Magnetotactic bacteria (MTB) are a diverse group of prokaryotes capable of sensing geomagnetic fields through intracellular, membrane-enveloped magnetic organelles known as magnetosomes. In Magnetospirillum gryphiswaldense MSR-1, the mamXY operon encodes MamY, MamX, MamZ, and FtsZ-like protein, which are associated with magnetosome maturation through coordinated protein assembly. In this study, we performed a comparative analysis of protein association patterns related to the MamXY complex in wild-type (WT) and ftsZ-like overexpressing (overexp-ftsZ-like) strains using a modified SDS/boiling method with BS2G-d0/d4 cross-linking and liquid chromatography–tandem mass spectrometry (LC–MS/MS). The MamXY-associated protein profiles exhibited more regular and coordinated abundance patterns in the overexp-ftsZ-like strain compared to WT, and FtsZ-like overexpression was also associated with alterations in basal metabolic pathways. Comprehensive relative protein abundance profiling across different magnetic response states identified 47 differentially abundant proteins with consistent trends between the two strains. Among these, 10 magnetosome membrane proteins showed strong association with MamXY-related magnetosome formation, particularly during the protein recruitment stage, as supported by proteotypic peptide analysis. Together, our results provide a protein association-level framework for understanding MamXY-associated magnetosome formation and highlight coordinated protein patterns that may contribute to magnetosome morphology and size, warranting further functional validation.

Multiple sclerosis (MS) is characterized by inflammatory demyelination, oxidative stress, and neurological dysfunction. Despite disease-modifying therapies, chronic active lesions and ongoing neurodegeneration remain largely untreated, highlighting the need for mechanistically informed translational targets. We analyzed a human single-nucleus RNA-sequencing dataset (GSE279180) including healthy controls and MS samples. Astrocyte subpopulations were examined using reclustering, pseudotime inference, co-expression network analysis, SLC7A11-high/low stratification, virtual knockout prediction, and gene set enrichment analysis. Key findings were further assessed in vivo using a cuprizone-induced demyelination mouse model. Broad cellular remodeling was observed in MS lesions, including altered neuroactive ligand-receptor interactions, calcium signaling, glutamatergic synapse, and synaptic vesicle cycle-related pathways. SLC7A11 was enriched in astrocytes and increased along later pseudotime stages. SLC7A11-high astrocytes were linked to impaired antioxidant defense (GSH/GPX4) and synaptic vesicle-related remodeling. CPZ-treated mice showed decreased SLC7A11/GPX4 colocalization and increased SYP–GFAP colocalization, along with behavioral deficits, demyelination, and inflammatory-redox imbalance. SLC7A11-associated astrocyte states are associated with redox imbalance and synaptic microenvironment remodeling in MS. These states may inform future biomarker development and therapeutic investigation. Our findings provide a glial-state framework linking neuroinflammation, oxidative stress, and synaptic dysfunction, highlighting potential avenues for translational strategies in MS.

Autoimmune Hepatitis (AIH) and Multiple Sclerosis (MS) are chronic inflammatory diseases with abnormal immune responses. This study aims to identify common biomarkers for AIH and MS using bioinformatics analysis. Gene expression data of AIH (GSE159676) and MS (GSE131279 and GSE131281) were obtained from the GEO database. Differentially Expressed Genes (DEGs) were identified using the limma package in R. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, Protein-Protein Interaction (PPI) network, and machine learning algorithm Least Absolute Shrinkage and Selection Operator (LASSO) regression were used to evaluate potential biomarkers. The common biomarker gene Zinc Finger Protein Y-linked (ZFY) was identified. KEGG analysis showed significant enrichment of the Phosphatidylinositol 3-Kinase/Protein Kinase B (PI3K/Akt) pathway in both diseases. LASSO regression identified ZFY as a potential diagnostic marker, with decreased expression in both AIH and MS groups. Single-gene immune infiltration analysis indicated a significant association between ZFY expression and immune cell infiltration levels. Experimental validation in ConA-induced hepatitis and CPZ-mediated demyelination model mice further verified the diagnostic potential of ZFY. This study reveals the potential of ZFY as a biomarker for AIH and MS, highlighting its role in the PI3K/Akt pathway and immune infiltration. These findings provide new insights into the common pathological mechanisms of AIH and MS and suggest potential targets for future therapeutic strategies.

