The Role Of Sulforaphane On Regulation Of CD4~+CD25~+Foxp3~+Tregs In EAE Mice Spleen

Objective: To explore the protective role of CD4⁺CD25⁺Foxp3⁺Tregsand Nrf2/ARE pathway in the pathogenesis of EAE and to confirm theprotective effect of sulforaphane on EAE.Methods:1Animal groupingThe18female8-10week-old C57BL/6mice weighing18-20g,wererandomly divided into two groups:EAE model group and sulforaphanegroup,each group had9mice.Sulforaphane group（n=9）:we used the method of MOG35-55+Mycobacterium tuberculosis H37Ra+PTX+CFA to immune mice andgave the intraperitoneal injection of sulforaphane for50mg·kg-1·d-1everytwo days since the first day after immunization until sacrificed.EAE group（n=9）:we used the method of MOG35-55+Mycobacteriumtuberculosis H37Ra+PTX+CFA to immune mice and gave theintraperitoneal injection of physiological saline for0.1ml/mouse at thesame time.2Induction of EAEImmunized all of the mice subcutaneously in the four points of backwith0.1ml emulsion.The emulsion was composed of the MOG as antigenwhich was diluted into6mg/ml with physiological saline and emulsified itwith an equal volume of complete Freund’s adjuvant（CFA）, and putMycobacterium tuberculosis H37Ra into it, which finally containedMycobacterium tuberculosis H37Ra4mg/ml（final concentation）. Pertussistoxin（PTX） was given in intraperitoneal way for0.5ml/mouse at0hour and48hour after immunized（1400ng/mouse）.3Treatment with sulforaphane Every Sulforaphane group mouse was given intraperitoneal injectionof sulforaphane for50mg·kg^-1·d^-1every two days since the first day afterimmunization until sacrificed. EAE group mice were given intraperitonealinjection of physiological saline for0.1ml/mouse at the same time.4Clinical score and weight changeSince the immune day,we used the double-blind way to weigh the miceand gave every mouse clinical score at8a.m. every day.It required two peopledoing the work together at the same time.In our study, we used the Weaver’s15-point disease score,which ranges from0to15and the sum of the state ofthe tail and all of the four limbs is the final score. A fully paralyzedquadriplegic animal equals14points and mortality equals15points.Weaver’s scoreTail:0:no signs1: a half paralyzed tail2: a fully paralyzed tailLimb:0: no signs1: a weak or altered gait2: paresis3: a fully paralyzed limb（The highest clinical score is the highest score in the pathogenesis of EAE）5Morbidity and the onset time of diseaseWe counted how many mice had have the disease, and compared theaverage morbidity in each group. We also observed when the disease hadoccured,and compared the average onset time in each group.6Spleen cell isolating and sample preparationOn day20,put all of the mice to death by breaking the neck and removedthe spleen. Spleen cells were prepared by mincing and homogenizing thespleen through the200-hole copper mesh and centrifuged, and finallyresuspended in physiological saline to make100-μl aliquots. 7Flow cytometryThe work such as adding the monoclonal antibodies,incubation,fixationand permeabilization,washing, suspension had been done to the sample,and ithad been analysed on the multi-color laser flow cytometry system,and finallyget the results:the percentage of CD4⁺CD25⁺Foxp3⁺Tregs in the CD4+T cellsin every mouse.We calculate the mean of the percentage of each group,andcompared the means between EAE group and Sulforaphane group.8Statistical analysisAll of the experimenatal data were statistically analyzed by SPSS18.0software. Measurement data were presented as mean±standard deviation,statistical significance was calculated using two-samples t-test（P<0.05）.Themorbidities were presented by percentages, statistical significance wascalculated using Chi-square test（P<0.05）.Results:1The incidence rate of EAE group was100%,and Sulforaphanegroup’s was44.4%,which was significantly lower than EAE group’s;themean time of onset of EAE group was13.44±1.59d, and the the meantime of onset of Sulforaphane group’s was17.25±1.71d, which wassignificantly later than EAE group’s;the mean highest clinical score ofEAE group of mice was8.00±2.60, and Sulforaphane group’s was3.50±2.65, which was significantly lower than EAE group’s;the mean weightdescend of EAE group was-2.62±0.87g, and Sulforaphane group’s was-0.60±0.96g, which was significantly lower than EAE group’s. All of thedifferences above were significantly statistical（P<0.05）.2The percentage of CD4⁺CD25⁺Foxp3⁺Tregs in the CD4+T cells of EAEgroup’s mice spleen were5.2%,4.2%,5.4%,4.9%,5.0%,3.8%,3.3%,4.5%,4.0%, and the mean percentage was （4.48±0.70）%；Sulforaphanegroup’s were6.2%,7.9%,9.3%,8.2%,6.6%,9.5%,6.0%,6.9%,7.9%, and the mean percentage was （7.61±1.28）%, which wassignificanly higher than EAE group’s and the difference was significantlystatistical（P<0.05）. Conclusion:1Sulforaphane could lower the morbidity of EAE，delay the time ofpathogenesis, and reduce the clinical severity of EAE,which indicate thatsulforaphane could play a protective role in the pathogenesis of EAE.2Sulforaphane could upregulate the percentage of CD4⁺CD25⁺Foxp3⁺Tregs in the CD4⁺T cells in the spleen of EAE mice,which could playa protective role in the pathogenesis of EAE.