| Objective:The destruction of integrated blood-brain barrier (BBB) is significant in the pathogenesis of experimental autoimmune encephalomyelitis (EAE) and multiple sclerosis (MS). In present study, to provide new theoretical support of immunopathogenesis of EAE and MS we utilize estradiol which is a kind of estrogen to treat ovariectomized Wistar rats with EAE. In addition, we hope to supply experimental evidence for clinical application of estrogen to treat MS and other CNS autoimmune disease.Methods:Wister rats which were ovariectomized were randomly divided into EAE group, E2 group and Complete Freund Adjuvant(CFA) group. Rats in E2 group were injected with E2 (250μg/kg) from day 0 to day 8 p.i.. Clinical symptoms and loss of body weight were evaluated among EAE, E2 and CFA group according to five forked method which is used internationally. The serum E2 level, CSF to serum albumin quotient (QA) were detected at different time points. The damage of BBB was evaluated by QA. The brain and spinal cords were removed on days from 8 to 17p.i. for detecting the expression of matrix metalloproteinase-9 (MMP-9) with Western blot. Meanwhile Transforming growth factor-β(TGF-β) was detected with immunohistochemical staining methods in different parts of CNS. Data were analyzed with SPSS 13.0 software.Results:1. The serum E2 level in EAE group decreased quickly in a short time (19days) after ovariectomization. It rised quickly through nine day's supplement of E2 (250μg/kg.d). It metabolized to the ovariectomized level fast (9days) afte the supplement of E2 was stopped. On days 8～14p.i., the serum E2 level in E2 group was increased when compared with EAE group. The diffference was statistically significant (F=1532.63, P<0.05) On day 17p.i., the serum level in E2 group decreased to the ovariectomized level.2. On day llp.i., the rats of EAE group began to show apparently clinical symptoms such as paralysis of tail, the weakness of hind limbs, the loss of body weight and so on. On day 14p.i. the symptoms reached the peak to display hindlimb paralysis, irritability, the rapid loss of weight and even in dying state. On day 17p.i., the severity began to relieve and clinical symptoms decreased and body weight began to increase. The differences of incidence, change of body weight, clinical symptom scores, the mean number of infectious foci and the mean onset date in E2 group were significantly decreased or delayed when compared with those of EAE group. The diffference were statistically significant(P<0.05).3. On day 8p.i., the QA value in EAE group has increased (0.36±0.04), then decreased gradually within nine days. The QA value in E2 group increased a little in the initial stage and the damage of BBB was slight. QA value increased generally and the peak (0.18±0.03) was later (on day 14 p.i.) than that in EAE group. The difference of QA was statistically significant between EAE group and E2 group on days 8～17 p.i. (F=21.26, P<0.05).4. MMP-9 expressed in spinal cords of rats in each group. Western blot showed 2 bands: one represented the enzymogen form of MMP-9, the relative molecular weight is 92KD; the other denoted the active form of MMP-9, the relative molecular weight is 83 KD. Western blot and fluorescent light immunohistochemistry showed that the expression of MMP-9 in spinal cords in EAE group increased obviously compared with those of adjuvant group (P<0.05). After intervention with E2, the expression and activation of MMP-9 decreased. The difference was significant (P<0.05). There is statistically significant correlation between activation of MMP-9 and clinical scores (r=0.87, P<0.05).5. Immunohistochemistry staining of TGF-P showed that surrounding lymphocyte which was effused out of blood vessel have different degree staining. The expression of TGF-βwas opposite with disease severity. On day 14 p.i., the average photodensity of TGF-βin E2 group reached maximum value, was statistically significant when compared with EAE group (3259.16±541.62 vs 1086.59±214.49, F=17.36, P<0.05). Our results suggest that the expression of TGF-βin E2 group was obviously higher than that in EAE group.Conclusion:1. The serum E2 level in EAE rats was low. It rised quickly following the supplement of E2 and metabolized to the ovariectomized level fast after the supplement of E2 was withdrawn.2. In the early stage of EAE, the injury of BBB was prior to appearence of clinical symptom and then gradually aggravated after the peak injury of BBB.3. The expression of MMP-9 was obviously upregulated obviously in the crest-time of EAE. The inhibiting mechanism of E2 is related to down-regulation of MMP-9 expression and activation, which may protect BBB from the damage of inflammation.4. The expression of TGF-βwas opposite with disease severity in EAE. E2 is a potential agent to inhibit EAE effectively. The inhibiting mechanism of E2 is related to up-regulation of TGF-β, which may promote the transdifferentiation of Th cells. |
PDF Full Text Request