Investigating The Mechanism Action Of The SOCS1-JAK2-STAT3Pathway In C57BL/6Mice EAE

Objective: To investigate the mechanism action of the SOCS1-JAK2-STAT3pathway in C57BL/6mice EAE, to detect the protective role of SOC-S1-JAK2-STAT3pathway on EAE, and to further ensure the therapeutic effectof SOCS1-JAK2-STAT3pathway on in patients with MS．Method:60Female C57BL/6mice aged6-8weeks, weight18to22g,were divided into three groups (EAE+AG490, EAE and Control) randomly.Induction of acute EAE C57BL/6mice were immunized with300ug SyntheticMOG35-55polypeptide dissolved in200ul phosphate buffered solution (PBS),Containing4mg/ml BCG with an equal amount of complete Freund’s adju-vant (CFA) fully mixed to oil-in-water emulsion, made complete antigen,bilateral lateral abdominal wall4:00subcutaneous injection. Both groups werein after immunization,0and48h after intraperitoneal injection of500ng per-tussis toxin, to establish a corresponding model immunized C57BL/6micePreparation EAE model, application AG490intervention. Blank control group,directly to200ul of PBS emulsified with an equivalent CFA immunized anim-al. Of EAE, Effects of AG490group on immunization given the first3,5,7,9,11,13,15,17,19day AG4901mg/DMSO100ul subcutaneous injection toput to death, the blank control group and the EAE model group given the first3,5,7,9,11,13,15,17,19day0.1ml saline mice were injected subcutaneously.Observe the impact of drugs on animals, clinical manifestations, withreference to the common clinical score of mice, neurological score.13daysafter immunization group were randomly drawn, denoted as before the onsetof the group, and recorded as peak group after immunization groups of about20days, were randomly drawn, recorded as the peak incidence group. Eachexperimental group of mice were sacrificed at different periods after thymusHE staining inflammatory cell infiltration, simultaneously immunohistoche- mical staining observed RORγt, IL-17, of SOCS1, P-STAT3positive cellsand counted, for comparison. SPSS16.0statistical software for statisticalanalysis, the incidence rate is expressed as a percentage, the groups werecompared using the chi-square test, measurement data are presented as mean±standard deviation (±S) said that multiple sets of measurement data tocompare the number applications ANOVA analysis of variancepairwisecomparisons between groups using the SNK test, P<0.05, the difference wasstatistically significant. When heterogeneity of variance (P<0.1), with multipleindependent samples nonparametric tests.Results:1The observation of morbidityThe mice are listlessness, fur unsmooth, decreased appetite, weight loss afterimmunization gradually. When onset tail feeble appear at first, then hind limbsand fore limbs, incontinence of urine and feces.(1) The comparison of morbidity timeThere is no morbidity in the control group, the EAE group is (11.0000±1.684)days, and the EAE+AG490group is (17.3333±0.750) days, there is statistica-lly significant(P<0.05).(2) The comparison of morbidity20mice morbidity in EAE group, the morbidity is100%,9rats morbidity inthe Edaravone group, the morbidity is45%, there is statistically significant(P<0.05).(3) The comparison of mean clinical scoresThe mean clinical score of the EAE group is (4.0000±0.12566）in immune afer20days, The mean clinical score of the EAE+AG490group is (2.0556±0.24216) in immune afer20days There is statistically significant of the two groups atthe same time (P<0.05).Animal incidence: EAE+AG490group, the average time of onset was signif-icantly lower than the EAE group, with a statistically significant (P<0.05);20days afer immuned,EAE+AG490group immune average score lower thanEAE group, a statistically significant (P<0.05); EAE+AG490group the incidence is lower than EAE group, a statistically significant (P<0.05).2The observation of histopathology(1) Clear demarcation of cortex and medulla of the control group, lymphocyt-eintensive nuclear bluish purple.(2) Immunity after13days EAE and EAE+AG490group thymocytes apopto-sis in thymus tissue begins to shrink, the the cortex started thinning, reducingthe number of lymphocytes, the cells loose nuclear chromatin has beenconcentrated, the nuclear start pyknosis.(3) Immunity after20days Apoptotic cells in EAE group, showing significa-nt atrophy of the thymus, the cortex was significantly thinner, reducing thenumber of lymphocytes, cells loose, an increase in the number of apoptoticcells, nuclear chromatin condensation, nuclear condensation, pathologicalmanifestations than the EAE+AG490groupserious.3The observation of immunohistochemical(IHC).3.1RORγtWidely expressed in lymphoid organs interval cells (CD4+T CD8+Tdoublepositive cells).(1)13days after immunization the EAE group immunoreactive cells numberof EAE+AG490treated group immunoreactive cell number increased significa-ntly, the two groups was statistically significant (P<0.05).(2)20days after immunization the EAE group of immunoreactive cell numb-ers of EAE+AG490-treated group immunoreactive cell number increased signi-ficantly, the two groups was statistically significant (P<0.05).(3)EAE group two time points:13days after immunization group20daysafter immunization with group was statistically significant (P<0.05).(4)EAE+AG490group two time points:13days after immunization group20days after immunization with group was statistically significant (P<0.05).3.2IL-17IL-17is a newly discovered by memory CD4+T lymphocytes, CD8+T cells,eosinophilic granulocytes and other cells secrete a cytokine.(1)13days after immunization the EAE group immunoreactive cells number of EAE+AG490treated group immunoreactive cell number increased signifi-cantly, the two groups was statistically significant (P<0.05).(2)20days after immunization the EAE group of immunoreactive cellnumbers of EAE+AG490treated group immunoreactive cell number incr-eased significantly, the two groups was statistically significant (P<0.05).(3)EAE group two time points:13days after immunization group20daysafter immunization with group was statistically significant (P<0.05).(4)EAE+AG490group two time points:13days after immunization group20days after immunization with group was statistically significant (P<0.05).3.3SOCS1The SOCS1protein widely expressed in a variety of human cells.(1)13days after immunization the EAE group immunoreactive cells numberof EAE+AG490treated group immunoreactive cell number increased sign-ificantly, the two groups was statistically significant (P<0.05).(2)20days after immunization the EAE group of immunoreactive cell number-s of EAE+AG490treated group immunoreactive cell number increased signif-icantly, the two groups was statistically significant (P<0.05).(3)EAE group two time points:13days after immunization group20daysafter immunization with group was statistically significant (P<0.05).(4)EAE+AG490group two time points:13days after immunization group20days after immunization with group was statistically significant (P<0.05).3.4p-STAT3The p-STAT3protein widely expressed in a variety of human cells.(1)13days after immunization the EAE group immunoreactive cells numberof EAE+AG490treated group immunoreactive cell number increased signif-icantly, the two groups was statistically significant (P<0.05).(2)20days after immunization the EAE group of immunoreactive cell number-s of EAE+AG490treated group immunoreactive cell number increased signi-ficantly, the two groups was statistically significant (P<0.05).(3)EAE group two time points:13days after immunization group20daysafter immunization with group was statistically significant (P<0.05). (4)EAE+AG490group two time points:13days after immunization group20days after immunization with group was statistically significant (P<0.05).Conclusions:1.AG490through inhibition of JAK, thereby inhibiting theSOCS-JAK-STAT signaling pathway, reducing the specific immune responseand decrease inflammation.2.SOCS-JAK-STAT signaling pathway play animportant role in experime-ntal autoimmune encephalomyelitis.