Genetic variation in ZmWAX2 confers maize resistance to Fusarium verticillioides.

Fusarium verticillioides (F. verticillioides) is a widely distributed phytopathogen that incites multiple destructive diseases in maize, posing a grave threat to corn yields and quality worldwide. However, there are few reports of resistance genes to F. verticillioides. Here, we reveal that a combination of two single nucleotide polymorphisms (SNPs) corresponding to ZmWAX2 gene associates with quantitative resistance variations to F. verticillioides in maize through a genome-wide association study. A lack of ZmWAX2 compromises maize resistance to F. verticillioides-caused seed rot, seedling blight and stalk rot by reducing cuticular wax deposition, while the transgenic plants overexpressing ZmWAX2 show significantly increased immunity to F. verticillioides. A natural occurrence of two 7-bp deletions within the promoter increases ZmWAX2 transcription, thus enhancing maize resistance to F. verticillioides. Upon Fusarium stalk rot, ZmWAX2 greatly promotes the yield and grain quality of maize. Our studies demonstrate that ZmWAX2 confers multiple disease resistances caused by F. verticillioides and can serve as an important gene target for the development of F. verticillioides-resistant maize varieties.

Sulfur dioxide improves the thermotolerance of maize seedlings by regulating salicylic acid biosynthesis.

Heat stress (HS) has become a serious threat to crop growth and yield. Sulfur dioxide (SO2) is being verified as a signal molecule in regulating the plant stress response. However, it is unknown whether SO2 plays a significant role in the plant heat stress response (HSR). Herein, maize seedlings were pretreated with various concentrations of SO2 and then kept at 45 °C for heat stress treatment, aiming to study the effect of SO2 pretreatment on HSR in maize by phenotypic, physiological, and biochemical analyses. It was found that SO2 pretreatment greatly improved the thermotolerance of maize seedlings. The SO2-pretreated seedlings showed 30-40% lower ROS accumulation and membrane peroxidation, but 55-110% higher activities of antioxidant enzymes than the distilled water-pretreated seedlings under heat stress. Interestingly, endogenous salicylic acid (SA) levels were increased by ∼85% in SO2-pretreated seedlings, as revealed by phytohormone analyses. Furthermore, the SA biosynthesis inhibitor paclobutrazol markedly reduced SA levels and attenuated SO2-triggered thermotolerance of maize seedlings. Meanwhile, transcripts of several SA biosynthesis and signaling, and heat stress-responsive genes in SO2-pretreated seedlings were significantly elevated under HS. These data have demonstrated that SO2 pretreatment increased endogenous SA levels, which activated the antioxidant machinery and strengthened the stress defense system, thereby improving the thermotolerance of maize seedlings under HS. Our current study provides a new strategy for mitigating heat stress damage for safe crop production.

Enhancing maize's nitrogen-fixing potential through ZmSBT3, a gene suppressing mucilage secretion.

Maize (Zea mays) requires substantial amounts of nitrogen, posing a challenge for its cultivation. Recent work discovered that some ancient Mexican maize landraces harbored diazotrophic bacteria in mucilage secreted by their aerial roots. To see if this trait is retained in modern maize, we conducted a field study of aerial root mucilage (ARM) in 258 inbred lines. We observed that ARM secretion is common in modern maize, but the amount significantly varies, and only a few lines have retained the nitrogen-fixing traits found in ancient landraces. The mucilage of the high-ARM inbred line HN5-724 had high nitrogen-fixing enzyme activity and abundant diazotrophic bacteria. Our genome-wide association study identified 17 candidate genes associated with ARM across three environments. Knockouts of one candidate gene, the subtilase family gene ZmSBT3, confirmed that it negatively regulates ARM secretion. Notably, the ZmSBT3 knockout lines had increased biomass and total nitrogen accumulation under nitrogen-free culture conditions. High ARM was associated with three ZmSBT3 haplotypes that were gradually lost during maize domestication, being retained in only a few modern inbred lines such as HN5-724. In summary, our results identify ZmSBT3 as a potential tool for enhancing ARM, and thus nitrogen fixation, in maize.

Evaluation and Identification of Resistance Lines and QTLs of Maize to Seedborne Fusarium verticillioides.