Protein function is inherently spatial: the same molecule can elicit distinct biological outcomes depending on its localization, interacting partners, and surrounding microenvironment. Spatial proteomics enables systematic in situ characterization of protein localization, abundance, and interactions across subcellular to tissue scales, surpassing the resolution and contextual information accessible to conventional bulk proteomics. Recent technological advances including DNA-barcoded multiplexing methods, cyclic fluorescence platforms, and mass spectrometry imaging have substantially increased multiplexing capacity, sensitivity, and spatial accuracy. These capabilities directly support clinically relevant applications, such as tumor immune microenvironment analysis, mapping of protein aggregation in neurodegeneration, growth factor dynamics during tissue repair, patient stratification, pharmacodynamic mapping, and target-engagement assessment. Computational innovations, including graph neural networks, self-supervised embeddings, and workflow management tools (e.g. Snakemake, Nextflow), further enhance cell segmentation, noise reduction, and multi-modal data integration, enabling extraction of robust, spatially resolved proteomic information from complex tissues. Future research will aim to standardize protocols, enable real-time clinical analysis, and develop 3D spatial proteome maps to advance spatial proteomics toward precision diagnostics and targeted therapies.

Iron response regulator (IrrB) in Magnetospirillum gryphiswaldense MSR-1, is a member of the ferric uptake regulatory (Fur) protein family and plays an important role in maintaining the balance between iron and oxygen metabolism in MSR-1. In this study, we conducted RNA-seq analysis to investigate the expression patterns of differentially expressed genes at various growth stages and under different iron conditions following irrB deletion. The RNA-seq analysis unveiled that IrrB exerts a more pronounced regulatory role under conditions of low iron availability compared to high iron levels. It primarily regulates cellular signal transduction, signal maintenance, and the metabolism of amino acids and inorganic ions for the maintenance of cellular homeostasis, as indicated by COG analysis. The ChIP-seq analysis revealed that IrrB has the ability to selectively bind to the promoter regions of 75 genes under low iron conditions. The integrated analysis of ChIP-seq and RNA-seq, coupled with in vitro validation using EMSA, demonstrated that IrrB exerts direct regulatory control over the genes MGMSRv2_0423, 2151, 2215, and irrC. Additionally, unlike Irr proteins in non-magnetic bacteria, MSR-1 IrrB also plays a role in processes such as amino acid metabolism and electron transport, while simultaneously regulating the expression of magnetosome island (MAI) genes. The results illustrate that within the framework of global OxyR regulation, MSR-1 IrrB works in conjunction with Fur and IrrC to finely regulate the balance between intracellular iron and oxygen. This process forms an integral component of a comprehensive regulatory network in MSR-1. This study exhibits the IrrB-OxyR-Fur regulatory network in MSR-1, which coregulates iron-oxygen homeostasis with magnetosome biosynthesis and metabolism, providing key insights for developing magnetosome-based biomedical nanomaterials.

The identical protein precursor encoded by the identical gene is capable of forming mature proteins with opposing phenotypes or distinct functions via different posttranslational modifications (PTMs), which alter its localization, interaction, and conformation. This substantially enhances the complexity and functionality of the proteome. Among these PTMs, glycosylation, being the most intricate and vital type, mediates the functional reversal of proteins from tumor suppression to tumor promotion through competitive modification and serves as the core regulatory factor of PTM-driven role reversal (PDRR). This functional reversal not only redefines disease subtypes but also creates an opportunity for precise treatment, presenting significant theoretical and clinical value. This article will focus on delineating the three levels of PDRR formation: the roles and specific regulatory instances of Topological Fate Switch (TFS), Biased Signaling Switch (BSS), and Phase/Oligomerization Switch (POS). Simultaneously, given the immense challenges in researching PTMs, it is essential to combine them with certain cutting-edge technologies to elevate the influence of PTMs on protein function from the theoretical level to technological highlights and integrate omics with spatial and single-cell analysis, live-cell imaging, and chemical biology. The relationship between PTMs and various physiological or pathological conditions is investigated at the level of individual cells or individual protein molecules. Owing to the crucial role of PTM enzymes in diseases, they have emerged as highly attractive drug targets, signifying the clinical translational value of the theory of PTMs and the three switches. That is, to link writer enzymes, eraser enzymes, and reader enzymes with measurable PTM profiles as drug targets and biomarkers, offering novel design and research concepts and directions for the development of new drugs.

BackgroundNeurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease, etc., are a group of complex and heterogeneous disorders characterized by progressive synaptic loss and pathological protein alterations. Mitochondria are the main source of energy produced by neurons and support the high energy consumption of the nervous system. Mitochondrial quality control, involving processes like mitophagy and mitochondrial biogenesis, is crucial for mitochondrial homeostasis, and mitochondrial dysfunction is closely related to neurodegenerative diseases pathogenesis, making targeting mitochondrial quality control a potential therapeutic strategy. Natural products offer benefits such as cost-effectiveness, fewer side effects, and other positive qualities, making them suitable choices as supplements or alternatives to traditional drugs for treating neurodegenerative diseases.MethodsA thorough search was conducted on many databases including Web of Science, PubMed, EMBASE, and MEDLINE to investigate the role of mitochondria in neurodegenerative diseases and the therapeutic effects of natural products.ResultsBy searching the relevant studies on neurodegenerative diseases and mitochondria in recent years, we observed a rise in the number of studies examining the functional characteristics and biological events of mitochondrial quality control systems in neurodegenerative diseases pathogenesis and the potential for natural products regulating mitochondrial quality control to improve neurodegenerative diseases.ConclusionThis review summarizes the functional characteristics and biological events of mitochondrial quality control systems in neurodegenerative diseases pathogenesis, and comprehensively analyzes the pharmacological mechanisms by which natural products regulate mitochondrial quality control to improve neurodegenerative diseases, aiming to provide a scientific basis for further research and new clinical drug development.

Bone marrow-derived mesenchymal stem cells (BMSCs) are promising candidates for treating autoimmune diseases like multiple sclerosis (MS) due to their ability to differentiate into multiple lineages and their immunomodulatory properties. However, the immunomodulatory capacity of BMSCs is highly adaptable, and primarily regulated by inflammatory factors. In this study, we evaluated the therapeutic effectiveness of BMSCs in dexamethasone (DEX)-pretreated experimental autoimmune encephalomyelitis (EAE) mice. Our results demonstrated a significant interaction between DEX and BMSCs. In contrast to their effect in non-pretreated mice, BMSCs administration in DEX-pretreated EAE mice resulted in a significant increase in infiltrating CD4+ T cells and a concomitant decrease in regulatory T (Treg) cell populations in the central nerves system, which likely resulted from DEX-induced changes in the peripheral immune microenvironment. Our findings in the EAE model indicate that the immune microenvironment established by DEX pretreatment is detrimental to the efficacy of BMSC therapy. This preclinical evidence suggests that evaluating the peripheral immune status may be a critical consideration for future clinical studies of MSC treatment in MS.

Oxidation of edible oil, especially those rich in polyunsaturated fatty acids, remains an inevitable problem. Since synthesis antioxidants may have some side effects, countries have been encouraging the development of natural alternatives. Polyphenols are natural compounds demonstrating notable potential in mitigating oil oxidation, but the effectiveness of polyphenols in inhibiting oil oxidation seems to be influenced by their antioxidant mechanisms, components, solubility, and application forms. To promote polyphenol application in oils, the present study aims to provide a comprehensive summary of the antioxidant mechanism of polyphenols in vitro, the common polyphenols employed to inhibit oil oxidation, and the pivotal technologies for incorporating polyphenols with low-fat solubility into oils including esterification modification, co-extraction of polyphenols and oils, nano-emulsion, microcapsules, and oleogels. In addition, a strengths, weaknesses, opportunities, and threats analysis of polyphenol application in oil was conducted. This review will provide a guidance for the application of polyphenols in oils.

