Therapeutic Effect And Mechanism Of Rapamycin On Experimental Autoimmune Myasthenia Gravis Rats

Objective To preliminarily study the therapeutic effect of rapamycin(RAPA)on experimental autoimmune myasthenia gravis(EAMG)rats,aiming to explore the relevant immune mechanism at the cellular and molecular level,and for clinical treatment Myasthenia gravis provides laboratory evidence.Methods(1)The EAMG rats model was constructed and divided into CFA group,EAMG group and RAPA treatment group [1 mg/(kg·d)].(2)The Lennon muscle strength scale was used to evaluate the clinical symptoms of rats in each group every other day and record the weight change.(3)Detect the level of anti-R97-116 antibody in rat serum by ELISA method.(4)Prepare splenocytes and stimulated with 5 μ g/m L Con A,10μg/m LR97-116,RPMI1640 culture solution respectively,and the proliferation activity of rat splenocytes was detected by CCK-8 assay.(5)Flow cytometry was used to detect the changes in the number of Th1 cells,Th17 cells,and Treg in rat splenocytes.(6)Western blot method was used to detect the expression of mTOR,p-mTOR,and HIF-1α proteins in the spleen.(7)The transcription levels of T-bet,RORγt,FOXP3,mTOR,HIF-1α,Glut1,PKM2 m RNA in the spleen was detected by RT-q PCR method.(8)The morphological and structural changes of rat gastrocnemius muscle tissue was observed by HE.(9)The expression of Ach R in rat gastrocnemius muscle tissue was detected by Immunofluorescence technology.Results(1)Compared with the CFA group,EAMG rats had dry hair,decreased weight,no improvement in muscle weakness,and a significant increase in clinical scores.Compared with EAMG,the body weight and muscle strength in the RAPA treatment group had significantly improved,indicating RAPA treatment can significantly improve muscle weakness symptoms of EAMG rats.(2)ELISA results showed that the level of anti-R97-116 antibody in the EAMG group was significantly increased,compared with the CFA group.the anti-R97-116 antibody level of the serum in the RAPA treatment group was significantly reduced,compared with the EAMG group.(3)CCK-8 results showed that the proliferation activity of lymphocytes in the EAMG group was significantly enhanced under the three stimulation of RPMI1640 medium,R97-116 and Con A,compared with the CFA group.However,the proliferation activity of lymphocytes in the RAPA group obvious reduced,compared with the EAMG group.(4)The results of flow cytometry showed that the number of Th1 and Th17 cells in the spleen of the EAMG group increased and the number of Treg decreased,compared with the CFA group.Compared with the EAMG group,the number of Th1 and Th17 cells in the spleen of the RAPA treatment group was significantly reduced,the number of Tregs increased.(5)RT-q PCR results showed that the m RNA expression level of Th1 and Th17-related cell transcription factors T-bet and RORγt in the spleen of the EAMG group increased significantly,while the m RNA expression level of the Treg-related transcription factors FOXP3 decreased,and compared with the CFA group.The expression level of T-bet and RORγt m RNA in the RAPA treatment group decreased,while the expression level of FOXP3 increased significantly,compared with the EAMG group.(6)The expression level of mTOR,p-mTOR,HIF-1α protein and m RNA in the spleen tissue were detected by Western Blot and RT-q PCR.The results showed that the expression of p-mTOR and HIF-1α protein in the EAMG group increased compared with the CFA group.Compared with the EAMG group,the expression of p-mTOR and HIF-1α protein in the RAPA group decreased,but the total mTOR protein did not change significantly.At the same time,the RT-q PCR results are consistent with the Western Blot results.(7)RT-q PCR was used to detect the m RNA expression level of Glut1 and PKM2 in spleen tissue.The results showed that the expression levels of Glut1 and PKM2 m RNA in the EAMG group increased,and compared with the CFA group.The m RNA expression levels of Glut1 and PKM2 in the spleen of the RAPA treatment group decreased,compared with the EAMG group.(8)HE staining results showed that atrophy of the muscle fibers occur,the connective tissue formes,and a large amount of inflammatory cell infiltration in the EAMG group.while there was still a small unmber of inflammatory cell infiltration in the RAPA treatment group,but the degree of inflammation was significantly reduced.(9)Immunofluorescence results showed that the Ach R content in the gastrocnemius muscle tissue of the EAMG group was significantly reduced,while the Ach R content of the gastrocnemius muscle tissue increased in the RAPA treatment group.Conclusion RAPA can effectively regulate the balance dysfunction of Th1/Th17/Treg cells in the spleen,thereby the clinical symptoms of EAMG rats were relieved.Its regulatory mechanism may be related to the inhibition of mTOR/HIF-1α-related metabolic pathways by RAPA.