Extracellular vesicles derived from Kaempferia Galanga L. show promise for targeted oral therapy in the treatment of ulcerative colitis

Abstract

Ulcerative colitis (UC) is characterised by chronic intestinal inflammation and its global prevalence is increasing. Although a variety of drugs have been approved for the clinical treatment of UC, their application is often limited by unsatisfactory long-term effects, side effects, and high treatment costs. Therefore, there is an urgent need for the development of effective drugs with fewer side effects. In this study, we found that the extracellular vesicles extracted and purified from rhizomes of Kaempferia galanga L. (KGEVs) exhibit promising therapeutic effects in treating UC disease. With an average diameter of 133.8 nm, KGEVs are rich in functional components, including lipids, proteins, and pharmacologically and immunologically active molecules. In vivo experiments revealed that KGEVs accumulated in the colorectal region 6 h after oral administration, demonstrating targeted enrichment at the site of enteritis. Moreover, we found that KGEVs effectively alleviate DSS-induced colitis in mice, as indicated by reductions in body weight loss, DAI score, spleen index and colon length shortening. Mechanistically, KGEVs may alleviate colitis by repairing the intestinal barrier, inhibiting oxidative stress and colonic inflammation regulating gut microbiota and inhibiting the polarisation of macrophages into pro-inflammatory M1 macrophages during the inflammatory response, indicating significant anti-inflammatory effects. These results suggest the potential of KGEVs as a promising, cost-effective, and efficient oral therapeutic agents for UC treatment.

Graphical abstract

Supplementary Information

The online version contains supplementary material available at 10.1186/s12951-025-03897-8.

Introduction

Ulcerative colitis (UC) is categorised as an idiopathic form of inflammatory bowel disease (IBD), primarily targeting the mucosal epithelium of the colon and rectum [1]. The condition is characterised by chronic inflammation and ulceration within the colonic mucosa, presenting with symptoms such as diarrhea, abdominal discomfort, rectal hemorrhaging, and distension [2]. Recently, the incidence of UC worldwide has been rising [3]. Current treatment approaches include pharmacological interventions, nutritional therapy, and surgical procedures [4, 5]. However, these treatments are associated with limitations, including inadequate efficacy, significant adverse effects, prolonged treatment durations, and high costs. These factors significantly contribute to the burden patients face. Therefore, there is a critical demand for the development of therapeutic agents that offer improved efficacy, reduced side effects, and reasonable cost-effectiveness. These agents are essential for effectively managing the symptoms and inflammation associated with UC, ultimately enhancing the overall treatment paradigm for this condition.

Anti-inflammatory and antioxidant strategies are crucial in the treatment of UC. The anti-inflammatory efficacy of medications can be achieved by inhibiting the production of pro-inflammatory cytokines such as interleukin-6 (IL-6), IL-1β, and tumour necrosis factor-α (TNF-α), thereby reducing the infiltration of inflammatory cells and the production of inflammatory mediators [6, 7]. Alleviating intestinal inflammation can be facilitated by promoting the production of anti-inflammatory cytokines, such as IL-10, which significantly affects the onset and progression of UC [8, 9]. Moreover, classically activated M1 macrophages are capable of producing large quantities of pro-inflammatory cytokines, including IL-1β、IL-6、IL-12、IL-23 and TNF-α [10, 11]. Hence, inhibiting the macrophage polarisation towards the M1 phenotype can also mitigate the inflammatory response. Oxidative stress is also a key factor in the pathogenesis of UC [12, 13]. The antioxidant effects of medications can treat UC by neutralising free radicals, protecting ular structures, inhibiting the production of inflammatory mediators, aiding the anti-inflammatory process, and promoting the repair and regeneration of damaged mucosal tissue [14, 15]. Therefore, compounds with dual anti-inflammatory and antioxidant properties are more advantageous for the treatment of UC. Increasingly, studies have shown that plant-derived extracellular vesicles (EVs) exhibit significant anti-inflammatory and antioxidant effects, offering advantages for the treatment of UC.

Nanoparticles capable of scavenging reactive oxygen species (ROS), modulating the gut microbiota, and targeting the intestine exhibit significant promise for the treatment of enteritis, as exemplified by mucoadhesive probiotic backpacks loaded with ROS nanoscavengers [16]. KGEVs similarly possess these key characteristics and thus hold significant therapeutic potential. In contrast to the aforementioned engineered nanoparticles, the distinct nature of KGEVs stems from their direct derivation from the natural plant Kaempferia galanga L (KG).

