Investigating Protection Effects And Mechanisms Of Metformin In Experimental Autoimmune Encephalomyelitis(EAE)

Multiple sclerosis(MS)is an autoimmune disease characterized by chronic inflammation and demyelination of the central nervous system(CNS). Although the precise etiology of MS remains unclear, immoderate immunoreaction myelin-specific T helper 17(Th17) cells and dysfunction of regulatory T(Treg) cells, which exert immune tolerance function, are indicated to play an important role in its pathogenesis.Therefore, a therapeutic approach that suppresses inappropriate inflammation while promoting Treg responses could benefit patients with MS.Stimulated T lymphocytes can differentiate into distinct subsets of T cells such as effector T(Teff) or Treg cells, which requires different metabolic pathways to support their specific immunological functions. Accumulating evidence indicates that to meet the increased biosynthetic demands and generate sufficient energy, the metabolic processes of activated effector T cells shift to aerobic glycolysis to enhance energy while the quiescent and Treg cells gain energy by oxidative phosphorylation.The mammalian target of rapamycin(mTOR) plays a central role in regulating the cellular size, growth, proliferation, survival, and metabolism, as well as integrating various signals that promote helper T cell activation and differentiation.mTOR functions as a major regulator of glucose metabolism in activated lymphocytesand the downstream signaling molecule, hypoxia-inducible factor(HIF)-1α, which is an important regulator of Teff cell differentiation and development. HIF-1α also regulates multiple metabolic genes such as glucose transporter 1(Glut1), pyruvate kinase isozyme M2(PKM2), which have been identified as encoding enzymes involved in glycolysis and differentiation of effector T cells.Adenosine monophosphate(AMP)-activated protein kinase(AMPK) is a highly conserved cellular energy sensor, which is regulated by various metabolic stressors. AMPK and mTOR pathways serve as a signaling nexus in regulating cellular metabolism and immunity with opposing roles. AMPK inhibits the mTOR pathway through multiple mechanisms involving phosphorylation of the tuberous sclerosis 2(TSC2) protein of the TSC1/2 complex, which is a negative regulator of the mTORC1 pathway. In addition, AMPK phosphorylates raptor, which is a component of the rapamycin-sensitive mTORC1. AMPK has been shown to be associated with Th17 cell suppression by inhibiting mTOR and signal transducer and activator of transcription 3(STAT3). Furthermore, AMPK activation increased the percentage of airway-infiltrating Treg cells in a mouse model of asthma.Metformin(MET) is a biguanide family member widely used for the treatment of patients with type 2 diabetes mellitus, and its protective mechanism is attributed to activation of AMPK.In addition to hypoglycemic effects, metformin appears to have pleiotropic activities including anti-inflammatory, antioxidant, and the ability to facilitate the transformation of classically-activated macrophages to the alternative-activated form, as well as therapeutic effects in various disease.In the present study, we investigated the regulation of Th17/Treg immune responses by MET in C57BL/6 mice immunized with a myelin oligodendrocyte glycoprotein(MOG) peptide to induce experimental autoimmune encephalomyelitis(EAE) and explore possible mechanisms of the protective effects of MET. Part one Metformin ameliorates the development of experimental autoimmune encephalomyelitis by regulating T helper 17 and regulatory T cells in miceObjective: In C57BL/6 mice EAE model, to explore the diffierence of morbidity and inflammatory cells in the spinal cords between EAE group and MET-treatment group and observe the changes of two groups of Th17 cells and Treg cell percents as well as the corresponding characteristics of inflammatory factors, transcription factors.Methods:1 Induction, assessment, and treatment of EAE mice: Female C57BL/6 wild-type mice were injected subcutaneously with 250 μg of the MOG35-55 peptide emulsified in complete Freund’s adjuvant containing 4 mg/mL of heat-killed Mycobacterium tuberculosis. On day 0 and 48 h post-immunization, the mice were injected with 500 ng of pertussis toxin, intraperitoneally.2 The mice were randomly divided into EAE model group and MET treatment group randomly, the mice in MET treatment group were treated intraperitoneally with 100mg/kg(body weight) MET from day 1 to the end of the study. Another two groups were injected with vehicle saline only. The mice were weighed and scored for clinical signs daily up to the day of euthanasia.3 To examine the infiltration of inflammatory cells in the spinal cords stained with HE in different groups.4 To explore the different percents of Th17 cells and Treg cells in each group with flow cytometry.5 To examine concentrations of IL-10, IL-17 A, and TGF-β in supernatants in each groups by ELISA.6 To examine RORγt, Foxp3, IL-17 A, IL-10, and TGF-β mRNA transcripts in spleen and spinal cord in each groups using qPCR.7 SPSS16.0 software was used for the statistical analysis. The data are presented as mean ± standard deviation(SD). The onset rates of EAE between groups were analyzed by chi-square test. The difference in scores between two groups of mice was analyzed using the Mann–Whitney U test. All other statistical comparisons among groups were examined using the Student’s t-test or an analysis of variance(ANOVA) followed by Student-Newman-Keuls(SNK)-q multiple comparison tests. A value of P< 0.05 was considered significant.Results: 1 MET attenuates severity of EAE. The clinical scores in MET-treatment mice were significantly lower than in EAE mice and there were numerous inflammatory cells in the EAE group compared to the MET-treatment group, which exhibited milder signs of EAE.2 