RESUMO
Myasthenia gravis (MG) is a prototypical antibody-mediated neurological autoimmune disease with the involvement of humoral immune responses in its pathogenesis. T follicular helper (Tfh) cells have been implicated in many autoimmune diseases. However, whether and how Tfh cells are involved in MG remain unclear. Here, we established and studied a widely-used and approved animal model of human MG, the rat model with acetylcholine receptor alpha (AChRα) subunit (R-AChR)-induced experimental autoimmune myasthenia gravis (EAMG). This model presented mild body-weight loss 10 days after the first immunization (representing the early stage of disease) and more obvious clinical manifestations and body-weight loss 7 days after the second immunization (representing the late stage of disease). AChR-specific pre-Tfh cells and mature Tfh cells were detected in these two stages, respectively. In co-cultures of Tfh cells and B cells, the number of IgG2b-secreting B cells and the level of anti-AChR antibodies in the supernatant were higher in the cultures containing EAMG-derived Tfh cells. In immunohistochemistry and immunofluorescence assays, a substantial number of CD4/Bcl-6 T cells and a greater number of larger germinal centers were observed in lymph node tissues resected from EAMG rats. Based on these results, we hypothesize that an AChR-specific Tfh cell-mediated humoral immune response contributes to the development of EAMG.
Assuntos
Animais , Feminino , Linfócitos B , Alergia e Imunologia , Modelos Animais de Doenças , Imunidade Humoral , Linfonodos , Alergia e Imunologia , Miastenia Gravis Autoimune Experimental , Alergia e Imunologia , Subunidades Proteicas , Alergia e Imunologia , Proteínas Proto-Oncogênicas c-bcl-6 , Alergia e Imunologia , Ratos Endogâmicos Lew , Receptor Cross-Talk , Receptores Colinérgicos , Alergia e Imunologia , Linfócitos T Auxiliares-Indutores , Alergia e ImunologiaRESUMO
Objective:To observe the effects of All-trans retinoic acid (ATRA) on the immune functions of AChR-specific lymphcytes via in vitro assays,and investigate the possibility of ATRA in the clinical treatment of myasthenia gravis (MG).Methods:CFA control group and EAMG experimental rats were established to obtain single lymphocytes suspension and cells were followed by AChR97-116 peptide with or without ATRA stimulation for 72 h,and then viable cell population,cell apoptosis,cell cycle and the distribution of Th cells were determined by flow cytometry.CCK-8 assay was selected to evaluate the effects of ATRA on proliferatory ability of lymphocytes.ELISA was used to detect the antibody secretion of B cells affected by ATRA.Results:Compared with CFA group,lymphocytes obtained from EAMG rats had higher ratios of living cells,and this ratio was obviously decreased after ATRA treatment,P＜0.001.Different concentrations of ATRA promoted the apoptosis of AChR-specific cells (P＜0.001),and the promoted effects were ATRA dose-dependent,however,cell cycles were not changed.ATRA markedly inhibited the proliferation of cells from both CFA and EAMG groups,moreover,AChR-specific cells were more sensitive to ATRA treatment (P＜0.01) than that of cells from CFA rats (P＜0.05).The ratio of AChR-specific CD4+T cells was reduced by ATRA (P＜0.01),and ATRA incubation significantly promoted the percentages of Th2,(PCD4+-4IL-4+＜0.001),Treg (PCD4+-Foxp3+＜0.001) cell types,but markedly inhibited the percentages ofThl7 (PCD4+-IL-17+＜0.05),Thl (PCD4+-IFN-γ+＜0.001) cells.ELISA data showed us that ATRA obviously down regulated the antibody secretion of AChR-specific B cells,P＜0.01.Conclusions:ATRA not only inhibited the functions of AChR-specific T cells,but also suppressed the roles of AChR-specific B cells,predicating a therapeutic effect of ATRA on myasthenia gravis therapy.
RESUMO
Objective:To observe the effect of “Warming Yang and Nourishing Qi” Acupuncture on the acetylcholine receptor (AchR)mRNA expression in neuromuscular junction (NMJ)of the rats with experimental autoimmune myasthenis gravis (EAMG), and to analyze the mechanism of acupuncture treatment for EAMG. Methods:AchRα1 129-145 peptide fragments were used to immune the 60 female Lewis rats to establish the EAMG rat models. 30 EAMG rats were selected randomly and divided into model control group,acupuncture treatment group and drug control group (n = 10).Another 10 SPF- “grade three”female Lewis rats,which were raised together with EAMG rats,were used as blank control group. The rats in model control group and blank control group had no treatment.The rats in acupuncture treatment group were treated with “Warming Yang and Nourishing Qi”Acupuncture.The rats in drug control group were treated with pyridostigmine bromide by gavage. The tissue samples in NMJ of gastrocnemius of the rats were collected,and the expression levels of AChR mRNA were detected with Real-time PCR method; the differences among various groups were analyzed quantitatively. Results:Compared with blank control group,the expression levels ofγ-AChR mRNA andε-AChR mRNA in NMJ of the rats in acupuncture treatment group drug control group and model control group were significantly increased (P <0.01).Compared with model control group,theγ-AChR mRNA andε-AChR mRNA in NMJ of the rats in drug acupuncture treatment group and control group were significantly increased (P < 0.01 ). Conclusion:“Warming Yang and Nourishing Qi”Acupuncture can effectively improve the expression level of AchR mRNA and it is an effective treatment method for myasthenia gravis.
