Study On The Therapeutic Effect Of Isoliquiritigenin On Experimental Autoimmune Encephalomyelitis

Background:Multiple sclerosis(MS)is an autoimmune neurodegenerative disease characterized by inflammatory infiltration of the central nervous system(CNS)and myelin loss.One of the most important factors of leading to neurological disability in middle-aged people is MS,which is bringing a heavy burden to the families and countries of its intractable.Experimental autoimmune encephalomyelitis(EAE),a classic animal model of MS,provides an important basis for the study of the pathogenesis and therapy of MS.Isoliquiritigenin(ILG),a flavonoid purified from the root of an ancient medicinal plant liquorice,has a excellent function of anti-inflammatory,antioxidant,neuroprotective and immunomodulatory effects.In previous animal studies,ILG can not only inhibit lps-induced inflammation,but also have therapeutic effects on alzheimer's disease(AD)and other central nervous degeneration.Objective:In this study,the EAE model successfully established by myelin oligodendrocyte glycoprotein(MOG)35-55 was used to explore the therapeutic effect of ILG on EAE,in order to provide the experimental basis for the clinical application of ILG in MS.Method:In this study,female C57BL/6 mice of 6-8 weeks clean grade were used to induce the EAE animal model by subcutaneous neck and back injection of MOG35-55 combined with Pertussis toxin(PT)intraperitoneal injection.Animals were randomly divided into 3 groups for 10 per group.The EAE+ILGprevent group was given ILG 20 mg/kg by gavage on the second day after modeling,the EAE+ILGtreatment group was given the drug at the onset of EAE,and the EAE+Vehicle group was given PBS with 2%DMSO.Two observers were assigned the behavioral score daily until animals were killed.Spinal cord tissues embedded in paraffin were sectioned and stained with hematoxylin-eosin(H&E)to reveal inflammatory cells infiltration and Luxol fast blue(LFB)to reveal demyelination.Flow cytometry was used to collect and analyze the changes of the proportion of Th1,Th17 and Treg cell subsets in the CNS and peripheral.And to explore the interaction between ILG and Th1 cell subsets also the relationship between ILG and astrocytes in vitro cell experiments.Results:(1)Compared with the EAE+Vehicle group,ILG significantly reduced the clinical behavior score(p<0.01 VS p<0.001),the total incidence score(p<0.05 VS p<0.01)and the maximum daily average score(p<0.05 VS p<0.001),and EAE+ILGprevent group was significantly better than the EAE+ILGtreatment group.(2)Compared with the EAE+Vehicle group,H&E staining illustrated that EAE+ILGprevent group significantly reduced CNS inflammatory cell infiltration(p<0.001),and LFB staining showed a significantly reduced of demylelination(p<0.001).(3)The results of flow cytometry analysis explained that in the CNS,the ratio of CD4+IFN?+ Th1 in EAE+ILGprevent group significantly lower(p<0.01)than EAE+Vehicle group,while the ratio of CD4+IL-17A+Th17increased(p<0.05).the ratio of CD4+CD25+Treg has no significant change.In the peripheral period,the proportion of CD4+IFN?+Th1 in EAE+ILGprevent group decreased significantly(p<0.01)and the proportion of CD4+IL-17A+Th17 not show a significant change.the ratio of CD4+CD25+Treg has no significant change,too.(4)It was found that after stimulate 24 hours under the present of Th1 polarization experiment and inflammatory factor existed,ILG gradually inhibited the proliferation and differentation of Th1 cells with the increase of dose,but had no effect on the apoptosis of Th1 cells.(5)The results of immunofluorescence staining revealed that in animal experiments,the EAE+ILGprevent group can promote the microglia and astrocytes activation(p<0.01).In cell experiments(vitro),ILG can activate astrocyte by the interaction between cells,rather than the direct effect,and ILG indirect effect can affect the expression of GFAP in reactive astrocytes,maybe the interaction between cells makes reactive astrocytes play an anti-inflammatory function at the present of ILG.Conclusion:(1)ILG can significantly reduce the severity of EAE,and reduce the degree of inflammatory cell infiltration and demylelination of CNS.(2)ILG can reduce the proportion of Th1 cells in CNS and peripheral in the EAE mouse.(3)ILG inhibited proliferation and differentiation.not apoptpsis of Th1 cells,In the polarization experiment of Th1 cells(vitro).(4)ILG can indirectly activate CNS"reactive astrocytes" and regulate its GFAP expression.