| Objective: Multiple sclerosis (MS) is a chronic inflammatorydemyelinating disease of the central nervous system (CNS), the pathogenesisis still unclear now. Recently many studies have shown that the oxidativestress which caused by exogenous and endogenous electron affinity substanceis one of the most important factors of MS brain injury. Intra-cellular oxidativesubstances have oxidation characteristics, which can lead to neuron death.Therefore, cell activates anti-oxidative stress system to protect the neuronfrom ischemia. Nuclear factor erythroid-2related factor2(Nrf2) is atranscription factor which can bind the antioxidant response element (ARE),and their combination can activate the endogenous antioxidant system forgenerating many antioxidant enzymes, thereby inhibiting injury caused byoxidative stress. Sulforaphane (SFN) is a kind of isothiocyanates, widelyexisting in the cruciferous vegetables in its precursor glucoraphenin forms. Abig number of evidences have indicated, sulforaphane is an importantactivation agent of Nrf2, which can promote Nrf2translocation to the nucleus,bind to ARE, and induce the antioxidant phase Ⅱ enzymes to have theantioxidant effect.We could speculate that active the Nrf2/ARE can protect theEAE. The protective effect of Sulforaphane to EAE has not been proved yet,and it can strongly induce Nrf2nuclear translocation, and it has little sideeffects, can pass through the blood-brain barrier,intraperitoneal injection canbe absorbed, so choose the SFN to test, and compared to the current clinicallyused drugs dexamethasone (DXM), it can verify whether SFN has aprotective effect and difference of strong and weak, furthermore to show thetherapeutic value of MS to SFN.An experimental autoimmune encephalomyelitis(EAE) mice model wasestablished successfully, measured at different time points in mice brain tissue Nrf2and malondialdehyde (MDA) expression level, application of SFN andDXM intervention, observed by SFN and DXM on neural function defectscore, expression influence in Nrf2and MDA, expression regulation of Nrf2and MDA and sulforaphane to the protective effect of EAE.Methods:120female C57BL/6mice (inbred strain,aged8-10weeks)wheighted18-20g were induced by MOG35-55to make EAEmodel.They were randomly divided into a healthy control group(30mice),anEAE group(30mice),a SFN(50mg/kg)group (30mice)and a DXM(0.07mg/kg)group(30mice).Each group was divided into three subgroups:13day subgroup(10mice),20day subgroup(10mice),30day subgroup(10mice).Diluted antigen MOG35-55by physiological saline into6mg/ml,according to the volume1:1, mixed Complete Freund's Adjuvant (finalconcentration of tubercle bacillus H37Ra is4mg/ml). After emulsion, eachexperimental rat was subcutaneously immunized in four points on the twoback sides with0.1ml, and gave peritoneal injection0.5mlPTX (1400ng/each)at0hour and at48hours. The healthy control group left untreated.SFN groupand DXM group were injected intraperitoneal respectively SFN50mg/kg,DXM0.07mg/kg on the first day of immunization,injected everyother day until they were and48hour. The healthy control group leftuntreated.SFN group and DXM group were injected intraperitonealrespectively SFN50mg/kg,DXM0.07mg/kg on the first day ofimmunization,injected every other day until they were sacrifice. The mice incontrol group and EAE group received equal volume of physiological salineevery other day.Two investigators assessed the clinical signs of mice twice aday.Scores were assigned on the basis of the following symptoms:0,noparalysis;1,tail weakness;2,hindlimb weakness;3,mild limbparalysis;4,hindlimb and forelimb paralysis;5,moribund or dead.During theexperiment,the mean weight and maximal score of mice at different time pointwere observed and scored.Mice were sacrificed after anesthesia injection at the13day,20day,30day.Tissues of the brain were fixed with4%paraformaldehde24hours.Then the tissues were embedded in paraffin and sectioned at5um thickness,and thenused the immunohistochemistry to analyse the expression of Nrf2under thelight microscopy.The experiment mice were sacrificed after anesthesia at13days,20days,30days.The animal was decapitated immediately,took the brain in the ice trayrapidly,then divested the cerebral dura mater.Removed thecerebellum,brainstem,frontal polar and the cortex of periventricular.Washedthe tissue in the0～4℃physiological saline,got rid of blood,bloted with thefilter paper and then weighed.According to1:9to join the ice-cold saline madehomogenate.Then ultrasonic with400ampere,5s/time,gaped10s repeated3to5times in the ice bath.2000r/min,4℃centrifugal15min.Taked thesupernatant,then stored in the-70℃refrigerator.Used the TBA method tomeasured the MDA.Results:1The morbidity of disease in the different groupThe morbidity of EAE group,SFN group and DXM group respectivelywas:100%,33.33%,27.78%. Morbidity of EAE group were significantlyhigher than SFN group and DXM group.The differences were statisticallysignificant(P <0.05).2EAE clinical profile in different groupThe mean clinical score of EAE group,SFN group and DXM grouprespectively was:2.38±0.52,1.33±0.50,1.25±0.50.Neurological deficitsscores of high dose of EAE group was significantly higher than SFN groupand DXM group.The differences were statistically significant(P <0.05).3Mental state and fur gloss observation:The EAE group mental was listless and fur was dark,and had ulceroccationally;The SFN group mental was normal and fur was smooth;TheDXM group mental was excited and fur was dark with ulcer.4The immunoposition cells of Nrf2in the brain tissue:The immunoposition cells of Nrf2in EAE group,SFN group and DXMgroup were all higher than control group.The immunoposition cells of Nrf2in control group,EAE group,SFN group and DXM group at the peak stagerespectively was:5.80±2.17,23.60±7.64,73.60±10.55,69.00±8.09.And thedifferences of EAE group,SFN group and DXM group were statisticallysignificant(P <0.05).5The MDA assay of brain tissue:The mean MDA concentration of control group,EAE group,SFN groupand DXM group at the peak stage respectively was:20.35±1.10,40.34±7.50,9.65±5.50,8.86±5.21, SFN group and DXM group were significantly lowerthan EAE group. The differences were statistically significant(P <0.05).Conclusions:1The expression of Nrf2in EAE group was up-regularted,but in SFN groupand DXM group the expression were more obvious.2Sulforaphane could reduce the morbidity,and reduce the nerve damage.3Sulforaphane played an important role on oxidative stress thoughup-regularting the expression of Nrf2.4Sulforaphane compared to dexamethasone,DXM is better than SFN in theneurological deficits scores and morbidity,but SFN has good safty,will notcause the mental change and can aviod the fur ulcers which is unaviodable ifuse the DXM longtime.SFN displays the advantage of good tolerance andsafety. |
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