Drug-resistant bacterial infection severely impedes skin wound healing, highlighting the urgent need for effective non-antibiotic therapies. Herein, we develop a carrier-free injectable supramolecular hydrogel (GA-GLA@Zn) through the co-assembly of gallic acid (GA) and glycyrrhizic acid (GLA) reinforced by Zn2+ coordination. The hydrogel exhibits excellent injectability, mechanical stability, and sustained Zn2+ release, providing a functional platform for infected wound treatment. GA-GLA@Zn shows potent antibacterial and antibiofilm activity against methicillin-resistant Staphylococcus aureus (MRSA) by disrupting bacterial membranes, while maintaining favorable biocompatibility and blood compatibility. In vitro studies demonstrate effective reactive oxygen species scavenging, suppression of inflammatory responses, and promotion of fibroblast proliferation and migration. In a MRSA-infected full-thickness wound model, GA-GLA@Zn markedly accelerates wound closure, reduces bacterial burden, alleviates excessive inflammation, and enhances collagen deposition and tissue remodeling. Mechanistically, integrated network pharmacology and molecular simulations identify signal transducer and activator of transcription 3 (STAT3) as a key molecular target of GA and GLA. In vivo results further reveal inhibition of STAT3 signaling and its downstream effectors Cyclin D1 and SOCS3, accompanied by enhanced angiogenesis via the HIF-1 alpha/VEGF pathway. Overall, this study elucidates a STAT3-centered mechanism and highlights GA-GLA@Zn as a promising antibiotic-free strategy for treating drug-resistant infected wounds.

Abstract The sustainable recovery of plant-derived polyphenols requires extraction systems that can simultaneously reduce organic solvent use, improve process efficiency, and provide mechanistic clarity. Herein, a computationally guided natural deep eutectic solvent (NADES) strategy was developed for efficient and sustainable polyphenol recovery from Dioscorea cirrhosa Lour. The conductor-like screening model for realistic solvents (COSMO-RS) was first employed to predict solvent-solute affinities among eight NADES candidates, identifying betaine-maleic acid (Bet-MA) as the most promising green solvent due to its favorable polarity distribution and hydrogen-bonding complementarity. The ultrasound-assisted extraction process was subsequently optimized using an artificial neural network coupled with a genetic algorithm. Under identical initial extraction conditions, Bet-MA achieved a 1.63-fold higher TPC yield than 70% ethanol. After ANN-GA optimization, the UABM process reached 38.14 mg GAE/g DW, corresponding to a 1.73-fold increase compared with the conventional UAE control. Mechanistic investigations combining FT-IR, SEM, and 100 ns molecular dynamics simulations revealed a synergistic dual mechanism: ultrasonic cavitation disrupted the compact plant cell wall architecture, while Bet-MA provided stronger thermodynamic stabilization of released phenolics through electrostatic and van der Waals interactions. UPLC-Q Exactive/MS profiling confirmed the enrichment of diverse phenolic acids and flavonoids, and antioxidant assays verified that the intensified process preserved the bioactivity of the recovered extract. The Bet-MA solvent retained approximately 62 and 49% of its initial extraction capacity after the fifth and sixth reuse cycles, respectively, suggesting its reuse potential under the tested conditions. An AGREEprep-based greenness assessment gave a score of 0.80 for the Bet-MA-based process, compared with 0.59 for conventional ethanol extraction, indicating a more favorable greenness profile. This work provides a rational framework for sustainable biomass valorization through computational solvent design, process intensification, molecular-level mechanism elucidation, and solvent reuse.

The sustainable recovery of bioactive phytochemicals is frequently hindered by the toxicity and separation bottlenecks of conventional volatile organic solvents. Herein, a novel amine-free temperature-responsive biphasic deep eutectic solvent (TRDES) system, composed of betaine, levulinic acid, and octanoic acid (BLevA-OA), was constructed for the integrated extraction and separation of polyphenols from Moringa oleifera Lam. (M. oleifera) leaves. This system exhibits reversible thermomorphic behavior, transitioning from a homogeneous single phase at 60 °C to maximize mass transfer, to a biphasic state at 25 °C for spontaneous phase-selective separation. To address the nonlinear complexity of the extraction process, a hybrid Gaussian Whale Optimization Algorithm-Support Vector Regression (GSWOA-SVR) model was developed, which precisely optimized the parameters to achieve a maximum total polyphenol content of 39.8 mg GAE/g DW, which is 1.7 times that of conventional ethanol extraction. Furthermore, multiscale computational analyses combining COSMO-RS and Molecular Dynamics (MD) simulations elucidated the microscopic mechanism: the superior extraction efficiency stems from a cooperative network of specific hydrogen bonding and hydrophobic interactions, while the temperature-triggered phase transition is driven by the kinetic modulation of component miscibility and diffusion. This work establishes a sustainable, closed loop strategy for the high efficiency recovery of polyphenols from M. oleifera leaves, offering a theoretical and practical foundation for the industrial application of smart green solvents in the circular bio-economy.

