Experimental Study On Ameliorating EAE By Acting PI3K/AKT/FoxO1 And IL-17 With BEZ235

Objective:To explore the mechanism of BEZ235 treating experimental autoimmune encephalomyelitis（EAE）by targeting PI3K/AKT/FoxO1.Methods:1.Sixty 4-week-old C57BL/6 female mice were used to induce EAE model with myelin oligodendrocyte glycoprotein（MOG35-55）and complete Freund’s adjuvant（CFA）.On the 14th day after immunization,mice were treated with PBS（5mg/kg/d）or BEZ235（5mg/kg/d）by gavage,mice treated with PBS were used as EAE group,and EAE mice treated with BEZ235 were used as EAE/BEZ235group.The clinical scores of mice in each group were observed,and the spinal cord was taken for histopathological section,H&E staining and Luxol fast blue（LFB）staining on the 46th day after immunization to observe the inflammatory cell infiltration and demyelination changes in the spinal cord tissue of mice.2.Western blotting was used to detect the expression of IL-17 and PI3K/AKT/FoxO1 in mouse spinal cord.3.On the 46th day after immunization,the spleen of mice in each group was made into a single cell suspension of spleen,and T cell apoptosis was detected by Annexin V and PI staining,Tregs ratio was detected by CD25/CD4/Foxp3 staining,initial T cell ratio was detected by CD62L staining,and effector T cell ratio was detected by CD44 staining.Seven days after stimulation with anti-CD3 or MOG_35-55,the proliferation of T cells were detected by Ki67 staining and the activation of T cells were detected by CD69 staining.Meanwhile,T cells were stained with TNF-αand IL-17,and the changes of cytokines TNF-αand IL-17 were analyzed by flow cytometry.The levels of cytokines TNF-α,IFN-γ,IL-17 and IL-10 in serum of mice in each group were detected by ELISA,and the levels of cytokines TNF-α,IFN-γ,IL-17 and IL-10 in supernatant of spleen cells stimulated by anti-CD3 or MOG_35-55were also detected.Results:1.On the 8th day after immunization,the neurological symptoms began to appear in the model mice,and the symptoms gradually worsened and the clinical score increased in the 10th to 23rd day.The PI3K inhibitor BEZ235 or PBS treatment（5.0 mg/kg/day）was administered,starting the 14th day and ending on the 46th day after immunization.Pathological sections of spinal cord were taken,and H&E staining and LFB staining showed that the spinal cord of mice in EAE group manifested obvious inflammatory infiltration and demyelination,while the inflammatory infiltration and demyelination decreased significantly after taking BEZ235 treatment.The scores of inflammatory infiltration and demyelination in EAE/BEZ235 group were significantly lower than those in EAE group（P<0.05）.2.Western blotting results showed that the protein levels of IL-17,p-PI3K and p-AKT in spinal cord of mice in EAE/BEZ235 group were significantly lower than those in EAE group,but the protein level of p-FoxO1 was significantly higher（p<0.05）.However,there was no significant difference between non-phosphorylated PI3K,AKT and FoxO1（P>0.05）.3.Flow cytometry analysis of spleen cells in EAE/BEZ235 group and EAE group showed that compared with EAE group mice,apoptosis of CD4 and CD8 T lymphocytes in spleen of EAE/BEZ235 group mice decreased significantly,and the proportion of Tregs increased,the proportion of initial T cells also increased,and the proportion of effector T cells decreased.The proliferation and activation of T lymphocytes decreased after anti-CD3 or MOG_35-55 stimulation for 7 days.TNF-α,IFN-γand IL-17 secreted by T lymphocytes in spleen of mice treated with BEZ235decreased significantly（P<0.05）.4.ELISA showed that the pro-inflammatory factors TNF-α,IFN-γand IL-17 in the peripheral blood plasma of EAE/BEZ235 group were significantly decreased,while the anti-inflammatory factor IL-10 was significantly increased.The cytokine content in the supernatant of spleen cells stimulated by anti-CD3 or MOG35-55 was consistent with the results in plasma.Conclusions:1.BEZ235 can effectively relieve symptoms and delay disease progression of treating EAE.2.BEZ235 can regulate the changes of PI3K/AKT/FoxO1 and IL-17;3.BEZ235 may inhibit the proliferation,activation and apoptosis of T cells,promote the differentiation of initial T cells towards Tregs,reduce the proportion of effector T cells,increase the proportion of initial T cells and Tregs,reduce the secretion of proinflammatory cytokines,increase the secretion of anti-inflammatory factors by regulating PI3K/AKT/FoxO1,and play an immunomodulatory role to ameliorate the process of EAE.