Regulation Of Cornel Iridoid Glycoside On Microglia Activation In Experimental Autoimmune Encephalomyelitis Mice And Its Underlying Mechanisms Exploration

ObjectiveMultiple sclerosis(MS)is a chronic autoimmune disease,which characterized by inflammatory demyelination in the white matter of central nervous system(CNS).Microglia,an unique type of myeloid cell,is the most abundant resident macrophage population in the CNS and play a key role in the formation of mainly immune defence.Cornel iridoid glycoside(CIG),the main component of Cornus Officinalis,was extracted by our department(Department of Pharmacology,Xuan Wu Hospital of Capital Medical University,Beijing).Previous studys showed that CIG has anti-inflammatory,immunoregulation and neurotrophic effects.In order to explore the underlying mechanisms of CIG on immunoregulation,the present study will examine the anti-inflammatory effect in mice experimental autoimmune encephalomyelitis(EAE)model and in vitro cell model.Our results suggest that CIG might have beneficial effects for the treatment of CNS demyelinating diseases such as MS and provide new clue for drug development and research.Methods1.EAE model was induced by immunization of 18-20 grams female C57BL/6J mice with synthetic myelin oligodendrocyte glycoprotein(MOG)35-55 peptides.All mice were divided randomly into the following four groups:control group,EAE group obtained distilled water,EAE mice with CIG 50 or 100 mg/kg administration.The disease incidence,death proportion,body weight and neurological score of each mouse were observed everyday.All mice were sacrificed on day 24 post-immunization and removed the whole brain.Myelin sheath(markered with myelin basic protein antibody)and microglia(markered with Iba-1 antibody)were determined by immunohistochemical staining.2.In vitro cell model was induced by LPS combined with IFN-? in murine BV2 microglia cells.There are seven groups including control groups,control cell incubated with CIG(100 ? g/mL),LPS/IFN-Y induced model group,and CIG(25,50,100,200 ? g/mL)pretreatment then induced by LPS/IFN-? group.Microglia cells were pretreatment with or without CIG for 30 min then stimulated with LPS and IFN-? for 24 h.IL-6,IL-6R a,gp130,p-JAK1,JAK1,p-JAK3,JAK3,p-STAT1(Y701),p-STAT1(S727),STAT1,p-STAT3(Y705),p-STAT3(S727),STAT3,NF-?B,COX2,iNOS and ICAM-1 were analyzed by western blot assay.3.Another microglia activation model was induced by the same dose of LPS and IFN-? in murine N9 cells.The following six groups including control group,normal N9 cells incubated with CIG(100 ?g/mL),LPS/IFN-?induced model group,and CIG(50,100,200 ? g/mL)pretreatment then induced by LPS/IFN-Y group.N9 cells were pretreatment with or without CIG for 30 min and then stimulated with LPS and IFN-? for 24 h.p-STAT1(Y701),p-STAT1(S727),STAT1,p-STAT3(Y705),p-STAT3(S727)and STAT3 were evaluated by western blot assay.In addition,we also examined whether CIG could interfere the combination of LPS or IFN-Y with their receptors through immunofluorescent confocal microscopy.Minocycline was used as a positive control.Results1.Our results demonstrated that C57BL/6J mice induced by MOG35-55 peptide could establish classical EAE model successfully.CIG(100 mg/kg)administration decreased the neurological deficit severity obviously.Immunohistochemical staining showed that microglia activation was increased obviously in brain of EAE mice without demyeliantion.CIG treatment could inhibit the microglia activation.2.LPS/IFN-Y stimulated BV2 microglia cells leading to NF-?B and JAK/STAT signaling pathway activated.The expression of COX-2,iNOS and ICAM-1 were also detected.(1)NF-? B,an important nuclear transcription factor,combined with its ? B inhibitors(I-?B)in the cytoplasm unstimulated cells.In response to inflammatory stimulation,NF-? B dissociated from ?-? B protein and entryed into nuclear involved in gene transcription.Our results showed that CIG couldn' t inhibit NF-?B activation induced by LPS/IFN-? stimulation.(2)JAK belongs to the family of non-receptor protein tyrosine kinases,which activated under the stimulation of inflammation.JAK phosphorylated then stimulate STAT phosphorylation and regulate downstream gene transcription.Our results demonstrated that LPS and IFN-? could induced increased expression of 2 types of IL-6 receptors including IL-6R? and gp130,phosphorylates of JAK1,JAK3,STAT1,STAT3.CIG at 100 or 200 ?g/mL could inhibit expressions of IL-6R? and gp 130,reduce p-JAK1,p-JAK3,p-STAT1(Y701),p-STAT1(S727),p-STAT3(Y705),p-STAT3(S727)phosphorylation.(3)COX-2 is an important protein in inflammatory response induced.by LPS and peptidoglycan.iNOS is induced in response to LPS and proinflammatory cytokines,which catalyzed nitric oxide production.ICAM-1 is an adhesion molecule and playing an important role in inflammatory reaction.Our results showed that the expressions of COX-2,iNOS and ICAM-1 were increased markedly after LPS and IFN-? stimulation and CIG could inhibit ICAM-1 expression significantly.CIG treatments have no effect on COX-2 and iNOS expression.3.LPS/IFN-? induction showed similar results of STAT1 and STAT3 phosphorylations in N9 microglia cells.CIG could decrease p-STAT1(Y701),p-STAT1(S727),p-STAT3(Y705),p-STAT3(S727)phosphorylation significantly.Confocal assay showed that CIG could inhibit LPS or IFN-?combined with their receptors.ConclusionBoth in vivo and in vitro results demonstrated that CIG could regulate microglia activation,the underlying mechanism may go through IL-6 and its receptor mediated JAK/STAT signaling pathway.CIG may be used as a novel potential therapeutic agent for neuroinflammatory disorders including MS.