The Dynamic Expression Of The Associated Cytokines Of Th17Differentiation In EAE And The Protective Effect Of Edaravone On EAE

Objective:To investigate the dynamic expression of CD4+Th17effectivecells and associated cytokines in the EAE, to detect the protective role ofEdaravone on EAE, and to further ensure the therapeutic effect of Edaravonein patients with MS．Method:175Female Wistar rats aged6-8weeks, weight180to220g, purchased fromHeBei Medical University Laboratory Animal Center, were divided into threegroups (Edaravone EAE and Control) randomly. Induction of acute EAEWistar rats were immunized with low dose guinea pig spinal cordhomogenate(GPSCH)(GPSCH/physiological saline=1/10), then GPSCHwas mixed with Freund’s complete adjuvant (CFA),the mixture,0.5ml／rat,was injected in the back and the four footpads respectively. Control wasimmunized with physiological saline/CAF. The method of Edaravone groupimmunity was the same as EAE. Edaravone group was interfered withEdaravone (10mg·kg-1·d-1) injected in the cavum abdominis until executed.EAE and Control were interfered with physiological saline (1ml/rat).Observing the effect to clinical sydrome with the drug, using the globalcriteria to evaluate the nervus function.After being immuned, the10th dayafter disease was as pre-diseaese group. After attacted, drawing materials atthe third day and the seventh day randomly as the third day after disease(Acute Phase) and the seventh day after disease (Remission Stage). Eachgroup rats were executed at different time point randomly, the spinalintumescentia lumbalis were embedded in paraffin and sectioned at3-5μmthickness, the sections were stained with HE staining andimmunohistochemistry of RORγt. Using RT-PCR to determine the content and to investigate the dynamic expression of CD4~+Th17associated cytokines(IL-17mRNA IL-23mRNA) and HO-1mRNA each group.Using ELISA todetermine CD4~+Th17associated cytokines (IL-6IL-17) in the peripheralblood. In all cases, a statistical analysis software package (Sta-tistical Programfor Social Sciences13.0; SPSS) was used，the incidence was statisticsed withpercentage(%), the contrast between groups use the chi-square test, themeasurement data use Mean±Standard deviation.the comparison of severalgroups use one-way ANOVA, the comparison between groups use SNK,p<0.05was considered significant throughout all analyses. The comparison ofseveral groups use K-Related-Samples when the Homogeneity of variance test(p>0.1).Results:1The animal attacted status: The mean attacted time of Edaravone grouplower than EAE group (P<0.05), Edaravone group of clinical neurologicalscore lower the EAE group (p<0.05) on the third day, the incidence of theEdaravone group lower the EAE group(P<0.05).2Histopathology Observation：2.1HE Staining:2.1.1Pre-disease: There have been few monocytes and lymphoidocytesinfiltrate in spinal cord of EAE group and Edaravone group in the gray andwhite matter, but no perivascular cuffings.2.1.2The third day after disease: EAE group indicated evidently white matterpuff, a few of monocytes and lymphoidocytes infiltrated and moreperivascular cuffings (mainly located in the juncture of gray and white matter).These are more serious than Edaravone group.2.1.3The seventh day after disease: The monocytes and lymphoidocytesinfiltrated in the tissue were decreased evidently and fewer perivascularcuffings in the tissue. These are obvious in the Edaravone group.2.2Immunohistochemistry of RORγ t2.2.1Pre-disease：EAE group, Edaravone group and Control have few postivecells infiltrate in the gray and white matter of spinal cord, but the EAE group is the most obvious.2.1.2The third day after disease: There are cavity formation and puff in theEAE group, the mean rank of postive cells higher than the Control andEdaravone group (P<0.05).2.2.3The seventh day after disease: The degree of white cavity and puff haveno evident changes, but the Edaravone group presents less sevious. The meanrank of postive cells higher than the Control and Edaravone group (P<0.05).2.2.4Dynamic alteration: Control group changes few among each time(p>0.05). In EAE group, the third day have more white cavity and puff,more postive cells than the other