| Objective: To investigate the association between CD4+CD25+Foxp3+T regulatory cells (CD4+CD25+Foxp3+Tregs) and HO-1 in the pathogenesis of EAE and the protective effect of Edaravone on EAE, and further ensure the efficacy of Edaravone on MS.Methods: Low dose (W: V=1:10) of guinea pig spinal cord homogenate (GPSCH) emulsified with complete Freund's adjuvant (CFA) was adopted to prepare EAE models.Edaravone was administrated for intervention. 171female Wistar rats were divided into three groups randomly: control group, EAE group and edaravone group.The rats of control group were immunized with physiological saline and CFA.The method of immunization for edaravone group was the same as EAE group's.The rats of edaravone group were given intraperitoneal injections of Edaravone for 10 mg·kg-1·d-1 since immunized until sacrificed.The rats of the other two groups were given intraperitoneal injections of physiological saline for 1ml/rat at the same time.The efficacy of Edaravone on the clinical manifestation was observed, and the clinical score was assessed in accordance with the standard generally used.Rats of each group were randomly sacrificed on the 10th day after immumization, 3 days and 7 days after the onset of EAE.The lumbar of the spinal cords were stained with HE to observe the inflammation in situ, and also stained with immunohistochemisty (IHC) to observe the distribution of the Foxp3 and HO-1 immunopositive cells.The mRNA contents of Foxp3 and HO-1 in both spinal cords and thymus were evaluated by real-time PCR.The concentrations of IL-10 and TGF-βin the peripheral blood were estimated by enzyme-linked immunosorbent assay (ELISA).All data were statistically analyzed by SPSS 13.0 software.The incidence rates were represented by percentage, the method for the comparison was chi square test.Measurement data were expressed as mean±standard deviation.The statistical method for comparisons of several groups was one-way ANOVA. LSD was used for the comparisons between groups. Linear correlation between Foxp3 and HO-1 was also evaluated.P value <0.05 was considered statistically significant.Results:1 Pathogenesis of different groups The incidence rate of edaravone group was 46.8%,which was significantly lower than EAE group's.The mean time for rats of edaravone group to develop disease was significantly later than EAE group's.The mean clinical score of edaravone group was significantly lower than EAE group's.The differences were all significantly statistical (P<0.05).No rat developed disease in control group.2 Histopathology of different groups in lumbar spinal cords(1) Pre-disease There were scattered inflammatory cells on sections of EAE group.There was no inflammatory cells on those of edaravone groups.(2) 3 days after onset There were diffuse inflammatory cells infiltrated and mass"blood vessel muffs"formed on sections of EAE group.There were less"blood vessel muffs"formed on sections of edaravone group and the inflammatory cells were mainly around small vessels.(3) 7 days after onset The"blood vessel muffs"and the inflammatory cells on sections of EAE group were diminished,while those of edaravone group were vanished.(4) There was no"blood vessel muff"or inflammatory cell on sections of control group.3 Expression of Foxp3 and HO-1 in the lumbar spinal cordsFoxp3 was immunopositive on inflammatory cells, HO-1 was immunopositive on neurons, glial cells and inflammatory cells.We mainly observe the two factors on inflammatory cells.(1) The means of two immunopositive cells in control group had no statistical difference among three different times (P>0.05).(2) Pre-disease In EAE group, the mean number of Foxp3 immunopositive cells was (20.57±3.31), and that of HO-1 was (19.28±3.56).In edaravone group, the mean number of Foxp3 immunopositive cells was (26.78±4.02), and that of HO-1 was (29.26±3.86), both of which were significantly more than EAE group's (P<0.05).All datas of the two groups were significantly higher than control group's (P<0.05).(2) 3 day after onset In EAE group, the mean number of Foxp3 immunopositive cells was (23.60±2.78), and that of HO-1 was (37.03±3.68).In edaravone group, the mean number of Foxp3 immunopositive cells was (44.05±2.68), and that of HO-1 was (50.50±3.67), both of which were significantly more than EAE group's(P<0.05).The mean of the two immunopositive cells in either group was significantly increased compared with the data pre-disease (P<0.05). The datas of the two groups were significantly higher than control group's (P<0.05).(3) 7 day after onset In EAE group, the mean number of Foxp3 immunopositive cells was (27.86±2.88), and that of HO-1 was (41.73±4.41), the datas above were significantly increased compared with those on 3rd day after morbidity (P<0.05).In edaravone group, the mean number of Foxp3 immunopositive cells was (33.36±3.19), and that of HO-1 was (45.82±3.71), both of which were significantly decreased compared with the data on 3rd day after morbidity, but were significantly more than EAE group's (P<0.05). The datas of the two groups were significantly higher than control group's (P<0.05).4 The mRNA contents of Foxp3 and HO-1 from spinal cords and thymuses4.1 In spinal cords(1) Pre-disease In EAE group, the mean RQ of Foxp3 was (0.213±0.060), and that of HO-1 was (0.346±0.020).In edaravone group, the mean RQ of Foxp3 was (1.142±0.027), and that of HO-1 was (1.353±0.034), both of which were significantly higher than EAE group's (P<0.05).The datas of the two groups were significantly higher than control group's(P<0.05).(2) 3 days after onset In EAE group, the mean RQ of Foxp3 was (1.058±0.031), and that of HO-1 was (1.446±0.025). In edaravone group, the mean RQ of Foxp3 was (3.650±0.031), and that of HO-1 was (4.444±0.024), both of which were significantly higher than EAE group's (P<0.05).The mean RQ of the two factors in either group was significantly higher than control group's and the data pre-disease (P<0.05).