Effect Of LL-37 On Experimental Autoimmune Encephalomyelitis By Inhibiting Microglia Activation And Neuronal Ferroptosis

ObjectivesMultiple Sclerosis(MS)is a global,highly relapsing and disabling inflammatory demyelinating and degenerative disease of the central nervous system.The long-term efficacy of immunomodified drugs in MS remission period to prevent recurrence and cumulative disability is still not ideal.There are no original drugs in our country.As an important member of human endogenous immune peptide,LL-37 has not only strong antibacterial effect,but also important immunomodulatory function.Recent studies have shown that the immunomodulatory effects of LL-37 in different diseases,tissues and microenvironments are double-sided.At present,the role and possible mechanism of LL-37 in central nervous system autoimmune diseases remain unclear.Therefore,this study aims to explore the role and mechanism of LL-37 on the experimental autoimmune encephalomyelitis(EAE),the classic animal model of MS,and provide new ideas for the immunomodified treatment of MS.Methods1.EAE mouse models were constructed and randomly divided into four groups:healthy control group,EAE model group,LL-37 preventive treatment group.2.The effects of LL-37 on the onset and neurological function of EAE mice were evaluated by recording the changes in body weight,morbidity and clinical scores of mice in each group.3.Hematoxylin-eosin staining(HE)was used to detect the infiltration of spinal cord and encephalitis cells,and Luxol fast blue(LFB)was used to evaluate the degree of demyelination of spinal cord and brain.To determine the effect of LL-37 on central inflammation and myelin sheath in EAE.The activation of microglia and the proportion of M1 and M2 subtypes in spinal cord tissues were detected by flow cytometry to determine the effect of LL-37 on the activation and polarization of microglia in EAE.4.Immunofluorescence co-localization and Western blotting(WB)were used to qualitatively and quantitatively evaluate the key proteins in the ferroptosis signaling pathway in the spinal cord tissue of mice in each group.Results1.LL-37 prophylaxis significantly reduced the clinical morbidity(P < 0.001)and clinical scores(P<0.001)of EAE model mice.2.Preventive administration of LL-37 significantly inhibited inflammatory cell infiltration and improved demyelination in the brain and spinal cord of EAE mice(P< 0.001).3.Preventive administration of LL-37 significantly inhibited the activation of microglia in the brain and spinal cord of EAE mice(P < 0.001)and induced the polarization of microglia from pro-inflammatory M1 to anti-inflammatory M2(P <0.01).4.LL-37 significantly reduced the lipid iron content and peroxide level in the spinal cord tissue of EAE mice(P < 0.001),and down-regulated the expression levels of GPX4 and SCL7A11 proteins(P < 0.05).ConclusionLL-37 may effectively improve EAE by affecting the activation and polarization of microglia in the central nervous system and inhibiting ferroptosis.