An Experimental Study Of Role Of T Helper Cell Subsets On Early Optic Neuritis In Experimental Autoimmune Encephalomyelitis Mouse And Effects Of β-elemene

IntorductionOptic neuritis(ON) is one of the most common clinical neuro-ophthalmology diseases. It is an acute or subacute inflammation involving the optic nerve. The most common type is primary or idiopathic demyelinated optic neuritis (IDON), which is closely related to the incidence of Multiple Sclerosis (MS). Both have a common pathological changes. Epidemiological studies show that there is evidence of ON in 70% of MS cases; 10 to 20% of MS cases first present with ON; The incidence of both Idiopathic Demyelinated Optic Neuritis and MS showed an upward trend year after year. However, the treatment of the status quo is not optimistic. EAE is the ideal animal model for researching the MS pathogenesis. P-elemene developed by our country with the role of immunoregulation is the non-cytotoxic antineoplastic agents in the second category. Clinical study found that elmene have exact efficacy to tumor, and easily pass through the blood brain barrier. In view of the pathological mechanisms of ON, It may be involved similarly with the immune regulation of tumor. Therefore, this study through the MOG EAE mouse model to explore the ON incidence of neuroimmunologic mechanism and the neuro-protective mechanism ofβ-elemene, which provide new methods and experimental evidence for basic research and clinical treatment of optic neuritis.Part oneStudy of early pathological changes and axonal injury of optic nerve in MOGEAE mouseObjectiveAdopting the experimental autoimmune encephalomyelitis(EAE) mouse model induced by recognized myelin oligodendrocyte glycoprotein peptide(MOG35-55), observing early pathological changes of the optic nerve in EAE mice, setting up the experimental platform for researching the demyelinated optic neuritis.MethodUsing the synthetic MOG(myelin oligodendrocyte glycoprotein)35-55 In the assist of pertussis toxin(PTX) and complete Freund's adjuvant(CFA), we immunize the female C57BL/6 mice to produce EAE model, and observe the model behavior s(body weight and clinical score), histology(optic nerve hematoxylin-eosin staining,electron microscope observation) and immunohistochemistry changes.Result1,Behavior change:about 14 days after immunization, mice are gradually emerging sport dysfunction, and score begin to rise. In the period of observation, mice body weight is upward in the general trend. When clinical symptoms obvious, the weight has a slight decline in volatility.2,Morphological changes:7,11,15,19 days after immunization, inflammatory response and axonal myelin injury of optic nerve is gradually appeared.3,Immunohistochemistry changes:7 days after immunization, the optic nerve axonal injury appeared(β-APP protein expression),11 days after immunization, the optic nerve myelin damage appeared(CNPase protein expression), in the peak of incidence, obvious axonal myelin damage appeared.Part twoThe time course of T helper cell subsets cytokine and their specific transcription factors in Optic nerve of MOGEAE mouse earlyObjectiveStudy the T helper cell subsets Thl, Th2, Treg, Th17 cytokine-specific transcription factors expression in the time of early incidence of demyelinated optic neuritis, In order to identify the immune response priming and sustain immune inflammatory mechanism of demyelinated optic neuritis in EAE mice.Method Though the immunohistochemistry,ELISA and the Real-time PCR in different points of time (7,11,15,19 days after immunization) to observe T helper cell subsets Thl, Th2, Treg, Th17 specific cytokines and their transcription factor's protein and mRNA expression.Result1,ELISA:the contents of IL-17 protein in the optic nerve are significant elevated at 11 days after immunization, and so are IFN-y protein contents at 19 days, the contents of IL4 protein in the optic nerve decline obviously at 19 days.2,Immunohistochemistry:the expression decline of FOXP3 appeared at 11 days after immunization,then keep the decline until 19days after immunization.3,Molecular Bilology:The mRNA expression of IL-17, IFN-y and the transcription factor RORyt, T-beta have the consistent with their protein expression. FOXP3 mRNA transcription decline at 11 days after immunization, GATA3 mRNA transcription decline at 19 days after immunization.Part threeThe neuroprotective effect and intervention target ofβ-elemene on early optic neuritis in MOGEAE mice.ObjectiveStudy the neuroprotective effect ofβ-elemene on optic neuritis axonal injury of MOGEAE mice. In order to identify theβ-elemene how to regulate the differentiation of T helper cell subsets and the transcription of their specific cytokines to implement the neuroprotective effect. And provide new treatment methods and experimental evidence on demyelinated optic neuritis.MethodObserving the neuroprotective effect of P-elemene on demyelinated optic neuritis of EAE mice through the techniques of behavior, morphology(optic nerve hematoxylin-eosin staining, electromicroscope observation)and immunohistochemistry. Futher observing the transcriptive factors and their regulatory factors affect the mRNA expression ofβ-elemene on specific cytokine of T helper cell subsets. Result1,Behavior changes:β-elemene can improve neurological functional score in EAE mice.2,Morphology changes:β-elemene can reduce axonal and myelin damage in EAE mice.3,Immunohistochemistry changes:β-elemene can reduce P-APP protein and increase CNPase protein expression in EAE mice.4,Molecular Biololgy changes:11 days after immunization,β-elemene can reduce the expression of cytokine IL-17, transcripive factor RORyt, differentiation regulatory factor IL-6, and IL-23p19, at the same time to increase the expression of FOXP3, Socs3 mRNA.19 days after immunization,β-elemene can reduce the expression of cytokine IFN-y, transcriptive factor T-beta, differentiation regulatory factor IL-12 p35, at the same time to increase the expression of GATA3 mRNA and keep the expression of FOXP3 mRNA higher.Conclusion1,Early axonal and myelin damage in optic nerve can be seen in the model of MOG EAE mouse,and axonal injury didn't completely result from the myelin damage2,Th17 cell subsets launch the specific immune response in the early stage of demyelinated optic neuritis. Thl cell substs play an important role in the process of sustaining the specific immune responses. Treg subsets inhibit the specific immune response not only at the early stage but also give a negative regulation to the specific immune response together with Th2 subsets at the peak stage.3,P-elemene can improve early demyelinated and axonal injury of demyelinated optic neuritis, and improve the clinical symptoms.4,P-elemene can reduce Th17 and Thl cytokine expression, and increase Treg cytokin expression, which lead to the shift of Thl7/Treg and Thl/Th2 polarization.β-elemene can impact the neuroprotective effect through the transcriptive facctors mRNA expression of specific cytokines and their regulatory factors of T helper cell subsets.