The H9N2 subtype of avian influenza virus remains a major threat to poultry production and public health, creating a need for effective vaccines that induce mucosal immunity. Oral immunization is attractive for poultry because it is convenient and suitable for mass application, but its efficacy is often limited by antigen degradation in the gastrointestinal tract and poor intestinal uptake. In this study, we developed a mannosylated chitosan-poly(lactic-co-glycolic acid) nanoemulsion loaded with Ganoderma lucidum triterpenes (MPG) as an oral mucosal adjuvant and delivery system, and subsequently incorporated inactivated H9N2 virus to prepare an oral inactivated vaccine. The MPG nanoemulsion showed a mean particle size of 439.7 ± 2.7 nm, with a negative zeta potential, good stability, protection under simulated gastric conditions, rapid release under intestinal conditions, favorable mucoadhesive properties and a promising immunostimulatory activity in vitro. Broiler chickens were orally immunized twice, and immune responses and protective efficacy were evaluated. Compared with the other groups, the MPG-adjuvanted vaccine significantly increased serum hemagglutination inhibition antibody titers and intestinal H9N2-specific secretory immunoglobulin A levels. After homologous challenge, vaccinated chickens showed reduced oropharyngeal viral shedding and milder lung lesions. In jejunal tissue, the MPG-adjuvanted vaccine increased the RNA expression of immunoglobulin A, polymeric immunoglobulin receptor, J-chain, IL-17, TNF-α, and IL-10, suggesting enhanced intestinal antibody transport and local immune responses. These results indicate that MPG improves the oral adjuvant activity of Ganoderma lucidum triterpenes and enhances both systemic and intestinal immune responses to an inactivated H9N2 vaccine. This strategy may provide a practical approach for oral mucosal vaccination against H9N2 in poultry.

The industrial extraction of bioactive compounds from plant matrices is increasingly shifting towards sustainable and green technologies for pharmaceutical, cosmetic, and functional food applications. In this study, a green and highly efficient strategy combining natural deep eutectic solvents (NADES) with ultrasound-assisted extraction (UAE) was developed to recover antioxidant polyphenols from Celastrus orbiculatus Thunb. To avoid empirical trial-and-error, the conductor-like screening model for realistic solvents (COSMO-RS) was first employed to predict the thermodynamic solubility of representative polyphenols, identifying the betaine-lactic acid (Bet-LA) system as the optimal solvent. Subsequently, an artificial neural network-genetic algorithm (ANN-GA) model was utilized to map and optimize the non-linear extraction parameters. Under optimal conditions (liquid-solid ratio of 33 mL/g, ultrasonic time of 36 min, and power of 520 W), the polyphenol yield reached 22.89 ± 0.41 mg GAE/g DW, representing a 1.86-fold increase over conventional 70% ethanol extraction. UPLC-Q Exactive/MS analysis revealed a significant enrichment of bioactive phenolics, predominantly epicatechin. Furthermore, scanning electron microscopy (SEM) and molecular dynamics (MD) simulations elucidated the extraction mechanisms: ultrasound-induced cavitation aggressively disrupted the plant cell walls, while the dense hydrogen-bonding network of Bet-LA provided superior solvation energy and affinity for the target polyphenols. Crucially, the green Bet-LA extracts exhibited robust in vitro antioxidant capacities (DPPH, ABTS, and FRAP). Overall, this study provides a theoretical and practical framework for the sustainable extraction of plant-derived polyphenols, highlighting their promising potential as natural antioxidants in various bio-based industries.

