Mechanism Underlying Oridonin Protection Against Experimental Autoimmune Encephalomyelitis

Background and ObjectiveMultiple sclerosis is a chronic autoimmune disease characterized by inflammation,demyelination of central nervous system（CNS）.This disease is clinically manifested as dysfunction of motor neuron that is becoming one of the most prominent cause of neurologic disability among young adults.The most significant animal model of MS is experimental autoimmune encephalomyelitis（EAE）which shares with MS pathological features of inflammatory infiltration,and demyelination.It has been widely used for the study of MS and to evaluate the efficiency of potential therapeutic approaches for MS.The etiology and pathogenesis of MS have not been fully elucidated,but it is generally believed that immunoinflammatory reaction is an important pathological factor of MS.Nowadays many approved modifying agents for MS are largely based on immune regulation and inflammation suppression.These drugs can reduce disease activity,but are only suitable for certain type of MS.Chinese medicine has been used in China for a long history.Many drug molecules have immune regulation function and alleviate side effects of hormone and immunosuppressive agents bring.ORI,an active diterpenoid that was first extracted from Isodon species,has been shown to possess immunosuppressive and anti-inflammatory activities widely used in traditional Chinese medicine for anti-inflammation therapy.ORI has an efficient therapeutic effect on ulcerative colitis（UC）mice by inhibiting T lymphocyte immune response,but whether it can be used to treat EAE has not yet been reported.Organ-specific EAE shares with UC mice T cell meditated inflammatory reaction.Therefore,it is suggested that ORI may be efficacious in EAE.In this study,ORI was intraperitoneally（i.p.）administered on EAE mice to investigate its preventive and therapeutic effects in EAE and explore its possible molecular mechanism and provide a reliable theoretical basis for the treatment of multiple sclerosis.Method1 Induction of EAE miceThe animal model of EAE was established by immunization with myelin oligodendrocyte glycoprotein(MOG_35-55)in C57BL/6 mice.2 Animal group and administration of ORIEAE mice were randomly divided into three groups:EAE vehicle group（Vehicle,n=24）,ORI prevention group（n=24）and ORI treatment group（n=12）.ORI（15mg/kg）or PBS was i.p.injected staring from 1 day postimmunization for the prevention protocol,and continued every other day until 5 days postimmunization.ORI（15mg/kg）or PBS was i.p.injected staring from 12 days postimmunization for the treatment protocol,and continuing daily until 30 days postimmunization.3 EAE evaluationEAE mice were evaluated daily and scored for disease day onset and clinical signs.4 HistologySpinal cords were removed from EAE vehicle or ORI prevention group mice and stained with H&E to observe pathological changes by light microscopy.5 Isolation of infiltrating MNCs in spleen and CNSMononuclear cells（MNCs）were derived from spleen and spinal cord of EAE vehicle or ORI prevention group mice and then counted by light microscopy.6 Flow cytometryMNCs derived from spleen and spinal cord of EAE vehicle or ORI prevention group mice were stained to detect the percentages of CD4⁺T、CD8⁺T、CD19⁺、CD11b⁺and CD4⁺Foxp3⁺Treg cells by flow cytometry.MNCs derived from spleen and spinal cord of EAE vehicle or ORI prevention group mice were cultured in the presence of MOG peptide（20μg/mL）.Cells were stained to detect the percentages of Th1 and Th17 cells by flow cytometry.7 MOG peptide specific T cell proliferationFor ex vivo study,splenocytes were derived from EAE vehicle or ORI prevention group mice and cultured in the presence of MOG peptide at the indicated concentrations.For in vitro study,splenocytes derived from vehicle-treatment mice were cultured in the presence or absence of MOG peptide（20μg/mL）with a series of concentrations of ORI.Cells were labeled with CFSE and then stained with fluorochrome-conjugated antibodies to CD4 and analyzed by Flow cytometry.8 Cytokines assaySplenocytes derived from EAE vehicle or ORI prevention group mice