Plant-derived EVs present a constellation of favorable attributes, such as their derivation from abundant sources, cost-effective production, low immunogenicity, high biocompatibility, substantial pharmacological efficacy, and minimal toxicity. Consequently, they are increasingly investigated as a promising nanocarrier platform for targeted drug delivery. In a parallel development within bionanotechnology, bacteria are being harnessed for therapeutic applications. Their distinctive physiological and genetic characteristics render them uniquely suited for integration with nanotechnologies to create novel bio-hybrid therapeutic systems [17].

Delivery platforms has composed primarily of lipids, proteins, and nucleic acids [18], plant-derived EVs play a role in intercellular communication. It has been confirmed that the plant-derived EVs can be utilised as therapeutic agents for a variety of diseases. For instance, EVs from plants such as tea leaves [19, 20], purslane [21], mulberry bark [22], strawberries [23], turmeric [24, 25], garlic [26, 27], cabbage [28], celery [29], asparagus [30], sesame leaves [31], and Artemisia annua [32] exhibit notable medicinal properties.

Plant-derived EVs hold significant research value. KG is a popular medicinal and edible plant. Previous studies have indicated that KG possesses antimicrobial, antioxidative, anti-inflammatory, analgesic, antitubercular, and antithrombotic properties. It is also used in the treatment of gastrointestinal disorders [33]. However, the preventive and therapeutic effects of KG-derived EVs (KGEVs) on colonic diseases remain unclear. This study is the first to isolate and extract high-purity EVs from fresh KG and to investigate their therapeutic effects on UC. Our findings reveal that KGEVs targeted and enriched at the damaged colorectal sites. They demonstrated significant anti-inflammatory effects, inhibited the polarisation of macrophages to M1 subtype, and markedly improved the symptoms of DSS-induced enteritis without causing adverse reactions.

Currently, synthetic nanoparticles have been extensively developed for the targeted delivery of drugs to tissues affected by colitis and colonic tumours [34, 35]. However, before their further application, it is necessary to ascertain their stability, specificity for targeting disease sites and biocompatibility. KGEVs are derived from edible plant, they are known to be stable and highly biocompatible [36]. KG is also a sustainable source for the large-scale supply of natural EVs. The use of KGEVs for drug delivery reduces the need for artificial synthesis. KGEVs also overcome various limitations of synthetic processes, thereby significantly enhancing the efficacy and economic benefits of targeted therapy for UC.

Materials and methods

Chemicals and reagents

Fresh KG was collected from Maoming (Guangdong, China). Dextran Sulphate Sodium Salt (DSS, MW 36–50 kDa) was purchased from Yeasen (China). Cell culture reagents including high glucose Dulbecco’s Modified Eagle’s Medium (DMEM), phosphate-buffered saline (PBS), and 0.25% trypsin-EDTA, were obtained from Biological Industries (Israel), and fetal bovine serum was purchased from ExCell (Uruguay). Cell culture plates or dishes, PCR strip tubes, and centrifuge tubes, centrifuge tube were purchased from NEST (China). DiD cell-labelling solution was purchased from Invitrogen (USA). Immunohistochemical (IHC) antibodies for ZO-1, Claudin and Occludin were purchased from Servicebio (China). The ROS Assay Kit was purchased from Beyotime (China), and flow cytometry antibodies (F4/80, CD11b, CD86, CD206) were purchased from Proteintech (USA). ELISA kits (TNF-α, IL-6, IL-1β, IL-10) were purchased from Boshen (China). The reverse transcription kit and SYBR GREEN were purchased from TOYOBO (Japan). CAT, GSH and SOD kits were purchased from Jiancheng (China). FITC-dextran was purchased from Aladdin (USA). Lipopolysaccharide (LPS) was purchased from Sigma-Aldrich (USA), and Bicinchoninic Acid (BCA) protein assay kits were purchased from Keygen Biotech (China).

Isolation and characterisation of KGEVs

The fresh KG rhizomes were washed with pure water and homogenised using a grinder. The juice was centrifuged at 200 g for 10 min, 2,000 g for 30 min, 12,000 g for 15 min, and 95,000 g for 30 min. Subsequently, the juice was filtered through 0.45 μm and then 0.22 μm microporous membranes using a vacuum extraction device. To collect the KGEVs, the filtered liquid was then centrifuged at 170,000 g for 3 h to collect the precipitate. Finally, the precipitate was resuspended in pre-cooled sodium PBS, the protein concentration was determined using a BCA protein assay kit, and the sample was stored at −80 °C for future use. For the characterisation of KGEVs, the size distribution was determined by NTA (Zetaview Particle Metrix, Germany). To observe the morphology of KGEVs, the sample was diluted in PBS buffer, placed on a copper mesh, stained with 2% uranyl acetate at room temperature, and imaged using transmission electron microscopy (TEM) (Hitachi H-7650, Japan).

