The Roles And Mechanism Of Arctigenin In Experimental Autoimmune Encephalomyelitis

Background:Arctigenin is a phenylpropanoid dibenzylbutyrolactone lignan extracted from Arctium lappa(L.),and was reported to exhibit some pharmacological actions,including antidiabetes,antitumor,antioxidant,and neuron protective activities.However,the effects of arctigenin on central nervous system inflammatory diseases are still unclear.Experimental autoimmune encephalomyelitis(EAE)is an animal model thought to be a T cell-mediated chronic inflammatory demyelination disease.It is generally accepted that overactivation of CD4~+T cells especially Th1 and Th17subpopulations,is the direct cause of this disease.Methods:In vivo study,EAE were induced with myelin oligodendrocyte glycoprotein peptides and treated with two concentration of arctigenin or DMSO.Clinical scores of EAE mice were observed and recorded daily after immunization.The spinal cord inflammation and demyelination were measured by immunohistochemical staining with hematoxylin-eosin and luxol fast blue.The inflammatory cells infiltrations were also evaluated by immunohistochemical procedures.The effects of arctigenin on T cells from spleen,lymph node and spinal cords were detected by flow cytometry.We isolate the single cell suspension from the spleen,lymph node,and the spinal cords of DMSO-or Arctigenin-treated EAE mice by grinding and filtrating the tissue through the cell strainer.Surface and intracellular markers of cells were detected by flow cytometer.The expression of specified cytokine m RNAs were evaluated by quantitative PCR.The relative signaling pathways involving AMPK,phosphorylated p38,PPAR-γ,and RORγt were detected by Western Blot.Results:Arctigenin-treated mice are resistant to EAE,the clinical score of arctigenin-treated mice(5mg/kg,10mg/kg)is significantly reduced,histochemistry assay of spinal cord sections also showed that arctigenin-treated mice(5mg/Kg)developed milder inflammation and demyelination than DMSO-treated mice.The absolute number of Th1 and Th17 cell were decreased in the spleen and lymph node of arctigenin-treated mice upon MOG immunization,while Th1 cell in CNS did not have significant changes.The expression of Th1 and Th17 cell proliferation and polarizing cytokines are inhibited in arctigenin-treated mice spleen and lymph node.We detected Th1 characteristic cytokine IFN-γand transcription factor T-bet,and Th17 characteristic cytokines IL-17A,IL-17F,and transcription factor RORγt,all of them are significantly suppressed.Which has been verified in vitro using Jurkat cell treated with different concentration(15,25,50μM)of arctigenin.Meanwhile,western blot demonstrated that arctigenin upregulates AMPK and inhibits phosphorylated p38,in addation,arctigenin also upregulates PPAR-γand suppress RORγt.Conclusions:Our findings demonstrated that arctigenin suppress mouse CD4~+T helper cells proliferation and differentiation by inhibiting MAPK/p38 upregulating PPAR-γand suppressing RORγt,which ameliorate the symptom of mice EAE.These suggest that arctigenin have properties of anti-inflammation and immunosuppression,which could be a potential therapeutic drug for MS and other autoimmune inflammatory diseases.