Self-adaptive nanozyme-loaded yeast microcapsules for multifunctional and precise therapy of colitis via intestinal microenvironment remodeling

Abstract

Inflammatory bowel disease (IBD) management remains a significant challenge due to its complex pathogenesis. While current therapies for IBD are effective in symptom management, a radical cure remains elusive. However, the high cost, adverse effects, and acquired drug resistance limit their long-term use, further highlighting the pressing need for safer, multifunctional, and precise therapeutic strategies. Herein, we developed a novel iron-based nanozyme (DHM-Fe) by coordinating dihydromyricetin (DHM) with iron ions. The DHM-Fe nanozyme confers antioxidant and anti-inflammatory effects by mimicking SOD/CAT activities and suppressing M1-like macrophage polarization. To improve gastric stability and targeting, the DHM-Fe nanozyme was encapsulated in yeast microcapsules (YM), forming the DF@YM oral delivery system. Oral administration of DF@YM exhibited good biocompatibility and alleviated DSS-induced colitis by suppressing pro-inflammatory responses, restoring barrier integrity, and modulating gut microbiota composition towards homeostasis. Metabolomic and molecular docking analyses revealed that DF@YM alleviates intestinal inflammation, potentially through activating the aryl hydrocarbon receptor (AhR)/IL22 pathway. Furthermore, direct visualization via mass spectrometry imaging confirmed that DF@YM markedly enhances the tissue-specific accumulation of DHM. As an oral delivery system for natural compounds, this multifunctional strategy enables precise targeting and synergistic regulation of the intestinal microenvironment, presenting a promising avenue for IBD treatment.

Graphical Abstract

Supplementary Information

The online version contains supplementary material available at 10.1186/s12951-026-04544-6.

Introduction

Inflammatory Bowel Disease (IBD) is a chronic, non-specific inflammatory disorder of the intestine, primarily comprising ulcerative colitis and Crohn’s disease [1]. The prevalence of IBD is rapidly increasing worldwide and has become a global disease [2, 3]. The onset and progression of IBD are significantly influenced by compromised intestinal barrier integrity, dysbiosis of the gut microbiome, and excessive immune response triggered by oxidative stress [4]. Currently, the clinical treatment strategies for IBD, include 5-aminosalicylic acid (5-ASA), glucocorticoids, immunosuppressants, and biologics (e.g., infliximab). While providing symptomatic relief, conventional medications rarely achieve a fundamental cure for IBD. Their long-term utility is further limited by the risks of adverse effects and acquired drug resistance, which stem primarily from the lack of selectivity and systemic distribution [5, 6]. Moreover, a single therapeutic modality rarely achieves optimal clinical outcomes. Therefore, a multifunctional and precisely targeted synergistic approach offers a promising strategy for IBD treatment.

Reactive oxygen species (ROS) drive many inflammatory diseases, including free radicals (e.g., superoxide anion (O₂•⁻) and hydroxyl radical (•OH)) and non-radicals (e.g., hydrogen peroxide (H₂O₂)) [7]. The pathophysiological mechanisms of IBD are intimately linked to the excessive production of ROS and reactive nitrogen species (RNS). During the activation of intestinal, ROS/RNS coupled with increased M1 macrophage infiltration, are key drivers of disrupted gut homeostasis in IBD. Excessive ROS/RNS induce intestinal tissue damage and degradation of critical gut proteins, ultimately compromising barrier integrity [8, 9]. Therefore, inhibiting oxidative stress by scavenging excessive ROS is pivotal in IBD therapy.

Nanozymes, a class of nanomaterials exhibiting enzyme-mimicking properties, offer unique advantages including low cost, high stability, adjustable catalytic activity, and enhanced scalability for mass production [10, 11]. Nanozymes serve as substitutes for natural enzymes with multienzyme-like activities, effectively scavenging ROS and demonstrating promising therapeutic potential in alleviating colitis [12]. Owing to their biocompatibility and ROS scavenging capabilities, iron-based nanozymes have attracted considerable interest for diagnostic and therapeutic applications [13–15]. Their unique capacity to mimic SOD/CAT activities ( conversion of O₂•⁻ into H₂O₂ by SOD, followed by the CAT-mediated decomposition of H₂O₂ into H₂O and O₂) enables on-demand conversion of ROS in inflammatory microenvironments.

Natural bioactive compounds demonstrate considerable promise for IBD treatment, owing to their low side effects, easy accessibility, and broad pharmacological activities [16, 17]. Dihydromyricetin (DHM), a polyphenolic flavonoid, exhibits potent antioxidant activity that protects tissues against oxidative damage. DHM demonstrates anti-inflammatory and antioxidant activities while modulating gut microbiota composition, as evidenced in multiple studies [18, 19]. Effective absorption and utilization of DHM are essential for its biological activity to manifest at the target site. However, due to the poor aqueous solubility, limited oral bioavailability, and rapid metabolism, DHM fails to attain therapeutic concentrations, limiting its efficacy in IBD therapy [20]. The distinctive chemical scaffold of polyphenols mediates their metal-chelating activity, primarily through electron pair donation from hydroxyl oxygen atoms, forming coordination bonds that enhance water solubility and confer catalytic properties [14, 21].

Oral nanoparticles selectively accumulate in inflamed colonic tissue by penetrating the intestinal mucosa, thereby bypassing rapid systemic clearance and avoiding diarrhea-related elimination. Despite enabling passive targeting to inflamed colonic tissue, nanoparticles suffer structural/functional compromise and rapid elimination in the harsh gastrointestinal milieu (e.g., pH fluctuations, enzymatic activity), leading to subtherapeutic drug accumulation in colonic lesions [22]. Yeast microcapsules (YM) are a natural drug delivery carrier with a rough, porous surface and hollow cores [23]. The β-1,3-D-glucan of YM is recognized by Dectin-1 receptors on macrophages and intestinal M cells, conferring high targeting specificity, excellent safety, and exceptional stability that protects drugs from gastric degradation [24, 25]. Meanwhile, the mannan component of YM serves as a prebiotic, promoting their proliferation, suppressing pathogenic bacteria, and modulating the gut microbiota in IBD patients [26, 27]. This enables targeted controlled release in the intestine, demonstrating significant promise for oral drug delivery systems.

In this work, a novel DHM-Fe nanozyme was successfully synthesized via the coordination between DHM and iron ions, which exhibits potent antioxidant and anti-inflammatory capabilities. The iron-based nanozyme, dihydromyricetin, and yeast microcapsule were not merely physically combined, but were constructed into an integrated system capable of cascade reactions and bidirectional regulation. Through the specific coordination between DHM and Fe³⁺, the DHM molecule itself was converted into part of the catalytic active center, achieving a chemical unity between the active molecule and the catalytic center. In vitro and in vivo studies demonstrated that DHM-Fe nanozyme mitigates oxidative stress by scavenging ROS at inflammatory sites, while simultaneously reducing inflammation through downregulation of pro-inflammatory cytokines and suppression of M1-like macrophage polarization. The DHM-Fe nanozyme was then encapsulated into YM to construct an oral delivery system (DF@YM) that enhances gastric stability and provides excellent biocompatibility. Following oral administration, DF@YM enables sequential drug delivery via a self-adaptive model in the intestinal inflammatory microenvironment, thereby achieving effective targeting and accumulation at inflammatory sites via binding to the Dectin-1 receptor. This “all in one” multifunctional strategy targeting the core pathogenic factors synergistically modulates the intestinal microenvironment, offering a new approach for treating IBD (Fig. 1).

Fig. 1.