Astrocytes play a key role in the occurrence and development of ischemic stroke. However, reactive astrocytes have both detrimental and protective roles in ischemic stroke. Regrettably, the stimulation signals associated with the transformation of astrocytes into different subclusters lack systemic analysis, and the mechanism by which astrocytes produce multiple effects is not entirely clear. We investigated the heterogeneity of mouse astrocytes 12 h after cerebral ischemia-reperfusion via Single-cell RNA sequencing and verified gene expressions by reverse transcription-polymerase chain reaction. We acquired astrocyte subclusters’ transcriptional characteristics involved in diversified functions. To explore what stimulus signals cause astrocyte heterogeneity, we present a blueprint for cellular communication between astrocyte subclusters and other surrounding brain cells 12 h after ischemia-reperfusion, and identified 9 genes which are potential and promising for being therapeutic targets and 6 genes were specific to astrocyte subcluster 2 that tend to resist ischemia-reperfusion injury. At 12 h after ischemia-reperfusion, each subcluster of astrocytes is characteristic in terms of function and communication with surrounding cells, which is based on the activation genes and transcription molecules that we have revealed with subcluster characteristics. Our results provide a basis for revealing the anti-injury response of astrocytes to cerebral ischemia-reperfusion, which involves coordination of different subclusters and the coordination of astrocytes with surrounding brain cells.

Abstract Background Toxoplasma gondii (T. gondii) is one of the most prevalent parasites worldwide. At present, the majority of drugs used for the treatment of toxoplasmosis target the tachyzoite stage of T. gondii and are largely ineffective against bradyzoites. Furthermore, these treatments are typically accompanied by adverse events. Consequently, there is an urgent need for the development of novel drugs that are both safe and effective against T. gondii. Methods A total of 20 flavonoids were preliminarily screened for their anti-T. gondii activity using microscopy. Next, the cell counting kit (CCK)-8 method was employed to assess the toxicity of glabridin (GLA) to host cells, while the RH strain of T.0gondii, which expresses β-galactosidase, was utilized to evaluate the inhibitory, anti-invasive, and antiproliferative effects of GLA on T. gondii. In addition, the Prugniaud (PRU) strain was employed to investigate the impact of GLA on the bradyzoites of T. gondii. Subsequently, the effect of GLA on the ultrastructure of T. gondii was examined via transmission electron microscopy (TEM), followed by an assessment of the influence of GLA on the autophagy and mitochondria of T. gondii through monodansylcadaverine (MDC), MitoTracker™ red CMXRos, and CM-HDCFDA and MitoSOX Red staining. Results Among the 20 flavonoids assessed, GLA exhibited the most potent anti-T. gondii activity. Indeed, it significantly inhibited both the invasive and proliferative abilities of T. gondii, thereby disrupting its lytic cycle. Moreover, GLA markedly reduced the number of bradyzoites and concurrently inhibited cyst growth. Meanwhile, ultrastructural analysis revealed that GLA induced mitochondrial swelling, membrane rupture, and autophagy in T. gondii. Finally, fluorescent probe staining provided further evidence that GLA triggers mitochondrial dysfunction and autophagy in this parasite. Conclusions Our findings collectively indicate that the flavonoid compound GLA exhibits significant activity against both T. gondii tachyzoites and bradyzoites. The underlying mechanism of action potentially involves the induction of autophagy and mitochondrial dysfunction and the disruption of the membrane of T. gondii, thereby offering new avenues for treating toxoplasmosis and establishing a theoretical reference for future research. Graphical Abstract

Walnut kernel pellicle, a by-product of the walnut industry, is rich in polyphenols. However, research on walnut kernel pellicle polyphenols (WPP) remains limited, and the composition and bioactivity of WPP are influenced by extraction methods, gastrointestinal digestion, and colonic fermentation. Therefore, this study aimed to optimize subcritical water extraction (SWE) and NKA-9 resin purification of WPP. Next, the changes in WPP throughout simulated gastrointestinal digestion and colonic fermentation were evaluated. The results demonstrated that SWE achieved higher polyphenol yield compared to ultrasound-assisted extraction, with ellagic acid and ellagitannins as the main components. Although digestion and fermentation reduced WPP contents and antioxidant activities, they facilitated the biotransformation of ellagic acid and ellagitannins into urolithins. Meanwhile, WPP modulated the gut microbiota by promoting beneficial bacteria. Additionally, WPP enhanced beneficial microbial metabolites, including short-chain fatty acids and indole derivatives. These findings highlighted the potential of WPP as a functional ingredient for gut health.