Compositional analysis of KGEVs

Samples for proteomic, lipidomic, and metabolomic analyses from KGEVs were submitted to Suzhou PANOMIX Biomedical Tech CO., Ltd. (Jiangsu, China). In brief, protein samples were enriched using Proreo Miner Protein Enrichment Kits (Bio-Rad, USA) and separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The samples were then subjected to enzymatic digestion using the MagicOmics-MMB8X Kit (QL Bio Beijing, China) and quantified by LC-MS/MS (Thermo Fisher Scientific, Bremen, Germany). Lipid compositions were extracted using a mixed solvent (chloroform: methanol, 2:1, v/v) and analysed by LC-MS (Thermo Fisher Scientific, USA) to separate and analyse the lipid compositions of KGEVs. The data were reported as percentages of total signals for the molecular species, determined after normalisation to internal standards of the same lipid class. In metabolomics, KGEVs were dissolved in a mixed solution (acetonitrile (ACN): methanol: H₂O, 2:2:1, v/v/v) and analysed by LC-MS (Thermo Fisher Scientific, Bremen, Germany) with gradient elution using different proportions of the mobile phase.

Animals and cell culture

Eight-week-old male C57BL/6 mice were purchased from SPF Biotechnology Co., Ltd (China). Mice received humane care under specific pathogen-free conditions and were maintained on a 12-hour light/dark cycle. The Institutional Animal Care and Use Committee of Southern Medical University approved all animal experimental procedures. RAW264.7, the murine macrophage cell line, was cultured in high-glucose DMEM containing FBS (10%, v/v) and penicillin/streptomycin (1%, w/v). The cells were cultured in a humidified atmosphere at 37 °C with 5% CO₂.

Biodistribution study of KGEVs

The distribution of KGEVs in the body after oral administration was examined using a staining method. For staining, KGEVs (3.5 µg/µl) were labelled with 1% DiD (v/v) for one hour at 37 °C and washed once with PBS by centrifuging at 170,000 g for 1.5 h at 4 °C. The labeled KGEVs were gavaged to healthy mice or DSS-induced acute colitis mice. After anesthesia and skin preparation, images of the mice were captured using the imaging system (In-Vivo FX PRO, Bruker, Germany and ABL X3, Tanon, China) at the predetermined time point. Subsequently, mice were sacrificed, and their colons, along with other major organs, were excised for ex vivo imaging.

The therapeutic efficacy of KGEVs on the DSS-induced ulcerative colitis mice

C57BL/6 male mice were randomly divided into five groups (n = 8): control (CTRL) group, the healthy control group; model (MOD) group, 5-Aminosalicylic acid (5-ASA) group, low dose KEGVs (L-KEGVs) group, and high dose KEGVs (H-KEGVs) group. After 7 days of adaptation, mice in the CTRL and MOD groups were gavaged with PBS, while the 5-ASA, L-KEGVs, and H-KEGVs groups were gavaged with 100 mg/kg 5-ASA, 75 mg/kg, or 100 mg/kg KGEVs, respectively, for 3 days. Thereafter, mice were treated with 3% DSS (w/v) in their drinking water continuously for 9 days. During the DSS treatment period, body weights, fecal characteristics, physical activity, and water intake were monitored daily, and feces were collected on day 4 for 16 S rRNA analysis. On day 9, mice were sacrificed to collect serum, colons, and major organs for further examination, and the colon length and spleen weight were recorded.

Intestinal permeability assay

Mice were fasted for 4 h before the experiment, then FITC-dextran (40 mg/mL) was gavaged to mice (600 mg/kg). After 3 h, mice were sacrificed, and blood was collected and allowed to stand at room temperature for 1 h. The blood was then centrifuged at 3000 rpm for 10 min, 12,000 rpm for 10 min at 4 °C. Supernatant (5 µl) was mixed with 95 µl of PBS and added to a 96-well plate. FITC-dextran with a concentration of 20 µg/ml (0, 2.5, 5, 10, 20, 30, 40, 50 µl) was added to the 96-well plate, and different volumes of PBS were added to each well to make up a total volume of 100 µl as a standard control. Serum from mice administered with PBS was used as the negative control. The concentration of FITC in the serum was measured using a Synergy H1 microplate reader (Biotek, Vermont, USA) with excitation at 485 nm and emission at 528 nm.