Internal fungal contamination in cereal grains may affect plant growth and result in health concerns for humans and animals. Fusarium verticillioides is a seedborne fungus that can systemically infect maize. However, few efforts had been devoted to studying the genetics of maize resistance to seedborne F. verticillioides. In this study, we developed a disease evaluation method to identify resistance to seedborne F. verticillioides in maize, by which a set of 121 diverse maize inbred lines were evaluated. A 160 F10-generation recombinant inbred line (RIL) population derived from a cross of the resistant (BT-1) and susceptible (N6) inbred line was further used to identify major quantitative trait loci (QTLs) for seedborne F. verticillioides resistance. Eighteen inbred lines with a high resistance to seedborne F. verticillioides were characterized and could be used as potential germplasm resources for genetic improvement of maize resistance. Six QTLs with high heritability across multiple environments were detected on chromosomes 3, 4, 6, and 10, among which was a major QTL, qISFR4-1. Located on chromosome 4 at the interval of 12922609-13418025, qISFR4-1 could explain 16.63% of the total phenotypic variance. Distinct expression profiles of eight candidate genes in qISFR4-1 between BT-1 and N6 inbred lines suggested their pivotal regulatory roles in seedborne F. verticillioides resistance. Taken together, these results will improve our understanding of the resistant mechanisms of seedborne F. verticillioides and would provide valuable germplasm resources for disease resistance breeding in maize.

A new adenylyl cyclase, putative disease-resistance RPP13-like protein 3, participates in abscisic acid-mediated resistance to heat stress in maize.

In plants, 3´,5´-cyclic adenosine monophosphate (cAMP) is an important second messenger with varied functions; however, only a few adenylyl cyclases (ACs) that synthesize cAMP have been identified. Moreover, the biological roles of ACs/cAMP in response to stress remain largely unclear. In this study, we used quantitative proteomics techniques to identify a maize heat-induced putative disease-resistance RPP13-like protein 3 (ZmRPP13-LK3), which has three conserved catalytic AC centres. The AC activity of ZmRPP13-LK3 was confirmed by in vitro enzyme activity analysis, in vivo RNAi experiments, and functional complementation in the E. coli cyaA mutant. ZmRPP13-LK3 is located in the mitochondria. The results of in vitro and in vivo experiments indicated that ZmRPP13-LK3 interacts with ZmABC2, a possible cAMP exporter. Under heat stress, the concentrations of ZmRPP13-LK3 and cAMP in the ABA-deficient mutant vp5 were significantly less than those in the wild-type, and treatment with ABA and an ABA inhibitor affected ZmRPP13-LK3 expression in the wild-type. Application of 8-Br-cAMP, a cAMP analogue, increased heat-induced expression of heat-shock proteins in wild-type plants and alleviated heat-activated oxidative stress. Taken together, our results indicate that ZmRPP13-LK3, a new AC, can catalyse ATP for the production of cAMP and may be involved in ABA-regulated heat resistance.

Linkage mapping and genome-wide association study reveals conservative QTL and candidate genes for Fusarium rot resistance in maize.

BACKGROUND: Fusarium ear rot (FER) caused by Fusarium verticillioides is a major disease of maize that reduces grain yield and quality globally. However, there have been few reports of major loci for FER were verified and cloned. RESULT: To gain a comprehensive understanding of the genetic basis of natural variation in FER resistance, a recombinant inbred lines (RIL) population and one panel of inbred lines were used to map quantitative trait loci (QTL) for resistance. As a result, a total of 10 QTL were identified by linkage mapping under four environments, which were located on six chromosomes and explained 1.0-7.1% of the phenotypic variation. Epistatic mapping detected four pairs of QTL that showed significant epistasis effects, explaining 2.1-3.0% of the phenotypic variation. Additionally, 18 single nucleotide polymorphisms (SNPs) were identified across the whole genome by genome-wide association study (GWAS) under five environments. Compared linkage and association mapping revealed five common intervals located on chromosomes 3, 4, and 5 associated with FER resistance, four of which were verified in different near-isogenic lines (NILs) populations. GWAS identified three candidate genes in these consistent intervals, which belonged to the Glutaredoxin protein family, actin-depolymerizing factors (ADFs), and AMP-binding proteins. In addition, two verified FER QTL regions were found consistent with Fusarium cob rot (FCR) and Fusarium seed rot (FSR). CONCLUSIONS: These results revealed that multi pathways were involved in FER resistance, which was a complex trait that was controlled by multiple genes with minor effects, and provided important QTL and genes, which could be used in molecular breeding for resistance.

Linkage mapping and genome-wide association reveal candidate genes conferring thermotolerance of seed-set in maize.

