Study On The Therapeutic Effect Of GPR97 Agonist BDP On EAE Mice And Its Mechanism

Multiple sclerosis (MS) is an inflammatory disease that is characterized by immune-mediated demyelination and neurodegeneration of the central nervous system (CNS). MS is considered as a complex disease depending on genetic, environmental and immune factors. Due to the cause of diversity, its exact pathogenesis remains unknown. At present, drugs currently used in MS are corticosteroids and interferon beta, the vast majority of drugs are proved to only have some effect. Experimental autoimmune encephalomyelitis (EAE) is a commonly used animal model that mimics the symptoms of MS in humans and EAE model has been extensively used to study the pathogenesis of MS and preliminary screening of clinical medicine. Many studies reported that GPCRs are related to the occurrence and development of MS or EAE, and can be used as drug targets of the disease. GPR97 belongs to adhesion GPCR family and expresses on many immune cells such as dendritic cell, neutrophil, eosinophil, basophil, mast cell, etc. Beclomethasone dipropionate (BDP), a glucocorticoid steroid, can act as an agonist of GPR97, and it can bind GPR97 and activate Gao to mediate the biological functions.Our previous results found that GPR97-knockout mice exhibited the aggravated EAE and the deficiency of GPR97 could enhance the function of DCs. To evaluate whether the agonist of GPR97 BDP has the therapeutic effect on EAE, EAE model was established by immunizing wild-type mice with MOG35-55 peptide and Freund’s adjuvant. The therapeutic research was performed by administering BDP subcutaneously from day 8 and the therapeutic effect was analyzed by the clinical score, histopathological analysis of spinal cord tissue and immunological indexes. The results showed that the highest clinical scores and the accumulated clinical scores in BDP-treatment group were lower than those in untreated group. Less infiltration of inflammatory cells and demyelination in the spinal cord and lower level of pro-inflammatory cytokines such as TNF-α、IL-17A and IL-6 in the cells from spleen and lymph node were detected in BDP-treatment mice. MOG-specific T cell proliferation, the number of Thl and Th17 and the expression level of surface molecules on DCs in the splenocytes from the treatment group was significantly lower than the untreated group. Taken altogether, these results suggest that BDP has therapeutic effect on EAE and the inhibition of the surface molecule expression on DCs was indicated.To further explore the possible mechanism of BDP on therapeutic effect of EAE, bone marrow-derived DCs were prepared and used as target cells, the biological functions of DCs were analyzed such as the cytokine secretion profile, surface molecule expression, the ability of antigen presenting and induction of Th cell differentiation, etc. The results detected by FQ-PCR and ELISA showed that less IL-6, TGF-β and IL-12 P40 but more IL-10 were detected in BDP-treated DCs. BDP-treated DCs had lower expression of surface molecule CD40 and CD86. Also, BDP could inhibit allogeneic T cell proliferation induced by DCs and antigen-presenting ability of DCs. Intracellular staining analysis indicated that BDP could inhibit the differentiation of CD4+ T cell into Thl and Th17 subsets induced by DCs.To understand the molecular basis that BDP affected the function of DCs, GPR97-deficient DCs were used. The results demonstrated that the cytokine secretion and surface molecule expression were not affected by BDP treatment, which showed BDP affected the function of DCs by GPR97. To further elucidate the molecular mechanism, the signal pathway of MAPK and NF-κB which were important in DC functions was analyzed. Western blot analysis indicated that the phosphorylation of Erk and NF-κB P65 in BDP-treated DCs was lower than those in untreated DCs, which showed that BDP affected the signal pathway of Erk and NF-κB via activating GPR97 and took impact on the biological functions of DCs finally.In conclusion, BDP had therapeutic effect on EAE to some extent, the inhibition of BDP on the biological function of DCs was mediated by activating GPR97 and affecting the signal pathway of Erk and NF-κB. The study lay the theoretical and experimental basis for the MS/EAE treatment using GPR97 as the therapeutic target.