The Role And Mechanism Of 4-octyl Itaconate In Experimental Autoimmune Encephalomyelitis

ObjectivesMultiple sclerosis(MS)is an immune-mediated demyelinating chronic inflammatory disease of the central nervous system(CNS).Experimental autoimmune encephalomyelitis(EAE)is a classic animal model of MS,now it is widely used in research of MS.Microglia are the innate immune cells in CNS and have two different phenotypes,namely M1 and M2 phenotypes.Microglia of the M1 phenotype is responsible for the production of pro-inflammatory cytokines(TNF-α and IL-1β),while the M2 phenotype is responsible for the production of anti-inflammatory cytokines(IL-4 and IL-10).Previous studies have shown that inducing the transformation of microglia from M1 to M2 phenotype can help to improve neuroinflammation and alleviate the clinical symptoms of EAE.Although treatment options for MS have been widely studied,the development of treatments to prevent or reverse disease progression has been slow.Therefore,it is particularly important to explore the pathogenesis of MS and to find new therapeutic drugs.As an important metabolite of the Tricarboxylic acid cycle(TCA),itaconic acid has anti-bacterial,antiviral,anti-oxidant and anti-inflammatory effects.Dimmethyl itaconate(DMI)and 4-octyl itaconate(4-OI)are one of the new metabolites of itaconic acid,and both of them have good membrane permeability.At present,it has been proved that DMI can reduce the incidence of EAE and delay disease progression.However,DMI cannot be metabolized into itaconic acid in cells because it is easily degraded.However,4-OI can overcome this limitation and can be hydrolyzed into itaconic acid by cytoplasmic esterase in cells.Therefore,this study aims to explore the effect of 4-OI on EAE and its possible mechanism.When macrophages were activated,the expression of immune response gene1(IRG1)was increased,which could catalyze the decarboxylation of cisaconic acid to produce increased itaconic acid.Similar to itaconic acid,IRG1 also plays an important role in immune and inflammatory processes.However,the expression level of IRG1 in demyelinating diseases of the central nervous system and the interaction of IRG1-itaconic acid need to be further explored.Therefore,this study also explored the expression level of IRG1 in neuroinflammation,as well as the influence of 4-OI intervention on the expression of IRG1.MethodsThis study was divided into two main parts: in vivo and in vitro experiments.In vivo experimental part1.The animal model of EAE was established,and different doses of prophylactic administration were used to intervene the mouse model of EAE.The efficacy of 4-OI prophylactic administration was evaluated according to clinical score and incidence,and the optimal dose was determined.After selecting the optimal dose,the mice were grouped again,and the spinal cord tissue samples were collected at the peak period for subsequent mechanism study.2.Hematoxylin-eosin staining(HE)and Luxol fast blue(LFB)were used to detect spinal cord inflammatory cell infiltration and demyelination.Real-time Polymerase Chain Reaction(RT-PCR)was used to detect the expression changes of spinal cord inflammatory factors to determine the anti-inflammatory effect of 4-OI on EAE.3.Immunofluorescence staining of microglia and astrocyte markers,RT-PCR of microglia subtypes in spinal cord,and flow cytometry of the frequecies of microglia subtypes in microglia in brain and spinal cord were used to clarify the effect of 4-OI on microglial hyperactivation and polarization of EAE.4.Western blotting(WB)and RT-PCR were used to clarify the protein and transcription levels of IRG1 at the peak of EAE.The effect of 4-OI intervention on IRG1 expression in EAE was further explored by intervening 4-OI in EAE.In vitro experiment part1.The effects of different concentrations of 4-OI on the viability of BV2 cells were detected by cell counting kit-8(CCK-8),and the appropriate concentrations of 4-OI was selected for subsequent mechanism study.2.To explore the influence of 4-OI on the inflammatory expression of BV2,the M1 polarization model of BV2 microglia was used to intervene BV2 microglia with different concentrations of 4-OI.The morphology of BV2 cells was observed by microscope,and the expression of inflammatory factors was detected by RT-PCR and ELISA.3.The expression changes of M1-type related indicators in microglia were detected by WB and RT-PCR to clarify the influence of 4-OI on the polarization state of BV2.4.To explore the mechanism of 4-OI inhibiting the polarization of BV2 toward M1 by WB and PCR detection of pathway-related proteins.5.To clarify the effect of 4-OI on oxidative stress of BV2,ROS expression level were detected by Reactive Oxygen Species(ROS)kit.6.LPS activation model was used to detect the expression changes of IRG1 by RT-PCR and WB to explore the expression of IRG1 in neuroinflammatory cells.By using different doses of 4-OI to intervene the M1 polarization model of BV2 microglia,the effect of 4-OI intervention on the expression of IRG1 in the M1 direction polarization of BV2 microglia was further explored.Results:1.Both 50 mg/kg/d and 100 mg/kg/d 4-OI prophylactic administration significantly reduced the morbidity,clinical scores and improved the symptoms of neurological deficits in EAE mice.The low dose group of 4-OI can effectively reduce the morbidity of EAE mice,but cannot delay the disease progression,while the high dose group of 4-OI can make up for this defect and delay the disease progression.2.4-OI can inhibit spinal cord inflammatory cell infiltration and myelin loss in EAE mice,and down-regulate the expression of inflammatory factors in the spinal cord tissue of EAE mice.3.4-OI can inhibit microglia activation and astrocyte proliferation in the spinal cord of EAE mice,reduce the m RNA expression levels of i NOS,CD86 in the spinal cord,and decrease the proportion of M1 microglia in the spinal cord and brain tissue of EAE mice.4.Different concentrations of 4-OI did not significantly inhibit the viability of BV2 cells,so the morphological observation results showed that 4-OI intervened in the BV2 microglia polarization model using different concentrations of 4-OI could cause a shift from the activated state to the resting state when BV2 microglia were polarized.5.4-OI intervention down-regulated inflammatory factor expression during BV2 microglia polarization.6.4-OI inhibits differentiation of BV2 microglia toward M1 polarization by promoting Nrf2 protein entry into the nucleus,thereby activating the Nrf2/HO-1pathway.7.4-OI downregulates ROS production during BV2 microglial cell polarization.8.The protein content and m RNA expression levels of IRG1 were significantly increased in both the peak EAE model and the LPS-stimulated activation model of BV2 microglia,and the m RNA expression levels of IRG1 decreased after the administration of 4-OI intervention in vivo and in vitro,respectively.Conclusion:1.4-OI can decrease the severity of EAE via significantly inhibiting the hyperactivation and polarization state of microglia,which suggest that 4-OI may become a novel drug for the treatment of MS/EAE.2.4-OI can exert anti-inflammatory and antioxidant effects on M1 polarization of BV2 microglia,and its specific mechanism may be through promoting Nrf2 protein entry into the nucleus,thus activating the Nrf2/HO-1 pathway.3.IRG1 expression was elevated during neuroinflammation,and 4-OI intervention could down-regulate its expression level,suggesting that IRG1 may be a potential target in neuroinflammation.