It is predicted that high-temperature stress will increasingly affect crop yields worldwide as a result of climate change. In order to determine the genetic basis of thermotolerance of seed-set in maize under field conditions, we performed mapping of quantitative trait loci (QTLs) in a recombinant inbred line (RIL) population using a collection of 8329 specifically developed high-density single-nucleotide polymorphism (SNP) markers, combined with a genome-wide association study (GWAS) of 261 diverse maize lines using 259 973 SNPs. In total, four QTLs and 17 genes associated with 42 SNPs related to thermotolerance of seed-set were identified. Among them, four candidate genes were found in both linkage mapping and GWAS. Thermotolerance of seed-set was increased significantly in near-isogenic lines (NILs) that incorporated the four candidate genes in a susceptible parent background. The expression profiles of two of the four genes showed that they were induced by high temperatures in the maize tassel in a tolerant parent background. Our results indicate that thermotolerance of maize seed-set is regulated by multiple genes each of which has minor effects, with calcium signaling playing a central role. The genes identified may be exploited in breeding programs to improve seed-set and yield of maize under heat stress.

Genetic analysis of cob resistance to F. verticillioides: another step towards the protection of maize from ear rot.

KEY MESSAGE: We lay the foundation for further research on maize resistance to Fusarium verticillioides cob rot by identifying a candidate resistance gene. Fusarium verticillioides ear rot is the most common type of maize ear rot in the Huanghuaihai Plain of China. Ear rot resistance includes cob and kernel resistance. Most of the current literature concentrates on kernel resistance, and genetic studies on cob resistance are scarce. We aimed on identifying the QTLs responsible for F. verticillioides cob rot (FCR) resistance. Twenty-eight genes associated with 48 single nucleotide polymorphisms (SNPs) were identified (P < 10-4) to correlate with FCR resistance using a whole-genome association study. The major quantitative trait locus, qRcfv2, for FCR resistance was identified on chromosome 2 through linkage mapping and was validated in near-isogenic line populations. Two candidate genes associated with two SNPs were detected in the qRcfv2 region with a lower threshold (P < 10-3). Through real-time fluorescence quantitative PCR, one candidate gene was found to have no expression in the cob but the other was expressed in response to F. verticillioides. These results lay a foundation for research on the resistance mechanisms of cob and provide resources for marker-assisted selection.

Phosphoproteomic analysis of the resistant and susceptible genotypes of maize infected with sugarcane mosaic virus.

Protein phosphorylation plays a pivotal role in the regulation of many cellular events. No information is yet available, however, on protein phosphorylation in plants in response to virus infection. In this study, we characterized phosphoproteomes of resistant and susceptible genotypes of maize (Zea mays L.) in response to Sugarcane mosaic virus (SCMV) infection. Based on isotope tags for relative and absolute quantification technology, TiO2 enrichment method and LC-MS/MS analysis, we identified 65 and 59 phosphoproteins respectively, whose phosphorylation level regulated significantly in susceptible and resistant plants. Some identified phosphoproteins were shared by both genotypes, suggesting a partial overlapping of the responsive pathways to virus infection. While several phosphoproteins are well-known pathogen response phosphoproteins, virus infection differentially regulates most other phosphoproteins, which has not been reported in literature. Changes in protein phosphorylation status indicated that response to SCMV infection encompass a reformatting of major cellular processes. Our data provide new valuable insights into plant-virus interactions.

Comparison of intracerebral transplantation effects of different stem cells on rodent stroke models.

In the present study, induced pluripotent stem cells (iPSCs), induced neural stem cells (iNSCs), mesenchymal stem cells (MSCs) and an immortalized cell line (RMNE6), representing different characteristics of stem cells, were transplanted into normal and/or injured brain areas of rodent stroke models, and their effects were compared to select suitable stem cells for cell replacement stroke therapy. The rat and mice ischaemic models were constructed using the middle cerebral artery occlusion technique. Both electrocoagulation of the artery and the intraluminal filament technique were used. The behaviour changes and fates of grafted stem cells were determined mainly by behaviour testing and immunocytochemistry. Following iPSC transplantation into the corpora striata of normal mice, a tumour developed in the brain. The iNSCs survived well and migrated towards the injured area without differentiation. Although there was no tumourigenesis in the brain of normal or ischaemic mice after the iNSCs were transplanted in the cortices, the behaviour in ischaemic mice was not improved. Upon transplanting MSC and RMNE6 cells into ischaemic rat brains, results similar to iNSCs in mice were seen. However, transplantation of RMNE6 caused a brain tumour. Thus, tumourigenesis and indeterminate improvement of behaviour are challenging problems encountered in stem cell therapy for stroke, and the intrinsic characteristics of stem cells should be remodelled before transplantation.

