Research On The Mechanism Of AXL-mediated Microglial Polarization In Experimental Autoimmune Encephalomyelitis And Screening Of Targeted Drugs

Background:The pathogenesis of multiple sclerosis（Multiple sclerosis,MS）is still unclear,and there is still a lack of effective drugs.At present,it has been proved that the polarization of microglia to the pro-inflammatory type aggravates the inflammatory demyelination injury,and the polarization to the neuroprotective type can promote the myelination of oligodendrocytes and reduce the axonal injury.AXL is a member of the TAM family of receptor tyrosine kinases.Studies have found that the binding of AXL and its ligand Gas6 initiates a downstream cascade reaction and regulates the function of microglial cells.However,the regulation of AXL on the polarization of microglial cells in MS has not yet been revealed.The previous study found that AXL was a negative immune regulator and plays a protective role in MS.The proportion of MG2 cells gradually increased during the recovery period of MS,suggesting that AXL can be a key regulator of MS microglial polarization.High-throughput drug screening is currently a widely used method in the development of targeted drugs,which has the advantages of a short research duration,simplified procedure,and practicality.The study intends to investigate the expression changes of AXL in MS in vivo and in vitro models and screen for drugs targeting AXL,to provide evidence of drug development and mechanism research for MS.Objective:（1）To explore whether AXL negatively regulates inflammation in the course of MS;（2）To explore whether AXL can regulate microglial polarization in vivo and in vitro and to clarify its molecular mechanism;（3）To screen drugs targeting AXL and alleviating MS based on FDA Drug library and explore the molecular mechanism.Methods:Part I:Exploring the effect of AXL on the polarization of microglia.EAE mice were constructed to simulate the course of MS in vivo.The key proteins differentially expressed in the EAE model were detected using DIA quantitative proteomics.The dynamic expression of AXL in the spinal cord was observed at the gene level in the course of EAE.The expression of AXL and MG1/MG2 phenotype in the spinal cord was localized by immunofluorescence.The expression of AXL in different polarization states of BV2 cells was observed.The effect of AXL on the polarization phenotype of microglia was assessed in vitro and in vivo,respectively.AXL^-/-mice were constructed and EAE was induced,and the effect of AXL gene knockout on the severity of EAE was observed.The signaling molecules differentially expressed in the spinal cord of AXL^-/-EAE mice were further mined by proteomic sequencing.Part II:Exploring the mechanism of AXL on microglia polarization.Using lentivirus transfection to construct BV2 cell lines stably overexpressing and knocking down AXL.The effects of AXL on the expression of inflammatory factors,MG1/MG2 phenotype,phagocytosis,and cell morphology in BV2 cells in vitro were observed.The differential protein expression downstream of AXL in omics results,and the effect of AXL on microglial polarization were verified.To explore the effect of AXL on microglial polarization in the EAE mice in vivo.AXL gene knockout mice were constructed to observe the effects of knockout AXL on neuroinflammation and demyelination in EAE mice,the effects of microglial polarization in spinal cord tissue,and the effects of differential protein expression in the downstream AXL in the spinal cord.The primary microglial cells of EAE mice were extracted,and the effect of AXL on the polarization of primary microglial cells was observed.The AXL ligand Gas6was used to simulate the overexpression of AXL,and the effects of activation and knockout of AXL on the degree of disease in EAE mice were observed in vivo.Part III:Screening the drugs that up-regulate AXL based on the FDA drug library to alleviate MS and exploring the curative effect.Using a high-throughput drug screening method,a BV2 cell drug screening model of the AXL fluorescent reporter gene was constructed,and compounds that could significantly up-regulate AXL and promote BV2 cell proliferation were screened from 2,769 FDA-approved compounds.Compare and preliminarily identify candidate drugs.The virtual molecular docking of the candidate drug with the AXL protein was carried out to observe its binding ability with AXL,and the compound with the best binding effect with AXL was selected for preliminary curative effect observation.The optimal concentration and intervention time of candidate drugs