were cultured in the presence of MOG peptide（20μg/mL）.Supernatants were harvested at48h to measure IFN-γ,IL-17,IL-4 and IL-10 level using ELISA assay kit.9 Westernblot1.Splenocytes from EAE vehicle or ORI prevention group mice were cultured for 24h in the presence of MOG peptide（20μg/mL）.2.Splenocytes from vehicle-treatment mice were cultured for 4h in the presence or absence of Oridonin（5μM）stimulated with or without MOG peptide（20μg/mL）.Cells were lysed and total protein was extracted.Level of relative protein were detected by westernblot.10 Statistical analysisStatistical analysis was performed using GraphPad Prism5.0 software.Clinical scores and percentages of lymphocyte were expressed as mean±SD.The differences among the results of the different groups were evaluated by one way analysis of variance and then analyzed by the student t test.A value of P<0.05 was considered to represent statistical significance.Result1.ORI efficiently alleviates symptoms of EAE mice:EAE mice were successfully induced.Mice with vehicle-treatment demonstrated clinical symptoms from 8⁹ days postimmunization.The initial clinical sign was tail tonus weakness that progressed to paralysis of the tail,followed by a progression up the body to affect the hind limbs and even the forelimbs and head.Intraperitoneally injection of ORI in mice staring from 1 day postimmunization significantly prolonged disease onset and relieved the clinical symptoms.ORI treatment staring from 12 days postimmunization effectively reduced the severity of EAE mice compared with vehicle-treated EAE.2.Histopathological changes:H&E staining of spinal cord tissue sections showed a large number of inflammatory cells infiltration in CNS of vehicle-treated EAE,and ORI application in the prevention protocol significantly reduced CNS inflammatory cells infiltration.3.Infiltrating MNCs in CNS:Infiltrating mononuclear cells of CNS were counted by light microscope.ORI application in the prevention protocol reduced the absolute number of MNCs of CNS.4.Cell population changes:Cell surface staining showed that the percentages of CD4⁺T cells derived from spleen and CNS were largely reduced.However,ORI application in the prevention protocol didn’t have a big influence on the percentages of CD11b⁺,CD8⁺and CD19⁺cells.To figure out which subsets were affected among CD4⁺T cells,cells were intracellular stained and results showed that ORI application in the prevention protocol markedly decreased the percentages of MOG-specific Th1and Th17 cells in spleen and CNS compared with those of vehicle-treated.However,the percentages of CD4⁺Foxp3⁺cells were not greatly altered by ORI administration.5.MOG-specific T cell proliferation:Flow cytometry analysis showed that ORI administration ex vivo or in vitro markedly decreased the percentages of MOG peptide induced proliferation of T cells in CD4⁺T cells,indicating that ORI administration significantly inhibited the proliferation of MOG-reactive encephalitogenic T cells.6.Cytokines production:Cytokines secreted by encephalitogenic T cells in response to MOG peptide challenge were detected and the results showed that ORI application in the prevention protocol efficiently decreased the production of pro-inflammatory cytokines characteristic of EAE（e.g.,IFN-γand IL-17）but did not have a marked regulation on the secretion of anti-inflammatory cytokines associated with EAE such as IL-4 and IL-10.7.Expression level of transcription factors of inflammatory signaling pathways:Westernblot analysis showed that ORI administration ex vivo or in vitro increased expression level of IκBα,and decreased expression level of p-IκBα,p-ERK,p-JNK,p-c-jun and c-fos compared with those of vehicle-treated mice.Conclusion1.ORI exerts efficiently preventive and therapeutic effects on EAE.2.ORI-presented protective effects on EAE may be associated with inhibition of proliferation of MOG-reactive T cells and differentiation of Th1 and Th17 cells.3.ORI has anti-inflammatory properties and exerts efficaciously preventive and therapeutic effects on EAE by suppressing Th1 and Th17 cells via targeting NF-κB and ERK/c-fos、JNK/c-jun signaling pathways.