Schematic illustration of the construction of DF@YM and its mechanism for IBD treatment. The DHM-Fe nanozyme was synthesized via the coordination between DHM and Fe³⁺ and was then encapsulated into YM to fabricate a robust oral delivery system (DF@YM). Encapsulation of the DHM-Fe nanozyme within YM confers gastric protection and enables macrophage-specific targeting via Dectin-1 binding for enhanced accumulation in inflamed intestines. By oral administration, DF@YM effectively alleviates oxidative stress, diminishes inflammatory responses, promotes the repair of the epithelial barrier, and remodels the gut microbiota. In addition, DF@YM regulated tryptophan metabolites, including indole-3-acetic acid (IAA) and kynurenic acid (KYNA). MSI confirmed that the DF@YM system significantly enhances the tissue-specific accumulation of DHM

Materials and methods

Materials

DHM was obtained from Yuanye Bio-Technology Co., Ltd (98%, Shanghai, China). Active baker’s Yeast was purchased from Angel Yeast Co., Ltd. (Yichang, China). Dulbecco’s Modified Eagle Medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco (California, USA). Calcein-AM/PI kit was purchased from Solarbio Technology Co., Ltd. (Beijing, China). Lipopolysaccharide (LPS) was purchased from Sigma-Aldrich, and the antibody CD86 was obtained from Biolegend (Nanjing, China). DSS was sourced from Yeasen (Shanghai, China). 2,7-dichlorofluorescein diacetate (DCFH-DA) was purchased from Beyotime (Shanghai, China). Annexin V-FITC apoptosis detection kit was obtained from Beyotime Biotechnology (Jiangsu, China). Anti-Claudin antibody (AF0127) was sourced from Affinity Biosciences( Jiangsu, China). Anti-CYPA1 antibody(13241-1-AP), anti-ZO-1 antibody (21773-1-AP), and anti-Occludin antibody (27260-1-AP) were purchased from Proteintech (Wuhan, China). Anti-MUC 2 antibody (AB272692) was obtained from Abcam. StemRegenin 1 was purchased from KKL Med Inc. (Shanghai, China).

Preparation of DHM-Fe

DHM-Fe was synthesized via a one-step assembly method. The pH of the aqueous DHM solution (3mmol/L) was adjusted to 8–9 with 1 M NaOH solution. The assembly of DHM-Fe was initiated by the dropwise addition of an aqueous iron (III) chloride solution (2.5mmol/L). The reaction was stirred at room temperature for 10 min. The resulting mixture was purified by centrifugation at 5000 g using a Millipore ultrafiltration centrifugal filter (Ultracel-3 K) to remove unreacted components. The nanoparticles retained in the inner tube of the Millipore centrifugal filter were collected for subsequent use.

Preparation of Rhodanmin (RDM)-labeled DHM-Fe and DF@YM

RDM-labeled DHM-Fe was prepared by stirring 20 µL of 10 mM RDM with 1 mg/mL DHM-Fe overnight, with a subsequent centrifugation at 5000 g using a Millipore ultrafiltration centrifugal filter (Ultracel-3 K). RDM-labeled DF@YM was prepared by combining RDM-labeled DHM-Fe with YM according to the same method as that used to prepare DF@YM earlier.

Preparation of DF@YM

15 g of yeast was dissolved in 150 ml of 1 M sodium hydroxide and then heated to 80 °C for 1 h. The insoluble material containing yeast cell walls was collected by centrifugation at 2000 g for 5 min. After being washed with water twice, the insoluble material was suspended in 150 ml of a 4% aqueous acetic acid solution and reacted at room temperature for 2–3 h. The insoluble residue was collected by centrifugation, followed by sequential washing with 200 mL of water (twice), 80 mL of isopropanol (three times), and 80 mL of acetone (twice). The resulting slurry was placed in a glass dish and dried at room temperature to yield a fine, off-white powder. Subsequently, 50 mg of YM was dissolved in 5 ml of pH 9.2 carbonate buffer solution and incubated at 37 °C for 30 min. Then, 100 mg of PEI aqueous solution was added and incubated at 37 °C overnight. The mixture was centrifuged at 2000 g and washed three times with ultrapure water. The precipitate was resuspended in water, and 10 mg of DHM-Fe aqueous solution was added. The mixture was stirred at 37 °C for 24 h. The sample was then centrifuged at 2000 g for 5 min to remove unencapsulated DHM-Fe. Finally, the collected DF@YM suspension was resuspended in ultrapure water and freeze-dried under vacuum to obtain DF@YM powder.

Characterization of DHM-Fe and DF@YM

UV-vis spectroscopy was performed on a DENOVIX UV–vis spectrophotometer (USA). The FTIR spectra of DHM-Fe were recorded by a Fourier-transform infrared spectrophotometer (Bruker, V70, Germany). XRD analysis was performed by a Smartlab-SE X-ray diffractometer (Smartlab-SE, Japan). The X-ray photoelectron spectroscopy (XPS, Thermo Fisher ESCALAB 250Xi, USA) was used to characterize the elemental content and valence states of DHM-Fe. The morphology of DHM-Fe and DF@YM was characterized by TEM ( HT7800, Japan). The EDS spector of DF@YM was performed by EDS ( JED-2300T, Japan).

In vitro drug release profile

The release profiles of DHM from different formulations were assessed via dialysis. Briefly, 5 mL of DF@YM was immersed in 125 mL of SGF or SIF for release profiling. Concurrently, another DF@YM was incubated in 125 mL SIF containing 5% β-glucanase. All samples were shaken at 37℃ with 100 rpm agitation. At predetermined intervals, all samples were centrifuged, and 1 mL aliquots of the supernatant were collected for measurement. Then, 1 mL of fresh release medium was added to the aliquots. Cumulative release (%) was quantified using UV-vis spectroscopy.

ROS scavenging ability test

CAT-like enzyme activity: The CAT-like activity of DHM-Fe was assessed by measuring O₂ produced at different reaction times using a portable dissolved oxygen meter. Typically, 2 ml DHM-Fe and 2 ml H₂O₂ were mixed, and the concentration of dissolved oxygen was monitored for 10 min.

SOD-like enzyme activity: The SOD mimetic activity of DHM-Fe was evaluated by employing the NBT photoreduction inhibition assay. In brief, methionine (0.13 M), riboflavin (1.2 mM), NBT (1 mg mL^− 1 ), EDTA∙Na₂ (0.1 M), and DHM-Fe were sequentially added to PBS. Following a 20-minute irradiation under LED light, the absorbance of the mixed solution was recorded at 560 nm.

RNS scavenging capability: RNS scavenging capability of DHM-Fe was detected using (1,1-diphenyl-2-picrylhydrazyl) DPPH assays. In the DPPH• assay, 2 mL of DHM-Fe at various concentrations was combined with 2 mL of a 0.04 mg/mL DPPH ethanolic solution. The mixture was incubated in the dark for 30 min, and the absorbance at 519 nm was recorded using a UV-Vis spectrophotometer.

Cell culture

RAW 264.7 and Caco-2 cells were cultured under standard conditions (37 °C, 5% CO₂) in DMEM containing 10% FBS and 1% penicillin– streptomycin.

In vitro cytotoxicity assay

The CCK-8 assay was employed to assess the cytotoxicity of DHM-Fe on the Raw 264.7 and Caco-2 cells. Firstly, cells were seeded in a 96-well plate at 10,000 cells/well and incubated overnight to adhere. Subsequently, gradient concentrations of DHM-Fe were added to the wells. After 24 h, the mediums were replaced with 100 µL of 10% CCK-8 followed by incubation for 2 h. Finally, CCK-8 absorbance was measured using a microplate reader at 450 nm.

Intracellular uptake assay

Raw 264.7 cells were cultured in a 6-well plate or in 35 mm confocal dishes. After adhesion, cells were co-cultured with the same concentrations of RDM-labeled DHM-Fe and DF@YM for 4 h. In the inhibitor group, cells were pretreated with the Dectin-1 inhibitor Laminarin. Cells were then washed and harvested for flow cytometry analysis. For confocal imaging, cells were washed and stained with DAPI, and the images were acquired and analyzed by confocal microscopy.

Mitochondria localization

Cells were seeded in confocal dishes and co-incubated with DF@YM for 4 h. Subsequently, a mitochondrial probe was added and stained for 30 min. After being washed with PBS, cells were stained with DAPI for 20 min, followed by confocal imaging analysis.

Intracellular antioxidant assays

RAW 264.7 and Caco-2 cells were seeded and co-incubated with DHM-Fe (10, 20, 40, and 80 µg/mL) and H₂O₂ (500 μM ) for 24 h. The relative cell viability was tested by CCK8 assays. Additionally, the cells were further stained with Calcein AM and PI for 20 min. After PBS washing, the cells were analyzed by fluorescence microscopy to evaluate the cytoprotective effect of DHM-Fe.