Quantitative trait locus mapping with background control in genetic populations of clonal F1 and double cross.

In this study, we considered five categories of molecular markers in clonal F1 and double cross populations, based on the number of distinguishable alleles and the number of distinguishable genotypes at the marker locus. Using the completed linkage maps, incomplete and missing markers were imputed as fully informative markers in order to simplify the linkage mapping approaches of quantitative trait genes. Under the condition of fully informative markers, we demonstrated that dominance effect between the female and male parents in clonal F1 and double cross populations can cause the interactions between markers. We then developed an inclusive linear model that includes marker variables and marker interactions so as to completely control additive effects of the female and male parents, as well as the dominance effect between the female and male parents. The linear model was finally used for background control in inclusive composite interval mapping (ICIM) of quantitative trait locus (QTL). The efficiency of ICIM was demonstrated by extensive simulations and by comparisons with simple interval mapping, multiple-QTL models and composite interval mapping. Finally, ICIM was applied in one actual double cross population to identify QTL on days to silking in maize.

Sex Differences in Cochlear Transcriptomes in Horseshoe Bats.

Sexual dimorphism of calls is common in animals, whereas studies on the molecular basis underlying this phenotypic variation are still scarce. In this study, we used comparative transcriptomics of cochlea to investigate the sex-related difference in gene expression and alternative splicing in four Rhinolophus taxa. Based on 31 cochlear transcriptomes, we performed differential gene expression (DGE) and alternative splicing (AS) analyses between the sexes in each taxon. Consistent with the degree of difference in the echolocation pulse frequency between the sexes across the four taxa, we identified the largest number of differentially expressed genes (DEGs) and alternatively spliced genes (ASGs) in R. sinicus. However, we also detected multiple DEGs and ASGs in taxa without sexual differences in echolocation pulse frequency, suggesting that these genes might be related to other parameters of echolocation pulse rather than the frequency component. Some DEGs and ASGs are related to hearing loss or deafness genes in human or mice and they can be considered to be candidates associated with the sexual differences of echolocation pulse in bats. We also detected more than the expected overlap of DEGs and ASGs in two taxa. Overall, our current study supports the important roles of both DGE and AS in generating or maintaining sexual differences in animals.

Construction and verification of a histone deacetylases-related prognostic signature model for colon cancer.

Histone deacetylases (HDACs) contribute significantly to the initiation, progression, and prognosis of colorectal adenocarcinoma (COAD). Additionally, HDACs regulate the tumor microenvironment, immune escape, and tumor stem cells, and are closely linked to COAD prognosis. We developed a prognostic model for COAD that incorporates HDACs to evaluate their specific roles. The COAD dataset containing clinical and mutation data was collected using the TCGA and GEO databases to obtain genes associated with HDAC. LASSO analysis and univariate and multivariate Cox regression analysis were used to determine the presence of prognostic genes. Multivariate Cox analysis was also used to determine risk scores for HDAC-related features. Furthermore, genomic alterations, immune infiltration, and drug response were compared between high- and low-risk groups. Cellular experiments validated the potential regulatory role of BRD3 on COAD proliferation, migration, and apoptosis. The median risk scores, calculated based on the characteristics, demonstrated a more significant prognostic improvement in patients in the low-risk group. Furthermore, HDAC-related features were identified as important independent prognostic factors for patients with COAD. Additionally, genomic mutation status, immune infiltration, and function, as well as response to immunotherapy and chemotherapy, were found to be associated with risk scores. Subgroup analyses indicate that anti-PD-1 therapy may be beneficial for patients in the low-risk group. Additionally, a decrease in risk score was associated with a decrease in immune infiltration. Finally, HCT116 and HT29 cells exhibited inhibition of BRD3 gene proliferation and migration, as well as promotion of apoptosis. In patients with COAD, HDAC-related characteristics may be useful in predicting survival and selecting treatment.

JUJUNCAO-Stem-Based Interfacial Solar-Driven Evaporator with Natural Two-Phase Composite Structures of Functional Partition and Inherent Ultralow Vaporization Enthalpy of Water for Stable and Efficient Steam Production.

