Therapeutic Effects Of Different Doses Of Methylprednisolone Intreating EAE And Correlation Study On Efficacy, Glucocorticoidreceptor Isoforms And SRp30c

Backgroud:Multiple sclerosis (MS) is one of a common autoimmune inflammatory demyelinating disease of the central nervous system (CNS).The main clinical presentation is relapses, relief, exacerbations or attacks in neurological dysfunction. Short-term, high-dose methylprednisolone (MP) treatment has been considered for the treatment of relapses, exacerbations or attacks of MS.At present, the utilizing of MP in dosage is still disagreement.Most doctors always choose the dosage by their experience,such as 0.5g/d, lg/d or 2g/d respectively. However, a few patients happened to be resistant to glucocorticoid (GC).The underlying mechanisms of GC resistance remain unclear. Nowadays, it has been widely accepted that the glucocorticoid receptor (GR) isoforms: GRa and GRβplay important roles in GC resistance.Hence, at present study, we aim to assess the therapeutic effects between high dose and low dose MP treatment in the animal model of MS,and investigate the correlation between GR isoforms expression and therapeutic efficacy, and finally explore the potential mechanisms of GC resistance.Objective:To induce the animal model of MS-experimental allergic encephalomyelitis (EAE), and study the treatment of different doses of MP in EAE, evaluate the therapeutic effects by analyzing clinical scores, counts of inflammatory focis in tissue section, scores of demyelination and the concentrations of cytokine, explore the relationship between MP dosage and therapeutic efficacy; To investigate the correlation between MP therapeutic efficacy and GR isoforms, SRp30c mRNA expression in the nervous tissues of rats,and the correlation between splicing factor SRp30c and GRβ,explore the potential mechanisms of GC resistance from the aspects of animal experiment.Methods:1.Antigen was prepared by the Guinea pig spinal cord homogenate (GPSCH) which was emulsified in an equal volume of complete Freund's adjuvant (CFA).EAE model was induced by female Wistar rats, which were inoculated with 0.4ml GPSCH emulsion in the four footpads, and were then injected subcutaneously in the left hind dorsum of foot with pertussis solution.2.The EAE rats were divided randomly into three groups:high-dose, low-dose and model control group.Five normal Wistar rats were fed at the same condition as normal control group.Steroid treatment was started at 12 days post-inoculation when EAE rats showed severe clinical symptoms.High-dose and low-dose groups were given MP intravenously at a dose of 100mg/kg and 25mg/kg respectively. Model control group and normal control groups were injected with equivalent volume of 0.9% saline solution. MP was injected into caudal vein once daily for five days.The rats were monitored daily by neurological score and were executed after five days therapy.3.The plasma concentrations of interleukin-2 (IL-2) and tumor necrosis factor-alpha (TNF-a) levels were measured by radio immunoassay; The counts of inflammatory focis in tissue section and scores of demyelination were assessed by HE staining and myelin staining respectively; Immunohistochemistry and RT-PCR were used to investigate the expression of GR isoforms and splicing factor SRp30c.4. The therapeutic effects of high dose and low dose of MP in EAE were evaluated comprehensively by analyzing clinical scores,counts of inflammatory focis,scores of demyelination and cytokine production; and the correlation between MP therapeutic effects and GR isoforms,SRp30c mRNA expression, and the correlation between SRp30c mRNA and GRβexpression was studyed. Results:1.The manifestations of our EAE rats in clinical signs, HE and myelin staining were all corresponding with the typical clinical and pathological feature of EAE.2.Mean clinical scores were significantly ameliorated in high-dose and low-dose group after MP treatment, while symptoms of model control group rats were aggravated. There was no significant difference of score changes before and after treatment between high-dose and low-dose group.3.There were no significant differences in inflammatory focis and demyelination scores between high-dose and low-dose group.4.There were no significant differences in IL-2 and TNF-a level between high-dose and low-dose group.5.High-dose and low-dose group had similar levels of GRa and GRβ;The individual clinical score changes before and after MP treatment had a positive and linear correlation with the ratio of GRa/GRp(r=0.550,P=0.027).6.There was no significant difference in SRp30c mRNA expression between high-dose and low-dose group;The individual clinical score changes before and after MP treatment had a negative and linear correlation with SRp30c expression(r=-0.583,P=0.018);There was a positive correlation between SRp30c mRNA and GRβexpression (r=0.488, P=0.008).Conclusion:In some effective dose range, there is no dependent correlation between dosage and therapeutic effects, to increase the dose of MP can not enhance the efficacy; The ratio of GRa/GRβin the nervous tissues of EAE was associated with GC sensitivity, higher ratios correlate with GC sensitivity, while lower ratios correlate with GC resistance; SRp30c may play an important role in promoting alternative splicing of GR pre-mRNA to generate GRβin EAE rats and correlate with decreased GC sensitivity.