Effects On The Prescription For Invigorating Spleen And Kidney On The Serum IL- 4 And TGF-β Of Rats With Experimental Autoimmune Myasthenia Gravis(EAMG)

Objective: To observe the improvements of the symptom of myasthenia of rats with experimental autoimmune myasthenia gravis(EAMG) incurred by the prescription for invigorating spleen and kidney as well as the changes of the serum interleukin-4(IL-4) and TGF-β(transforming growth factor-β) to explore the immunological mechanism during the process of the occurrence of myasthenia gravis(MG) and thus provide meaningful basis of basic experiment for treating MG with traditional Chinese medicine.Methods: 60 healthy SPF-classed C57BL/6 female rats at the age of 8 to 10 weeks were randomly divided into two groups, that is, the normal control group(10 rats) and the model group(50 rats). The subunit 97-116 peptide fragment of the synthetic rat acetyl choline receptorαwas taken as the immunogen to establish EAMG model for the rats in the model group. During the primary immunization, 200μL of antigen emulsion containing 50 ug of the subunit 97-116 peptide fragment of the synthetic rat acetyl choline receptorαwas injected to the two shoulders and be under the skin of the two foot pads, that is, 50μL was injected to every part. Intensified immunization was conducted again to the same parts and in the same dosage of the rats in the model group thirty and fifty days after the primary immunization. Normal saline(NS) was injected to be under the skin of the rats in the normal control group in the same dosage as that in the model group. Model evaluation was conducted seventy days after the primary immunization, the euzymelinked immunosorbent assay(ELISA) was taken to examine the titer of the ACh RAb acetycholine receptor antibody(Ach R-Ab) in the serum of the rats, neostigmine test was conducted, comprehensive indexes like scoring the symptoms of myasthenia were observed to determine the standards for the model, and the changes of body weight were recorded every two days during the period of model construction. Finally altogether 26 rats were integrated into the model, and the rats having been successfully modelled in the model group were randomly divided into group of 8 rats traditional Chinese medicine prescription for invigorating spleen and kidney(4.5g/kg?d), group of 9 rats hormone(9mg/kg?d), and the group of 9 rats normal saline(0.2ml), finally same as 10 rats in the normal control group during the period of model construction(0.2ml),Since the date of the grouping, intragastric administration was conducted for one time every day, and the therapeutic process lasted for two weeks, during which the scores of the symptoms of myasthenia and body weight changes were recorded. Euzymelinked immunosorbent assay(ELISA) was taken to examine the contents of interleukin-4(IL-4) and TGF-β(transforming growth factor-β) before and after the treatment.Results: Finally altogether 26 rats were integrated to the model group according to the standards for determination of the model with the model establishment rate as about 52%. After the successful modelling, the rats in the model group had significant symptoms of myasthenia, which was specifically manifested as slow action, reduced diet volume, and slow increasing of body weight. In the normal control group the action and diet were both normal and no symptom of myasthenia appeared. Compared to the normal control group, the antibody titer and score of symptom of myasthenia in the model group were of significant statistical meaning(P<0.01). After the neostigmine test was conducted to the rats in the model group the improvements on the symptom of myasthenia was of significant statistical meaning after the administration of drug compared to that before the administration of drug(P<0.01). The model group was randomly divided into three groups and two weeks after the treatment the symptoms scores and body weight in the group of traditional Chinese medicine were of significant statistical meaning compared to those in the group of normal saline(P<0.01), but improvement degrees in the group of traditional Chinese medicine was lower than that in the the group of hormone, and statistical differences could be seen between the two groups(P<0.01). As to the changes of IL-4 and TGF-β titers in the serum of rats after being treated for two weeks, those of the group of traditional Chinese medicine and the group of hormone were of significant statistical meaning compared to the group of normal saline(P<0.01),, but titer changes in the group of traditional Chinese medicine was lower than that in the the group of hormone(P<0.01).Conclusion:(1) The rats with immune C57BL/6 of subunit 97-116 peptide fragment of the synthetic rat acetyl choline receptorαcould be taken to establish successful EAMG model with rather higher successful rate for model construction.(2) After the model has been successfully established, the scores of the symptoms of EAMG could be significantly reduced through treatment with prescription for invigorating spleen and kidney in addition that the clinical symptoms of myastheniacould be relieved,indicating that the prescription for invigorating spleen and kidney could be effective for the treatment on EAMG rats.(3) The immunological mechanism of the prescription for invigorating spleen and kidney affecting the EAMG rats is reducing the degree of the activation and added value of T cells and B cells through reducing the expression of the IL-4 level in serum or raise that of the TGF-β level so as to inhibit the reaction of autoimmune response and thus achieve the result of having the symptom of myasthenia improved.