| To investigate rituximab(RTX) on experimental allergicencephalomyelitis(EAE) in rats the incidence of central nervous systempathological changes in peripheral blood B-cell levels and brain tissue ofinterleukin-4(IL-4)、interleukin-10(IL-10)、interleukin-17(IL-17)、gamma-interfeon(IFN-gamma) content and serum myelin basic protein(MBP), myelinbasic protein antibody(Anti-MBP) in the content of the impact of changes toexplore the RTX treatment of EAE and its possible immunological mechanismof RTX treatment of multiple sclerosis(MS) to find the adequate theoreticalbasis. Method:1. Grouping, modeling and administration: The crude myelinbasic protein(CMBP) and complete Freud’s adjuvant(CFA) subcutaneousinjection of established EAE model of EAE model is divided into RTX high,medium, low-dose treatment group and EAE control group, n=10; normalcontrol group of10EAE model group injected with saline and the CFA. Fromthe date of the modeling, administration, RTX, low-dose treatment byintraperitoneal injection of RTX, dose, respectively100mg/kg/d,50mg/kg/d,25mg/kg/d, continuous treatment for3days, the normal control group and EAEby intraperitoneal injection of normal saline2ml/d. In the EAE control group,RTX treatment group for3consecutive days symptom score no aggravating orlimbs, paralysis, incidence of rats were sacrificed as EAE peak incidence ofdeath and dying rats were sacrificed when there are heart rate, body temperature, the rest to8weeks the end of all death.2. Changes in the incidence of rats, theincubation period, the progress of, and the peak of neurological dysfunctionscores and mortality were observed.3. By light microscopy to observe thepathological changes of rat brain and spinal cord tissue.4. Flow cytometry wasused to count each group of rat peripheral blood B cell count.5． Byenzyme-linked immunosorbent assay(ELISA) to detect each rat brain tissuehomogenates of IL-4, IL-10, IL-17and IFN-γ content.6. Detected by ELISA inserum MBP and Anti-MBP levels.7. EAE group and RTX each treatmentgroup indicators, the correlation analysis.8. All data SPSS17.0software forstatistical analysis. Results:1. Rats the incidence of:(1) Clinical symptoms:normal control rats, no abnormal; of EAE in the control group and RTXtreatment group, all rats were incidence, incubation period is10-30days, theincidence of rat has to eat to reduce weight loss, the dispirited, hind limbweakness, self-injury hind legs, hind legs completely unable to performance,severe cases, quadriplegia, convulsions and even death, clinical symptoms ofRTX in each treatment group were significantly reduced EAE control group, inwhich high-dose group reduce the most obvious.(2) latency change: of RTXincubation period of the treatment groups than extend the EAE controlgroup(P<0.01); of RTX high-dose group extended incubation period comparedwith the the RTX low dose group(P<0.01) of RTX high-dose treatment grouplatency extend than RTX in the dose group(P<0.01).(3) the progress of change:of RTX treatment group advanced the EAE control group was significantly decreased(P<0.01, P<0.05); of RTX high-dose treatment group progressshortened compared with the RTX low-dose group(P<0.01P<0.05) of RTXhigh-dose treatment group advanced reduced compared with RTX dosetreatment group(P<0.01).(4) changes in the peak incidence of neurologicaldysfunction score: RTX treatment group peak incidence of neurologicaldysfunction score compared with EAE was significantly decreased(P<0.01,P<0.05); of RTX high-dose treatment group peak incidence of neuraldysfunction score lower than the the RTX low-dose treatment group(P<0.01,P<0.05), RTX high-dose treatment group, peak incidence of neurologicaldysfunction score lower than the RTX dose treatment group(P<0.05).(5)mortality rate: high dose of RTX treatment group mortality than EAE controlgroup decreased significantly, the difference was statistically significant(P<0.05); of RTX low-dose group mortality than EAE control group, but thedifference was not statistically significance(P>0.05), and RTX in mortalitybetween low-dose treatment group showed no statistical difference (P>0.05).(2)changes of the brain and spinal cord pathology: normal control group of ratbrain and spinal cord pathological changes; brain and spinal cord of EAEcontrol group and RTX treated rats EAE typical pathological changes, smallperivascular inflammatory cell infiltration was the change in the "sleeve-likeperivascular demyelination of white matter change; of RTX each treatmentgroup demyelination and inflammatory cell infiltration compared with the EAEcontrol group to reduce; of RTX high-dose treatment group compared the low dose of RTX treatment reduce the group, the pathological changes in thelightest of RTX high-dose group. Rat brain and spinal cord HE staining,inflammatory cell infiltration score: of EAE control group and RTX treatmentgroup in the rat brain tissue, inflammatory cell infiltration score compared withnormal control group was significantly increased(P<0.01); of RTX treatmentgroup, inflammatory cell infiltration score compared with the EAE controlgroup decreased(P<0.01); of RTX high-dose treatment group brain tissueinflammatory cell infiltration score in the low the RTX low-dose treatmentgroup(P<0.01, P<0.05) of RTX high-dose group of brain tissue inflammatorycell infiltration score in RTX-dose treatment group(P<0.05).3rats in eachgroup the number of peripheral blood B cells change: of EAE in the controlgroup, and RTX low dose treatment group rat peripheral blood B cells thannormal controls were significantly higher(P<0.01, P<0.05); of RTX high dosetreatment of rats peripheral blood B cells than in normal control group wassignificantly lower(P<0.01); of RTX in all treatment groups in rat peripheralblood B cells were lower than the EAE control group(P<0.01, P<0.05); of RTXhigh in dose group rat peripheral B cells compared with the the RTX low-dosetreatment group(P<0.01) of RTX high-dose treatment group were peripheralblood B cells compared with RTX dose group(P<0.01).