RAW 264.7 cells were co-incubated with DHM-Fe and LPS, and stained with a DCFH-DA probe (20µM) for 30 min. The cell suspension was collected, and the fluorescence intensity was quantified by flow cytometry. Likewise, RAW 264.7 cells were seeded in confocal dishes. Cells were treated according to the above methods and steps. After staining with DAPI, the fluorescence intensity was observed using fluorescence microscopy.

RAW 264.7 and Caco-2 cells were seeded and co-incubated with DHM-Fe and H₂O₂ for 24 h. Cells were stained with JC-1 for 30 min and then subjected to imaging analysis.

Caco-2 cells were seeded and co-incubated with DHM-Fe (40 µg/ml) and H₂O₂ for 24 h. Following a 15-minute incubation with Annexin V-FITC/PI at 25 °C in the dark, apoptosis was quantified by flow cytometry.

Intracellular anti-inflammatory effect

Though termed M1 polarization in pharmacological studies, acute LPS primarily triggers a pro-inflammatory activation state that shares key M1 effector functions. To study the anti-inflammatory activity of DHM-Fe, Raw 264.7 cells were incubated with DHM and DHM-Fe (40 µg/mL) and LPS (1 µg/mL). After 24 h of incubation, the supernatants were collected and analyzed for pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) using ELISA kits according to the manufacturer’s instructions. To assess CD86 expression, LPS-induced cells were harvested, washed, and stained with an anti-CD86 antibody for 30 min at 4℃, and finally analyzed by flow cytometry.

Intestinal colonization of DF@YM

The ICG-labeled DHM-Fe and DF@YM were prepared using the same methodology as previously described. ICG-labeled DHM-Fe and DF@YM at the same concentration were orally administered to either healthy mice or mice with colitis. To assess the differences in fluorescence intensity among the various groups, in vivo fluorescence imaging was conducted at 3, 6, 12, and 24 h. After 24 h, the mice were euthanized, and the fluorescence signals in various tissues were quantified. The distribution differences of the orally delivered agents in vivo and across tissues were compared following the same experimental procedure.

Animals

C57/BL6 male mice (8 weeks old) from the Lanzhou Veterinary Research Institute (Chinese Academy of Agricultural Sciences) were used in this study.

Hemolytic test

A hemolytic test was conducted using fresh mouse blood. The blood (1 mL) was diluted in 10 mL of PBS and centrifuged at 1000 rpm for 15 min to isolate red blood cells (RBCs), followed by PBS washing until the supernatant became clear. The diluted blood was added to each test tube and incubated at 37 °C for 60 min. After incubation, a 20 µL aliquot from each group was examined using microscopy for morphological changes, while the remainder was centrifuged again under the same conditions. The absorbance of the final supernatant was measured at 540 nm, and the hemolysis percentage was calculated.

In vivo biocompatibility evaluation of DF@YM

Mice received a daily oral dose of 50 mg/kg for 10 and 30 days. Following this period, the mice were euthanized, and blood samples were collected for biochemical and hematological analyses. Major organs, including the heart, liver, spleen, lungs, kidneys, stomach, and colon, were harvested, fixed with 4% paraformaldehyde (PFA), and stained with hematoxylin and eosin for histological examination.

ELISA

To quantify the levels of inflammatory factors in serum, the samples were collected. The levels of TNF-α, IL-1β, IL-6, IL 22 and CYP1A1 were quantified with commercial ELISA kits according to the manufacturers’ protocols.

In vivo therapeutic effect

The DSS-induced colitis mouse model was used to evaluate the therapeutic efficacy of DF@YM. Following a one-week acclimatization period, male C57BL/6 mice were randomly assigned to groups and subjected to either a treatment or a delayed-treatment protocol. Body weight changes and disease activity index (DAI) scores were recorded daily. After euthanasia, colons were excised, and the colon length was measured and photographed. Colon tissue samples were collected and prepared for histological evaluation.

Immunohistochemical analysis

Tissue sections were deparaffinized, rehydrated, and subjected to heat-induced antigen retrieval in citrate buffer. After blocking endogenous peroxidase and non-specific sites, the sections were probed with primary antibodys overnight at 4 °C. Antigen binding was detected using an HRP-conjugated secondary antibody and a DAB chromogen, followed by hematoxylin counterstaining. All sections were imaged under a light microscope following dehydration and mounting.

16S rRNA Sequencing

Fecal samples were collected from control, DSS-induced, and DSS + DF@YM-treated mice to assess the influence of DF@YM on gut microbiota composition via 16 S rRNA sequencing. Genomic DNA was extracted with the MagPure Soil DNA LQ Kit (Magan), followed by quality assessment. Library sequencing was carried out on an Illumina NovaSeq 6000, and the resulting data were processed for alpha and beta diversity analyses within the QIIME 2 framework.

Untargeted metabolome

Metabolites were extracted from the collected mouse fecal samples using a methanol-water solution. The extracts were then subjected to chromatographic separation on a Waters ACQUITY UPLC I-Class plus system coupled to a Thermo Q Exactive HF high-resolution mass spectrometer equipped with a heated electrospray ionization (HESI) source. Data were acquired in both positive and negative ionization modes to profile the metabolome. The raw LC-MS data were processed with the XCMS software (v4.5.1) for baseline filtering, peak identification, peak integration, retention time correction, and alignment. The data matrix was analyzed by PCA using R to visualize the global sample distribution and assess the analytical workflow stability. Orthogonal partial least squares-discriminant analysis (OPLS-DA) and partial least squares-discriminant analysis (PLS-DA) were employed to discriminate metabolic profiles between groups. Metabolites with variable importance in projection (VIP) scores > 1.0 from the OPLS-DA model and p-values < 0.05 from two-tailed Student’s t-tests were identified as significantly altered.

Mass spectrometry imaging of colon

Colon tissues were collected and stored frozen at -80 °C. The frozen tissues were sectioned into 15 μm thick slices at -20 °C. After being deposited onto indium tin oxide (ITO)-coated glass slides, the tissue sections were allowed to dry before being coated with a matrix layer of α-cyano-4-hydroxycinnamic acid (CHCA). MALDI-MSI data were acquired every 40 μm using a Shimadzu iMScope QT MS imaging system, with 100 laser shots (repetition rate 1000 Hz, laser diameter 2 μm, intensity 65) applied to each section surface. The detection voltage of the microchannel plate detector was set at 2.15 kV, and the pixel pitch was 25 × 25 μm². Positive ion detection mode was used to measure ion signals (m/z range of 280–380) for MS analysis to determine the MSI characteristics of DHM.

Statistical analysis

All the results were statistically analyzed by GraphPad Prism 8.0 software. Student t-test or one-way ANOVA was used for comparison. Data were presented as mean ± standard deviation (SD). P -value < 0.05 indicated a statistically significant difference.

Results and discussion

Synthesis and characterization of DHM-Fe and DF@YM

The precise etiology of IBD has yet to be fully elucidated, and its occurrence and development are associated with dysregulation of mucosal immunity, dysfunction of the intestinal barrier, and dysbiosis of gut microbiota triggered by oxidative stress [28]. Given the complex pathogenesis of IBD, we have developed a ROS regulator based on yeast-derived microcapsule encapsulation (DF@YM). Compared with existing reports, this system has achieved the direct coordination of DHM with Fe³⁺, forming smaller and more stable nanostructures that are more conducive to tissue penetration and retention in the inflamed intestinal tract [29, 30]. DHM-Fe was synthesized via a one-step assembly method. The pH of the aqueous DHM solution was adjusted to 8–9 with 1 M NaOH solution. The assembly of DHM-Fe was initiated by the dropwise addition of an aqueous iron (III) chloride solution. The formation of the DHM-Fe complex effectively improved the dispersion and solubility of DHM in water (Fig. S1A). Transmission electron microscopy (TEM) analysis revealed that DHM-Fe possessed a uniform morphology and had a size of approximately 35.89 ± 8.08 nm (Fig. 2A-B).

Fig. 2.