The present study aimed to investigate the potential role and molecular mechanism of procyanidin B2 (PCB2) in the treatment of multiple sclerosis (MS), with the hope of providing novel strategies for the precision treatment of MS. The target genes of PCB2 and the disease-related target genes of MS were gathered from databases like GEO, GeneCards, OMIM, and DisGeNET. R language was employed for the identification of differentially expressed genes (DEGs), unsupervised clustering analysis, immune infiltration analysis, weighted gene co-expression network analysis (WGCNA), Gene Ontology (GO) analysis, and gene set enrichment analysis (GSEA). Eight machine learning algorithms were employed to screen key genes, and nomograms and ROC curves were constructed to assess the value of the screened biomarker genes in MS diagnosis. Through these analyses, four key genes closely associated with the pathogenesis of MS were selected, namely potassium ion channel subfamily H member 2 (KCNH2), prostaglandin-endoperoxide synthase 1 (PTGS1), estrogen receptor 1 (ESR1), and vascular endothelial growth factor A (VEGFA). These genes were significantly enriched in biological processes related to oxidative stress and served as potential targets for PCB2 in treating MS. Moreover, a key gene-transcription factor (TFs)-microRNA (miRNA) regulatory network was established to preliminarily explore the upstream regulatory mechanisms of these genes. Meanwhile, molecular docking and single-gene GSEA enrichment analysis were carried out to verify the interaction between PCB2 and these key genes. Finally, a CPZ (cuprizone)-induced mouse model of MS was set up, and the effects of PCB2 in MS treatment were verified by means of behavioral tests, pathological staining, immunofluorescence staining, ELISA, RT-PCR, and Western blot detection methods. PCB2 significantly improved behavioral performance in CPZ-induced MS mice, including enhanced motor coordination, reduced anxiety and hyperactivity, and improved spatial learning and memory. Additionally, PCB2 could promote myelin repair and decrease the levels of inflammatory response and oxidative stress in the mouse brain. Specifically, the expression levels of inflammatory factors such as TNF-α, IL-1β, and IL-6 decreased significantly, while the expression level of the anti-inflammatory factor IL-10 increased, and the activities of antioxidant enzymes like CAT, SOD, and GSH-Px increased notably. More importantly, PCB2 could upregulate the expression levels of the KCNH2, PTGS1, ESR1, and VEGFA genes and their related proteins, which may played significant roles in biological processes related to oxidative stress. This study disclosed the potential role of PCB2 in MS treatment by regulating the expression of key genes, providing a theoretical basis and potential therapeutic targets for personalized treatment of MS. PCB2 demonstrates good application prospects in MS treatment. Future research will further validate the clinical application value of these key genes and deeply explore the specific mechanism of PCB2 in MS treatment, aiming to develop more effective treatment regimens for MS.

BACKGROUND:Multiple sclerosis (MS) is marked by inflammatory demyelination in the central nervous system (CNS), involving diverse glial populations. This pathological process is associated with inflammation and oxidative stress. Proanthocyanidin B2 (PCB2), with its potent antioxidant properties, has been shown to alleviate demyelination in the cuprizone (CPZ) mouse model. It attenuates neuroinflammation and oxidative stress in both the cerebral microenvironment and astrocytes (AS). The xCT/GSH/GPX4 axis is a key regulatory pathway for oxidative stress. Therefore, we hypothesize that PCB2 can alleviate CPZ-induced demyelination by regulating the xCT/GSH/GPX4 axis in AS. METHODS:The study utilized forty C57BL/6 mice, randomly allocated into four groups of ten: a control group, a control group supplemented with PCB2 (60 mg/kg/day), a CPZ-exposed group, and a CPZ-exposed group supplemented with PCB2 (60 mg/kg/day). The control groups received a standard diet, whereas the CPZ groups were given the same diet supplemented with 0.2% CPZ for 6 weeks. From the fifth week onwards, the control and CPZ groups were administered physiological saline via intraperitoneal injection, whereas the PCB2-supplemented groups received PCB2 for 2 weeks. Immunofluorescence staining, Western blot, and ELISA elucidated the cellular/molecular mechanisms of PCB2 targeting the xCT/GSH/GPX4 axis in AS to alleviate demyelination in vivo and in vitro. RESULTS:In this study, PCB2 markedly regulated the xCT/GSH/GPX4 axis in AS, ameliorated the behavioral performance in CPZ mice, reduced inflammation, oxidative stress, lipid peroxidation, and the damage to oligodendrocytes (OLs), and inhibited demyelination. CONCLUSION:PCB2 can regulate the entire xCT/GSH/GPX4 axis of AS to reduce CPZ-induced OL injury and demyelination, which may be a potentially effective drug for the treatment of multiple sclerosis.

