Study Of Thymopentin On Experimental Autoimmune Encephalomyelitis In Rats

ObjectiveMultiple sclerosis(MS)is an inflammatory demyelination disease of the central nervous system(CNS)that causes relapsing and progressive neurological impairment.The disease-modifying agents currently used in the treatment of MS are not completely effective.It is our main aim that we investigate the pathogenesis of MS and seek effective therapeutic scheme for MS by setting up an animal model.Thymopentin(TP-5)is a synthetic pentapeptide corresponding to amino acids 32-36 of the thymic polypeptide thymopoietinâ…¡,which contains 49 amino acids.TP-5 has the whole physiological function of thymopoietinâ…¡.TP-5,as an immunomodulator,has been shown to induce T lymphocyte differentiation and activation,reduce the production of the autoantibodies,normalize the level of CD4⁺/CD8⁺ and regulate the immune reconstitution.The effect of TP-5 is significantly in some autoimmune diseases such as rheumatoid arthritis(RA) and systemic lupus erythematosus(SLE).In our study,we administrate TP-5 to the rats of experimental autoimmune encephalomyelitis(EAE)-an animal model of MS,in order to investigate the therapeutic potential in treatment of neuroinflammatory diseases like MS.MethodsSeventy-eight healthy female Wistar rats weighing 180～200g were divided randomly into five groups:normal control group(6 rats),EAE group(18 rats),dexamethasone(DXM) group(18 rats),low dose of TP-5 group(18 rats)and high dose of TP-5 group(18 rats).EAE group and treatment groups were divided three groups:7 day group,14 day group,21 day group, separately.All experimental rats were immunized subcutaneously in the Wistar rats four foot pads by fresh guinea pig spinal cord homogenate(GPSCH)and complete Freund's adjuvant(CFA).Clinical signs of EAE were assessed the mean of twice daily by two observations.Scores were assigned on the basis of the following symptoms:1,tail weakness;2,tail weakness plus limb asthenia;3,mild limb paralysis;4,severe limb paralysis;5,moribund/dead.EAE group,DXM group,low dose of TP-5 group and high dose of TP-5 group were injected intraperitoneal respectively NS 0.5mL/d,DXM 5 mg·kg^-1·d^-1,TP-5 0.05 mg·kg^-1·d^-1,TP-5 0.25mg·kg^-1·d^-1.This treatment was started on the first day of immunization and continued daily for the duration of the experiment.During the experiment,the mean maximal score of animals at different time point and the incidence of disease were observed as results.Rats were sacrificed after anesthesia with intraperitoneal injection.Tissues of the brain and spinal cord were fixed with 4 %formalin,then the tissues were embedded in paraffin and sectioned at 4μm thickness.Some of the sections were stained with HE to assess lymphocyte infiltration and inflammation.The expression of cytokines IFN-γand IL-4 mRNA in the brain of rats was measured by semiquantitative reverse-transcription polymerase chain reaction(RT-PCR)on days of 7,14 and 21 post immunization(p.i.).Results1 The morbidity of disease in different groupMorbidity of low dose of TP-5 group and DXM group were significantly lower than those of EAE group and high dose of TP-5 group(P＜0.01).2 Clinical profile of EAE in different groupNeurological deficits scores of low dose of TP-5 group and DXM group were significantly lower than those of EAE group and high dose of TP-5 group(P＜0.01).3 Neuropathological findingsThe results demonstrated that some monocytes infiltration was observed in the tissues of brains and spinal cords before the clinical signs emerging.The degree of infiltration was associated with the severity of EAE.The extent of inflammation of low dose of TP-5 group and DXM group were significantly lower than those of EAE group and high dose of TP-5 group(P＜0.01).4 Expression of IFN-γand IL-4 mRNAIn low dose of TP-5 group,the expression of IFN-γmRNA significantly decreased(P＜0.01)and that of IL-4 mRNA significantly increased on days of 14 and 21 post immunization (P＜0.01).Conclusion1 TP-5 reduces the morbidity,and protects the rats from the severity of the disease.2 TP-5 can reduce the expression of IFN-γmRNA,and then reduces the lymphocyte infiltration and inflammation in the CNS of EAE rats.3 TP-5 can increase the expression of IL-4 mRNA,which is associated recovery of EAE rats.4 The effect of TP-5 on the EAE is not dose-dependent.5 TP-5 has good safty,and tolerance compared with DXM. These results suggest that TP-5 may be a useful immunomodulator for demyelinating diseases without suppressing immune response.