Characterization of DHM-Fe and DF@YM and RONS scavenging activities. (A) TEM images and diameters of DHM-Fe. (B) Size distributions of DHM-Fe. (C) UV–vis-spectra of DHM-Fe. (D) FTIR spectra of DHM-Fe. (E) Wide-range XPS patterns of DHM-Fe. C1 s (F), O1 s (G), and Fe 2p (H) high-resolution XPS spectra of DHM-Fe. (I, G) TEM image of YM and DF@YM. (K) EDS spectra of DF@YM. (L, M) Particle sizes and zeta potentials of YM and DF@YM. (N)The quantitative analysis of DPPH scavenging rate by DHM-Fe at different concentrations. (O) Relative SOD-like activity at different DHM-Fe concentrations. (P) Relative CAT-like activity at different DHM-Fe concentrations. (Q) Relative CAT-like activity of DHM-Fe (100 µg/ml) at different H₂O₂ concentrations. (R) Schematic diagram of the ROS-scavenging mechanism of DHM-Fe

The amorphous structure of DHM-Fe was further confirmed by X-ray diffraction (XRD) analysis. (Fig. S1B). The UV–vis spectroscopy of DHM-Fe displayed a maximum absorption peak at 292 nm, consistent with that of DHM, and possessed a characteristic peak corresponding to FeCl₃. (Fig. 2C). Fourier-Transform Infrared spectroscopy (FTIR) of DHM-Fe revealed the coordination of iron ions with the C = O and HO–C groups of DHM, which was evidenced by intensity changes in the regions of 1643–1637 cm⁻¹ (C = O stretching band) and 3354–3426 cm⁻¹ (HO-C stretching band), respectively. (Fig. 2D). Additionally, X-ray photoelectron spectroscopy (XPS) was used to analyze the elemental composition of DHM-Fe, confirming the presence of C, O, and Fe. (Fig. 2E). High-resolution XPS (HR-XPS) spectra showed peak fitting for C 1s, O 1s, and Fe 2p, offering detailed insights into the chemical bonding environment within DHM-Fe (Fig. 2F–H). The HR-C 1s spectrum was assigned to C–C, C-O–C, C–O, and C = O bonds, while the O 1s spectra confirmed the existence of metal oxides, alongside the expected C–O and C = O in DHM-Fe. Notably, the Fe 2p spectrum demonstrated the coexistence of both Fe²⁺ and Fe³⁺ oxidation states.

Next, a hollow YM was prepared mainly according to a previously reported method [31, 32]. TEM revealed that YM has an elliptical spherical morphology (Fig. 2I). Subsequently, DHM-Fe was further loaded into the hollow YM via electrostatic adsorption and pore entrapment. TEM coupled with energy-dispersive spectroscopy (EDS) was employed to verify the successful encapsulation of DHM-Fe within YM. TEM images showed high-contrast DHM-Fe within the core of YM, demonstrating successful encapsulation (Fig. 2J). The presence of iron signals, as detected by EDS, confirmed the successful encapsulation of DHM-Fe in YM (Fig. 2K and Fig. S1C). Particle size measurements showed that YM and DF@YM had diameters of 4662.8 ± 363.6 nm and 4946.8 ± 419.0 nm, respectively. (Fig. 2L). DF@YM exhibited a modest increase in particle size compared to YM, suggesting the loading of DHM-Fe. The zeta potential of DHM-Fe, YM, and DF@YM were − 24.48 ± 1.00 mV, -18.17 ± 0.21 mV, and − 20.1 ± 0.20 mV, respectively (Fig. 2M). Collectively, all these results indicated the successful synthesis of DF@YM. We further investigated the release profile of DF@YM in the gastrointestinal environment. As shown in Fig. S2, after incubation in simulated gastric fluid for 2.5 h, the drug release rate of DF@YM remained below 5%, indicating its good stability in the gastric environment. In simulated intestinal fluid, the drug release gradually increased and reached a plateau, with a cumulative release rate of 30% at 6 h. Notably, in simulated intestinal fluid containing β-glucosidase, the drug release was further enhanced to 40%. These results demonstrated that DF@YM remained structurally stable in the gastric environment with minimal drug release, but once reaching the intestine, it achieved gradual release under the specific intestinal conditions, thereby improving therapeutic efficacy.

Enzymatic activities of DHM-Fe

Accumulating evidence suggests that various inflammatory mediators ultimately lead to the generation of large amounts of ROS at the site of IBD through highly active enzymes (such as iNOS, NOX, and MPO), thereby driving the progression of the disease [33]. The high oxidative activity of ROS induces lipid peroxidation and disrupts the intestinal epithelial barrier. Additionally, excessive ROS leads to gut microbiota dysbiosis in IBD by disrupting the intestinal barrier and severely altering the gut microenvironment [34]. Therefore, scavenging excess ROS is critical for the treatment of IBD. Given the ROS-scavenging capacity of iron-based nanozymes [13] and the antioxidant properties of DHM [35], we first evaluated the catalytic activity of DHM-Fe nanoparticles against RNS, O₂•-, and H₂O₂. DPPH, a stable nitrogen-centered radical, is widely used in free radical scavenging assays. The DPPH radical assay was utilized to evaluate the RNS scavenging capability of DHM-Fe nanoparticles. Notably, DHM-Fe exhibited dose-dependent scavenging of RNS, and nearly 70% DPPH was oxidized at the concentration of 6.25 µg/mL, indicating the effective RNS scavenging ability (Fig. 2N and Fig. S3A). The SOD-like catalytic activity (O₂•⁻ scavenging) of DHM-Fe nanoparticles was studied using the NBT method, The O₂•⁻ scavenging assay demonstrated that DHM-Fe acted in a dose-dependent manner, effectively eliminating over 90% of O₂•⁻ within 20 min at the concentration of 100 µg/mL (Fig. 2O and Fig. S3B). H₂O₂ is produced through posed into O₂•⁻ disproportionation and can be enzymatically decomposed into H₂O and O₂ by CAT (Fig. 2R). The CAT-mimicking activity of DHM-Fe was assessed by using a dissolved oxygen meter to measure its catalysis of O₂ release from H₂O₂. As illustrated in Fig. 2P and Q, DHM-Fe effectively catalyzed O₂ generation. The H₂O₂ scavenging capacity was concentration-dependent. Moreover, the generated O₂ of DHM-Fe increased with an increasing concentration of H₂O₂. Collectively, these results demonstrated that DHM-Fe exhibited excellent enzymatic activities, which enable it to effectively scavenge ROS and RNS to protect cells from oxidative stress.

Anti-oxidant and anti-inflammatory effects of DHM-Fe

Biocompatibility is a prerequisite for nanomedicine. To validate the in vitro biocompatibility of DHM-Fe, cytotoxicity was investigated through CCK-8 assays. DHM-Fe showed no significant cytotoxicity towards RAW 264.7 and Caco-2 cells even at high concentrations (up to 320 µg/mL). In contrast, DHM already exhibited obvious cytotoxic effects at a concentration of 160 µg/mL (Fig. S4A, 4B). Macrophages play a crucial role in the onset and progression of IBD [36]. Targeting macrophages is a feasible strategy for treating IBD. Given that the YM surface expresses β-glucan, which is specifically recognized by the Dectin-1 receptor of macrophages, DF@YM can better target macrophages in the intestine. This was verified by drug uptake assays in macrophages. To better visualize the binding effect of nanoparticles within macrophages, Rhodamine B (RhB) -labeled DHM-Fe and DF@YM were used to assess the uptake ability of RAW 264.7. As presented in Fig. 3A, the uptake of DF@YM by RAW 264.7 cells was significantly higher than that of DHM-Fe after 4 h of incubation. However, when RAW 264.7 cells were pretreated with the Dectin-1 inhibitor laminarin, the DF@YM group showed reduced red fluorescence intensity in CLSM images, whereas no notable change was observed in the DHM-Fe group, which aligned with flow cytometry results (Fig. 3B). These findings indicated that DF@YM effectively targeted RAW 264.7 cells by binding to the surface Dectin-1 receptor.

Fig. 3.