Histopathological analysis

To evaluate histopathological changes, colorectal tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. For hematoxylin and eosin (HE) staining, sections were deparaffinised in xylene, rehydrated through a graded ethanol series, and stained with hematoxylin. After washing, the sections were counterstained with eosin, dehydrated through a graded ethanol series, cleared in xylene, and mounted. For Alcian Blue-Periodic Acid-Schiff (AB-PAS), sections were deparaffinised and rehydrated, then stained with Alcian blue solution to detect acidic mucopolysaccharides. Following rinsing to remove excess dye, sections were counterstained, dehydrated, cleared, and mounted for microscopic examination.

Measurement of inflammatory factors and oxidative stress parameters in vivo

Precision weighing of 30 mg of colonic tissue is followed by homogenisation with pre-chilled PBS at a 1:9 (w/v) ratio. Homogenisation was performed at reduced temperature to ensure a uniform mixture, followed by centrifugation at 4 °C and 10,000×g for 5 min. The resulting supernatant was carefully collected, and the protein concentration of each specimen was quantified using a BCA kit. The sample loading volume was meticulously adjusted in accordance with the determined protein content to maintain uniform protein concentrations across the samples, which were subsequently stored at −80 °C for future analytical purposes. Measurements of inflammatory factors and oxidative stress were performed according to the manufacturer’s guidelines.

Immunohistochemistry and Immunofluorescence

For IHC staining, the slides were dewaxed and rehydrated, antigen retrieval was performed using heat methods, endogenous peroxidase activity was blocked, the slides were incubated with primary antibodies specific to each protein, the slides were washed to remove unbound antibodies, and secondary antibodies conjugated to enzymes such as horseradish peroxidase (HRP) were applied. The colorurimetric signal was developed using a chromogenic substrate such as such as DAB, counterstained with hematoxylin, washed, dehydrated, and cleared in xylene. For immunofluorescence staining, frozen colon sections were fixed in 4% paraformaldehyde for 15 min at room temperature, followed by washing with PBS with Tween-20 (PBST). After blocking with 5% bovine serum albumin (BSA) for one h, sections were incubated overnight at 4 °C with primary antibodies against Claudin, ZO-1, and Occludin (1:200). After washing, sections were incubated with fluorescent secondary antibodies for one hour, followed by DAPI counterstaining. The sections were then mounted and imaged using a microscope.

Uptake of KGEVs by RAW264.7 cells

To evaluate the kinetics of KGEVs uptake, DID-labelled KGEVs (12.5 ng/ml) were co-incubated with RAW264.7 cells for 0, 1, 3, and 6 h. Subsequently, the cell membranes and nuclei were stained with DiO and Hoechst 33342, respectively. Following incubation, the cells were analysed at each time point. The efficiency of internalisation was quantified by flow cytometry (SA3800, Sony, Japan), while the spatial distribution of the vesicles within cells was examined using structured-illumination confocal microscopy (LSM800, Carl Zeiss, Germany).

Anti-inflammatory activity of KGEVs in vitro

The in vitro experiment was divided into four groups: CTRL, LPS, KGEVs, and LPS + KGEVs. RAW 264.7 macrophages in the absence of KGEVs and LPS were used as a negative control, while LPS- stimulated cells were treated as a positive control. RAW 264.7 macrophages were seeded in 24-well plates at a density of 2.5 × 10⁵ cells/well overnight and incubated with KGEVs (protein concentration was detected by BCA kit, 12.5 ng/mL) or LPS (100 ng/mL). After incubation for 3 h, cells were collected, and total RNA was extracted using a TRIzol reagent (AG, Hunan, China). Using a reverse transcription reagent kit to synthesise cDNA. After mixing the cDNA with SYBR Green Master Mix and specific primers, qRT-PCR was performed on Light Cycler Real-Time PCR System (Roche, Switzerland). The reference gene 18 S was used to normalize the relative expression levels.

Isolation of cells from mesenteric lymph nodes and FACS analysis of macrophage population

After treatments, mesenteric lymph nodes were harvested from the mice which was passed through a 70-mesh sieve to disaggregate the tissue. The resulting cell suspension was then centrifuged at 1500 rpm for 5 min at 4 °C to pellet the cells, and the supernatant was discarded. The cell pellet was resuspended and incubated with Fc receptor blocking buffer on ice for 10 min. Subsequently, the cells were stained with a panel of fluorophore-conjugated antibodies against surface markers (CD11b, F4/80, CD86, and CD206) on ice for 30 min in the dark. For compensation controls, single-stained samples were prepared in parallel using the corresponding individual antibodies. After incubation, the cells were washed with PBS and centrifuged again at 1500 rpm for 5 min. The cell pellet was then fixed with 200 µL of 2% paraformaldehyde for 20 min at room temperature in the dark. Finally, the cells were washed, resuspended in PBS, and analysed using a flow cytometer (FACS Aria III, Becton, Dickinson and Company, USA).

