The Prevention And Treatment Research Of Tofacitinib On Experimental

Objective To investigate the effects of tofacitinib on experimental autoimmune encephalomyelitis(EAE)rats and its possible immunological mechanisms Methods Total fifty female Wistar rats were assigned into normal control group,EAE control group,low-dose,median-dose and high-dose tofacitinib group(10 for each)randomly.The rats were given subcutaneous injection of the myelin basic protein(MBP)of guinea pigs spinal cord emulsified with complete Freund's adjuvant(CFA)to induce EAE model.The low-dose,median-dose and high-dose tofacitinib group rats were given by gavage with tofacitinib 1mg,2mg and 4mg /(kg·d)for 10 days from three days before.The normal and EAE control groups were given with the same volume of saline.Afterward,rats were executed at the peak of onset(clinical score was not exacerbation for 3 days,all limbs paralyzed or died),whereas rats free of disease were executed after 8 weeks from the beginning of experiment.The incubation period,progressive stage and neurological dysfunction score(NDS)at the peak of onset were recorded.The pathologic changes of rat brain tissue were observed.By immunohistochemistry,using anti-glial fibrillary acidic protein(GFAP)antibody to detect the active astrocytes in each group and calculated anti-GFAP average optical density values(IOD)for each rats;using anti-myelin myelin protein(MBP)antibody to stain demyelination of braintissue,and calculated the IOD of anti-MBP.The proportions of Tfh cells(CD4+CXCR5+ICOS+T cells)and Tfr cells(CD4+Foxp3+CXCR5+ICOS+T cells)in spleen was detected by flow cytometry.The levels of CXCL13 and TGF-?1 in the cerebral homogenates and the levels of IL-10 and IL-21 in serum were evaluated by Enzyme-Linked Immunosorbent Assay(ELISA).Results In each tofacitinib group,the incubation period extended,progressive stage shortened and neurological dysfunction score(NDS)reduced at the peak of onset.The proportion of Tfh cells and the level of CXCL13 were significantly decreased,the proportion of Tfr cells,the ratio of Tfr/Tfh and the level of TGF-?1 were significantly increased compared with EAE control group(P<0.01,P<0.05).(1)Clinical manifestation:None of rats in the normal control group exhibited symptoms;however,all rats in the EAE control group exhibited symptoms,and some rats in the tofacitinib groups exhibited symptoms.The morbidity of low-dose,median-dose and high-dose tofacitinib group were 90%?60% ? 40% respectively.The sick rats started to exhibit energy loss,weight reduction,low activity,and loss of appetite from days 10 to 12 after modeling in most situations.Conditions of rats worsened gradually and reached the peak of severity from days 15 to 20 after modeling,as characterized by emaciation and weakness,powerlessness in single hind legs or bilateral hind limbs,dragging of limbs across the ground,ataxia,paralysis,and even death.? The incubation period:In contrast with the EAE group,the incubation periods of all tofacitinib groups were extended,the higher the dose,the more obvious the extension of incubation period,and incubation period of the high dose tofacitinib group wasthe longest.The difference between the high dose tofacitinib group and the low dose tofacitinib group was statistically significant(P<0.01).The difference between the median dose tofacitinib group and the low dose tofacitinib group was statistically significant(P<0.01).The difference between the high dose tofacitinib group and the median dose tofacitinib group was statistically significant(P<0.01).? The progressive stage : compared with EAE control group,the progressive stages of all tofacitinib groups were shortened.Compared with the low-dose tofacitinib group,the progressive stages of median-dose and high-dose tofacitinib groups were shortened(P<0.01),the progressive stagesof high-dose tofacitinib group were shortened when compared with the median-dose tofacitinib group,with significant difference(P<0.01).?The neurological dysfunction score:compared with EAE control group,the neurological dysfunction scores at the onset of the peak were shortened(P<0.01).Compared with the low-dose tofacitinib group,the neurological dysfunction scores of median-dose and high-dose tofacitinib groups were shortened(P<0.01,P<0.05),the neurological dysfunction scores of high-dose tofacitinib group were shortened compared with the median-dose tofacitinib group(P > 0.05).(2)Pathologic changes of rat brain tissue in all groups:compared with EAE control group,the pathologic scores of each tofacitinib group were decreased,with significant difference(P<0.01).the inflammatory cell infiltration of the high dose tofacitinib group decreased the most obviously,with significant difference among each group(P<0.01,P<0.05)).(3)The immunohistochemiscal results in brain tissue of each group rats at the peak incidence:(1)astrocytesactivation:normal control group has no activated astrocytes;the activation of astrocytes in EAE control group showed diffuse distribution.Compared with EAE group,the anti-GFAP positive cells average optical density(IOD)in brain tissue of different doses of tofacitinib treated group were lower than those in EAE group(P<0.01).Compared with low-dose tofacitinib group,the mean optical density(IOD)value of anti-GFAP positive cells significantly reduced in brain tissue of high-dose and median-dose tofacitinib group(P<0.01).Compared with median-dose tofacitinib group,the mean optical density(IOD)value of anti-GFAP positive cells significantly reduced in brain tissue of high-dose tofacitinib group(P<0.01).(2)Demyelination changes: Myelin intact in normal control group,while the positive expression of MBP average optical density(IOD)significantly decreased in EAE control group and each tofacitinib group compared with normal control group(P<0.01).Compared with low-dose tofacitinib group,the average optical density of MBP positive expression in median-dose and high-dose tofacitinib groups increased significantly(P<0.01).Compared with median-dose tofacitinib group,the average optical density of MBP positive expression significantly increased in high-dose tofacitinib group(P<0.01).