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Objective:To investigate the immune mechanism of Shengxian decoction in experimental autoimmune myasthenia gravis(EAMG) rats. Methods:Lewis rats were immunized with the rat sequence 97-116 of the AChRαsubunit(Rα97-116) in CFA, 25 of which were successful. They were randomly divided into 5 groups:EAMG model group,prednisone group(5. 4 mg/kg),Shengxian decoction low, medium, high dose groups ( dosage 2. 6 g/kg, 5. 2 g/kg, 10. 4 g/kg ) . Clinical symptoms, weight, and the decrement percentage of RNS(5 Hz) were evaluated,and ELISA were adopted to determine the titers of AChR Ab,TGF-β,IFN-γ,IL-2,IL-4 and IL-17 in serum. Results:After molding,the percentage of decrement of RNS in each group noticeably increased by more than 10% in comparison with that in the CFA control group ( P<0. 01 or P<0. 05 ) . At the same time, they were also subjected to progressive decreasing weight and typical myasthenia symptoms,showing the successful molding. With medication,the decrement percentage of RNS of rats in the groups with low,medium and high dose of Shengxian decoction were all on obvious decline with alleviated weight decrease (P<0. 01),testifying to the symptom improvement. Compared with the CFA control group,the groups with low,medium and high dose of Shengxian decoction were coupled with decreasing AChR Ab content(P<0. 05),rising TGF-βlevel and reducing IFN-γ,IL-2,IL-4 and IL-17 level(P<0. 01 or P<0. 05). Conclusion: Shengxian decoction can turn the decrement percentage of RNS around,improve the progressive weight decrease in EAMG rats and increase the weight gains. By up-regulating the TGF-βlevel,lowering IFN-γ,IL-2,IL-4 and IL-17 level,preventing B cells from producing AChR Ab and reducing the content of AChR Ab in serum,it will soothe the damage of NMJ to AChR and cure EAMG.
RESUMO
Objective:To study the influence of “Warming Yang and Nourishing Qi”Acupuncture in the expression levels of acetylcholine receptor protein (AchR)in gastrocnemius neuromuscular junction (NMJ)of the rats with experimental autoimmune myasthenia gravis (EAMG),and to clarify the mechanism of “Warming Yang and Nourishing Qi”Acupucture in the treatment myasthenia gravis (MG).Methods:The female Lewis rats were used to establish the EAMG rat models immuned with AchRα1 129-145 polypeptide fragments.30 EAMG rats were selected randomly and divided into model control group, drug control group, and acupuncture treatment group (n=10),and another 10 rats purchased at the same period were used as blank control group.The rats in drug control group were treated with 1 8.5 mg · kg-1 · d-1 pyridostigmine by gavage. And the rats in acupuncture treatment group were treated with irritating by“Warming Yang and Nourishing Qi”Acupuncture.The rats in blank control group and model control group were not treated .The Lennon symptom scores of the rats in various groups were recorded.After treatment,the tissues of gastrocnemius NMJ of the rats were taken,and the AchR protein expression levels in NMJ of the rats in various groups were detected by Western blotting method. Results:Compared with blank control group,after successfully modelling,the Lennon symptom scores of the rats in model control group, drug control group and acupuncture treatment group before treatment were significantly increased (P<0.01).Compared with model control group,the Lennon symptom scores of the rats in drug control group and acupuncture treatment group after two treatment course were significantly increased (P<0.01 ). Compared with blank control group, the AchR protein expression level in gastronemus NMJ of the rats in model control group was significantly decreased (P<0.01). Compared with model control group, the AchR protein expression levels in gastronemus NMJ of the rats in acupuncture treatment group and drug control group after treatment were significantly increased (P<0.01).Conclusion:“Warming Yang and Nourishing Qi”Acupuncture has good therapeutic effect on the EAMG through increasing the NMJ AchR protein expression level of the EAMG rats.