Due to the increasing prevalence of drug-resistant Eimeria strains, there is an urgent need to develop novel, eco-friendly, and safe alternatives for controlling chicken coccidiosis. This study aimed to investigate the combined anticoccidial efficacy of eucalyptus oil (EO) and mangosteen extract (ME) and develop an advanced delivery system via microencapsulation to enhance their therapeutic potential. In a broiler chick model of Eimeria tenella (E. tenella) infection, the combination of EO (100 mg/kg body weight, oral gavage) and ME (250 mg/kg feed) exerted the optimal effect against E. tenella with an anticoccidial index (ACI) of 169, indicating moderate anticoccidial activity. This combined efficacy appears to be multifaceted. It may involve (i) preservation of intestinal barrier integrity, (ii) suppression of oxidative stress and pro-inflammatory cytokine cascade, and (iii) restoration of cecal microbiota homeostasis. EO exhibits poor water solubility, posing challenges for its clinical administration. To address this, the sodium alginate-calcium chloride (CaCl₂) cross-linking method was used to prepare eucalyptus oil microcapsules (EOM). The results showed that the combined in-feed administration of EOM (50 mg/kg feed) and ME (250 mg/kg feed) yielded an ACI of 171. Furthermore, this treatment regimen alleviated cecal tissue damage and mitigated the inflammatory response. In conclusion, the combination of EO and ME may represent a potent, multi-target natural therapy for coccidiosis, and microencapsulation is a viable strategy to facilitate its practical application as a novel feed additive.

To address the poor water solubility and high volatility of eugenol (EU), this study developed an EU nanoemulsion (NanoEU) and evaluated its anti-Salmonella enterica serovar Typhimurium (S. Typhimurium) activity and protective effects against S. Typhimurium infection in vitro and in vivo. NanoEU showed a uniform milky-white appearance, an average particle size of 137.53 ± 5.07 nm, and a PDI of 0.23 ± 0.03. It exhibited in vitro antibacterial activity, with MIC and MBC values of 512 μg/mL and 1024 μg/mL, respectively. No hemolysis was observed in chicken erythrocytes at 128-1024 μg/mL, indicating preliminary biosafety. NanoEU also maintained stable appearance and unchanged antibacterial activity during 30 days of storage at 4 °C and 25 °C. A chicken model of S. Typhimurium infection was established to assess the protective roles of NanoEU. The results showed that NanoEU partially alleviated impaired body weight gain, reduced bacterial loads in the cecum, liver, and spleen, and mitigated histopathological damage. In addition, NanoEU improved cecal mucus secretion, upregulated tight junction-related gene and protein expression, alleviated local oxidative stress and inflammatory responses. In conclusion, NanoEU exhibited anti-S. Typhimurium activity, preliminary biosafety, and favorable storage stability. More importantly, it exerted both anti-infective and host-protective effects in vivo, suggesting its potential application in controlling S. Typhimurium infection and reducing antibiotic use in poultry.

Eimeria tenella (E. tenella), a pathogenic protozoan parasite, poses a significant threat to the global poultry industry due to its severe impact on intestinal health and growth performance. Given the limitations of conventional anticoccidial drugs, such as the development of drug resistance and chemical residues, it is imperative to explore natural alternative strategies for effective control. In this study, we evaluated the synergistic anti-coccidial effect of pomegranate peel extract (PPE) and Bacillus subtilis (BS). PPE exhibits antioxidant and anti-inflammatory properties, whereas BS, contributes to maintaining intestinal microbial balance. Broiler chickens were challenged with E. tenella and treated with 400mg/kg PPE in feed and 400mg/L BS in drinking water, using diclazuril (DIC) as a positive control. The results demonstrated that the PPE+BS combination significantly alleviated cecal lesions, reduced oocyst it mainly parasitizes on the mucosa of the cecum output and mortality, lowered organ indices, suppressed inflammatory responses, and restored body weight gain. The anti-coccidial index (ACI) reached 163, indicating moderate efficacy. Gut microbiota analysis revealed that PPE+BS restored microbial homeostasis by increasing the abundance of Firmicutes, reducing Escherichia coli and Enterococcus, and restoring Micrococcus to levels comparable to uninfected controls. To further explore the potential of the intestinal probiotics, Lactobacillus salivarius (LS) and Lactobacillus curvatus (LC), isolated and identified from chicken feces, achieved comparable anti-coccidial efficacy when combined with PPE, confirming the role of specific probiotics in the observed synergy. In conclusion, the combination of PPE and BS represents an effective and sustainable strategy for controlling avian coccidiosis, primarily through modulation of the gut microbiota and enrichment of beneficial probiotics.