The cellular internalization, anti-oxidant, and anti-inflammatory effects of DHM-Fe and DF@YM. (A) Cellular uptake of DHM-Fe and DF@YM in RAW 264.7. (B) Intracellular fluorescence in RAW 264.7 was detected by flow cytometry. (C, D) Fluorescence images and flow cytometry quantification of DCFH-stained RAW 264.7 cells after different treatments. (E) Fluorescence images of RAW 264.7 cells stained with Calcein-AM/PI. (F, G) Relative viabilities of RAW 264.7 cells with different treatments. (H) Co-localization fluorescence images of DF@YM and mitochondria. (I)The PCC and MCC analysis of DF@YM with mitochondria. (J) The plot profile analysis of DF@YM with mitochondria. (K) JC-1 staining showing mitochondrial membrane potential changes in RAW 264.7 cells. (L) The apoptosis analysis of Caco-2 cells by flow cytometry in different groups. (M-O) Measurement of TNF-α, IL-1β, and IL-6 in cell supernatants of RAW 264.7 cells by ELISA. (P) Flow cytometric analysis of M1 macrophage in each group. Data are presented as means ± SD (n = 3; *P < 0.05, **P < 0.01, ***P < 0.001)

Excessive generation of ROS is a key driver of many inflammatory diseases, resulting in lipid peroxidation, protein oxidation, and DNA damage. To evaluate the intracellular ROS scavenging capacity of DHM-Fe, the 2’, 7’-dichloro-dihydrofluo-.

rescein diacetate (DCFH-DA) fluorescent probe was employed. LPS was used to stimulate RAW 264.7 cells for intracellular ROS production, followed by treatment with DHM-Fe nanoparticles. Assessment of intracellular ROS levels was performed by means of fluorescence imaging and flow cytometry. After LPS treatment, strong green fluorescence was observed, indicating elevated ROS levels. By contrast, treatment with DHM and DHM-Fe led to a decrease in green fluorescence intensity (Fig. 3C). Flow cytometry analysis further confirmed that DHM and DHM-Fe notably decreased ROS levels, with DHM-Fe exhibiting superior scavenging capacity (Fig. 3D).

Exposure to oxidative stress, like high concentrations of H₂O₂, can lead to oxidative damage and cell death. To evaluate the protective effect of DHM-Fe against H₂O₂-induced oxidative damage, cell viability was measured using the CCK-8 assay. DHM-Fe nanoparticles attenuated H₂O₂-induced cell death in a concentration-.

dependent manner (Fig. 3F-G), whereas DHM alone showed minimal protective effect (Fig. S4C-E). Further assessment of cell status using calcein-AM/PI staining confirmed that H₂O₂ stimulation induced cell death due to oxidative stress. In contrast, treatment with DHM-Fe significantly reduced the number of dead cells, demonstrating its strong protective activity (Fig. 3E).

Mitochondria are the primary organelles responsible for generating ROS in cells. Therefore, we examined whether DF@YM could scavenge ROS by co-localizing with mitochondria. Fluorescence images showed that RhB-labeled DF@YM exhibited co-localization with mitochondria (Fig. 3H). The extent of colocalization between the RhB fluorescent signal and Mito-Tracker Green was evaluated using the Pearson correlation coefficient (PCC) and Manders’ colocalization coefficient (MCC). A PCC value of 0.95 confirmed the colocalization of DF@YM with mitochondria to some extent (Fig. 3I ). The result of the PCC analysis was consistent with the MCC, which was further validated by plot profile analysis. The observed correlation between the red (DF@YM) and green (Mitochondria) peaks served as visual evidence for their colocalization (Fig. 3J). The above results demonstrated that DF@YM could scavenge ROS by co-localizing with mitochondria.

High levels of ROS can oxidize lipids in the mitochondrial membrane, leading to a loss of structural integrity and subsequent proton leakage, ultimately resulting in a decrease in membrane potential. The effect of DHM-Fe on H₂O₂-induced changes in mitochondrial membrane potential (ΔΨm) was investigated. As shown in Fig. 3K and Fig. S5, exposure to H₂O₂ triggered a marked rise in green fluorescence intensity, whereas treatment with DHM-Fe increased the red fluorescence, demonstrating the efficacy of DHM-Fe in H₂O₂ scavenging. This result suggested that DHM-Fe can alleviate the decrease in ΔΨm induced by oxidative stress. Next, the protective effect of DHM-Fe against cell apoptosis under excessive oxidative stress was further evaluated. Flow cytometric analysis using Annexin V-FITC/PI staining indicated that DHM-Fe treatment suppressed H₂O₂-induced apoptosis, which consequently enhanced cell viability (Fig. 3L). To evaluate the effect of DHM-Fe on the repair capacity of intestinal epithelial cells, we employed a scratch wound healing assay. As shown in Fig. S6, in the H₂O₂ group, the scratches closed very slowly over 24 h. In contrast, the scratches in the DHM-Fe group closed faster, indicating that DHM-Fe significantly enhanced the migratory capacity of Caco-2 cells, thereby accelerating the “wound” healing process in vitro. This study demonstrated that DHM-Fe can significantly promote the migration and proliferation of intestinal epithelial cells Caco-2. The above results indicated that DF@YM could co-localize with mitochondria, stabilized the ΔΨm by scavenging excess ROS, thereby preventing cell apoptosis and promoting cell proliferation.

Macrophage polarization is critically involved in the pathogenesis of numerous diseases. Disruption of the homeostatic balance between M1 and M2 macrophage polarization contributes to the development of pathological microenvironments and chronic inflammation [37]. As a major driver of IBD, M1 macrophages exacerbate the progression through their pro-inflammatory effects. LPS promotes the polarization of macrophages towards the M1 phenotype and stimulates the production of key pro-inflammatory cytokines by activating the TLR4 receptor on macrophages. In this.

study, we utilized LPS to induce an acute inflammatory state in RAW 264.7 cells. The relative expression of critical inflammatory cytokines (TNF-α, IL-1β, and IL-6) released by M1 macrophages was quantified. LPS stimulation markedly increased the expression of these cytokines; this effect was significantly reversed by both DHM and DHM-Fe. Notably, the DHM-Fe group exhibited a more potent suppressive effect on cytokine expression (Fig. 3M-O and Fig. S7) Additionally, the M1 marker CD86 on macrophages was measured by flow cytometry to further investigate whether DF@YM can effectively modulate phenotypic changes in macrophages. As shown in Fig. 3P and Fig. S8, CD86 expression was significantly increased after LPS induction. DHM-Fe was able to modulate macrophage polarization, leading to a notable decrease in CD86. To further illustrate the reprogramming capacity of DHM-Fe on macrophages, morphological changes in RAW 264.7 cells were recorded via microscopy. Microscopic observation revealed that cells in the control group exhibited a rounded morphology, whereas the LPS-treated group displayed heterogeneous shapes accompanied by a notable decrease in round cells. The cellular morphology in the LPS group resembled that of M1 macrophages. On the contrary, cells in the DHM-Fe treatment group predominantly maintained a rounded shape, indicating suppression of cell polarization (Fig. S9). These findings demonstrated that DF@YM exerted anti-inflammatory effects by suppressing macrophage polarization.

Biocompatibility evaluation of DHM-Fe and DF@YM

A comprehensive biocompatibility assessment was performed to validate the oral safety of DF@YM, and the hemolysis test was utilized to evaluate the biocompatibility. As shown in Fig.S3A and 4B, the 1% Triton X-100 treatment group (positive control) demonstrated a significantly higher hemolysis rate compared to other groups (p < 0.001). Conversely, the hemolysis rates of DHM-Fe and DF@YM were measured at (0.4 ± 0.4)% and (0.6 ± 0.3)%, respectively. No statistically significant difference was observed compared to the PBS group (negative control) (p > 0.05), and both were significantly below the safety threshold of 5%. The in vivo biocompatibility of DF@YM was further assessed. Body weight was monitored after oral administration of DF@YM (50 mg/kg) for 10 and 30 days. As illustrated in Fig. 4C and Fig. S10A, no significant body weight difference was observed between the two groups.

Fig. 4.

Biocompatibility evaluation of DF@YM. (A) Observation of hemolysis under the microscope after different drug treatments, (B) Hemolysis rate in different treatment groups. (C) Body weight changes in mice within 10 days after oral administration of DF@YM. (D) H&E staining of vital organs was conducted to assess the systemic toxicity of DF@YM. (E) Analysis of complete blood panel and blood biochemistry in treated mice. Data are presented as means ± SD (n = 3)

H&E staining-based histopathological examination of major organs showed no detectable histological changes or cellular damage in DF@YM-treated mice compared to the healthy control group. Additionally, the hepatorenal toxicity of DF@YM was subsequently evaluated. Key markers of liver function, key markers of renal function, and MDA/GSH were measured [38]. Analysis of serum biochemical and hematological parameters in mice administered DF@YM showed no notable deviations from those of the healthy control group (Fig. 4D-E and Fig. S10B-E). Taken together, these findings demonstrated the favorable biocompatibility of DF@YM, making it suitable for in vivo research.