The effect of KGEVs on RAW 264.7 macrophage polarisation

To investigate the effect of KGEVs on macrophage polarisation, LPS was used to stimulate RAW 264.7 cells to generate M1 macrophages. Cells were divided into four groups (n = 3): CTRL group, LPS group, KGEVs group, and LPS + KGEVs group. The cells were seeded into 12-well plates at a density of 5 × 10⁵ cells/well and incubated for 24 h with PBS, KGEVs (12.5 ng/mL) or LPS (100 ng/mL). After 24 h of treatment, the cells were harvested, blocked, and stained with an antibody combination targeting M1 surface markers (CD86) or M2 surface markers (CD206) before flow cytometry analysis.

Statistical analysis

The statistical evaluation of the data was conducted utilising GraphPad Prism 8.0 and SPSS software. Data are presented as mean values ± standard error of the mean (SEM). Comparisons between two groups were performed using the Student’s t-test. For comparisons among three or more groups, one-way ANOVA followed by the LSD post-hoc test was used for inter-group comparisonsa. The sequencing data were subjected to specific statistical methodologies as delineated in the figure legends. The levels of significance are denoted as follows: *p < 0.05, **p < 0.01, and ***p < 0.001, indicating statistical significance, whereas ns (no significant difference) indicates a lack of statistical significance.

Results

Characterisation of KGEVs

KGEVs were isolated from fresh KG using differential centrifugation and then further purified by microporous membranes with varying pore sizes through a negative pressure system filter (Fig. 1A). Characterisation of the purified KGEVs revealed a mean particle diameter of 133.8 nm as determined by Nanoparticle Tracking Analysis (NTA) (Fig. 1B). Transmission Electron Microscopy (TEM) confirmed that the vesicles were spherical nanoparticles possessing an intact lipid bilayer structure (Fig. 1C). The stability of KGEVs in a simulated gastric environment was evaluated over time (0, 0.5, 1, 2, and 4 h), with particle diameters measuring 133.8 nm, 128.5 nm, 134.1 nm, 149.6 nm, and 151.6 nm, respectively. This demonstrates that KGEVs maintain structural stability in gastric fluid (Fig. S1). Collectively, these analyses, including the uniform particle size and intact morphology, indicated that the KGEVs were successfully isolated with high quality and were suitable for subsequent functional experiments.

Fig. 1.

Isolation and characterisation of Kaempferia galanga L.-derived extracellular vesicles. (A) Schematic of the KGEVs isolation procedure. (B) Particle size of the KGEVs was measured using zeta view (NTA). (C) Transmission electron microscopy (TEM) images of KGEVs were captured at a magnification of ×15,000 and ×40,000 revealing intricate structural details. (D) The distribution of molecular weights of identified proteins. (E) Protein gel electrophoresis of KGEVs. (F) The lipid profiles of KGEVs. (G) The LC-MS spectrum of KGEVs. (H) The metabolomic profiles of KGEVs

Multi-omics profiling of KGEVs

To comprehensively characterise the molecular composition of KGEVs, integrated proteomic, lipidomic, and metabolomic analyses were undertaken. Proteomic analysis by LC-MS/MS identified 552 proteins, with the vast majority (81.65%) having molecular weights between 10.6 and 136.6 kDa (Fig. 1D, E). Subsequent lipidomic profiling via LC-MS revealed a complex lipid composition. Among the 10 most abundant components, the primary lipid classes are triglycerides (TG, 1%–47% of total lipids), phosphatidylglycerol (PG, 24%), N-acylethanolamine (AEA, 1%), (Fig. 1F, Fig. S1A). Furthermore, metabolomic analysis show that KGEVs are predominantly composed of carboxylic acids, quinolines, and organonitrogen compounds (Fig. 1G, Table S1B). The most abundant metabolites identified included 3-hexanoly-NBD Cholesterol, citric acid, 11-Aminoundecanoic acid, 8-Methoxykynurenate, Diosmin (Fig. 1H, Table S1B). These findings establish a detailed molecular profile of KGEVs, revealing a rich repertoire of structural, energetic, and bioactive components.