time groups (p<0.05). The tendency ofEdaravone group was the same as EAE group.3RT-PCR3.1The concentration of IL-17in the peripheral blood3.1.1Pre-disease: In EAE group, the concentration of IL-17higher than theother groups (p<0.05).3.1.2The third day after disease: In EAE group, the concentration of IL-17higher than the other groups (p<0.05); the concentration of Edaravone grouphigher than the Control group (p<0.05).3.1.3The seventh day after disease: The concentration of IL-17in EAE grouphigher than the other groups (p<0.05).3.1.4Dynamic alteration: Control group changes few among each time(p>0.05). In EAE group, the concentration of IL-17at third day point higherthan the seventh day point (p<0.05). In Edaravone group, the concentration ofIL-17at third day point higher than the seventh day point and pre-disease(p<0.05).3.2The concentration of IL-6in the peripheral blood3.2.1Pre-disease: In EAE group, the concentration of IL-6higher than theother groups (p<0.05).3.2.2The third day after disease: The concentration of IL-6in the three groupshave no satistically significant.3.2.3The seventh day after disease: The concentration of IL-6in the three groups have no satistically significant.3.2.4Dynamic alteration: Control group changes few among each time(p>0.05)，In EAE group, the mean content of IL-6at pre-disease point higherthan the other groups (p<0.05). In Edaravone group, the concentration of IL-6at pre-disease point higher than the seventh day point (p<0.05).4The expression of IL-17mRNA IL-23mRNA HO-1mRNA in the spinal cord.4.1The expression of IL-23mRNA4.1.1Pre-disease: In EAE group, the expression of IL-23mRNA higher thanthe other groups (p>0.05).4.1.2The third day after disease: In EAE group, the expression ofIL-23mRNA higher than the other groups (p<0.05).4.1.3The seventh day after disease: In EAE group, the expression ofIL-23mRNA higher than the other groups (p>0.05).4.1.4Dynamic alteration: Control group changes few among each time(p>0.05). In EAE group, the expression of IL-23mRNA at third day pointhigher than the seventh day point (p<0.05). The tendency of Edaravone groupwas the same as EAE group (p<0.05).4.2The expression of IL-17mRNA4.2.1Pre-disease: In EAE group, the expression of IL-17mRNA higher thanthe other groups (p<0.05).4.2.2The third day after disease: In EAE group, the expression ofIL-17mRNA higher than the other groups (p<0.05); the expression ofEdaravone group higher than the Control group (p<0.05).4.2.3The seventh day after disease: The expression of IL-17mRNA in theEAE group and Edaravone group higher than the Control group (p<0.05).EAE group contrast with Edaravone group (p>0.05).4.2.4Dynamic alteration: Control group changes few among each time(p>0.05). In EAE group, the expression of IL-17mRNA at third day pointhigher than the seventh day point and pre-disease (p<0.05). The tendency ofEdaravone group was the same as EAE group.4.3The expression of HO-1mRNA 4.3.1Pre-disease: In Edaravone group, the expression of HO-1mRNA higherthan the other groups (p<0.05).4.3.2The third day after disease: In Edaravone group, the expression ofHO-1mRNA higher than the other groups (p<0.05); the expression of EAEgroup higher than the Control group (p<0.05).4.3.3The seventh day after disease: The expression of HO-1mRNA in theEdaravone group higher than the other groups (p<0.05). The EAE grouphigher than Contrast group (p<0.05).4.3.4Dynamic alteration: Control group changes few among each time(p>0.05). In EAE group, the expression of HO-1mRNA at third day pointhigher than pre-disease (p<0.05), the seventh day higher than the third day(p<0.05). The tendency of Edaravone group was the same as EAE group(p<0.05).Conclusions:1Edaravone could delay the peak time, reduce the clinical severity of EAEevidently, lower the incidence of EAE effectively.2Edaravone could lower specific immune response, reduce the inflammatoryreaction.3In the peripheral blood, Edaravone might impress the content of IL-6, andthus inhibit development and differentiation of Th17, reduce the content ofIL-17and alleviate the clinical syndrom.4In spinal cords, Edaravone might promote the expression of HO-1mRNA,and down the expression of IL-23mRNA and IL-17mRNA.