IntroductionBlood proteomics offers a powerful approach for identifying disease-specific biomarkers. However, no reliable blood markers are currently available for the diagnosis stroke. Nervonic acid (NA), a vital long-chain monounsaturated fatty acid found in mammalian nervous tissue, shows promising therapeutic potential in neurological disorders. This study aimed to develop a reliable methodology for whole blood proteomics to identify early warning biomarkers and evaluate drug treatment efficacy.MethodsAfter modeling via the classic thread embolization method, whole blood samples were collected from the rats. Morphological assessments of brain tissue indicated that NA significantly mitigated brain and neuronal damage in rats. The differential protein expression profile was analyzed using Liquid Chromatography—Tandem Mass Spectrometry (LC-MS/MS) whole blood proteomics.ResultsZZZGene Ontology (GO) analysis revealed that, compared to ginkgo biloba extract (EGb), the proteins differentially expressed under NA intervention were predominantly involved in oxidative stress response and calcium-dependent adhesion processes. Key targets of NA in the treatment of middle cerebral artery occlusion (MCAO) models included ENO1, STAT3, NME2, VCL, and CCT3.DiscussionThis whole blood proteomic approach provides a comprehensive understanding of protein profiles associated with disease states, offering valuable insights into potential therapeutic targets and enabling the evaluation of NA and EGb intervention efficacy. Our findings underscore the protective effects of NA against cerebral ischemia-reperfusion injury and highlight its potential as a treatment for stroke.

Background: In our previous studies, we have identified Gsk-3β as a crucial target molecule in response to Danhong injection for cerebral ischemia intervention. Furthermore, it can serve as a molecular imaging probe for medical diagnosis. Bacterial magnetic particles (BMPs), synthesized by magnetotactic bacteria, are regarded as excellent natural nanocarriers. Methods: In this study, we utilized biological modification and chemical crosslinking techniques to produce a multifunctional BMP known as "RVG29-BMP-FA-Gsk-3β-Ab", which exhibits both magnetic properties and brain-targeting capabilities. Then, a combination of analytical techniques was used to characterize the properties of the multifunctional BMPs. Finally, we evaluated the cell targeting ability of the RVG29-BMP-FA-Gsk-3β-Ab. Results: The multifunctional BMPs were observed to possess uniform size and shape using TEM analysis, with a particle size of 70.1±7.33 nm. Zeta potential analysis revealed that the nanoparticles exhibited a regular and non-aggregative distribution of particle sizes. Relative fluorescence intensity results demonstrated that the complex of 1mg of RVG29-BMP-FA-Gsk- 3β-Ab could bind to FITC-RVG29 polypeptide at a concentration of 2189.5 nM. Cell viability analysis indicated its high biocompatibility and minimal cytotoxicity. The RVG29-BMP-FAGsk- 3β-Ab was observed to possess active targeting towards neuronal cells and fluorescence imaging capabilities in vitro, as evidenced by fluorescence imaging assays. The complex of RVG29-BMP-FA-Gsk-3β-Ab exhibited favourable properties for early diagnosis and efficacy evaluation of traditional Chinese medicine in treating cerebral ischemia. Conclusion: This study establishes a fundamental basis for the prospective implementation of multimodal imaging in traditional Chinese medicine for cerebral ischemia.