Objective We aimed to investigate the therapeutic effects of Moringa oleifera leaf polysaccharide (MOLP) in ulcerative colitis (UC) and the underlying mechanism involving macrophages polarization. Methods Dextran sodium sulfate (DSS)-induced colitis mouse model, as well as lipopolysaccharide (LPS) and interferon-γ (IFN-γ) stimulated macrophages (PMA-differentiated THP-1 cells) were used for investigations. Therapeutic effect of MOLP was evaluated by measuring disease activity index (DAI), myeloperoxidase (MPO) activity and histological morphology in mice, with 5-aminosalicylic acid as positive control. Macrophages were determined by flow cytometry and immunofluorescence staining. Glycolysis activity was determined by measuring lactic acid content, extracellular acidification rate (ECAR) and the uptake of fluorescent deoxyglucose analog 2-NBDG. Results MOLP effectively alleviated colitis symptoms of DSS-induced UC mice, as demonstrated by reduced DAI score and MPO activity, alleviation of colon shortening, and mitigated colonic histopathological damage. Besides, MOLP decreased the proportion of M1 macrophages and the mRNA expression of their specific factors (TNF-α, IL-1β and iNOS), while increasing M2 macrophages and their specific factors (CD206, Arg1 and Chil3). Based on LPS and IFNγ-stimulated macrophages, MOLP treatment suppressed M1 macrophages polarization by inhibiting the glycolysis activity, which was confirmed by the glycolysis inhibitor 2-DG. Mechanistically, HMGB1 was down-regulated by MOLP, and recombinant HMGB1 protein treatment could offset the curative effects of MOLP on UC. Furthermore, HMGB1 interacted with HAVCR2, and the latter mediated macrophage glycolysis by PI3K/Akt/HIF-1α pathway, thereby regulating M1 macrophages polarization. Conclusion MOLP could alleviate UC by regulating macrophages M1 polarization through HMGB1/HAVCR2 mediated glycolysis.

Introduction: Crohn's disease (CD) is an inflammatory bowel disease characterized by chronic inflammation of the entire digestive lining. Although the pathogenesis of CD remains unclear, multiple factors, especially altered microbiota, are among its causes. Methods: In this study, an experimental CD model was established by trinitrobenzene sulfonic acid (TNBS) enema. Then the dynamic changes of colonic tissue lesions, tight junctions, inflammation response, and oxidative stress are respectively tested by hematoxylin and eosin staining, immunofluorescence staining, and commercial kits. 16S rRNA and ITS sequencing of colonic feces were applied to analyze the composition and diversity of the microbiome and mycobiome for lasting 5 weeks. Results: As a result, despite TNBS being applied only once time, the stimuli-caused injury reached a peak in the second week (the most severe period), after which symptoms began to gradually return to the normal stage. Additionally, consistent with the TNBS-caused colonic damage, deaths were also concentrated within 2 weeks after modeling, with only one death occurring in the subsequent period despite ongoing inflammation and other typical symptoms. In terms of gut bacteria, microbiome diversity decreased significantly while mycobiome diversity increased, along with the enrichment of harmful microbiota and shrinkage of probiotic microorganisms. Conclusion: Therefore, the data suggested that TNBS-induced CD can be roughly divided into two phases: the acute inflammatory phase (weeks 1-2) and the chronic inflammatory phase (weeks 3-5). However, the microbiome and mycobiome dysbiosis did not return to normal within the trial period. Hence, our findings may facilitate a better comprehension of the dynamic progress of experimental TNBS-induced CD.

