Therapeutic Effect And Mechanism Of Anti-human T Lymphocyte Porcine Immunoglobulin On EAE Rats

Background:Multiple sclerosis(MS),which is a classic-type of autoimmune diseases in the central nervous system(CNS).The main pathological features of MS include inflammatory cell infiltration,inflammatory demyelinating and axonal injury.At this stage,T lymphocytes play an important role in the autoimmune response of MS.Anti-lymphocyte/thymocyte globulin is a kind of biological immunomodulatory drug.It ues human lymphocytes or thymus as antigen,then immunized the healthy horses,rabbits,pigs and other animals.After that,the animals'anti-serum was separated and purificated,and finally obtained an immunoglobulin which could target T lymphocyte.At present,in the Phase II clinical trial of MS hematopoietic stem cell transplantation therapy,anti-lymphocyte/thymocyte globulin combined with cyclosporine(CsA)has been reported as a pretreatment related drug before transplantation,but no anti-lymphocyte/thymocyte cell has been reported.Proteins are directly applied to the study of MS and it`s animal models.Since the early 70s of last century,China began to develop such drugs.After full comparison of various types of immune antigens and immunized animals,the anti-human T lymphocyte Porcine immunoglobulin(Anti-human T Lymphocyte Porcine)was successfully developed in 1976.Immunoglobulin,ALG).Compared with others,ALG has a series of advantages such as high potency,less drug side effects,and low manufacturing cost.This study will investigate the therapeutic effects of anti-lymphocyte/thymocyte globulin in an animal model of MS-an experimental autoimmune encephalomyelitis(EAE)rat.The molecular mechanism of immunosuppression provides theoretical and experimental evidence for the direct application of anti-lymphocyte/thymocyte globulin in the treatment of MS.Objective:In this study,anti-human T lymphocyte immunoglobulin(ALG)was applied to the acute onset of experimental autoimmune encephalomyelitis(EAE)rats.In order to providetheoreticalandexperimentalevidencefortheapplicationof anti-lymphocyte/thymocyte globulin in the treatment of MS,we investigated the therapeutic effects of ALG on acute EAE rat.Methods:1.ALG improved neurological function in rats with EAE:A behavioral score curve howed that ALG can effectively alleviate the clinical behavior of EAE rats.The esults of HE staining and LFB staining showed that there were obvious emyelinating plaques in the white matter region of spinal cord of rats in AE+IgG group,and the demyelinated plaques in the spinal cord of EAE+ALG ats were significantly smaller than those in EAE+IgG group,and the lesions were urrounded.Infiltrating inflammatory cells were also significantly less than the AE+IgG group.These results demonstrate that ALG can effectively alleviate the eurobehavioral manifestations and pathological inflammatory changes in EAE ats.2.Evaluation of ALG targeting on T lymphocyte:Peripheral blood mononuclear ells(PBMC)were collected from healthy volunteers and normal rats and the cells ere randomly divided into ALG-treated group and IgG-treated group.The argeting effects of ALG on PBMCs was evaluated by laser confocal microscopy;ther cells were divided into Control group,PMA+Ionomycin group,MA+Ionomycin+IgG group and PMA+Ionomycin+ALG group treated with ifferent concentrations of ALG randomly.The corresponding interventions were iven 1 h before PMA+Ionomycin stimulation and 24 hours later by ELISA.The ontent of cytokines IL-2,IFN-?,and IL-17 in the cell culture supernatant was easured.3.Evaluation of ALG immunomodulatory effect on T lymphocytes:The PBMCs which collected from healthy volunteers and normal rats were divided into ontrol group,PMA+Ionomycin group,PMA+Ionomycin+IgG group,and MA+Ionomycin+ALG group randomly.The effect of ALG on the activation of T ymphocytes was assessed by flow cytometry.The effect of ALG on the ifferentiation of activated Na?ve T lymphocytes was assessed by flow cytometry.The effects of ALG on the proliferation and apoptosis of activated T lymphocytes ere evaluated by CSFE,Annexin-V/PI double staining and CCK-8.4.Evaluation of ALG biosafety:Cell viability of primary rat neuronal cells,microglia,astrocytes,and oligodendrocytes after 48 h treatment with different concentrations of ALG;Hematoxylin-Eosin(HE)staining was used to evaluate the pathology of heart,liver,spleen,lung and kidney in each group.Results:1.The result of behavioral score showed that the behavioral score of the rats in the administration group was lower than that of the model control group The results show that:ALG can effectively alleviate the clinical behavior of EAE rats.The results of HE staining and LFB staining showed that:ALG can effectively improve the spinal cord myenteric demyelination in EAE rats.The ELISA data showed that that:ALG can improve the inflammatory infiltration of EAE model mice and reduce the expression of inflammatory cytokines IL-2,IFN-?and IL-17 level in.the cerebrospinal fluid and serum of the EAE rats.2.ALG-targeted T-lymphocytes inhibit the expression of inflammatory cytokines:Laser confocal imaging results demonstrate that ALG-specific binding to human and rat CD3~+T cells;ELISA results show that ALG pretreatment is dose-dependent Reduced expression of PBMC inflammatory cytokines IL-2, IFN-?,and IL-17 after activation.3.ALG inhibited the differentiation of Na?ve T lymphocytes into Th1/Th17 subpopulation:flow cytometry results showed that ALG pretreatment had no effect on the expression of CD69 and CD25 on activated T lymphocytes,but inhibited the expression of IL-2.CSFE,Annexin-V/PI double staining and CCK-8 experiments proved that ALG had no effect on the proliferation and apoptosis of activated T lymphocytes;flow cytometry test confirmed that IFN-?~+in cells of PMA+Ionomycin+ALG group.CD4~+and IL-17~+and CD4~+T cells were significantly lower than those of PMA+Ionomycin+IgG group,that is,ALG pretreatment could reduce the proportion of proinflammatory Th1/Th17 subset T helper T lymphocytes in activated T lymphocytes.4.ALG has no obvious toxicity on rats:CCK-8 results showed that after treatment with different concentrations of ALG for 48 hours,the primary rat neurons,microglia,astrocytes,and oligodendrocytes.There was no significant difference in the cellular activity of the plasma cells.HE staining showed no abnormalities in heart,liver,spleen,lung and kidney tissues in the three groups.Conclusion:ALG can inhibit the inflammatory response in the CNS and effectively relieve demyelination in EAE rats.Furthermore,we found that the immunoregulatory function of ALG may be through that ALG targeting T lymphocytes and inhibition of helper T lymphocyte polarization from Naive T cells to Th1 and Th17 subsets.