The interfacial solar-driven evaporation has been deemed as an environmentally friendly approach for freshwater generation. Nevertheless, there is still a challenge to obtain solar evaporators with efficient vapor production from low-cost and renewable biomass through a simple preparation process. Herein, the JUJUNCAO stem was selected as the substrate material, and a kind of interfacial solar-driven evaporator with natural two-phase composite structures and inherent ultralow water vaporization enthalpy was constructed by a dip-coating process. The natural two-phase composite structures were utilized as independent functional partition: the low-tortuosity and hydrophilic vascular bundles served as hierarchical channels for rapid water transportation and continuous steam escape, and the honeycomb-like parenchyma cells were considered natural heat insulators for effective thermal management. Furthermore, the JUJUNCAO stem exhibited inherent ultralow water vaporization enthalpy which was only 1.15 kJ g-1. Benefiting from the natural two-phase composite structures of functional partition and inherent ultralow water vaporization enthalpy, the C-Js evaporator could achieve an evaporation rate of 2.77 kg m-2 h-1 with an efficiency of 85.6% under 1 sun illumination. Meanwhile, the C-Js exhibited a stable and ideal evaporation performance and metal ion rejection behavior in the actual brine desalination process. Owing to the cost-effective and simple pretreatment process, the C-Js evaporator has the potential for freshwater generation in undeveloped areas.

Photothermal-responsive lignin-based polyurethane with mechanically robust, fast self-healing, solid-state plasticity and shape-memory performance.

In light of the depletion of petrochemical resources and increase in environmental pollution, there has been a significant focus on utilizing natural biomass, specifically lignin, to develop sustainable and functional materials. This research presents the development of a lignin-based polyurethane (DLPU) with photothermal-responsiveness by incorporating lignin and oxime-carbamate bonds into polyurethane network. The abundant hydrogen bonds between lignin and the polyurethane matrix, along with its cross-linked structure, contribute to DLPU's excellent mechanical strength (30.2 MPa) and toughness (118.7 MJ·m-3). Moreover, the excellent photothermal conversion ability of DLPU (54.4 %) activates dynamic reversible behavior of oxime-carbamate bonds and hydrogen bonds, thereby endowing DLPU with exceptional self-healing performance. After 15 min of near-infrared irradiation, DLPU achieves self-healing efficiencies of 96.0 % for tensile strength and 96.3 % for elongation at break. Additionally, DLPU exhibits photocontrolled solid-state plasticity as well as an excellent phototriggered shape-memory effect (70 s), with shape fixity and recovery ratios reaching 98.8 % and 95.3 %, respectively. By exploiting the spatial controllability and photothermal-responsiveness of DLPU, we demonstrate multi-dimensional responsive materials with self-healing and shape-shifting properties. This work not only promotes the development of multi-functional polyurethanes but also provides a pathway for the high-value utilization of lignin.

Transcriptomic Insights into Metabolism-Dependent Biosynthesis of Bacterial Nanocellulose.

Bacterial nanocellulose (BNC) is an attractive green-synthesized biomaterial for biomedical applications and various other applications. However, effective engineering of BNC production has been limited by our poor knowledge of the related metabolic processes. In contrast to the traditional perception that genome critically determines biosynthesis behaviors, here we discover that the glucose metabolism could also drastically affect the BNC synthesis in Gluconacetobacter hansenii. The transcriptomic profiles of two model BNC-producing strains, G. hansenii ATCC 53582 and ATCC 23769, which have highly similar genomes but drastically different BNC yields, were compared. The results show that their BNC synthesis capacities were highly related to metabolic activities such as ATP synthesis, ion transport protein assembly, and carbohydrate metabolic processes, confirming an important role of metabolism-related transcriptomes in governing the BNC yield. Our findings provide insights into the microbial biosynthesis behaviors from a transcriptome perspective, potentially guiding cellular engineering for biomaterial synthesis.

Polystyrene microplastics aggravate acute pancreatitis in mice.

Microplastics (MPs) with a diameter of < 5 mm are emerging as a new type of environmental pollutants. With the discovery of MPs in human tissues, the health risks of MPs have attracted considerable attention in recent years. In this study, we aimed to investigate the impact of MPs on acute pancreatitis (AP). We exposed male mice to 100 and 1000 µg/L polystyrene MPs for 28 days, then intraperitoneally injected mice with cerulein to develop acute pancreatitis (AP). The results demonstrated that MPs dose-dependently exacerbated pancreatic injuries and inflammation in AP. High-dose MPs significantly increased intestinal barrier disruption in AP mice, which may be partly responsible for the aggravation of AP. Moreover, through tandem mass tag (TMT)- based proteomics of pancreatic tissues, we screened 101 differentially expressed proteins (DEPs) between AP mice and high-dose MPs-treated AP mice. Gene Ontology and KEGG Pathway analysis revealed that the DEPs were mainly implicated in the molecular events including cytoskeleton organization, acute inflammatory response, arginine metabolism, etc. These mechanisms may also contribute to the aggravating AP effects of MPs. Collectively, our data provide new evidence for the harmful potential of MPs.