Innate and adaptive immunity are intricately linked to the pathogenesis of ulcerative colitis (UC), with dysregulation of the Treg/Th17 balance and M2/M1 macrophage polarization identified as critical factors. Artesunate (ARS) has previously been shown to alleviate UC by inhibiting endoplasmic reticulum stress (ERS). To further investigate the regulatory effects of ARS on immune dysregulation associated with colitis and the role of ERS in this process, an experimental colitis model was established using dextran sulfate sodium (DSS). Flow cytometry was employed to assess changes in the Th17/Treg cell ratio in the spleen and macrophage polarization in the intestine, while RT-qPCR was used to quantify the transcription levels of relevant genes in colonic tissues. ARS treatment significantly mitigated DSS-induced pathological damage, reduced the proportion of CD4+Th17 cells, and downregulated the mRNA expression of IL-17A, IL-17F, and RORγt, while concurrently increasing the proportion of CD4+Treg cells and upregulating TGF-β expression. Additionally, ARS restored the DSS-induced decline in the M2/M1 macrophage ratio and enhanced the transcription of Arg-1 and IL-10, while suppressing the expression of pro-inflammatory markers, including iNOS, IL-1β, IL-6, and TNF-α. Notably, co-treatment with 4-phenylbutyric acid (4-PBA, ERS inhibitor) augmented the immunoregulatory effects of ARS, whereas 2-deoxy-D-glucose (2-DG, ERS agonist) co-treatment counteracted its protective activity against UC. These findings suggest that ERS plays a crucial role in mediating the therapeutic effects of ARS on UC, particularly by modulating Th17/Treg balance and macrophage polarization. This study provides further insights into the mechanistic basis of ARS in UC treatment offering a potential avenue for therapeutic intervention.

The excessive utilization of antibiotics gives rise to the development of bacterial resistance, the deterioration of animal immune functions, the increase in mortality rates, and the undermining of human immunity. Therefore, there is an urgent necessity to explore new antimicrobial agents or alternatives to tackle bacterial resistance. We investigated tea tree oil (TTO), a pure natural plant essential oil extracted from Melaleuca leaves, which exerted efficient antibacterial activities. However, the poor solubility and high volatility of TTO limited the clinical application. Therefore, tea tree oil and Tween 80 were formulated into a stable nanoemulsion (Nano TTO). We attested that Nano TTO, as an antibiotic adjuvant, enhanced the antibacterial activity of amoxicillin (AMX) against methicillin-resistant Staphylococcus aureus (MRSA) and inhibited the formation of biofilms. Mechanistic studies proved that the Nano TTO potentiation effect on AMX was primarily the result of inhibition of the Agr expression by targeting the accessory regulator AgrA. Furthermore, Nano TTO effectively boosts the efficacy of amoxicillin in the mouse septicaemia model and mouse skin wound infection model. Overall, these results revealed the potential of Nano TTO as an adjuvant to evade multidrug-resistant bacterial pathogens and improve treatment outcomes for drug-resistant infections.

A novel method for extracting polyphenols from Moringa oleifera Lam. (M. oleifera) using natural deep eutectic solvents (NADES) has been developed, with extraction conditions optimized via an artificial neural networkgenetic algorithm (ANN-GA) to maximize efficiency. Traditional extraction methods for polyphenols from M. oleifera leaves, such as ethanol extraction, are often associated with low efficiency, environmental concerns, and high operational costs. Furthermore, the empirical selection of solvents and extraction conditions is timeconsuming and lacks precision. In this study, 28 NADES were employed to predict the solubility of polyphenol molecules through a conductor-like screening model for realistic solvents (COSMO-RS) model. Based on the results, eight highly effective NADES combinations were selected and experimentally verified. The betainemaleic acid (Bet-MA) combination with the ultrasonic-assisted extraction showed the highest extraction rate of M. oleifera leaves polyphenol (MOLP), which was 1.56 times that of ultrasonic-assisted ethanol solvent. Simplex centroid mixture design (SCMD) and two-level factor experiments were conducted to optimize the ratio of betaine, maleic acid, and water and identify the most influential extraction factors on MOLP yield. The ANN-GA model was further applied to refine the extraction conditions and analyze their interactions on MOLP content. Additionally, antioxidant assays demonstrated that MOLP extracted with the Bet-MA solvent displayed significant antioxidant activity. In conclusion, this study demonstrates that COSMO-RS and ANN-GA may serve as powerful tools for solvent selection and extraction process optimization, offering a green and efficient approach for polyphenol extraction from M. oleifera leaves. The findings highlight the potential of NADES-based extraction methods for broader applications in the extraction of various bioactive substances, contributing to the development of sustainable and environmentally friendly extraction technologies.

