Voskuhl. Estrogen and Testosterone Therapies in Multiple Sclerosis[J].Prog Brain Res,2009; 175: 239–251.
[2] Subramanian S,Matejuk A,Zamora A, et a1.Oral feeding with ethinyl estradiol suppresses and treats experimental autoimmune encephalomyelitis in SJL mice and inhibits the recruitment of inflammatory cells into the central nervous system [J]. Immunology, 2003,170(3):1548—1555。
[3] Seema Tiwari-Woodruff, Laurie Beth J,Morales.et al. Differential neuroprotective and antiinflammatory effects of estrogen receptor ERαand ERβligand treatment[J]. Proc Natl Acad Sci U S A. 2007 September 11; 104(37): 14813–14818.
[4]Lang TJ. Estrogen as an immunomodulator[J]. Clin Immunol, 2004,113(3): 224-230.
[5]Drew PD, Chavis JA, Bhatt R. Sex steroid regulation of microglial cell activation: relevance to multiple sclerosis[J]. Ann N Y Acad Sci,2003, 1007: 329-334.
[6] Drew PD, Chavis JA, B hatt R. Sex steroid regulation of microglial cell activation: relevance to multiple sclerosis[J]. Ann N Y Acad Sci,2003, 1007: 329-334.
[7] Sicotte NL, Liva SM, Klutch R, et al. Treatment of multiple sclerosis with the pregnancy hormone estriol [J]. Ann Neurol, 2002, 52 (4):421-428.
[8] Matejuk A, Dwyer J, Zamora A, et al. Evaluation of the effects of17beta-estradiol (17beta-e2) on gene expression in experimental autoimmune encephalomyelitis using DNA microarray [J].Endocrinology, 2002, 143(1): 313-319.
[9] Kim S, Liva S, Dalal M, et al. Estriol ameliorates autoimmune demyelinating disease: implications for multiple sclerosis [J].Neurology, 1999, 52(6): 1230-1238.
[10] Karen MP, Liu HB, Loo KK, et al. Estriol treatment ameliorates disease in males with experimental autoimmune encephalomyelitis implications for multiple sclerosi [J]. J Neurosci, 2004, 149 (1-2):84-89.
[11] Laurie Beth J. Morales, Kyi Kyi Loo.etal.Treatment with an Estrogen ReceptorαLigand Is Neuroprotective in Experimental Autoimmune Encephalomyelitis[J]. The Journal of Neuroscience, June 21, 2006, 26(25):6823-6833.
[12] ]Xiao BG,Liu X,Link H.Antigen -specific T cell functions are suppressed over the estrogen - dendritic cell - indoleamine 2 ,3 -dioxygenase axis [ J ].Steroids.2004 ,69 ( 1 0 ) :6 5 3 - 6 5
[13]MoralesLB,LooKK,LiuHB,eta1.Treatment with an estrogen receptor alpha ligand is neuroprotective in experimental autoimmune encephalomyelitis [J].J Neurosci,2006,26(25):6823-6833.
[14] McClain MA,Gat son NN,Powell ND ,et al . Preg2 nancy suppresses experimental auto-Immune encephalomyelitis through immunoregulatory cytokine production [J ] .J Immunol , 2007,179 ( 12):8146—8152.
[15] Hong J ,ZangY C,Ni e H et al. C D 4+ regulatory T cell responses induced by T cell vacci nation i n patients with multiple sclerosis [ J ]. Pro Natl acad Sci US A,2006:1 0 3 ( 1 3 ):502 4 - 5 029.
[16] Offner H,Polanezyk M.A potential role for estrogen in experimental autoimmune enceph- alomyelitis and multiple cslerosis[J].Ann N Y Acad Sci,2006;l089:343-372.
[17] Okasha SA,Ryu S,Do Y e t al.Evidenceforestradiol -induced apoptosi and dysregulated T cell matttration in the thymus [ J ].Toxicology, 2 0 0 1 163:49-62
[18] Hoffman-Goetz L, Cherie F, McCutcheon D, et al. Effects o17bate-estradiol and voluntary exercise or lymphocyte apoptosis in mice[J]. Physiol Behave, 2001, 74(4-5): 653-658.
[19]宋向凤,孙翔,王辉.雌孕激素对T淋巴细胞生长的调节作用[J].细胞与分子免疫学杂志, 2005, 21(2): 249-253.
[20] Urich E,Guteher I,Prim M et a1.Autoantibndy-mediated demyelination depends on complement activation but not activatroy Fc-receptors[J].Proc Natl Acad Sci USA,2006;103(49):18697-18702.
[21] Magdalena J,Polanczyk,Richard E,et a1.T lymphocytes do not directly mediate the protective effect of estrogen on experimental autoimmune encephalomyelitis[J].Am J Pathol,2004,165(6):2069—2077.
[22] Pettersson A,Ciumas C,Chirsky V,et a1.Dendritic cells exposed to estrogen in vitro exhibit therapeutic effects in ongoing experimental allergicencephalomyeliris[J].J Neuroimmunol,2004,156(1-2):58—65。
[23] Xiao B G,Liu X,Link H.Antigen-spcific Tcell functions are suppressed over the estrogen-dendritic cell-indoleamine 2,3- dioxygenaseaxis [J].Steroids,2004;69(10):653-659
[24] Janis K,Hoeltke J,Nazareth M et al.Estrogen decreasese xpression of chemokine receptors,and suppresses chemoke bioactivity in murine monocytes[J].Am J Reprod Immunol,2004;51(1):22—31.
[25] Vlotides G,Gaertner R,Angstwurm M W.Modulation of monocytic lipopolysaccharide– induced tissue factor expression and tumor necrosis factor alpha release by estrogen and calcitrol [J].Hormones (Athens),2007;6(1):52-61
[26] Bruce-Keller AJ,KeelingJL,KellerJN,et a1.Anti-inflammatory effects of estrogen on microglial activation[J].Endocrinology,2000,141(10):3646-3656
[27]Baker A E,Brautigam V M,Watters JJ.Estrongen modulatesmicroglial inflammatory mediator production via interaction with estrogen receptorbeta[J].Endocrinology,2004;145(11):5021-5032
[28]Nilsson N,Carlsten H.Estrogen inducessuppression ofNatural Killer cell cytotoxicitiy and augmentation of Bcell activation[J].Cell Immunol,1994,158(1):131-139
[29] Takao T,Flint N,Lee L etal.17beta-estradiol protects oligodendrocytes from cytotoxicity induced cell death[J].J Neurochem,2004;89(3):660-673.
[30] Comabella M,Pericot L,Goertsches R,et al. Plasma osteopontin levels in multiple sclerosis[J]. Neuroimmunol,2005;158(1/2):231-239.
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