In vivo biodistribution evaluation

Oral administration for treating intestinal inflammation represents an effective and convenient therapeutic strategy for IBD. This approach requires that the oral drug delivery system efficiently targets the inflamed regions and achieves prolonged retention. By enriching β-1,3-D-glucan on its surface, YM can specifically target macrophages at inflammatory sites, making it an ideal carrier. Furthermore, the polysaccharide components and the irregular surface structure of YM contribute to an extended residence time in the intestinal tract, thereby enhancing their local therapeutic efficacy. Targeting gut-enriched macrophages is a crucial therapeutic strategy for IBD. We have successfully demonstrated that DF@YM can target the Dectin-1 receptor on macrophages in vitro. Using near-infrared (NIR) fluorescence imaging, we further evaluated the targeting and retention capabilities of DF@YM in vivo in DSS-induced colitis. Mice in the healthy group and the DSS group were administered ICG-labeled DHM-Fe and DF@YM, respectively. To track the distribution of DF@YM in mice, in vivo fluorescence imaging was conducted after oral administration. In the healthy group, DF@YM exhibited a trend of higher fluorescence intensity than DHM-Fe at each time point, although no statistically significant difference was observed in the average fluorescence intensity between the two groups. In the colitis mouse model, DF@YM exhibited excellent retention, with a statistically significant difference in the average fluorescence intensity between the two groups. Following euthanasia at the experimental endpoint, major organs and colons were harvested for ex vivo fluorescence imaging. Imaging revealed no discernible fluorescence signal in the major organs. In the DSS group, the colon exhibited significantly higher fluorescence intensity compared to the control group, and the difference was statistically significant (Fig. 5A-D). These results confirmed that DF@YM was able to target and accumulate at the intestinal inflammatory sites, demonstrating great potential for treating IBD.

Fig. 5.

Targeting and retention capability of DF@YM in the Inflamed Colon. (A) Fluorescence images of normal mice, DSS-induced colitis mice, organs, and colon tissues after oral DHM-Fe and DF@YM. (B, C) Fluorescence intensity analysis in normal mice and colitis mice at various time points after oral administration of different drugs. (D) Fluorescence intensity analysis of the mouse colon at the endpoint. (E) Immunofluorescence staining of colon tissue. Data are presented as means ± SD (n = 3,*P < 0.05)

To further examine the retention of DF@YM within the inflamed colon, colon tissue frozen sections were obtained after 24 h of oral administration and subjected to immunofluorescence staining for F4/80. The green fluorescence (representing F4/80) was observed in the colon tissue, which provided confirmation of macrophage (Fig. 5E). Meanwhile, the red fluorescence signal corresponding to DF@YM was mainly distributed in the mucosal layer. The distribution demonstrated that the YM encapsulation strategy significantly enhanced drug accumulation in macrophages and prolongs its retention at the site of inflammation. The fluorescence microscopic observation provided further evidence that DF@YM effectively targeted and accumulated in inflammatory regions, supporting its potential as a strategy for IBD treatment.

Preventive Effects of DF@YM on DSS-induced colitis

Based on the advantages of DF@YM in intestinal epithelial protection and biosafety, we hypothesized that it may exert a preventive effect before the onset of colitis. Mice received 2.5% DSS to induce colitis, with drug treatments administered simultaneously. Based on the biosafety assessment, the dosage of DHM, DHM-Fe, and DF@YM was set at 50 mg·kg⁻¹. To evaluate the efficacy of DF@YM, 5-ASA was selected as the positive control (Fig. 6A). The control group showed a gradual increase in body weight, with no significant change in the disease activity index (DAI). Compared to the DSS group, DF@YM treatment was most effective in ameliorating body weight loss (Fig. 6B) and significantly suppressed the increase in DAI scores (Fig. 6C), as well as prevented colon shortening and the elevation of the spleen index (Fig. 6D-E, and Fig. S11). Inflammatory cytokines are the primary drivers of the onset, progression, and exacerbation of colonic inflammation. They dysregulate intestinal immune homeostasis, promoting a persistent and excessive inflammatory response that directly contributes to tissue damage and clinical symptoms. Levels of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) were increased in the DSS group, while DF@YM treatment effectively lowered these levels (Fig. 6F-H).

Fig. 6.

Protective effect of DF@YM against DSS-induced colitis. (A) Schematic representation of the experimental process for treating the DSS-induced colitis model. (B-C) Body weight change and DAI of mice within 8 days. (D) colon images of mice in different treatment groups. (E) The length of the colon. (F-H) The levels of TNF-α, IL-1β, and IL-6 in colon tissues measured by ELISA. (I) DHE staining of colon tissues. (J) H&E staining of colon tissues. (K, L) Immunohistochemical image of occludin and ZO-1 in the colon. (M) WB analysis of Claudin, Occludin, and MUC2 protein expression in colon tissues. Data are presented as means ± SD; *P < 0.05, **P < 0.01, ***P < 0.001, representing different statistical significances

To evaluate intestinal inflammation, immunofluorescence staining and SOD activity in colon tissues was conducted. As shown in Fig. 6I and Fig. S12, the results showed that DF@YM treatment significantly reduced ROS accumulation and increased SOD activity in the colonic tissue of DSS-induced mice. H&E staining revealed inflammatory features such as mucosal structural disruption and cellular damage in DSS group (Fig. 6J). The tight junction, composed of specific proteins, serves as the core structural basis for the physical barrier function of the intestinal epithelium. The integrity and functional stability of tight junctions are key determinants of intestinal barrier permeability. Dysfunction of tight junctions is a central event in intestinal barrier impairment under various pathological conditions, serving as a key molecular link between etiology and disease manifestation [39, 40]. The tight junction proteins were assessed by immunohistochemical staining for Occludin and ZO-1. The expression of both proteins was significantly down-regulated in the DSS group. While DHM intervention resulted in a moderate increase, the protein levels in the DF@YM group were closer to those of the control group (Fig. 6K, L). This indicated that DF@YM can repair intestinal barrier integrity via the upregulation of tight junction proteins. We also examined the protein levels of Occludin, Claudin, and MUC2 by Western blot (WB). The results demonstrated that DF@YM treatment up-regulated the expression of tight junction proteins and mucin, which is consistent with the immunohistochemical findings (Fig. 6M). Additionally, DSS suppressed epithelial cell proliferation, as indicated by fewer Ki-67⁺ cells at the base of the colon crypt. DF@YM partially restored epithelial cell proliferation, as evidenced by an increase in Ki-67⁺ cell numbers compared to the DSS group, which demonstrated a reversal of the suppression (Fig. S13). These results indicated that DF@YM treatment not only restored colon length but also mitigated tissue damage. Overall, DF@YM showed comparable or superior efficacy to 5-ASA, effectively reducing inflammatory symptoms and enhancing barrier protection, demonstrating a protective effect on colonic tissue.

Modulatory effect of DF@YM on gut microbiota

Dysbiosis of the gut microbiota is a major contributor to the development and progression of IBD. Excessive production of ROS disrupts intestinal barrier integrity, facilitating bacterial invasion into the gut wall and triggering an imbalanced immune-inflammatory response [41]. High-throughput 16 S rRNA gene sequencing was performed to evaluate the modulatory effect of DF@YM on the composition and abundance of the gut microbiota. As demonstrated in Fig. 7A, DSS treatment led to a significant increase in the abundance of harmful microorganisms (e.g., Proteobacteria) at the phylum level versus control. In contrast, DF@YM treatment significantly elevated the levels of beneficial microbes (such as Bacteroidetes) [42] and modulated the microbial composition closer to that of the control group. Consistent trends were observed at the family level (Fig. 7B). These beneficial microorganisms play a key role in maintaining intestinal homeostasis by inhibiting pathogen growth and modulating mucosal immune function. To assess α-diversity, the Shannon index (reflecting the disorder of species distribution) and the Simpson index (representing niche differentiation) were estimated. The results indicated that the DSS group exhibited a significant decrease in microbial diversity relative to the control; in contrast, the DF@YM group was comparable to the control (Fig. 7C, D). Subsequently, changes in microbial community composition were assessed through β-diversity analysis. Both principal coordinate analysis (PCoA) and non-metric multidimensional scaling (NMDS) showed a distinct separation between the DSS and control groups, revealing a disrupted microbiota structure. Notably, samples from the DF@YM group clustered more closely with the control group, suggesting that DF@YM could modulate the gut microbiota structure in the colitis model and promote its restoration towards a normal state (Fig. 7E, F).