Moringa oleifera (MO) is a versatile non-traditional feed supplement rich in bioactive compounds. The objective of this study was to examine the effects of MO leaf (MOL) polysaccharide (MOLP) intake as a natural product on broiler chicken production and antioxidant indices. A polysaccharide with a molecular weight of 182.989 kDa was isolated from MOL in a previous study. Broiler chickens were allocated at random into four groups receiving varying doses of MOLP (0, 0.1, 0.2 and 0.4 g/kg feed) for three weeks. Feed intake (FI), average daily feed ingestion (ADFI), feed conversion ratio (FCR), and body weight gain (BWG) were monitored. Serological markers, including total protein (TP), albumin (ALB), globulin (GLO), albumin-to-globulin ratio (ALB/GLO), creatinine (CREA), as well as the activities of total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-Px), total antioxidant capacity (T-AOC) and the concentrations of malondialdehyde (MDA) were assessed. Results from days 21 to 28 demonstrated that the high dose of MOLP significantly enhanced BWG, ADFI, liver and bursa indices compared to the control group. Additionally, TP and GLO, T-SOD, GSH-Px, T-AOC and MDA levels were elevated (p < 0.05). In conclusion, MOLP supplementation, particularly at 0.4 g/kg feed, positively impacted broiler chicken growth performance and antioxidant indices, suggesting its potential as a valuable feed additive.

Fel d1 is the most important allergen secreted by cats, which can trigger asthma in sensitive individuals. Our objective was to knock-out the Fel d1 gene in the fetal fibroblasts of cats through CRISPR-Cas9 technology with two sgRNAs and to determine the impact of such mutations on the antigenicity of the Fel d1 protein. DNA samples from 38 domestic cats were collected and amplified by PCR to obtain the complete sequence of the Fel d1 gene. Throughout evolution, Fel d1 polypeptide chain 1(CH1) has proven to be much more conserved than Fel d1 polypeptide chain 2(CH2); therefore, we targeted CH2 and designed two single-guide RNAs (CH2-sgRNA-1 and CH2-sgRNA-2) for this region. Using these constructed sgRNAs, we performed gene knock-out in fetal fibroblasts, resulting in two mutations within the target gene. Following this, DNA was extracted and the target site product was cloned using TA cloning via PCR, and a single colony from this process was sequenced to analyze the physicochemical properties, antigenic sites, and three-dimensional structure of the mutated protein. The results revealed that there were 12 and 51 polymorphic loci (single-nucleotide polymorphisms, or SNPs) found in the CH1 and CH2 sequences, respectively, with most loci located in the GC-rich intron 2, while others were found in exon 2, intron 3, and exon 3. These SNPs guided sgRNA design by identifying conserved regions in the CH2 gene. The gene editing efficiency for the CH2 region, with this dual CRISPR system, was 40%, with 35% attributed to Type 1 mutation and 5% to Type 2 mutation. In conclusion, CH1 is significantly more conserved than CH2, and the antigenicity of the Fel d1 CH2 gene in domestic cats can be effectively reduced through CRISPR-Cas9 gene editing.

INTRODUCTION:Artesunate (ARS) has demonstrated therapeutic potential in experimental ulcerative colitis (UC) through immunomodulatory activities. However, the role of macrophages and the underlying mechanisms remain unclear. METHODS:In this study, a DSS-induced UC model was established, along with macrophage depletion, polarized macrophage reinfusion, and Transwell coculture systems. The typical colitis characteristics, including weight change, disease activity index, inflammation, and tissue damage, were systematically assessed. Key proteins in the mTORC1/HIF-1α pathway were analyzed. SPR-MS was employed to identify ARS targets in M1 macrophages. RESULTS:This study indicated that ARS markedly suppressed DSS-enhanced M1 polarization and macrophage infiltration. In a Caco2-BMDM coculture system, ARS reduced macrophage migration and inflammation. Macrophage depletion significantly attenuated the anti-colitis effect of ARS. Compared with M1 reinfusion, ARS-pretreated M1 cells alleviated UC symptoms. ARS also inhibited LPS- or DSS-induced M1 polarization and proinflammatory cytokine upregulation in vivo and in vitro, linked to mTORC1/HIF-1α signaling, as confirmed by the agonist Leucine (LEU). Moreover, ARS protected intestinal barrier function by preventing tight junction loss and permeability increases via suppression of M1 polarization. SPR-MS and molecular docking revealed that ARS directly activated INSR, inhibiting mTORC1/HIF-1α-driven glycolytic M1 polarization. CONCLUSIONS:Overall, macrophages are essential for ARS-mediated protection in UC. ARS alleviates UC by reprogramming macrophage polarization via the INSR/mTORC1/HIF-1α axis, providing new mechanistic insights and potential clinical applications.