In vivo biodistribution and colon-targeting of KGEVs

To determine the in vivo biodistribution and colon-targeting capability of KGEVs, mice were orally gavaged with DiD-labelled KGEVs (100 mg/kg). A time-course study in healthy mice shows that whole-body fluorescence peaked at 1 h post-gavage and declined thereafter. Ex vivo imaging of the gastrointestinal tract showed that KGEVs predominantly accumulated in the colon, with peak localisation observed at the 6-hour time point (Fig. 2A-C). Therefore, 6 h post-administration was selected as the optimal time point for subsequent analyses.

Fig. 2.

In vivo biodistribution and colon-targeting of orally administered KGEVs. (A) Whole-body fluorescence imaging at 1, 3, 6, 12, and 24 h post-oral gavage (n = 3). (B) Ex vivo imaging of the gastrointestinal tract and major organs (heart, liver, spleen, lung, kidney) at the corresponding time points (n = 3). (C) Quantification of fluorescence intensity in major organs at each time point (n = 3). (D) Representative bright-field image of the excised major organs. (E, F) Ex vivo image and heat map showing the fluorescence intensity of KGEVs in each organ over time. Targeted accumulation of KGEVs in the inflamed colon (n = 3). (G) Colon imaging of CTRL, MOD, H-KGEVs mice at 6 h post-gavage (n = 4). (H) Quantification of fluorescence intensity in the colon from CTRL, MOD, H-KGEVs mice (n = 4). Statistical comparisons were performed using One-way ANOVA followed by the post hoc LSD tests. *p <0.05, **p < 0.01, ***p < 0.001

The targeting potential of KGEVs in a dextran sulphate sodium (DSS)-induced colitis model was then investigated. All groups of mice received DiD-labelled KGEVs, and organs were imaged at the 6-hour time point. Strikingly, fluorescence intensity in the colon was significantly higher in the MOD group than in the CTRL and H-KGEVs group, demonstrating targeted accumulation in inflamed tissue (Fig. 2G-H). Furthermore, analysis of major organs confirmed low off-target accumulation. Fluorescence intensity is highest in the liver, followed by the lungs, kidneys, spleen and heart, but the signal in all these organs was negligible compared to that in the inflamed colon (Fig. 2D-F).

In summary, orally administered KGEVs preferentially accumulate in the colon and exhibit enhanced targeting to inflamed regions. This specific biodistribution profile supports their potential as a therapeutic agent for colitis.

KGEVs alleviated symptoms associated with DSS-induced acute colitis

To evaluate the therapeutic potential of KGEVs against UC, a DSS-induced acute colitis model in mice (Fig. 3A) was established. UC is characterised by clinical signs such as weight loss, a high disease activity index (DAI), colonic shortening, and increased intestinal permeability. Mice in the MOD group exhibited progressive weight loss, dropping to 83.7% of their initial body weight by day 9. In contrast, CTRL mice remained stable (Fig. 3B). Treatment with KGEVs effectively mitigates this weight loss in a dose-dependent manner. The L-KGEVs and H-KGEVs groups retain 85.1% and 92.5% of their initial weight, respectively, outperforming the positive control, 5-ASA (82.4%). Similarly, the DAI score is significantly elevated in the MOD group but is markedly reduced by both KGEVs treatments (Fig. 3C).

Fig. 3.

KGEVs alleviate DSS-induced acute colitis and restore intestinal barrier integrity. (A) Protocol for DSS treatment and administration of KGEVs and 5-ASA. (B) Body weight change expressed as the percentage of the day-zero weight (n = 8). (C) Disease activity index (n = 8). (D) Representative colonic morphology in each group. (E) Colon length (n = 8). (F) Spleen weight (n = 8). (G) Fluorescence intensity (n = 4). (H) HE-stained colon sections. (I) Histological scores derived from HE-stained colons (n = 8). (J) AB-PAS-stained colon sections. (K) Immunohistochemical staining of Claudin, ZO-1, and Occludin in frozen sections of colon (n = 8). (L) Immunofluorescence staining of Claudin, ZO-1, and Occludin in frozen sections of colon (n = 4). Each point represents the mean ± standard error of the mean (S.E.M.). Statistical comparisons were performed using One-way ANOVA followed by the post hoc LSD tests. *p < 0.05, **p < 0.01, ***p < 0.001

Colonic shortening, a hallmark of colitis, is also attenuated by KGEV treatments. The average colon length is 5.01 cm in the MOD group compared to 6.52 cm in the CTRL group. Treatment with 5-ASA, H-KGEVs, and L-KGEVs restored colon length to 5.60 cm, 6.35 cm, and 5.50 cm, respectively, with all treatments showing significant improvement over the MOD group (Fig. 3D, E, S2). Furthermore, spleen weight, an indicator of systemic inflammation, is lower in the H-KGEV group than in the MOD group (Fig. 3F).