MET inhibited Th17 response in EAE. The percentage of Th17 cells in EAE mice was significantly higher than MET-treatment mice as determined by flow cytometry. IL-17 A and RORγt mRNA levels were increased in EAE mice compared with healthy control mice and that was attenuated by treatment with MET. The concentrations of IL-17 and its mRNA transcripts in antigen-stimulated splenic T cell suspensions and spleen tissues were reduced by MET treatment and the levels of IL-17 A and RORγt mRNA slao were reduced in the spinal cord of EAE mice by treatment with MET.3 MET promoted Treg response in EAE. The percentage of Treg cells in MET-treatment mice was significantly higher than EAE mice as determined by flow cytometry. IL-10,TGF-β 和 Foxp3 mRNA levels were increased in MET-treatment mice compared with EAE mice. The concentrations ofIL-10,TGF-β and their mRNA transcripts in antigen-stimulated splenic T cell suspensions and spleen tissues were increased by MET treatment and the levels of IL-10, TGF-βand Foxp3 m RNA slao were increased in the spinal cord of EAE mice by treatment with MET. Part two The characteristics of molecules involved in the metabolism of AMPK/mTOR signaling pathways in experimental autoimmune encephalomyelitisObjective: In different states of disease, to explore the characteristics of AMPK、HIF-1α、Glut1、PKM2 expressions in EAE model.Methods:1 Induction, assessment, and treatment of EAE mice: Female C57BL/6 wild-type mice were injected subcutaneously with 250 μg of the MOG35-55 peptide emulsified in complete Freund’s adjuvant containing 4 mg/mL of heat-killed Mycobacterium tuberculosis. On day 0 and 48 h post-immunization, the mice were injected with 500 ng of pertussis toxin, intraperitoneally.2 The mice were randomly divided into EAE model group and MET treatment group randomly, the mice in MET treatment group were treated intraperitoneally with 100mg/kg(body weight) MET from day 1 to the end of the study. Another two groups were injected with vehicle saline only. The mice were weighed and scored for clinical signs daily up to the day of euthanasia.3 In different states of EAE, to examine AMPK、qAMPK、HIF-1α proteins expression by WB and HIF-1α、Glut1、PKM2、IL-17、RORγt、Foxp3 mRNA transcripts by qPCR in spleen of EAE mice.4 SPSS16.0 software was used for the statistical analysis. The data are presented as mean ± standard deviation(SD). All other statistical comparisons among groups were examined using the Student’s t-test or an analysis of variance(ANOVA) followed by Student-Newman-Keuls(SNK)-q multiple comparison tests. A value of P< 0.05 was considered significant.Results: The alterations in the expression of critical molecules involved in cellular metabolism during the different states of EAE were examined. In the spleen tissues, RORγt and IL-17 mRNA transcripts increased in EAE mice compared with healthy control mice, and the expression of HIF-1α was higher in EAE mice than healthy control mice, achieving maximum level during the peak period of disease, and Glut1 and PKM2 mRNA transcripts increased in EAE mice compared with healthy control mice. Howere AMPK activity was reached peak during the remission stage, the transcription factor Foxp3, which regulates Treg cell differentiation, showed a similar pattern of mRNA levels. Part three The protection mechanism of metformin is inhibition of mTOR/HIF-1α signal pathwayObjective: To explore the alterations of molecules in mTOR/HIF-1α signal pathway in EAE group and MET treatment group.Methods:1 Induction, assessment, and treatment of EAE mice: Female C57BL/6 wild-type mice were injected subcutaneously with 250 μg of the MOG35-55 peptide emulsified in complete Freund’s adjuvant containing 4 mg/mL of heat-killed Mycobacterium tuberculosis. On day 0 and 48 h post-immunization, the mice were injected with 500 ng of pertussis toxin, intraperitoneally.2 The mice were randomly divided into EAE model group and MET treatment group randomly, the mice in MET treatment group were treated intraperitoneally with 100mg/kg(body weight) MET from day 1 to the end of the study. Another two groups were injected with vehicle saline only. The mice were weighed and scored for clinical signs daily up to the day of euthanasia.3 To examine AMPK、qAMPK、S6K1、qS6K1、HIF-1α、IL-17 proteins expression by WB and HIF-1α、Glut1、PKM2 mRNA transcripts by qPCR in spleen of EAE mice in EAE group and MET treatment group.4 SPSS16.0 software was used for the statistical analysis. The data are presented as mean ± standard deviation(SD). All other statistical comparisons among groups were examined using the Student’s t-test or an analysis of variance(ANOVA) followed by Student-Newman-Keuls(SNK)-q multiple comparison tests. A value of P< 0.05 was considered significant.Results: Treatment with MET enhanced the phosphorylation of AMPKα and subsequently blunted the phosphorylation of S6K1. Moreover, MET-treatment mice had lower expression levels of HIF-1α, Glut1, and PKM2 compared to EAE mice.Conclusions:1 MET attenuates severity of EAE. The clinical scores in MET-treatment mice were significantly lower than in EAE mice and there were numerous inflammatory cells in the EAE group compared to the MET-treatment group, which exhibited milder signs of EAE.2 Metformin ameliorates the development of experimental autoimmune encephalomyelitis by regulating T helper 17 and regulatory T cells in mice. MET inhibited Th17 response and promoted Treg response in peripheral immune organ as well as in central nervous system in EAE.3 The alterations of molecules in mTOR/HIF-1α signal pathway were associated with the progression of EAE. MET inhibited the phosphorylation of S6K1, and then suppress the HIF-1α、Glut1、PKM2 mRNA transcriptions. A decrease in mTOR activity and its downstream target molecules likely mediated the observed alterations in the proportion of Th17/Treg cell populations.