The complex composition of traditional Chinese medicines (TCMs) has posed challenges for in-depth study and global application, despite their abundance of bioactive compounds that make them valuable resources for disease treatment. To overcome these obstacles, it is essential to modernize TCMs by focusing on precise disease treatment. This involves elucidating the structure-activity relationships within their complex compositions, ensuring accurate in vivo delivery, and monitoring the delivery process. This review discusses the research progress of TCMs in precision disease treatment from three perspectives: spatial multi-omics technology for precision therapeutic activity, carrier systems for precise in vivo delivery, and medical imaging technology for visualizing the delivery process. The aim is to establish a novel research paradigm that advances the precision therapy of TCMs.

The treatment of immunomodulation in multiple sclerosis (MS) can alleviate the severity and relapses. However, it cannot improve the neurological disability of patients due to a lack of myelin protection and regeneration. Therefore, remyelinating therapies may be one of the feasible strategies that can prevent axonal degeneration and restore neurological disability. Natural product icariin (ICA) is a flavonol compound extracted from epimedium flavonoids, which has neuroprotective effects in several models of neurological diseases. Here, we attempt to explore whether ICA has the potential to treat demyelination and its possible mechanisms of action using lipopolysaccharide-treated BV2 microglia, primary microglia, bone marrow-derived macrophages, and cuprizone-induced demyelination model. The indicators of oxidative stress and inflammatory response were evaluated using commercial kits. The results showed that ICA significantly reduced the levels of oxidative intermediates nitric oxide, hydrogen peroxide, malondialdehyde, and inflammatory cytokines TNF-α, IL-1β, and increased the levels of antioxidants superoxide dismutase, catalase, glutathione peroxidase, and anti-inflammatory cytokines IL-10 and TGF-β in vitro cell experiments. In vivo demyelination model, ICA significantly alleviated the behavioral abnormalities and enhanced the integrated optical density/mm 2 of Black Gold II and myelin basic protein myelin staining, accompanied by the inhibition of oxidative stress/inflammatory response. Immunohistochemical staining showed that ICA significantly induced the expression of nuclear factor erythroid derived 2/heme oxygenase-1 (Nrf2/HO-1) and inhibited the expression of toll-like receptor 4/ nuclear factor kappa B (TLR4/NF-κB), which are two key signaling pathways in antioxidant and anti-inflammatory processes. Our results strongly suggest that ICA may be used as a potential agent to treat demyelination via regulating Nrf2/HO-1-mediated antioxidative stress and TLR4/NF-κB-mediated inflammatory responses.

Multiple sclerosis is characterized by demyelination and neuronal loss caused by inflammatory cell activation and infiltration into the central nervous system. Macrophage polarization plays an important role in the pathogenesis of experimental autoimmune encephalomyelitis, a traditional experimental model of multiple sclerosis. This study investigated the effect of Fasudil on macrophages and examined the therapeutic potential of Fasudil-modified macrophages in experimental autoimmune encephalomyelitis. We found that Fasudil induced the conversion of macrophages from the pro-inflammatory M1 type to the anti-inflammatory M2 type, as shown by reduced expression of inducible nitric oxide synthase/nitric oxide, interleukin-12, and CD16/32 and increased expression of arginase-1, interleukin-10, CD14, and CD206, which was linked to inhibition of Rho kinase activity, decreased expression of toll-like receptors, nuclear factor-κB, and components of the mitogen-activated protein kinase signaling pathway, and generation of the pro-inflammatory cytokines tumor necrosis factor-α, interleukin-1β, and interleukin-6. Crucially, Fasudil-modified macrophages effectively decreased the impact of experimental autoimmune encephalomyelitis, resulting in later onset of disease, lower symptom scores, less weight loss, and reduced demyelination compared with unmodified macrophages. In addition, Fasudil-modified macrophages decreased interleukin-17 expression on CD4+ T cells and CD16/32, inducible nitric oxide synthase, and interleukin-12 expression on F4/80+ macrophages, as well as increasing interleukin-10 expression on CD4+ T cells and arginase-1, CD206, and interleukin-10 expression on F4/80+ macrophages, which improved immune regulation and reduced inflammation. These findings suggest that Fasudil-modified macrophages may help treat experimental autoimmune encephalomyelitis by inducing M2 macrophage polarization and inhibiting the inflammatory response, thereby providing new insight into cell immunotherapy for multiple sclerosis.