| Objective:1. To verify the feasibility of rat-derived 97-116(Rα97-116) peptide of the acetylcholine receptor( AchR) a-subunit inducing experimental autoimmune myasthenia gravis(EAMG) ,in order to provide a simple and available animal models for futher study of myasthenia gravis (MG.).2. To verify the therapeutic efficacy of nasal tolerance with Rα97-116(V108A) on the EAMG and explore the clinical application value of nasal tolerance in MG.3. To explore the expression changes of T-bet and GATA-3 during normal control group,EAMG group,nasal tolerance group and the pathogenesis of MG as well as the theoretical basis of nasal tolerance .Methods:Female Lewis rats(6-8 weeks) were injected subcutaneously at four spots with the synthetic peptide Rα97-116 in complete Freund's adjuvant(CFA) and boosted on day 30 and 60 with the same peptide in incomplete Freund's adjuvant(IFA) to iduce rat models of EAMG , or only with phosphate buffer saline(PBS) in control group. Clinical manifestation was evaluated by measurement of body weight and Lennon clinical score. Disease was further confirmed by 3,5 Hz repetitive nerve stimulation (RNS) for positive decremental response and ELISA for sera AchR-Ab(IgG) titers. Modeling Lewis rats were divided into EAMG group and nasal tolerance group , and then PBS and Rα97-116(V108A) was respectively droped in bilasteral nasal cavity on days 71 to 80 once daily , Clinial Lennon score and body weight were evaluated for 24 days since nasal administration. All experiment rats were sacrificed on day 95. The expression levels of transcription factor T-bet/GATA-3mRNA in peripheral blood mononuclear cells(PBMCs) were measured by reverse transcription polymerase chain reaction(RT-PCR).IL-4,IFN-γ,AchR-Ab(IgG) in plasma were detected by ELISA.Results:1. Signs of EAMG occurred in 75% of Lewis rats ,which was confirmed by the Lennon clinical score>1 grade, the positive decremental response D5 >10% of 3,5 Hz RNS and the ratio >2.1 of the plasma AchR-Ab(IgG) titers to the normal control group. The achievement ratio of EAMG model was 75%.2. The 15th day after Lewis rats receiving nasal tolerance with Rα97-116(V108A) , the body weight of Lewis rats in the nasal tolerance group were increased significantly compared with the EAMG group(P﹤0.05).3. The 10th day after Lewis rats received nasal tolerance with Rα97-116(V108A), average clinical score of nasal tolerance group was significant better than EAMG group (P<0.05).4. Compared with the EAMG group , the amount of anti-AChR IgG in nasal tolerance group decreased (P<0.05) after the 25th day nasal tolerance with Rα97-116(V108A).5. The expression levels of IL-4 and IFN-γin EAMG group were much higher than those in normal control group (P<0.05);while IL-4 and IFN-γexpression were obviously decreased after nasal tolerance with Rα97-116(V108A) (P<0.05),there were not significant difference between nasal tolerance group and control group(P>0.05).6. The expression levels of T-bet/GATA-3 mRNA in EAMG group were much higher than those in control group (P<0.05);while T-bet/GATA-3 mRNA expression were obviously decreased after nasal tolerance with Rα97-116(V108A) (P<0.05),there were not significant difference between nasal tolerance group and control group (P>0.05).Conclusions:1. Immunization with the synthetic Rα97-116 peptide could induce EAMG in Lewis rats .The achievement ratio of EAMG model was 75% . This animal models provided a simple and available research materials for MG.2. Nasal tolerance with Rα97-116(V108A) could ameliorate muscular weakness and improve related Lab indexes of EAMG rats, which will provide a new way of treament for MG.3. T-bet and GATA-3 may play a critical role in pathogenesis of MG, nasal tolerance with Rα97-116(V108A) could surpress ongoing EAMG, which may be related to the decrease of T-bet/GATA-3 expression and downregulation of AChR-specific B-cell responses and AChR-reactive T-cell function.
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