The Prevention And Treatment Research Of Leflunomide On Experimental Allergic Encephalomyelitis

Objective: Observed leflunomide (LEF) for the preventionand treatment of experimental allergic encephalomyelitis (EAE). Explorethe immunological mechanisms of LEF on the preventive effect of EAE.Methods:60female Wistar rats were randomly divided into five groups:blank control group, the control group of EAE and small, medium, largedoses of LEF treatment group (n=12). The crude myelin basic protein(MBP) and complete Freund’s adjuvant (CFA) immunizing antigen,hypodermic production EAE model, the control group only injection ofthe same amount of complete Freund’s adjuvant. Self-modeling of threedays before, small, medium and large doses of LEF treatment group wereto be LEF0.5mg/kg/d the2mg/kg/d gavage8mg/kg/d, blank controlgroup EAE control group each saline2ml orally for10days. RecordWistar rats the incidence of the incubation period, the progress and thepeak incidence of neurological dysfunction score. When the peakincidence of rats were killed, not the disease and the control group weresacrificed after4weeks. Specimens from rat brain spinal cord, thymusand orbital venous blood. HE staining in the rat brain and spinal cordtissue processing, peripheral blood mononuclear cells (PBMC) secretionof IL-4and IFN-γ levels measured by enzyme-linked immunosorbent assay (ELISA), brain tissue was detected by radioimmunoassay cytokineIL-2, IL-6, TNF-α content, measured by flow cytometry of peripheralblood T cell subsets distribution and thymocyte apoptosis rate. Results:(1) pathogenesis of Wistar rats: control rats and disease EAE controlgroup, the incidence rate of100%; small, medium, large doses of LEFcontrol group part of the animal disease, incidence rates were83.33%,66.67%,58.33%. EAE control group, the incubation period of11.00±1.28days, small, medium, large doses of LEF treatment groupincubation period were17.10±0.88,20.63±1.06,22.71±0.95days, LEFtreatment group incubation period than the head of the EAE control(P<0.01), dose-dependent relationship is obvious. Advances in thepathogenesis of EAE control group of6.83±0.72days, a small incidenceof advanced, large doses of LEF treatment group were5.20±0.79days,4.00±0.76days,3.00±0.82days, LEF prevention group the incidenceadvanced than EAE be shortened (P<0.01), dose-dependent manner. EAEcontrol group, the peak incidence of disease score was4.00±1.54points,small, medium, large doses of LEF treatment group peak incidence ofneurological dysfunction score of2.67±1.83,1.83±1.80points,1.17±1.34,large, medium dose of LEF treatment group compared with the EAEcontrol score (P<0.01), a small dose of LEF control group with EAEcontrol group, the difference was not statistically significant (P>0.05);peak incidence of neurological dysfunction score large doses of LEF treatment group The peak incidence of disease score the smaller dosesLEF control group, the difference was statistically significant (P<0.05), indoses of LEF treatment group peak incidence of disease score smallerdoses LEF control group, the difference was not statistically significant(P>0.05); LEF control group of high-dose peak incidence of neurologicaldysfunction score low of LEF treatment group compared to the middledose, but the difference was not statistically significant (P>0.05).(2)EAE control group and the small, medium, large doses of LEF treatmentgroup Wistar rat brain and spinal cord pathological changes: blank controlgroup, no abnormal brain tissue in rats. EAE control group and LEFcontrol group rat brain parenchymal perivascular inflammatory cellinfiltration, which lymphocytic infiltration accompanied by a smallnumber of mononuclear cells, typically form a "cuff kind of change. LEFtreatment group inflammatory cell infiltration severity of EAE comparedto the control group pathological changes reduce the dose the greater thedegree of reduction is more obvious.(3) LEF EAE rats peak incidence ofbrain tissue IL-2, IL-6, TNF-α content: the incidence of EAE controlgroup the peak of brain tissue in IL-2, IL-6, TNF-α content than thecontrol group was significantly higher (P<0.01), LEF treatment grouppeak incidence of brain tissue IL-2, IL-6, TNF-α content than EAEcontrol group was significantly lower (P<0.01), large, medium dose LEFprevention and treatment of rats peak incidence of brain tissue IL-2, IL-6, TNF-α content in smaller doses LEF treatment group group reduced(P<0.01).