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Objective To investigate the effects of Polygoni Cuspidati Rhizoma et Radix (PCRR) and its ingredient resveratrol (Res) on experimental autoimmune myasthenia gravis (EAMG). Methods EAMG was induced in Lewis rats by the immunization of a synthetic peptide corresponding to region 97–116 of the rat acetylcholine receptor (AChR) α subunit (R97-116). EAMG rats were randomly divided into PCRR group, Res group, and control (C) group, and were ig administered respectively with PCRR (2 g/kg), Res (20 mg/kg), and DMSO (0.4 mL/kg) every day from day 5 after immunization to day 42. Clinical evaluation, lymphocyte proliferation, cytokines, and anti-97-116 antibodies were performed for examination of their therapeutic effects. Results Treatments with PCRR and Res significantly ameliorated clinical symptoms, down-regulated TNF-α and up-regulated IL-10 in serum and culture supernatants of lymphocytes stimulated with R97-116, and decreased levels of anti-R97-116 IgG1 and IgG2a in serum compared with C group. Unexpectedly, PCRR but not Res inhibited lymphocyte proliferation compared with C group. Conclusion PCRR and Res ameliorating EAMG is associated with suppressing immune response, and indicates a therapeutic potential for EAMG and even human myasthenia gravis (MG). Res may be the main effective ingredient from PCRR ameliorating EAMG, but further experiments are necessary.
RESUMO
Objective:To observe the influence of the “Warming yang and Nourishing qi” acupuncture treatment in the serum interleukin-12 (IL-12) and interleukin-18 (IL-18) expression levels,in the rats with experimental autoimmune myasthenis gravis (EAMG) and to analyze the mechanism of acupuncture treatment to myasthenis gravis (MG).Methods: AchRα1 129-145 peptide fragments were used to immune the female Lewis rats to establish EAMG rat models.30 successful EAMG model rats were randomly selected and divided into acupuncture treatment group ,drug control group and model control group ( n=10 );the unmodelled 10 rats purchased at the same period were used as blank control group.The rats in acupuncture treatment group were treated by “Warming yang and Nourishing qi” acupuncture treatment,30 min/time,once a day,7 d for a period,1 d rest between two periods,two periods of continuous treatment.The rats in drug control group were treated by the pyridostigmine lavaged treatment , 18.5 mg/( kg · d ) , continuous treatment of 15 d.The rats in the other two groups had no any special treatment.After treatment,the blood samples from the vein of tail root of the rats in 4 groups were collected ,and the serum expression levels of IL-12 and IL-18 were detected with ELISA method.Results:After treatment,compared with model control group ,acupuncture treatment and drug control group the serum IL-12 and IL-18 expression level of the EAMG rats were significantly decreased ( P0.05 ).Conclusion:“Warming yang and Nourishing qi” acupuncture treatment can reduce the serum IL-12 and IL-18 expression levels of EAMG rats to treat MG ,and the effect of treatment was similar to pyridostigmine.
RESUMO
Objective:To investigation the effect of receptor associated protein (Rapsyn) in acetylcholine receptor (AChR) of end plate of experimental autoimmune myasthenia gravis (EAMG).Methods:Made and purified the pcDNA Rapsyn with genetic engineering technique and injected the muscle of vastus lateralis of left leg of Lewis rats, and then stimulated with electro square porator. EAMG induced by passive transfer mAb35 injected intraperitoneally, and monitor body weight and assessment clinical signs score. Sacrificed animal and removed the muscle of vastus lateralis of Lewis rats after 48 hour of induction of EAMG, measurement the AChR with radioimmunoassay (RIA) and calculated the AChR loss rate.Results:There were lower AChR loss rate found in muscle pretreated with pcDNA Rapsyn then muscle pretreated with Vector(as negative control).Conclusion:Rapsyn protein can protect the AChR of EAMG model. [
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Objective: To explore cytokine mechanism of Zhongjiling Tablet therapy to experimental autoimmune myasthenia gravis (EAMG). Methods: Lewis rats were immunized with N2-AChR(from Torpedo's electrical organ)in CFA. ELISA were adopted to determine IFN-?,IL-4,TGF-? levels in serum and surpernatant fluids and anti-N2-AChR total IgG,IgG1,IgG2 titers in serum of EAMG on week 7 post immunization and treatment.Results:In serum and surpernatant fluids:IFN-?,IL-4 level in model group were dramatically higher than CEA group ( P
RESUMO
Objective; To explore mechanism of Zhongjiling tablet treats experimental autoimmune myasthenia gravis on rats. Methods:Normal mice, immunosuppressive mice and EAMG rats were used to investigate the mechanism. Results:Zhongjiling tablet enhanced T and B cells proliferation of normal mice and immunosuppressive mice induced by ConA or LPS, and increased the production of IL-2. T cell proliferation of EAMG rats was improvement stimulated by AChR and decline by ConA. Zhongjiling tablet inhibited the improvement by AChR and enhanced the decline by ConA, and reduced the expression of IFN-? and IL-4 mRNA. Zhongjiling tablet also induced the apoptosis of CD4 + T lymphocyte stimulated by AChR. Conclusion: Zhongjiling tablet increased immunity of normal mice, immunosuppressive mice and EAMG rats. But it suppressed specific T lymphocyte proliferation induced by AChR and reduced the expression of IFN-? and IL-4 mRNA. It was probably one of mechanisms Zhongjiling tablet induced the apoptosis AChR specific CD4+T lymphocyte.