| Objective: The aim of this study was to investigate the prophylaxis and treatment effects of alpha-melanocyte-stimulating hormone (alphα-MSH) and possible mechanism. of action in Guinea pig model of experimental autoimmune encephalomyelitis (EAE). To seek new medicine for treating multiple sclerosis Method: 40 female Guinea pigs were included and were assigned randomly to four groups: namely, the normal group, the EAE contral group, the low dose treating EAE group and the high dose treating EAE group. Seven days before the mould to fastigium, Guinea pigs in the normal group and the EAE control group were intraperitoneal injected with PBS or saline water 1ml/kg each day, the low dose treating EAE group with alphα-MSH 0.25mg/kg, the high dose treating EAE group with alphα-MSH 1mg/kg. Guinea pigs in the EAE contral group, the low dose treating EAE group and the high dose treating EAE group were subcutaneously injected with antigen of whole spinal cord homogenate (WSCH) by the rear feet by the dose of 0.2ml each, the normal group with saline water. After the EAE was in operation, the low dose treating EAE group and the high dose treating EAE group were intraperitoneal injected with alphα-MSH for the purpose of protection in the same way as former, the normal group, the EAE control group with PBS until the end. The delitescence, the progression and the maximal disease score, mortality of each group were compared. Observing the brain and spinal cord tissue for pathological examination. Detection the proportion of CD4+CD25+ FOXP3+T lymphocyte and Th17 cell were checked by FCM; the level of IL-4,IL-17,IFN-γsecreted by peripheral blood mononuclear cells (PBMC) and content of IL-23 were detected by ELISA. Results:①The incidence and severity of all group: There was no incidence of EAE in normal group but various incidence occurred in the EAE control group, the low dose treating EAE group and the high dose treating EAE group. Through statistics analysis, we found that the delitescence of the low dose treating EAE group (13.4±1.7days)was longer than that of the EAE control group(10.8±1.6 days) (p<0.01) and the delitescence of the high dose treating EAE group (17.0±1.3 days) prolonged significantly compared with that of the EAE control group and the low dose treating EAE group (p<0.01). The progression also differed among groups, The low dose treating EAE group (4.1±1.1days) had shorter progression than high dose treating EAE group (3.5±0.7 days) and that had much shorter progression than EAE control group (5.9±0.9 days). The neurological disturbances score declined with the sequence of the EAE control group (3.4±0.6 marks), the low dose treating EAE group (2.7±0.7 marks) and the high dose treating EAE group (2.1±0.6 marks).②The pathological changes of all group: we found that Varying degrees of demyelination and inflammatory cell infiltration in EAE control group and treating groups. But the high dose treating group showed the lightest pathological changes.③The percentages of CD4~+CD25~+FoxP3~+Treg of all groups: the percentages of CD4~+CD25~+FoxP3~+Treg of EAE control group(4.21±0.57%) obvious drop than the low dose treating group(4.64±0.41%) and the high dose treating group(5.52±0.44%)(P<0.05,P<0.01)Difference also exists between the high dose treating group and low dose treating group(P<0.05).④The percentages of Th17 cell of all groups: the percentages of Th17 cell of EAE control group(2.63±0.44%) is much higher than the normal conrol group(1.39±0.48%)(P<0.01).The low dose treating group and the high dose treating group separately for the proportion of (1.97±0.89%, 1.42±0.66%),than the EAE control group significantly decreased (P <0.05, P <0.01),but the obvious decreased in the high dose treating group((P <0.05).⑤Peripheral blood mononuclear cell secretion of IFN-γ,IL-4,IL-17 levels and peripheral blood IL-23 levels of all all groups: The levels of IFN-γ,IL-4,IL-17,IL-23 in peripheral blood in EAE control group were higher than those in the normal group(P<0.01), IL-4 the opposite (P<0.01); The levels of IFN-γ, IL-17, IL-23 ofα-MSH high dose treating and low dose treating group were lower than the EAE control group(P<0.01); IL-4 also the opposite (P<0.01). but the significantly changes exsist in the high dose treating group.⑥The ratio of IFN-γ/IL-4 for all groups: the ratio of IFN-γ/IL-4 in EAE control group was significantly increased than the normal control group(P<0.01);the ratios of IFN-γ/IL-4 in high dose treating group and low dose treating group were decreaced than the EAE control group(P<0.01).⑦Correlative analysis: the proportion of peripheral Th17 cells,IL-17 levels,IL-23 levels,IFN-γlevels,the ratio of Th1/Th2 were negatively correlated with delitescence in EAE control group and all treating groups, positively correlated with nerve dysfunction score and progression ;but the The percentages of CD4~+CD25~+FoxP3~+Treg and the level of IL-4 were the opposite; The levels of IL-17 and IL-23 were positively with the ratio of IFN-γ/IL-4;Furthermore,the negatively correlation between the ratio of CD4~+CD25~+FoxP3~+Treg and Th17cell has observed. Conclusion:(1) In the EAE mould by subcutaneous administrating WSCH with the method of multi-point single subcutaneous injection, neurological disfuntion of EAE Guinea pigs were remarkable. Perivascular inflammatory cell infiltration and demyelination in white matter were clearly showed.(2) immune imbalance exists.in EAE: The previous experiments revealed that EAE was caused by Th1 overstimulation and Th2 understimulation and Th cell polarization to Th1 cells in the direction of drift; the immune balance of CD4~+CD25~+FoxP3~+Treg/Th17 drifted to the side of Th17 cells.(3) Th17 cells and Th1 cells exert effects in the process of cooperation; CD4~+CD25~+FoxP3~+Tregs and Th17 cells in the function of mutual antagonism, differentiation on the inter-linkages,which through secret different cytokines to affect the Th1/Th2 cell balance;meanwhile Th1/Th2 cells aslo effect the balance of CD4~+CD25~+FoxP3~+Treg/Th17cell.(4)α-MSH has protective effect over the incidence of EAE because it can prolong the delitescence, shorten he progression, alleviate neurological disturbances of EAE Guinea pigs and lower mortality in maximal, which is positively correlated with the dose ofα-MSH.(5)α-MSH on the incidence of EAE in guinea pigs protective mechanism:α-MSH through regulation of Th1/Th2 and CD4~+CD25~+Tcell/Th17cell imbalance to exert its immunosuppressive function. |
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