| Objective: To investigate the therapeutic effect of JAK2 inhibitor to MS and the function to Th17/Treg balance pathway.Method:60 Female C57BL/6 mice aged weight 18 to 22g,6-8 weeks,were di-vided into three groups(EAE,AG490干预and Control)randomly.C57BL/6 mice were immunized via 200ul phosphate buffered solution(PBS)involving 300ug synthetic MOG35-55 polypeptide,then completely mixed with an equal amount of complete Freund’s adjuvant(CFA)containing 4 mg/ml BCG to oil-in-water emulsion,which made complete antigen,subcutaneous injected to bilateral lateral abdominal wall 4:00,to induce acute EAE.EAE and EAE+AG490 groups were in after immunized,0 and 48 h after intraperitoneal in-jection of 500 ng pertussis toxin,to establish a EAE model.Blank control group,directly to 200ul PBS emulsified with an equivalent CFA immunized animal.EAE+AG490 group immunized with,in the first 3,5,7,9,11,13,15,17,19 day,AG490 1mg/DMSO 100ul subcutaneous injection until death.The blank control group and the EAE group were correspondingly given saline 0.1 ml/d,mice were injected subcutaneously.Observe the impact ion of drugs on an-imals,clinical manifestations.group of 13 days after immunization were ran-domly drawn,denoted as before the onset group,and recorded aniamals of af-ter immunization about 20 days as peak group,what were randomly drawn.Each group of mice were sacrificed at different periods after spinal HE staining inflammatory cell infiltration,immunohistochemical staining ob-served simultaneously IL-17,Foxp3+,p-STAT5 positive cells and counted,for comparison.SPSS13.0 statistical software for statistical analysis,measurement data are presented as mean±standard deviation(±S)The date were statistical compared via one-way ANOVA after rank case when non-normality(P<0.05).SNK was used for the comparisons between groups,P<0.05,the difference was statistically significant.Results: 1 Histopathology of different groups in spinal cords.(1) There were no “blood vessel muffs”an sparse inflammatory cells on sections of control group of different times(2) 13 days after onset There were diffuse inflammatory cells infiltrated and mass “blood vessel muffs” formed on sections of EAE group.There were rare “blood vessel muffs”and less inflammatory cells were founded on sec-tions of EAE +AG490 group.(3) 20 days after onset There were more inflammatory cells infiltrated and mass “blood vessel muffs” formed on sections of EAE group and EAE +AG490 group than sections of 13 days after onset.There were less inflam-matory cells and “blood vessel muffs”on sections of EAE +AG490 group comparing sections of EAE group. 2 Immunohistochemistry of different groups in spinal cords. 2.1(1)IL-17,secreted by CD4+Th17 cells,was extensively related to autoim-mune disease and multiple sclerosis.It was primarily immunopositive in cyto-plasm.(2) Foxp3 was characteristic transcription factor of CD4+CD25+Treg cell what was therapeutic to MS by inhibiting inflammation. It was mainly immunopositive in cytoplasm.(3)P-STAT5 was phosphorylated STAT-5 which is one of STATs fami-ly.JAK2-STAT5 signing pathway regulate the development of CD4+CD25+Treg cells.P-STAT5 was principally immunopositive in cytoplasm and nuclei. 2.2.1 13 day after onset(1)IL-17 The mean number of EAE group immunopositive cells(44.89±10.43) significantly more than EAE +AG490 group’s(16.00±6.80)(P<0.05).Both the mean immunopositive cells mean number of EAE group and EAE +AG490 group were significantly more than control group’s(8.22±3.00)(P<0.05).(2)Foxp3+ The mean number of EAE group immunopositive cells(32.67±11.12) significantly less than EAE +AG490 group’s(61.00±11.54)(P<0.05).Both the mean immunopositive cells mean number of EAE group and EAE +AG490 group were significantly more than control group’s(7.56±3.68)(P<0.05).(3)p-STAT5 The mean number of EAE group immunopositive cells(28.56±12.80) significantly less than EAE +AG490 group’s(64.89±23.28)(P<0.05).Both the mean immunopositive cells mean number of EAE group and EAE +AG490 group were significantly more than control group’s(11.22±6.10)(P<0.05). 2.2.2 20 day after onset(1)IL-17 The mean number of EAE group immunopositive cells(57.33±13.36) significantly more than EAE +AG490 group’s(20.56±5.45)(P<0.05).Both the mean immunopositive cells mean number of EAE group and EAE +AG490 group were significantly more than control group’s(8.22±3.00)(P<0.05).(2)Foxp3+ The mean number of EAE group immunopositive cells(22.67±6.89) significantly less than EAE +AG490 group’s(61.00±11.54)(P<0.05).Both the mean immunopositive cells mean number of EAE group and EAE +AG490 group were significantly more than control group’s(7.56±3.68)(P<0.05).(3)p-STAT5 The mean number of EAE group immunopositive cells(17.56±7.84) significantly less than EAE +AG490 group’s(43.44±8.14)(P<0.05).Both the mean immunopositive cells mean number of EAE group and EAE +AG490 group were significantly more than control group’s(8.78±2.11)(P<0.05). 2.2.3 comparing between 13 day after onset and 20 day after onset within same group(1)IL-17 The mean number of 13 d immunopositive cells significantly less than 20 d group’s in EAE group.(P<0.05).There were not significantly difference between different times in EAE +AG490 and control group.(P>0.05).(2)Foxp3+ The mean number of 13 d immunopositive cells significantly more than 20 d group’s in EAE and EAE +AG490 group(P<0.05).There were not significantly difference between different times(P>0.05).(3)p-STAT5 The mean number of 13 d immunopositive cells signifi-cantly more than 20 d group’s in EAE and EAE +AG490 group(P<0.05).There were not significantly difference between different times(P>0.05).Conclusion:AG490 reduced the secretion of IL-17,while increased levels of Foxp3+ and p-STAT5,therefore AG490 and other JAK2 inhibition were therapeutic to EAE or MS. |
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