(3) 7 days after onset In EAE group, the mean RQ of Foxp3 was (2.894±0.068), and that of HO-1 was (3.058±0.031), both of which were significantly higher than the datas on the 3rd day after morbidity (P<0.05). In edaravone group, the mean RQ of Foxp3 was (3.585±0.053), and that of HO-1 was (3.861±0.020), both of which were significantly lower than the datas on the 3rd day after morbidity,but were significantly higher than EAE group's (P<0.05). The mean RQ of the two factors in either group was significantly higher than control group's (P<0.05).(4) The mean RQ of the two factors in control group had no statistical difference among three different times (P>0.05).4.2 In thymuses(1) Pre-disease In EAE group, the mean RQ of Foxp3 was (1.853±0.030), and that of HO-1 was (0.551±0.035), both of which were significantly lower than control group's (P<0.05).In edaravone group, the mean RQ of Foxp3 was (3.451±0.027), and that of HO-1 was (0.957±0.021), both of which were significantly higher than EAE group's and control group's (P<0.05).(2) 3 days after onset In EAE group, the mean RQ of Foxp3 was (3.553±0.029), and that of HO-1 was (1.556±0.025). In edaravone group, the mean RQ of Foxp3 was (4.446±0.024), and that of HO-1 was (2.165±0.021), both of which were significantly higher than EAE group's (P<0.05).The mean RQ of the two factors in either group was significantly higher than control group's and the data pre-disease (P<0.05).(3) 7 days after onset In EAE group, the mean RQ of Foxp3 was (1.856±0.021), and that of HO-1 was (0.545±0.024), both of which were significantly lower than control group's (P<0.05). In edaravone group, the mean RQ of Foxp3 was (4.348±0.035), and that of HO-1 was (1.255±0.025), both of which were significantly higher than control group's and EAE group's (P<0.05).The mean RQ of the two factors in either group was significantly lower than the data on the 3rd day after morbidity (P<0.05).(4) The mean RQ of the two factors in control group had no statistical difference among three different times (P>0.05).5 The amount of immunopositive cells of Foxp3 and HO-1 in spinal cords, and also the mRNA contents of the two factors in spinal cords and thymuses in the same group at the same time had significant positive correlation (P<0.05, r>0).6 The concentration of IL-10 and TGF-βin the peripheral blood(1) Pre-disease In EAE group, the mean concentration of IL-10 was (17.72±0.23), and that of TGF-βwas (80.64±0.21), both of which were significantly lower than control group's (P<0.05). In edaravone group, the mean concentration of IL-10 was (19.39±0.21), and that of TGF-βwas (86.59±0.21), both of which were significantly higher than EAE group's and control group's (P<0.05).(2) 3 days after onset In EAE group, the mean concentration of IL-10 was (20.39±0.28), and that of TGF-βwas (86.52±0.26).In edaravone group, the mean concentration of IL-10 was (21.43±0.27), and that of TGF-βwas (88.42±0.33), both of which were significantly higher than EAE group's.The mean concerntration of the two factors in either group was significantly higher than control group's and the date pre-disease (P<0.05).(3) 7 days after onset In EAE group, the mean concentration of IL-10 was (17.40±0.24), and that of TGF-βwas (80.77±0.19), both of which were significantly lower than control group's (P<0.05).In edaravone group, the mean concentration of IL-10 was (20.45±0.36), and that of TGF-βwas (87.42±0.29), both of which were significantly higher than EAE group's and control group's (P<0.05).The mean concerntration of the two factors in either group was significantly lower than the data on the 3rd day after morbidity (P<0.05).(4) The mean concentration of the two factors in control group had no statistical difference among three different times (P>0.05).Conclusion: 1 Edaravone could lower the incidence rate of EAE, delay the time of pathogenesis, and alleviate the clinical severity of EAE.2 The expression and the content of mRNA of Foxp3 and HO-1 in spinal cords were increased with the course of EAE, which suggested that the two factors were could play immunoregulation and antioxidation following the course of EAE.After treated with edaravone, the datas above were increased, which suggested that edaravone could promote the expression and renovation of Foxp3 and HO-1 and had therapeutical effect on EAE.3 The content of mRNA of Foxp3 and HO-1 in the thymuses were high at crest-time and low before onset of EAE and at recovery stage, which suggested that the low regeneration of nTregs and HO-1 from thymus resulting low immunoregulation and antioxidation was the important factor for the developing and relapsing of EAE.After treated with edaravone, the content of mRNA of the two factors were increased and higher than normal, which suggested that edaravone could promote the renovation of nTregs and HO-1 from thymus and had therapeutical effect on EAE.4 The amount of immunopositive cells of Foxp3 and HO-1, and also the content of mRNA of Foxp3 and HO-1 had significant positive correlation, which suggested that mainly HO-1 on the surface of CD4+CD25+Foxp3+Tregs changed in the course of EAE and play immunoregulation with Foxp3. Edaravone may promote the development and function of CD4+CD25+Foxp3+Tregs via upregulating the expression of HO-1, and play immunoregulation.5 The concentration of IL-10 and TGF-β, which were the effectors of CD4+CD25+Foxp3+Tregs rised at first and declined later following the course of EAE in the peripheral blood of EAE rats.After treated with edaravone, the concentration of the two factors rised at the same time, which suggested that edaravone could promote CD4+CD25+Foxp3+Tregs playing immunoregulation via cytokine.
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