KGEVs restore intestinal barrier integrity

To evaluate the therapeutic potential of KGEVs in restoring the intestinal barrier, a comprehensive analysis was undertaken that included intestinal permeability assays, histology, immunohistochemistry, and immunofluorescence. Intestinal permeability assay shows a significant increase in serum FITC-dextran levels in the DSS group compared with the CTRL group, indicating compromised intestinal barrier function. Conversely, treatment with H-KGEVs markedly reduced this permeability, demonstrating a protective effect against DSS-induced barrier dysfunction (Fig. 3G).

This functional restoration was corroborated by histological analysis. HE staining showed that DSS administration causes severe disruption of colonic architecture and extensive inflammatory cell infiltration, which are significantly ameliorated by KGEV treatment (Fig. 3H, I).

The integrity of the mucus barrier, a critical physical defence, was then considered. AB-PAS staining demonstrates that DSS-induced colitis is characterised by severe crypt damage and a profound loss of goblet cells. KGEVs treatment effectively restores both goblet cell numbers and the integrity of the mucus layer (Fig. 3J).

At the molecular level, the expression of tight junction proteins, key regulators of paracellular permeability, was investigated. Immunohistochemical and immunofluorescence analysis revealed that Claudin, ZO-1 and Occludin expression were significantly downregulated in the DSS group. Notably, KGEVs treatment robustly reversed this downregulation, leading to a substantial increase in the expression of these tight junction proteins, with a particularly pronounced effect on Occludin (Fig. 3K, L).

Collectively, these findings demonstrate that KGEVs restore intestinal barrier integrity through a multi-level mechanism: preserving histological structure, replenishing the protective mucus layer, and upregulating the expression of critical tight junction proteins.

KGEVs inhibit M1 macrophage polarisation and exert anti-inflammatory effects

To evaluate the impact of KGEV internalisation on immune cell populations, we performed flow cytometric analysis on cells isolated from mesenteric lymph nodes. Consistent with the induction of an inflammatory state, the frequency of CD86 + cells, a marker for M1 macrophages, was significantly elevated in the model (MOD) group (19.3%) compared to the control (CTRL) group (10.9%). Notably, administration of KGEVs at a dose of 100 mg/kg markedly reduced this percentage to 13.9% (Fig. 4A, B). In contrast, no significant differences were observed in the proportions of B cells (B220+) or T cell subsets (CD4 + and CD8a+) among the CTRL, MOD, and KGEV-treated groups (Fig. S3).

Fig. 4.

KGEVs suppress M1 macrophage polarisation in vitro and attenuate systemic inflammation in colitis. (A) Flow cytometric analysis of the percentage of CD86 + M1 macrophages of mesenteric lymph nodes (n = 3). (B) Quantification of the M1/M2 macrophage ratio (n = 3). (C–F) Serum levels of cytokine IL-6, IL-1β, TNF-α, IL-10 (n = 8). (G) Time-dependent uptake of DiD-labeled KGEVs by RAW264.7 cells. Representative fluorescence images captured at 0, 1, 3, and 6 h post-incubation. (H) Representative histograms of DiD fluorescence at 0, 1, 3, and 6 h. (I) Flow cytometric analysis of the percentage of CD86 + M1 macrophages of RAW264.7 (n = 3). (J) Quantification of the M1 and M1/M2 macrophage ratio of RAW264.7 (n = 3). (K–N) Relative mRNA expression of IL-6, IL-1β, TNF-α, and CD86(n = 6). Each point represents the mean ± standard error of the mean (S.E.M.). Statistical comparisons were performed using One-way ANOVA followed by the post hoc LSD tests. *p < 0.05, **p < 0.01, ***p < 0.001

To investigate the impact of KGEVs internalisation on RAW264.7 macrophages, we assessed their effect on polarisation and anti-inflammatory potential using an LPS-stimulated in vitro model. Flow cytometric analysis revealed that LPS treatment substantially increased the proportion of M1 macrophages (CD86+) from 0.39% to 13.9%. Co-treatment with KGEVs effectively reversed this polarisation, reducing the CD86 + population to 4.41%. Quantification of the M1/M2 ratio further confirmed that KGEVs significantly mitigated the LPS-induced shift towards a pro-inflammatory state (Fig. 4I, J).

