| Objective:Multiple sclerosis(MS)is a chronic inflammatory demyelinating immune-mediated disease of the central nervous system(CNS).Experimental autoimmune encephalomyelitis(EAE)is MS's commonly accepted animal model.Nowadays,the pathogenesis of MS is unclear.The possible pathogenic mechanism is immune response to myelin antigens conducted by CD4+T cells.Abundant production of cytokines activated by immune cells infiltrate into the blood brain barrier and damage neurons and oligodendrocytes,including interleukin-12(IL-12),IL-6,IL-17,IL-21,IL-23,et al.The Janus Kinase/Signal Transducer and Activator of Transcription(JAK/STAT)signaling pathway mediates the biological activities of these cytokines and is essential for the development and regulation of immune responses.Dysregulation of the JAK/STAT pathway contributes to numerous autoimmune diseases,including MS/EAE.The JAK/STAT pathway is aberrantly activated in MS/ EAE because of excessive production of cytokines,loss of expression of negative regulators such as suppressors of cytokine signaling proteins,and significant enrichment of genes encoding components of the JAK/STAT pathway,including STAT3.Specific JAK/STAT inhibitors have been used in numerous preclinical models of MS and demonstrate beneficial effects on the attenuation of innate and adaptive immune responses[1].JAK/STAT signal pathway emerges as an important determinant of MS/EAE's disease progress.Nowadays,there's no effective therapeutic for MS.Ruxolitinib is a selective inhibitor of JAK1 and JAK2 that was developed for the treatment of myelofibrosis.Its therapeutic mechanism is reducing STAT3 phosphorylation.Ruxolitinib has been shown to be a potent immunomodulator which affects circulating cytokine levels and regulates theactivation of dendritic cells and T lymphocytes.Ruxolitinib's effect on MS/EAE is unclear.In this study,we used ruxolitinib as the therapeutic and observed the clinical symptoms,expression of inflammatory cytokines and the percentage of CD4+CD25+FoxP3+T cells in control,EAE,and ruxolitinib treatment groups to explore ruxolitinib's influence on immune mechanism of EAE.Methods:80 female C57BL/6 mice(8 to 10 weeks old)of 18 g to 20 g body weight,were randomly divided into 4 equal groups: group control,group EAE,high dose(HD)ruxolitinib group and low dose(LD)group,20 mice each groups.Then EAE model was established.Treatments started on the day when EAE model were established(recorded as 0 day).Ruxolitinib was diluted in a 1:1solution of ethanol:PBS for the intraperitoneal(ip)injection.For high dose treatment group,mice received 10ml/Kg high concentration solution(0.5mg/ml)every other day;Low dose treatment mice received 10ml/Kg low concentration solution(0.25mg/ml)every other day;EAE and control group mice received 1:1 ethanol:PBS solvent 10ml/Kg every other day till executed.Their weight and clinical score were recorded every morning.The relative mRNA levels of IL-6,IL-17,STAT3,TNF-?,FoxP3+ in spleen were detected by qrtPCR procedures.The expression levels of IL-6 and TNF-?in serum were evaluated by ELISA procedures.CD4~+CD25~+Foxp3~+ regulatory T cells' percentage was assessed by flow cytometry.Results:1 25 days post the immunization,compared with group EAE,the mean value of clinical score of group ruxolitinib decreased remarkably(P<0.05).Morbidity ofhigh dose treatment group reduced significantly compared with EAE group(P<0.05),while there was no significant difference between low dose treatment group and EAE group(P > 0.05).The latent period of group EAE was(15.22±1.72)d,the latent period of ruxolitinib high dose treatment group was(18.17±1.85)d,and the latent period of low dose treatment group was(17.75±1.49)d;2 Relative mRNA levels of IL-6,IL-17,STAT3,TNF-? and FoxP3+ in spleen: 25 days post the immunization,compared with group control,relative mRNA levels of IL-6,IL-17,STAT3,and TNF-? remarkably elevated(P<0.01)in the EAE mice.While there's no significant difference between EAE and control groups' expression of FoxP3+(P>0.05).The mRNA translation levels of IL-6,IL-17,STAT3,and TNF-?in the high dose ruxolitinib treated-mice were significantly lower than that in the EAE mice(P<0.01).The relative levels of FoxP3+ mRNA transcripts in the high dose treatment group were significantly higher than that in the EAE mice(P<0.01).The mRNA translation levels of IL-6,STAT3,and TNF-? in the low dose treated-mice were significantly lower than that in the EAE mice(P<0.01).The relative levels of FoxP3+ mRNA transcripts in the low dose treatment group were significantly higher than that in the EAE mice(P<0.01).The relative levels of IL-17 mRNA transcripts in the low dose treatment group has no statistically significance versus the EAE mice(P > 0.05);3 The expression of IL-6 and TNF-? in mice serum of each group: 25 days post the immunization,significantly higher levels of IL-6 were detected in the EAE mice and ruxolitinib-treated mice,as compared with that in the control group(P<0.01).High dose ruxolitinib treatment effectively reduced the expression levels of IL-6,related to that in the EAE mice(P<0.05).While there was no significant difference in the levels of IL-6 expression between low dose treated mice and EAE mice(P>0.05).As to the expression levels of TNF-?,notably increase existed in the group of low dose treated mice and EAE mice,compared with that in the normal mice(P < 0.01).There was no obvious difference in the expression levels of TNF-? between the control and high dose-treated groups(P > 0.05).Both ruxolitinib subgroups had notably reduction in the levels of TNF-? compared with EAE mice;4 The proportion of CD4~+CD25~+Foxp3~+ regulatory T cells in each group: 25 days post the immunization,compared with normal mice,the percentage of CD4~+CD25~+Foxp3~+ regulatory T cells in the CD4+T cells obviously reduced in the EAE mice(P < 0.05).There was no significant difference in the percentage of CD4~+CD25~+Foxp3~+ regulatory T cells between ruxolitinib-treated mice and normal mice.Ruxolitinib treatment significantlyincreased the proportion of CD4~+CD25~+Foxp3~+T cells,compared with that in the EAE mice.Conclusions:1The onset of EAE are associated with the release of inflammatory cytokines and activation of JAK/STAT pathway.And the proportion of regulatory T cells decreases in EAE mice.2 Ruxolitinib can alleviate the neurological dysfunction of EAE,and reduce the morbidity.3 Ruxolitinib notably decreases the levels of inflammatory cytokines in spleen and serum of EAE mice.Furthermore,the extent of reduction has a positive correlation to the drug dose.The expression of FoxP3+ can be elevated by the treatment that is beneficial to autoimmune tolerance.4 Ruxolitinib can increase the percentage of CD4~+CD25~+Foxp3~+T cells,which plays a protective role in disease development.5 Ruxolitinib can mitigate EAE via suppressing JAK/STAT pathway. |
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