(4)The proportions of Tfh cells and Tfr cells,and the ratio of Tfr/Tfh from spleen in all groups:(1)The proportions of Tfh cells:Compared with the normal control group,the proportion of Tfh cells from rat spleens increased significantly in the EAE control group(P<0.01),while the proportion of Tfh cells decreased significantly in each tofacitinib group compared with that of EAE control group.Compared with low-dose tofacitinibgroup,the proportion of Tfh cells decreased significantly in high-dose and median-dose tofacitinib group(P<0.01,P<0.05).Compared with median-dose tofacitinib group,the proportion of Tfh cells decreased significantly in high-dose tofacitinib group(P<0.01,P<0.05).(2)The proportions of Tfr cells:Compared with the normal control group,the proportions of Tfr cells decreased in the EAE control group at the peak of onset(P<0.01,P<0.05),while the proportion of Tfr cells increased significantly in each tofacitinib group compared with that of EAE control group(P<0.01,P<0.05).Compared with low-dose tofacitinib group,the proportion of Tfr cells increased significantly in high-dose and median-dose tofacitinib group(P<0.01).Compared with median-dose tofacitinib group,the proportion of Tfr cells increased significantly in high-dose tofacitinib group(P<0.05).(5)The ratio of Tfr/Tfh:Compared with the normal control group,the ratio of Tfr/Tfh decreased significantly in EAE control group(P<0.05),while the ratio of Tfr/Tfh increased significantly in each tofacitinib group compared with that of EAE controlgroup(P<0.05).Compared with low-dose tofacitinib group,the ratio of Tfr/Tfh increased significantly in high-dose and median-dose tofacitinib group(P<0.05).Compared with median-dose tofacitinib group,the ratio of Tfr/Tfh increased significantly in high-dose tofacitinib group(P<0.05).(6)Levels of CXCL13 and TGF-?1 in cerebral homogenate in all groups: Compared with the normal control group,the level of CXCL13 in the cerebral homogenates of EAE control group and each tofacitinib group rats increased significantly(P<0.01).Compared with EAE control group,the level of CXCL13 in the cerebral homogenates in each tofacitinib groupdecreased significantly(P<0.01,P<0.05).Compared with low-dose tofacitinib group,the level of CXCL13 decreased significantly in median-dose and high-dose tofacitinib group.(P<0.01,P<0.05).Compared with median-dose tofacitinib group,the level of CXCL13 decreased significantly in high-dose tofacitinib group(P<0.05).Compared with the normal control group,the level of TGF-?1in the cerebral homogenates of EAE control group and each tofacitinib group rats decreased significantly(P<0.01).Compared with EAE control group,the level of TGF-?1 in the cerebral homogenates in each tofacitinib group increased significantly(P<0.01,P<0.05).Compared with low-dose tofacitinib group,the level of TGF-?1 increased significantly in median-dose and high-dose tofacitinib group(P<0.01).Compared with median-dose tofacitinib group,the level of TGF-?1 increased significantly in high-dose tofacitinib group(P<0.01).(7)Levels of IL-21 and IL-10 in serum in all groups: Compared with the normal control group,the level of IL-21 in the serum of EAE control group and each tofacitinib group rats increased significantly(P<0.01).Compared with EAE control group,the level of IL-21 in the serum in each tofacitinib group decreased significantly(P<0.01).Compared with low-dose tofacitinib group,the level of IL-21 decreased significantly in median-dose and high-dose tofacitinib group.(P<0.01).Compared with median-dose tofacitinib group,the level of IL-21 decreased significantly in high-dose tofacitinib group(P<0.01).Compared with the normal control group,the level of IL-10 in the serum of EAE control group and each tofacitinib group rats decreased significantly(P<0.01).Compared with EAE control group,the levelof IL-10 in the serum of each tofacitinib group increased significantly(P<0.01).Compared with low-dose tofacitinib group,the level of IL-10 increased significantly in median-dose and high-dose tofacitinib group(P<0.01).Compared with median-dose tofacitinib group,the level of IL-10 increased significantly in high-dose tofacitinib group(P<0.01).(8)Correlation analysis:(1)The incubation period in EAE control group and each dose tofacitinib group,were significantly positive correlated with the proportions of Tfr cells,the ratio of Tfr/Tfh from spleen,the level of TGF-?1 in the cerebral homogenates and the level of IL-10 in serum(P<0.01),while significantly negative correlated with the proportions of Tfh cells from spleen,the level of CXCL13 in the cerebral homogenates and the level of IL-21 in serum(P<0.01).(2)The progressive stage in EAE control group and each dose tofacitinib group,were significantly positive correlated with the proportions of Tfh cells from spleen,the level of CXCL13 in the cerebral homogenates and the level of IL-21 in serum(P<0.01),while significantly negative correlated with the proportions of Tfr cells,the ratio of Tfr/Tfh from spleen,the level of TGF-?1 in the cerebral homogenates and the level of IL-10 in serum(P<0.01).(3)The neurological dysfunction score at the peak of onset were significantly positive correlated with the proportions of Tfh cells from spleen,the level of CXCL13 in the cerebral homogenates and the level of IL-21 in serum(P<0.01),while significantly negative correlated with the proportions of Tfr cells,the ratio of Tfr/Tfh from spleen,the level of TGF-?1 in the cerebral homogenates and the level of IL-10 in serum(P<0.01,P<0.05).Conclusion Tofacitinib may have dampening effect on the severity andprogression of EAE in a dose-dependent manner.This inhibiting effect of EAE may be associated with the down-regulation of the proportion of Tfh cells and expression of CXCL-13,the up-regulation of the proportion of Tfr cells and expression of TGF-b 1.Tofacitinib may modulate the balance of Tfr/Tfh and induce the balance to shift to Tfr.