Genome Sequence of a Heavy Metal-Detoxifying Actinomycete, Microbacterium proteolyticum ustc.

A complete genome is presented for Microbacterium proteolyticum ustc, a member of the Gram-positive order Micrococcales of the phylum Actinomycetota that is resistant to high concentrations of heavy metals and participates in metal detoxification. The genome consists of one plasmid and one chromosome.

Portable Comestible-Liquid Quality Test Enabled by Stretchable and Reusable Ion-Detection Photonic Papers.

Currently, there have been widespread investigation conducted into responsive photonic crystal hydrogels (RPCHs) characterized by high selectivity and sensitivity for colorimetric indicators and physical/chemical sensors. In spite of this, it remains challenging to use RPCHs for sensing due to their limited mechanical property and molding capability. In the present study, a double-network structure is proposed to design highly stretchable, sensitive, and reusable ion-detection photonic papers (IDPPs) for assessing the quality of visual and portable comestible liquids (e.g., soy sauce). It is constructed by integrating polyacrylamide and poly-methacryloxyethyl trimethyl ammonium chloride with highly ordered polystyrene microspheres. The double-network structure improves the mechanical properties of IDPPs with their elongation at break increasing from 110 to 1600%. Meanwhile, the optical properties of photonic crystals are retained. The IDPPs achieve a fast ion response by applying control on the swelling behavior of the hydration radius of the counter ions through ion exchange. Given a certain concentration range (0.01-0.10 M), chloride ions can be detected fast (3-30 s) by exchanging ions with a small hydration radius through an IDPP, which is clearly observable. Due to the improvement of mechanical properties and the reversible exchange of ions derived from IDPPs, their reusability is significantly enhanced (>30 times). Characterized by a simple operation, high durability, and excellent sustainability, these IDPPs are promising for practical application in food security and human health assessment.

Integrating Network Pharmacology and Experimental Validation to Decipher the Anti-Inflammatory Effects of Magnolol on LPS-induced RAW264.7 Cells.

INTRODUCTION: Magnolol is beneficial against inflammation-mediated damage. However, the underlying mechanisms by which m+agnolol exerts anti-inflammatory effects on macrophages remain unclear. OBJECTIVE: In this study, network pharmacology and experimental validation were used to assess the effect of magnolol on inflammation caused by lipopolysaccharide (LPS) in RAW264.7 cells. MATERIALS AND METHODS: Genes related to magnolol were identified in the PubChem and Swiss Target Prediction databases, and gene information about macrophage polarization was retrieved from the GeneCards, OMIM, and PharmGKB databases. Analysis of protein-protein interactions was performed with STRING, and Cytoscape was used to construct a component-target-disease network. GO and KEGG enrichment analyses were performed to ascertain significant molecular biological processes and signaling pathways. LPS was used to construct the inflammatory cell model. ELISA and qRT‒PCR were used to examine the expression levels of inflammationassociated factors, immunofluorescence was used to examine macrophage markers (CD86 and CD206), and western blotting was used to examine protein expression levels. RESULTS: The hub target genes of magnolol that act on macrophage polarization were MDM2, MMP9, IL-6, TNF, EGFR, AKT1, and ERBB2. The experimental validation results showed that magnolol treatment decreased the levels of proinflammatory factors (TNF-α, IL-1ß, and IL-6). Moreover, the levels of anti-inflammatory factors (IL-10 and IL-4) were increased. In addition, magnolol upregulated the expression of M2 markers (Agr-1, Fizzl, and CD206) and downregulated M1 markers (CD86). The cell experiment results supported the network pharmacological results and demonstrated that magnolol alleviated inflammation by modulating the PI3k-Akt and P62/keap1/Nrf2 signaling pathways. CONCLUSION: According to network pharmacology and experimental validation, magnolol attenuated inflammation in LPS-induced RAW264.7 cells mainly by inhibiting M1 polarization and enhancing M2 polarization by activating the PI3K/Akt and P62/keap1/Nrf2 signaling pathways.