Amelioration Of Experimental Autoimmune Myasthenia Gravis By Suppressing Humoral And Cellular Immunity And Protecting AChR: Polygonum Cuspidatum And Its Ingredient Resveratrol

Background and objectivesMyasthenia gravis (MG) is a T cell-dependent antibody-mediated autoimmune diseasein which acetylcholine receptor (AChR) at the neuromuscular junction (NMJ) is the majorautoantigen. Its animal model, experimental autoimmune myasthenia gravis (EAMG), is adependable and useful model for the study of pathogenic mechanisms and therapeuticstrategies.Polygonum cuspidatum (PC), a traditional Chinese medicine (TCM), also known asHu-Zhang, whose property and flavor are a little bitter and cold, has effects ofheat-clearing and detoxicating, damp-excreting, blood-activating, and phlegm-eliminating,one of the effective ingredients in which is resveratrol that is a naturally occurring poly-phenol. They possess many pharmacological properties including anti-inflammation andimmunoregulatory efects as well as antioxidant, anti-cancer and lipid metabolism im-provement. What’s more, it was reported that PC could treat snakebite and n-butanol ex-tract of it could up-regulate protein expression of nAChR in SH-SY5Y (human neuroblas-toma cell line) cells in vitro.Unfortunately, the study of TCM about EAMG is less, and multi-component herbs arepredominating, while the single “bitter and cold” kind of TCM is much less. In our currentdocuments, we have not yet found the study of PC or Res about EAMG. This study is thefirst time and its purpose is to explore whether PC and Res have therapeutic effects or notand elucidate the possible mechanisms of action.Materials and Methods1. Materials 1.1Animals: female Lewis rats,6-8weeks of age,140-160g in body weight.1.2Antigen: a synthetic peptide corresponding to region97-116of the rat acetyl-choline receptor α subunit (R97-116).1.3Drugs: PC (granula), Res (Sigma-Aldrich)2Methods2.1Induction and clinical evaluation of EAMGLewis rats were subcutaneously immunized with R97-116twice to establish a modelof EAMG. Weights of all rats were recorded daily and their clinical symptoms were gradedbetween0and4according to the Lennon criteria with minor modification every day.2.2Administration of PC and Res to EAMG ratsRats were randomly divided into PC group, Res group and control group,5rats re-spectively. Both treatment groups were intragastric administration respectively with PC (2g/kg), Res (20mg/kg)(dissolving in DMSO aqueous solution) every day from day5afterthe first immunization, until day42(the morbidity peak period), and control group ratswere given the same concentration of DMSO (0.4ml/kg) aqueous solution in the sameway.2.3Lymphocyte cell proliferation assay by CCK-8Inguinal lymph nodes were excised from all of the rats and made into LMNC suspen-sions respectively, which were used to conduct lymphocyte cell proliferation assay byCCK-8.2.4Detection of cytokine levels in serum and lymphocyte cell culture supernatants byElisa including TNF-α, IFN-γ, IL-4, IL-10, IL-172.5Determination of anti-AChR α subunit R97-116IgG antibodies in rat serum in-cluding IgG, IgG1, IgG2a and IgG2b2.6Immunofluorescence analysis of AChR at the neuromuscular junction2.7Statistical AnalysisThe SPSS16.0was used for statistical analysis. One-Way ANOVA was used to deter-mine the significance of the results among3independent samples, and LSD-t test was em-ployed between two arbitrary groups. If the data didn’t meet the homogeneity of variance,Kruskal-Walli（sK-W）test would be applied. Differences were considered to be statisticallysignificant when the P value was <0.05. All results were shown as means±SD（standarddeviation）. Results1. Both PC and Res treatments ameliorated the clinical manifestation of EAMG.2. PC inhibited lymphocyte cell proliferation under the condition of the stimulusPBS or R97-116, and Res had a suppressive trend in spite of no statistical significance.3. Both PC and Res down-regulated expression of TNF-α in serum and drainingLMNC, while up-regulating IL-10in that, and had no statistical significance of IFN-γ, IL-4,IL-17.4. Both PC and Res reduced the levels of anti-AChR α subunit R97-116IgG1andIgG2a, and there was no statistical significance of IgG and IgG2b.5. AChRs in the treatment groups were relatively dense and integrated, while in thecontrol group incomplete and in disorder, which demonstrated that PC or Res protectedAChR at NMJ by increasing protein expression of AChR and/or decreasing the damage tothe receptor possibly.ConclusionsBoth polygonum cuspidatum and its ingredient resveratrol could ameliorate EAMG.And there may be three aspects of mechanisms about it. On the one hand, they could inhi-bit the production of anti-R97-116IgG1and IgG2a in serum by down-regulating expres-sion of TNF-α and up-regulating IL-10. On the other hand, they might eliminate an-ti-R97-116IgG1and IgG2a in serum. What’s more, they might increase the expression ofAChR at NMJ.Resveratrol could be the main effective component of polygonum cuspidatum sup-pressing EAMG, but further experiments are necessary.