Fig. 7.

DF@YM modulates gut microbiota composition in DSS-induced colitis. (A, B) Relative abundance of gut microbiota at the phylum and family level. (C, D) α-diversity metrics displayed as Shannon index and Simpson index. (E, F) β-diversity displayed as principal coordinate analysis and on-metric multidimensional scaling (NMDS) plot. (G, H) Linear discriminant analysis effect size (LEfSe) and cladogram based on LEfSe analysis. (I) Significant relative abundance changes at.genus levels

Linear discriminant analysis effect size (LEfSe) can be used to identify and interpret high-dimensional biological data, revealing statistically significant taxonomic differences following intergroup comparisons. To further investigate the differential characteristics of the microbial composition, LEfSe analysis was performed. Following the identification of significantly different species through intergroup comparisons, linear discriminant analysis (LDA) was employed to assess the effect of abundance changes across taxa. The LDA scores illustrated the dominant microbial taxa and their effects at various taxonomic levels, ranging from phylum to genus, with higher scores indicating greater enrichment in their respective groups. Under homeostatic conditions, the fecal microbiota is predominantly composed of obligate anaerobic bacteria, specifically the class Clostridia and the class Bacteroidia. In contrast, dysbiosis associated with inflammatory bowel disease (IBD) is characterized by an increase in facultative anaerobic Enterobacteriaceae (phylum Proteobacteria) and a concomitant reduction in Clostridia [43]. The results showed a marked increase in pathogenic Escherichia-Shigella and Enterobacteriaceae in the DSS group, while the beneficial Bacteroidota, Lachnospiraceae, and Clostridia were markedly increased in the DF@YM group (Fig. 7G, H).

Furthermore, changes in the abundance of microbiota at the genus level were analyzed. DF@YM intervention significantly reduced the abundance of two potentially harmful bacteria (Escherichia-Shigella and Helicobacter). Notably, an increase in Escherichia-Shigella has been demonstrated to be strongly linked to the induction of gut microbiota dysbiosis and the onset of colitis [44]. It is well-established that the beneficial role of Muribaculaceae in protecting intestinal health [45]. It is positively correlated with short-chain fatty acids [46]. The correlation analysis between bacterial abundance and colitis-related indicators revealed that both Clostridia_UCG-014 and Muribaculaceae showed significant positive correlations with colon length, body weight, and gut barrier index, while exhibiting negative correlations with the DAI and histopathological score [47]. Importantly, a significant increase in the abundance of Muribaculaceae and Clostridia_UCG-014 was observed after DF@YM treatment, compared to the DSS group (Fig. 7I). In summary, the therapeutic effect of DF@YM on colitis is partially attributable to gut microbiota modulation, with consequent amelioration of inflammation and restoration of barrier function.

Modulation of gut microbial metabolism by DF@YM

Gut microbial metabolites are key mediators of host-microbiota interactions. Based on its efficacy in modulating the gut microbiota, we next evaluated the effect of DF@YM on colitis-associated microbial metabolites in mice. The metabolite profile was severely disrupted in the DSS group compared to the control group, suggesting that DSS induction altered the metabolites of the gut microbiota. Strikingly, DF@YM administration significantly reversed the alteration (Fig. 8A). PCA showed a clear separation in metabolite profiles. The DF@YM group showed a clear separation from the DSS group, with its samples closer to the normal group, indicating a strong therapeutic effect. (Fig. 8B). Volcano plot analysis identified 254 upregulated and 2,363 downregulated metabolites in the DSS-induced colitis group compared to the normal (Fig. 8C). DF@YM treatment reversed this trend, resulting in 1,315 upregulated and 199 downregulated metabolites versus the DSS group (Fig. 8D). Gut bacteria modulate host metabolism and immune function by producing a broad spectrum of chemically diverse metabolites, notably those derived from amino acids, as revealed by metabolomic studies. KEGG enrichment analysis of the differentially expressed metabolites between the DSS and control groups revealed significant alterations in amino acid metabolism pathways. The downregulation of key pathways, particularly arginine and proline metabolism and tryptophan (Trp) metabolism, is known to exacerbate IBD progression [48]. The bar chart and bubble chart of KEGG enrichment analysis showed that, arginine and proline metabolism and Trp metabolism were downregulated in the DSS group, and DF@YM treatment significantly upregulated the Trp metabolism pathway in colitis mice (Fig. 8E-H). The gut microbiota metabolizes aromatic amino acids to produce a range of active compounds, such as indole and its derivatives. These microbial metabolites help maintain intestinal homeostasis not only by promoting the repair and regeneration of epithelial cells, thereby restoring and enhancing barrier integrity, but also by modulating the activity of local immune cells to suppress inflammation [49, 50].

Fig. 8.

DF@YM alters gut microbiota-derived tryptophan metabolism in DSS-induced colitis. (A) Number of metabolites. (B)The principal component analysis. (C) Volcano plot of DSS versus Control. (D) Volcano plot of DF@YM versus DSS. (E-H) Bar plot and bubble plot of KEGG pathway enrichment analysis. (I) Enrichment analysis of metabolites related to the tryptophan metabolic pathway. (J, K) Relative content of indole-3-acetic acid and kynurenic acid. (L) Western blot analysis of CYP1A1 in colon tissues. (M) ELISA analysis of IL 22 in colon tissues. Data were expressed as the mean ± SD (n = 3 or 4). *P < 0.05, **P < 0.01, and ns represents no statistical difference

The activation of aryl hydrocarbon receptor (AhR) is widely recognized as playing a critical protective role in maintaining intestinal homeostasis and suppressing inflammation. It is reported that a range of Trp metabolites produced by the gut microbiota mediate protective and anti-inflammatory effects on the epithelium, primarily through the activation of AhR signaling [51]. Enrichment analysis of metabolites related to the Trp metabolic pathway revealed changes in the abundance of 17 metabolites across groups. The heatmap illustrated that the abundance of kynurenic acid (KYNA) and indole-3-acetic acid (IAA) in the DSS group was significantly downregulated versus the control. DF@YM administration normalized the levels of these metabolites (Fig. 8I). The depletion of beneficial bacteria capable of metabolizing tryptophan, resulting from DSS-induced dysbiosis, causes a decline in gut-health essential metabolites, including indole and its derivatives such as IAA and indole-3-lactic acid [52]. The tryptophan metabolite KYNA serves as a key mediator of immunoregulation through its activation of the AhR. A comparative analysis of relative abundance indicated that KYNA and IAA were markedly reduced in the DSS group compared to the control (P < 0.01, Fig. 8J, K). This reduction was reversed by DF@YM treatment, which led to an increase in their levels compared to the DSS group. Research indicates that the severity of intestinal inflammation is inversely associated with the levels of KYNA and IAA, which activate the AhR signaling pathway as its ligands [53]. Therefore, we hypothesize that the protective role of DF@YM against colitis is attributed to its ability to modulate tryptophan metabolism, leading to elevated levels of KYNA and IAA and subsequent activation of the AhR signaling pathway. We also predicted the interaction between DHM and AhR through molecular docking. The results indicated that DHM directly binds to AhR via residues such as Phe220, Met237, and Cys222, with a binding energy of − 7.976 kcal mol^{− 1} (Fig. S14). To investigate whether the AhR pathway was activated, we analyzed the protein expression of CYP1A1, a classic biomarker of AhR activation [54], by WB. As shown in Fig. 8L, CYP1A1 expression was barely detectable in the DSS-treated group. In contrast, DF@YM treatment markedly increased its protein levels, indicating a potent activation of the AhR signaling pathway. Evidence indicates that AhR plays a key role in stimulating the production of interleukin-22 (IL-22). IL-22 promotes the repair of the intestinal wall by enhancing the expression of tight junction proteins and antimicrobial peptides, thereby helping to control intestinal inflammation [55]. Changes in the IL-22 levels across different groups were measured by ELISA. The results showed that the IL-22 level in the DSS group was significantly decreased, while it was slightly increased in the DHM group. However, the DF@YM intervention reversed this change induced by DSS (Fig. 8M). The above results revealed the key pathways and major metabolites involved in the treatment of colitis by DF@YM, indicating that its therapeutic effect may be closely linked to tryptophan metabolism and AhR/IL-22 pathway activation. To confirm this hypothesis, we exposed DSS-treated mice to an AhR inhibitor (StemRegenin 1, 50 mg/kg) to determine whether DF@YM exerts protective effects through modulation of the AhR/IL-22 pathway. The AhR antagonist largely abolished the protective effects of DF@YM against IBD, including the improvement in colonic histology, the reduction of TNF-α and IL-6 levels, and the upregulation of AhR downstream targets CYP1A1 and IL-22 in the tissue. However, the observed modest improvements in body weight and colon length suggested that DF@YM might alleviate disease symptoms through other pathways in addition to AhR activation (Fig. S15A-K).