for upregulating AXL and promoting BV2 cell proliferation were identified,and the effects of candidate drugs on the BV2 cell cycle,apoptosis,mitochondrial morphology,inflammatory factor expression,polarized phenotype,phagocytosis,and cell morphology were observed.Two candidate drugs with definite in vitro effects were selected for preliminary in vivo curative effect observation.According to the curative effect observation results in vivo,a drug with the most significant curative effect was finally selected as the target drug,and the effect of the target drug on up-regulating AXL and promoting the polarization of BV2 to MG2 was explored.Using Gas6 in vivo to simulate the overexpression of AXL in vivo,compare the effect of the target drug and Gas6 on improving EAE,and the target drug can play a corresponding role after knocking out AXL.Results:Part I:AXL promotes MG2-type polarization of microglia.The EAE model can significantly simulate the inflammatory demyelination changes of MS.Proteomics results showed that AXL was significantly differentially expressed in the EAE model.With the progression of EAE,the expression of AXL gradually increased.Immunofluorescence results showed that the expression trend of AXL and MG2markers in spinal cord tissue was consistent.In vitro,studies showed that the expression of AXL in BV2 microglia decreased in MG1 type and increased in MG2type,and AXL could significantly promote the transformation of microglia from pro-inflammatory MG1 to anti-inflammatory MG2.Knocking out AXL in vivo can significantly aggravate the pathological changes of EAE,and SOCS3 and JAK2/STAT1 signaling pathways are key signaling pathways in AXL^-/-EAE models.Part II:AXL promotes microglial polarization in vitro and in vivo.In vitro,studies showed that AXL could significantly up-regulate the anti-inflammatory factors TGF-βand IL-10 in BV2 microglial cells,and down-regulate the expression of pro-inflammatory factors TNF-αand IL-1β.AXL can respectively promote the polarization of BV2 microglia and microglia in the spinal cord tissue of EAE model mice to MG2 type,and enhance their phagocytosis.The results of proteomics showed that SOCS3,JAK2,and STAT1 downstream of AXL had a significant differential expression,and AXL could reduce the inflammatory response of BV2 cells by up-regulating SOCS3 and inhibiting JAK2/STAT1 signaling.The results of in vivo studies showed that knocking out AXL significantly aggravated the neuroinflammation and demyelination changes in EAE mice,inhibited the polarization of microglia to MG2 in the spinal cord of EAE mice,and activated JAK2/STAT1 signaling by inhibiting SOCS3.AXL can significantly promote the polarization of primary osteoblasts in EAE mice to the MG2 type,and the activation of AXL simulated by Gas6 in vivo can significantly reduce the clinical symptom score,neuroinflammation,and demyelination changes of EAE.However,AXL knockdown can antagonize the protective effect of Gas6.Part III:Betulin is a compound that can significantly up-regulate AXL and play a negative regulatory role in inflammation.Four compounds were preliminarily identified in the primary screening and re-screening,and a drug with poor binding to AXL was excluded by molecular docking.Finally,Betulin and Clofibric were selected for efficacy observation.Both Betulin and Clofibric can promote BV2 cell proliferation,prolong the S phase,inhibit apoptosis,improve mitochondrial function,improve inflammatory response,promote MG2-type polarization and morphological transformation,and enhance phagocytosis.The results of in vivo studies showed that Betulin was better than Clofibric Acid in improving EAE.Betulin promotes the MG2polarization of BV2 cells in a concentration-dependent manner within 1μM,inhibits the expression of pro-inflammatory factors,promotes the expression of anti-inflammatory factors,and enhances the phagocytic function of BV2 cells.And play the above role by up-regulating AXL/SOCS3 to inhibit JAK2/STAT1 signaling pathway.Mechanism studies in vivo showed that Betulin can up-regulate AXL to promote the transformation of microglial cells into MG2 type and alleviate EAE,and AXL gene knockout can antagonize the above-mentioned effects of Betulin.Conclusions:（1）AXL is a key molecular target in the treatment of MS;（2）AXL can inhibit the JAK2/STAT1 signaling pathway by up-regulating SOCS3,promote the MG2 polarization of microglial cells,and reduce inflammation in EAE model mice Demyelinating lesions;（3）Betulin can significantly upregulate AXL in microglia and improve EAE,which is a promising compound in MS clinical research.