(4) EAE control group and the peak incidence of LEF controlrats PBMC secretion of IL-4and INF-γ capacity and the ratio of: EAEcontrol rats peak incidence of PBMC secretion of IL-4capability,IL-4/INF-γ value than the control group (P<0.01) reduction in enhancedsecretion of INF-γ capacity than the control group (P<0.01); LEFtreatment group PBMC secretion of IL-4capability, IL-4/INF-γ. EAEcontrol group was significantly higher (P<0.01), secretion of INF-γability than EAE control group was significantly lower (P<0.01)., Indose LEF control rats peak incidence of PBMC ability to secrete IL-4, theIL-4/INF-γ value smaller doses LEF treatment group was significantlyhigher (P<0.01or P<0.05), secretion of INF-γ ability smaller doses ofLEF treatment group (P<0.01) lower. Large doses of LEF control ratspeak incidence of PBMC ability to secrete IL-4, IL-4/INF-γ valuecompared to the dose LEF group was significantly higher (P<0.01),reduced secretion of INF-γ ability LEF treatment group compared to themiddle dose (P<0.01).(5) EAE control group and LEF each preventiongroup the onset of the peak of T cell sub-group of the distribution change:EAE group incidence peak of peripheral blood T cell sub-group of CD4+,CD8+distribution ratio than the control group was significantly reduced(P<0.01), CD4+/CD8+value was significantly increased (P<0.01); LEFeach prevention group the onset of the peak period of peripheral blood CD4+, CD8+distribution ratio compared with EAE control group wassignificantly increased (P <0.01), CD4+/CD8+value was significantlyreduced (P<0.01); big, middle dose LEF prevention group rats incidencepeak of peripheral blood CD4+, CD8+distribution ratio smaller dosesLEF group was significantly increased (P<0.01), CD4+/CD8+value wassignificantly reduced (P<0.01), high-dose LEF prevention group Thepeak incidence of peripheral blood CD4+, CD8+distribution ratiocompared to the dose LEF group was significantly higher (P<0.01),CD4+/CD8+value was not changed significantly (P>0.05).(6) EAEcontrol group and LEF treatment group peak incidence of thymocyteapoptosis rate of change: EAE control group thymocyte apoptosis ratewas significantly higher than normal control group (P<0.01), LEFtreatment group peak incidence of thymocytes apoptosis rate than EAEcontrol group (P<0.01) lower;, in doses of LEF treatment groupthymocyte apoptosis rate of the smaller dose LEF control group (P<0.01),high-dose of LEF treatment group thymus cell apoptosis rate. The middledose LEF control group (P<0.01).(7) correlation analysis: EAE controlgroup and LEF each prevention group incidence incubation period withthe onset of the peak of IL-2, IL-6, TNF-α, CD4+/CD8+and thymocyteapoptosis was negative related (P<0.01or P<0.05), with IL-4/INF-γvalue was positively correlated (P<0.01or P<0.05); EAE control groupand the LEF prevention group the incidence of progress and the peak incidence of IL-2, IL-6, TNF-α, CD4+/CD8+thymocyte apoptosis waspositively correlated (P<0.01or P<0.05), with IL-4/INF-γ value wasnegatively correlated (P<0.01or P<0.05); EAE control group and LEFtreatment group incidence peak of disease score and incidence peak ofIL-2, IL-6, TNF-α, CD4+/CD8+and thymocyte apoptosis was positiverelated (P<0.01or P<0.05), and IL-4/INF-γ value was negativelycorrelated (P<0.01or P<0.05). Conclusion:1. Wistar rats by injection ofguinea pig spinal cord homogenate successfully produced EAE model.2.EAE rats the peak of brain tissue inflammatory cytokines increasedimbalance of Th1/Th2ratio of Th0differentiation to Th1drift, abnormaldistribution of T lymphocyte subsets, abnormal increase thymocyteapoptosis.3.LEF can reduce the incidence of EAE rats, prolong theincubation period, shorten the period of progress, while reducing the peakof clinical symptoms, reduce encephalomyelitis disease cell infiltration,LEF has a preventive effect on the incidence of EAE rats.4.LEF byinhibiting the inflammatory cytokines of the EAE correct Th1/Th2imbalance, regulating T lymphocyte subsets anomaly, inhibition of theEAE rat thymus cells apoptosis play a role in the prevention andtreatment of EAE.