This study aimed to develop a straightforward and efficient method for screening natural deep eutectic solvent (NADES) with high extraction yield of polysaccharides from Moringa oleifera Lam. (M. oleifera) leaves and to elucidate the solubilization mechanism of this eco-friendly extraction solvent. Traditional extraction methods are often limited by low efficiency, long processing times, and environmental hazards, which hinder the advancement of polysaccharide applications. In this study, the conductor-like screening model for realistic solvents (COSMO-RS) was adopted to prognosticate the solubility of biomolecules, and the M. oleifera polysaccharide (MOLP) solubility of 32 NADESs was appraised. Based on the solubility and activity coefficient results, we meticulously selected eight highly efficient NADESs combinations and conducted practical verification. The Bet-LA (Betaine- Lactic acid) combination demonstrated the highest extraction efficiency for MOLP, achieving an ultrasonic-assisted extraction rate 2.67-fold higher than that of the conventional ethanol solvent. The dissolution mechanism was further elucidated using frontier molecular orbital (FMO) and reduced density gradient (RDG) analyses. Antioxidant assays demonstrated that polysaccharides extracted by Bet-LA exhibited superior biological activity. These findings establish an efficient NADES screening protocol and provide mechanistic insights for optimizing M. oleifera leaves polysaccharide extraction.

In a previous study, Eugenol (EU) has been demonstrated to alleviate DSS-induced experimental colitis, due to its anti-inflammatory, anti-oxidant, and immune regulatory efficacy, but its underlying molecular mechanism remains unknown. In this study, EU applications were combined with peroxisome proliferator-activated receptor-[Formula: see text] (PPAR-[Formula: see text]) agonist (rosiglitazone) and inhibitor (GW9662) in order to clarify the role of PPAR-[Formula: see text] in EU against UC by testing NF-κB and Nrf2 signaling pathway activation and the salient features of colitis. The binding activity and adjusting effect of EU on inflammation and oxidative stress were further investigated in vitro. Similar to rosiglitazone, the results illustrated that EU remarkably reversed DSS-induced weight loss, reversed colonic shrinkage and integrity damage, and inhibited the DAI scores increase, excessive inflammatory response, and oxidative stress. However, the combination with GW9662 noticeably restrained the protective effect on mice. Additionally, molecular docking and a surface plasmon resonance assay evidenced the direct binding activity of EU with PPAR-[Formula: see text]. EU's anti-oxidant and anti-inflammation bioactivities were evidenced again in vitro. Overall, the above results further demonstrated the molecular mechanism of EU's defensive effect, which is directly dependent on PPAR-[Formula: see text] activation, on experimental colitis. Therefore, this study may facilitate a better understanding of EU's protective action against UC.

Moringa oleifera (MO)is a perennial tropical deciduous plant, its leaves (MOL) have both medicinal and edible values. Ulcerative colitis (UC) is characterized by recurrent and nonspecific intestinal inflammatory disease, and developing new treatment is still urgently needed. This study focused on the main composition and anti-colitis efficacy of crude ethanol and aqueous extraction of MOL (EEMOL & AEMOL). The results showed that EEMOL and AEMOL had similar contents and monosaccharide composition, while EEMOL had over three times more flavonoid content than AEMOL. Both EEMOL and AEMOL noticeably alleviated dextran sodium sulfate-induced murine UC by reducing the serious weight loss, disease activity index scores, colon shortening, and bloody diarrhea. EEMOL and AEMOL significantly inhibited the TLR4/NF-κB signaling pathway activation, reversed the expression of pro-inflammatory cytokines mRNA and anti-apoptotic proteins, while safeguarded the intestinal structure and tight junctions. In addition, MOL extract restored gut microbiota dysbiosis to varying degrees in the colitis model. Overall, above findings revealed the protective effect of EEMOL and AEMOL on UC is mainly achieved via inhibiting inflammation and apoptosis, maintaining the intestinal barrier, and reversing the microbiota dysbiosis. These data illustrated that both EEMOL and AEMOL have the potential to be an alternative to UC.