Protective Effect Of Estrogen On Experimental Allergic Encephalomyelitis In Rats

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.