(4) brain tissuehomogenates of IL-4, IL-10, IL-17, IFN-gamma content of change: of EAEcontrol group of RTX treatment group rat brain tissue homogenates of IL-4,IL-10levels compared with normal control group was significantly lower(P <0.01); of RTX in all treatment groups in brain tissue homogenates of IL-4,IL-10levels were significantly higher than those in the EAE control group(P<0.01); of RTX high-, medium-dose treatment brain tissue homogenates of IL-4,IL-10concentration was significantly higher than the RTX low-dose treatmentgroup(P<0.01); of RTX high-dose treatment group, brain homogenates of IL-4,IL-10levels in dose of RTX in the treatment group(P<0.01, P<0.05); of EAEcontrol group of RTX treatment group rat brain homogenates, IL-17, IFN-γcontent compared with the normal control group was significantly increased(P<0.01); of RTX in all treatment groups in brain tissue homogenates of IL-17,IFN-gamma levels were significantly lower than the EAE control group(P<0.01, P<0.05); of RTX high-dose treatment group brain homogenates of IL-17,IFN-γ content of less than RTX low-dose treatment group(P<0.01); of RTXhigh-dose treatment group, brain homogenates of IL-17, IFN-gamma dosegroup content is lower than in the RTX(P<0.01, P<0.05). Content changes inserum MBP and Anti-MBP: of EAE in the control group, RTX treatment group,serum levels of MBP and the Anti-MBP levels compared with the normalcontrol group was significantly higher(P<0.01); RTX treatment group, serumMBP and Anti-MBP content was significantly lower than that of the EAEcontrol group(P<0.01); of RTX high-dose treatment serum MBP and Anti-MBPlevels below the the RTX low-dose treatment group(P<0.01); of RTXhigh-dose treatment group, serum MBP and Anti-MBP levels lower than thedose of RTX in the treatment group(P<0.01).6. Correlation analysis:①of EAE in the control group and each treatment group in RTX rats peripheralblood B cell count, brain homogenates of IL-17, IFN-gamma content and MBPand Anti-MBP in the serum content of the incubation period was negativelycorrelated(P<0.01); of EAE control group and RTX treatment group rat brainhomogenates, IL-4, IL-10content of the incubation period was significantlycorrelated(P<0.01).②of EAE in the control group and each treatment group inRTX in rat peripheral blood B cells, the number of brain homogenates of IL-17content and serum IFN-γ in MBP and Anti-MBP content and progress ofsignificant positive correlation(P<0.01, P<0.05); of EAE control group andeach treatment group in RTX in rat brain tissue homogenate IL-4, IL-10levelsin advanced stage was a significant negative correlation(P<0.01).③of EAE inthe control group and each treatment group in RTX rats peripheral blood Bcells, brain tissue homogenates of IL-17, IFN-gamma content and serum MBPand Anti-MBP content with the peak of the nerve dysfunction score waspositively(P<0.05, P<0.01); of EAE control group and each treatment group inRTX in rat brain homogenates of IL-4, IL-10content and the peak of the nervedysfunction score was negatively correlated(P<0.01).(4) of EAE control groupand the treatment groups in RTX rat peripheral blood B cell count, brainhomogenates of IL-17, IFN-gamma content and serum MBP and Anti-MBPlevels and inflammatory cell infiltration score was significantly positively(P<0.05, P<0.01); of EAE control group and each treatment group in RTX in ratbrain homogenates of IL-4, IL-10levels and inflammatory cell infiltration score score was negatively correlated(P<0.01). Conclusion:1. The Wistar ratsinduced by multi-point the subcutaneous WSCH+CFA to establish EAEmodel, the clinical symptoms of EAE rat brain tissue pathological changetypical, indicating that the EAE’s model is success, the model is stable andreliable.2.B cells and MBP、Anti-MBP to promote the role of EAE occurred;its mechanism of action may be: B cells present antigen and produce antibodiesthat presenting MBP and produce anti-MBP to promote the occurrence of EAE.3. IL-4, IL-10can inhibit the occurrence of EAE development; IL-17and IFN-γon EAE onset catalytic role.4. RTX can be extended to EAE rats incubationperiod and reduce the incidence of advanced、 reduce the EAE neurologicaldisorders and reduce the mortality of EAE animals and reduce the EAE ratbrain pathology of the preventive effect of EAE onset．5RTX may reduce thenumber of EAE rat peripheral blood B cells, peripheral blood B cell countdepletion, B cell antigen-presenting and produce antibodies weakened, and thusplay the role of prevention and control of EAE.6. RTX can inhibit the synthesisof IL-17and IFN-γ by promoting the secretion of cytokines IL-4and IL-10, toplay the role of prevention and treatment of EAE. |
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