Therapeutic Efficacy of DF@YM in DSS-induced colitis

To evaluate the therapeutic effect of DF@YM on colitis, a DSS-induced colitis model was established by orally administering 2.5% DSS for seven days. Starting from the fourth day, different drug interventions were administered to assess their therapeutic effects. The evaluation system included dynamic monitoring of body weight and DAI, measurement of colon length, histopathological examination, and detection of relevant inflammatory cytokine levels. A schematic outlining the experimental procedure was provided in Fig. 9A. It was found that the mice of DSS exhibited weight loss and an increased DAI. All treatment groups showed reductions in both weight loss and DAI scores compared to the DSS group. Notably, the DF@YM group showed significantly less weight loss and lower DAI scores than the other treatment groups (Fig. 9B-C). DF@YM treatment ameliorated colon shortening, thus demonstrating its protective effect against colitis-induced tissue damage (Fig. 9D-E).

Fig. 9.

Therapeutic effect of DF@YM against DSS-induced colitis. (A) Schematic representation of the experimental process for treating the DSS-induced colitis model. (B-C) Body weight change and DAI of mice within 10 days. (D, E) Colon images and the length of the colon in different treatment groups. (F) The spleen index in different treatment groups. (G-I) The levels of TNF-α, IL-1β, and IL-6 in colon tissues measured by ELISA. (J) H&E staining of colon tissues. (K, L) ImmunoHisto-chemical image of Claudin and ZO-1 in the colon. (M) Analysis of DHM content in serum. (N) Mass spectrometry imaging of colon tissue analysis at m/z 359.25. Data are presented as means ± SD; *P < 0.05, **P < 0.01, ***P < 0.001, representing different statistical significances

Further studies demonstrated that DF@YM treatment significantly reduced the spleen index and levels of key inflammatory cytokines, including TNF-α, IL-1β, and IL-6 (Fig. 9F-I). HE of colon tissue further confirmed the efficacy of DF@YM. Compared with the DSS, YM, and DHM groups, the DF@YM treatment exhibited a significant reduction in histopathological damage, indicating that DF@YM promotes the repair of colonic tissue and maintains its integrity (Fig. 9J). Impaired intestinal barrier function is a hallmark of colitis. Therefore, we further examined the expression of two tight junction proteins, Claudin and ZO-1, which are critical for maintaining epithelial integrity [56]. DSS treatment markedly decreased the levels of both proteins in colonic tissue. In contrast, DF@YM treatment significantly restored their expression, indicating its reparative capacity (Fig. 9K-L). Collectively, these findings suggested that DF@YM had therapeutic potential to restore intestinal homeostasis in the colitis model, which was attributable to its anti-inflammatory properties and ability to repair the intestinal epithelial barrier.

The spatial distribution of a drug within target tissues is a critical determinant of its therapeutic efficacy. The therapeutic efficacy of DHM is compromised by its low bioavailability. To address this issue, we constructed a novel delivery system for enhanced drug targeting and enrichment at colonic lesion sites by coordinating DHM with metal ions and loading it into YM, thereby improving pharmacokinetic profile of DHM. To validate the efficacy of this strategy, we quantitatively analyzed the concentration of DHM in mouse blood. Analysis of the DHM concentration in blood demonstrated that its level in the DF@YM group was significantly higher than that in the free DHM group (Fig. 9M). Mass spectrometry imaging (MSI) has emerged as an indispensable tool for the label-free, multiplexed mapping of molecules on tissue surfaces, making it an indispensable technique for visualizing drug distribution in situ [57]. Here, Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) was used to investigate whether DF@YM could enhance the colonization and sustained release of DHM specifically in the inflamed colon. Colon tissue sections were analyzed by monitoring the signal at m/z 359.25 to determine the relative abundance and distribution of DHM in different experimental groups. MSI data indicated that compared with DHM, the level at m/z 359.25 in the DF@YM tissue sections was significantly elevated and primarily enriched in the mucosal layer of the colon, confirming that DF@YM treatment promoted the accumulation of DHM in colitis tissues (Fig. 9N). This finding demonstrated that DF@YM effectively increased the abundance of small molecules in tissues, consistent with the in vivo fluorescence imaging results. Overall, these results demonstrated that the delivery system, engineered through metal coordination and YM loading, enhanced the tissue-specific accumulation of DHM, thereby partially improving its therapeutic efficacy. However, a complete pharmacokinetic analysis was not performed in this study, and the current conclusions are primarily based on tissue distribution evidence. Future complete pharmacokinetic studies will help confirm the extent of improvement in bioavailability. Taken together, DF@YM might exert a therapeutic effect on IBD by inhibiting proinflammatory factors, improving the intestinal barrier integrity and facilitating the restoration of colonic structure.

Conclusion

In summary, this study confirms that oral microcapsules DF@YM can effectively treat IBD through their antioxidant, anti-inflammatory, gut microbiota-regulating, and intestinal barrier-repairing activities. The microcapsules achieve sequential drug delivery and prolong retention time in a self-adaptive manner through electrostatic interactions and precise targeting at the inflammatory site, thereby enhancing the tissue-specific accumulation of DHM and improving the therapeutic efficacy against colitis. The DHM-Fe polyphenol nanozyme, the core component of DF@YM, exhibits strong ROS scavenging capacity, effectively alleviating oxidative stress and protecting colonic epithelial cells from ROS-induced damage. Both in vitro and in vivo experiments demonstrated that DF@YM effectively alleviates oxidative stress, diminishes inflammatory responses, and promotes the repair of the epithelial barrier, showing a significant therapeutic effect against colitis. Furthermore, 16 S rRNA sequencing analysis revealed that DF@YM remodels the gut microbiota. Moreover, metabolomic analysis and molecular docking suggested that the protective effect of DF@YM against intestinal inflammation may be partially mediated by the activation of the AhR/IL 22 pathway. Finally, MSI confirmed that the DF@YM system significantly enhances the tissue-specific accumulation of DHM, which is achieved through its specific enrichment in target tissues. Although DF@YM exhibited promising therapeutic efficacy against colitis in murine models, further investigations are needed to precisely delineate the predominant role at different stages of colitis. As a promising multifunctional therapeutic strategy, DF@YM provided important evidence for the development of a novel and effective synergistic strategy for IBD treatment.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Wen Yin: Writing–original draft, Visualization, Methodology, Investigation, Data curation. Shuangjiao Hou: Methodology, Formal analysis, Validation. Yaohui Ma: Software, Investigation, Data curation. Songyang Yao: Methodology, Validation. Jinwu Wei: Data curation, Visualization. Dan Pu: Investigation,. Xu Fu: review & editing, Funding acquisition. Dekui Zhang: Writing–review & editing, Supervision, Funding acquisition.

Funding

This work was supported by grants from the National Natural Science Foundation of China (No. 82360115; 82260135), the Key Research and Development Program of Gansu Province (No. 22YF7WA087), the Cuiying Scientific and Technological Innovation Program of Lanzhou University Second Hospital (No. CY2022-YB-A02) and Science and Technology Program Funding of Chengguan District, Lanzhou (No. 2025-zd-6).

Data availability

All data generated or analyzed during this study are included in this published article and its supplementary information files.

Declarations

Ethics approval and consent to participate

All animal experiments were conducted in compliance with the Guidelines for Care and Use of Laboratory Animals of the Second Hospital & Clinical Medical School of Lanzhou University, and were approved by the Animal Ethics Committee.

Consent for publication

All authors agree to be published.

Competing interests

The authors declare no competing interests.