Study Of Rapamycin Impacted Regulatory T Cells And Relative Cytokines In Mice With Improved EAM

Backgroud:Human's Idiopathic inflammatory myopathies (IIM) are a heterogeneous group of skeletal muscle inflammatory diseases characterized by the presence of muscle weakness and inflammatory infiltrates in the skeletal muscles. Previous studies have shown that it is a group of autoimmune diseases. However, the etiology and pathogenesis of the disease are still poorly understood And the treatments of corticosteroids and immunosuppressive are partially efficacious but have many side effects.This is partly because there has been no good animal model for studis.To overcome this problem, the establishment of an ideal animal model has been exploring. In previous studis, they had reported that animal model of experimental autoimmune myositis (EAM) generated by immunization with muscle homogenates, partially purified skeletal myosin, native C-protein and recombinant of skeletal C-protein′s fragment 2(SC2) respectively. And animal model was induced in transgenic mice. In their studies, there were shortcomings of method complexity, time-consuming, mild inflammation,poor reproducibility, and low success rate in varying degrees. Thus, in this study, we modified traditional method of building animal model.by increaseing immunization dose of myosin and injection times of. Pertussis toxin (PT). We have successfully induced time-consuming and reproducible animal model.IIM is an autoimmune disease. Regulatory T cells are crucial for negatively regulating immune responses. Enhancement of the numbers and function of Treg cells may protect individuals from autoimmune diseases. It has been reported that glucocorticoids and rapamycin (rapa) could expand the murine naturally occurring Treg cells. Methylprednisolone is effective in IIM. Thus, compare with Methylprednisolone group, we assess the effect of Rapa in IIM.Objectives:To establish a method of time-saving, efficient, and reproducible experimental autoimmune myositis (EAM) animal model. Then it may be an experimental tool for study human idiopathic inflammatory myopathies. To evaluate the therapeutic effect of rapamycin and observe the change of regulatory T cell in experimental autoimmune myositis. We study the pathogenesis of IIM to provide new therapeutic strategies.Methods:Experiment one: Inducing experimental autoimmune myositis of animal model with myosin by two methods. And comparing the two models. Forty-eight BALB/c mice were randomly divided into modified model group (M-EAM), traditional model group (EAM) and adjuvant control group (CON). Each group has sixteen mice. The EAM group were immunized four times with containing 1 mg myosin emulsified with an equal volume of complete Freund's adjuvant (CFA, Sigma). Pertussis toxin (PT,Sigma) was injected intraperitoneally (500ng in 200 ul saline) one time. The M-EAM group were immunized two times by 1.5 mg myosin emulsified with equal volume of CFA. However, PT was injected intraperitoneally two times. For CON group, saline/CFA and PT were injected according to the same protocol of the M-EAM group. At ten days after the last immunization, we compared differences among groups in weight, muscle strength, creatine kinase ,electromyogram (EMG), Pathology and immunohistochemistry.Experiment two: Twenty BALB/c mice were randomly divided into four groups: normal group, EAM group,methylprednisolone group and rapamycin group. Each group has five mice. The normal group did not do any interference. The EAM group was induced according to the same protocol of the M-EAM group. Ten days after the last immunization mice were Observed. The methylprednisolone group and the rapamycin group were injected respectively and intraperitoneally for continuous fourteen days after induced successfully animal model.On the fifteenth day, compared the following aspects among groups: muscle strength, the length of spleen, pathological and immunohistochemical staining,cytokine of serum, the percentage of Tregs in splenocyte, Foxp3 mRNA expression in spleen and muscle.Results:Experiment one: Three groups of muscle strength′score were the EAM 55.05±7.27 seconds, the M-EAM 22.81±9.28 seconds and CON 98.16±13.79 seconds(among three groups P≤0.01); An elevation in the levels of creatine kinase was found in our model group. EAM was 2526.77±930.22 u/L and M-EAM was 2618.19±640.35 u/L. For CON 1161.69±267.98 u/L ( between model groups P>0.05, model v control P≤0.01). Measurement of the EMG, the model group was significantly shorter duration and lower amplitude than the adjuvant control group. (data not shown, Figure 1) The histological score of EAM, M-EAM and CON group were 2.45±0.73, 2.51±0.61, 0.47±0.31( between model groups P>0.05, model v control P≤0.01). Immunohistochemical staining of model groups showed: MHC class I overexpression was observed in some fibers but was not diffuse. CD8-positive T cells were mainly located in the endomysium and some infiltrated muscle fibers. Relatively small number of CD4-positive T cells infiltrated muscle fibers although there were many CD4-positive T cells in the endomysium. These findings exists only in model groups and control group did not find.Experiment two: Four groups of muscle strength′score were Nomal group>30min, EAM 20.50±2.27 S, Methylprednisolone group 88.13±4.77 S and Rapa group 102.20±7.83 S(among groups P≤0.05). Normal spleen was small, smooth and rosy. EAM spleen was large and unsmooth. Two treatment groups were middle. The histological score of Nomal group, EAM, Methylprednisolone group and Rapa group were 0,2.22±0.12,1.72±0.16 and 1.28±0.19 respectively(among groups P≤0.05). Flow cytometric analyses revealed that the percentages of Tregs in four groups′splenocyte were 3.7±0.34%, 12.52±1.71%, 5.65±0.83% and 7.08±0.56% respectively (Nomal group and Methylprednisolone group P>0.05; Methylprednisolone group and Rapa group P>0.05 ). TGF-B1 and IL-10 cytokine in serum were respectively measured by lumine were 1046.75±82.01 and 4.58±0.93, 12.52±1.71%, 5.65±0.83% and 7.08±0.56% , 2087.00±74.27 and 15.36±0.50, 2238.50±134.29 and 2.82±0.33, 2501.75±329.11 and 2.39±0.32 (TGF-B1: Nomal group and Methylprednisolone group P>0.05; Methylprednisolone group and Rapa group P>0.05; IL-10: between two treat groups P>0.05); Foxp3 mRNA expression in muscle and spleen were respectively 2.76±0.47 and 94.94±7.13, 0.38±0.10 and 16.44±2.19, 2.46±0.36 and 100.85±8.75, 0.99±0.02 and 233.24±8.17.(between two treat groups P>0.05, other groups among P≤0.01).Conclusion:By increasing the dose of myosin and inject times of pertussis toxin, we have successfully induced reliably experimental autoimmune myositis in animal model . In the basic pathology, EAM is similar to human's PM and DM. In this study, we spent half the time of traditional methods, while the Inflammatory reaction is better. The animal model provides useful tools for idiopathic inflammatory myopathy in the pathogenesis and specific treatment.Rapamycin is a immunomodulator. When mice with EAM administered rapamycin for 2 weeks,the mice were improved significantly in muscle strength and histologic score. We found that it is more effective than methylprednisolone in the treatment of EAM. Therefore rapamycin is expected to become a candidate treatment drugs for IIM.Rapamycin can increase the mRNA expression of Foxp3 in spleen. At the same time,it increased TGF- ?1 and maintain IL-10 in serum. However , number of Treg in outer peripheral lymphoid organs (spleen) has declined 2 weeks after treatment. In the pathogenesis of myositis, rapamycin may affect the mechanism of Treg. How effects myositis and Treg about Rapamycin need research for long-term.