| Abstract:Objective:To investigate the effect of leptin on the pathogenesis of experimental autoimmune encephalomyelitis and its role in regulating the balances of T cells. Methods:45 healthy female C57BL/6 mice were randomly divided into groups:normal control group, EAE control group, the leptin treated group,15 mice in each group. EAE control group and the leptin treated group were injected with MOG35-55 and complete Freund’s adjuvant (CFA) as an immunogen to make the EAE model, normal control group was given saline and CFA. Inject 0.2ml pertussis toxin solution into abdominal cavity of each mouse at the first day and 48 hours after the establishment of the model. Since the day of modeling, the leptin treated group was injected with recombinant leptin (0.5μg/g) into abdominal cavity twice daily (9:00 and 18:00) while normal control group and EAE control group were given intraperitoneal injection with equivalent saline twice daily for 10 consecutive days. Mice in EAE control group and leptin treated group appeared quadriplegia, and the symptoms score did not increase for three days or the mice were death can be considered as the onset of the peak, kill the mice at that time; mice in normal control group and unaffected mice were killed after fed for 4 weeks, leave spinal cord and brain tissue of mouse, orbital venous blood and spleen.1.Record the main symptoms, the morbidity, the incubation period, progressive stage and neurological dysfunction scores of all groups.2.HE staining to observe the pathological changes of brain and spinal cord of mice.3.Detect the ratio of CD4+CD25+Foxp3+Treg and Th17 cell in CD4+T cell of splenocyte suspension by flow cytometry. 4.Detect the contents of serum IFN-y, IL-4, IL-17, TGF-01 by ELISA. 5.The correlation of each index in EAE control group and the leptin treated group were analyzed. Results:1.The changes of the main symptoms, the incubation period, progressive stage and neurological dysfunction scores of all groups:normal control group without the disease, there were 13 mice get sick in EAE control group and all of mice get sick in the leptin treated group; Compared with EAE control group, the main symptoms were worse, the incubation period was shorter (P＜0.01), the progressive stage was longer(P<0.01), the peak of neurological dysfunction score increased in the leptin treated group (P<0.05).2.The pathological changes of brain and spinal cord of all groups:Brain and spinal cord of normal control group had no pathological changes, EAE group and the leptin treated group showed that the brain and spinal cord tissue loose swelling, peripheral vascular especially venules surrounded by inflammatory cell infiltration. It showed a typical "cuff like change, obvious demyelination change in peripheral vascular white matter; Compared with EAE control group, the pathological score of leptin treated group mice brain and spinal cord tissue increased, there was a significant difference (P＜0.01).3.The contents of serum IFN-γ and IL-4 and the ratio of IFN-y/IL-4 in all groups:Compared with the normal control group, the content of serum IFN-y significantly increased, the content of serum IL-4 decreased, and the ratio of IFN-y/IL-4 increased in both EAE control group and the leptin treated group, there were significant differences (P＜0.01); Compared with EAE control group, the content of serum IFN-y significantly increased, the content of serum IL-4 decreased, and the ratio of IFN-y/IL-4 increased in the leptin treated group, there were significant differences (P＜0.01); 4.The contents of serum IL-17 and TGF-β1 in all groups:Compared with the normal control group, the content of serum IL-17 in EAE control group and the leptin treated group increased, the content of serum TGF-β1 significantly decreased, there were significant differences (P<0.01); Compared with EAE control group, the content of serum IL-17 in the leptin treated group significantly increased, and the content of serum TGF-β1 significantly decreased, there were significant differences (P＜0.01); 5.The ratios of CD4+CD25+Foxp3+Treg and Th17 cell in CD4+T cell in splenocyte suspension of all groups:Compared with the normal control group, the ratio of CD4+CD25+Foxp3+Treg in CD4+T cell of EAE control group and the leptin treated group mice decreased significantly, the ratio of Th17 cell in CD4+T cell increased, there were significant differences (P<0.01); Compared with EAE control group, the ratio of CD4+CD25+Foxp3+Treg in CD4+T cell of the leptin treated group mice decreased, the ratio of Th17 cell in CD4+T cell increased, there were significant differences (P＜0.01).6.Correlation analysis:①The incubation period of mice in EAE control group and the leptin treated group were positive with serum IL-4, TGF-β1 and the ratio of CD4+CD25+Foxp3+Treg in splenocyte suspension (P<0.01); They were negative with serum IFN-β, IL-17 and the ratio of Th17 cell in splenocyte suspension (P<0.01). ①The progressive stage of mice in EAE control group and the leptin treated group were negative with serum IL-4, TGF-β1 and the ratio of CD4+CD25+Foxp3+Treg in splenocyte suspension (P<0.01); They were positive with serum IFN-y, IL-17 and the ratio of Th17 cell in splenocyte suspension (P<0.01). ③The peak of neurological dysfunction score of mice in EAE control group and leptin treated group were negative with serum IL-4, TGF-β1 and the ratio of CD4+CD25+Foxp3+Treg in splenocyte suspension (P<0.01); They were positive with serum IFN-y, IL-17 and the ratio of Th17 cell in splenocyte suspension (P<0.01). σThe pathological score of mice in EAE control group and leptin treated group were negative with serum IL-4, TGF-β1 and the ratio of CD4+CD25+Foxp3+Treg in splenocyte suspension (P<0.01); They were positive with serum IFN-y, IL-17 and the ratio of Th17 cell in splenocyte suspension (P<0.01). Conclusion:1.C57BL/6 mice were immunized with MOG35-55 to establish the model of EAE, EAE mice showed a significant nerve dysfunction, and the pathological sections showed obvious inflammatory cell infiltration around blood vessels and demyelination in white matter in both brain and spinal cord. That indicated the model was successful.2.EAE mice have T cells imbalance. The increase in the content of serum IFN-γ, IL-17 and the ratio of Th17 cell in splenocyte suspension, the decrease in the content of IL-4, TGF-β1 and the ratio of CD4+CD25+Foxp3+Treg in splenocyte suspension indicated an imbalance of Th1/Th2 cells and an imbalance of Th17/Treg cells in EAE mice.3.Leptin can shorten the incubation period of mice, prolong the progression and increase nerve dysfunction score, aggravate the inflammatory cell infiltration in brain and spinal cord, which indicated that leptin contributes to the pathogenesis of EAE mice. 4.Leptin can promote the differentiation of Th1 and Th17 in EAE mice, improve the expression of inflammatory factors IFN-γ and IL-17, inhibit the differentiation of Th2 and Treg and the secretion of anti-inflammatory cytokines IL-4, TGF-β1, making the balance of Th1/Th2 shifting to Th1, the balance of Th17/Treg shifting to Th17, that contributes to an aggravation of inflammation in EAE. |
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