To corroborate these findings at the transcriptional level, we measured the expression of key M1 macrophage markers. As expected, LPS stimulation significantly upregulated the mRNA levels of the surface co-stimulatory molecule CD86 (Fig. 4N). Consistent with the flow cytometry data, KGEV co-treatment markedly attenuated this LPS-induced transcriptional activation (Fig. 4I). Notably, KGEVs alone did not elicit any inflammatory response, confirming their non-stimulatory nature. Collectively, these data demonstrate that KGEVs exert potent anti-inflammatory effects by inhibiting M1 macrophage polarisation and the associated cytokine production, highlighting their therapeutic potential for inflammatory diseases.

Time-dependent uptake of KGEVs by RAW264.7

KGEVs are efficiently internalised by RAW264.7 macrophages in a time-dependent manner, a critical prerequisite for their biological activity. To visualise and quantify this process, RAW264.7 cells were incubated with DID-labelled KGEVs and analysed at 0, 1, 3, and 6 h. Internalisation was rapid, with detectable fluorescence within the first hour. The uptake kinetics accelerated over time, with the vast majority of cells showing positive staining by three hours and prominent intracellular accumulation evident at six hours (Fig. 4G, H). This efficient uptake by the inflammatory cell model underscores the potential of KGEVs to modulate key cellular functions, such as metabolism and proliferation.

KGEVs attenuate systemic inflammation in colitis by modulating cytokine profiles

To validate the anti-inflammatory efficacy of KGEVs in vivo, ELISA analyses on serum samples from mice were undertaken. Pro-inflammatory cytokines, such as IL-1β, IL-6, and TNF-α, are pivotal mediators of UC pathogenesis, and their serum levels reflect the systemic inflammatory burden. As expected, DSS-induced colitis mice exhibited a dramatic elevation in the serum levels of IL-1β, IL-6, and TNF-α compared to the control group (Fig. 4C-E). Notably, treatment with 100 mg/kg KGEVs significantly reversed this pro-inflammatory trend substantially reducing cytokine levels. Conversely, the serum concentration of the anti-inflammatory cytokine IL-10 was suppressed in the DSS group but is effectively restored upon KGEVs administration (Fig. 4F).

To validate the anti-inflammatory efficacy of KGEVs in vitro, we established an inflammatory model using LPS-stimulated RAW264.7 macrophages, with LPS treatment serving as the positive control. The mRNA expression levels of pro-inflammatory cytokines (IL-6, IL-1β, and TNF-α) were quantified by quantitative real-time PCR (qRT-PCR). As expected, LPS significantly upregulated the expression of all these genes. Notably, this LPS-induced upregulation was significantly attenuated upon co-treatment with KGEVs (12.5 ng/mL) (Fig. 4K-M).

These findings collectively demonstrate that KGEVs can modulate the inflammatory milieu in colitis by suppressing pro-inflammatory signals while promoting anti-inflammatory pathways, thereby restoring immune homeostasis.

KGEVs mitigate oxidative stress in colitis

Oxidative stress, driven by the overproduction of reactive oxygen species (ROS), is a pivotal factor in the pathogenesis of UC, amplifying inflammation and exacerbating tissue damage. Therefore, scavenging excessive ROS represents a key therapeutic strategy for UC.

To evaluate the in vivo ROS-scavenging capacity of KGEVs, DCF fluorescence imaging was used to measure ROS levels in colonic tissues. As shown in Fig. 5A, the green fluorescence in the MOD group is significantly more intense than that in the CTRL group. Quantitative analysis (Fig. 5B) reveals a significant reduction in DCF fluorescence intensity in the KGEV group compared to the MOD group, confirming the adequate clearance of colonic ROS by KGEVs.

Fig. 5.

KGEVs mitigate oxidative stress in colitis. (A) Representative DCF fluorescence images of ROS in colonic tissues. (B) Quantitative analysis of DCF fluorescence intensity in colonic tissues (n = 4). (C) Fluorescence microscopy images of intracellular ROS in RAW264.7 cells. (D-F) Activities of CAT, SOD, and GSH in colonic tissues (n = 8). (G) Dose-dependent DPPH radical scavenging activity of KGEVs (n = 6). Each point represents the mean ± standard error of the mean (S.E.M.). Statistical comparisons were performed using One-way ANOVA followed by the post hoc LSD tests. *p < 0.05, **p < 0.01, ***p < 0.001

The body’s antioxidant defence system is mediated by key enzymes, including catalase (CAT), superoxide dismutase (SOD), and glutathione (GSH). The activities of these enzymes were assessed and found to be significantly suppressed in the DSS-induced colitis model (Fig. 5D-F). Following KGEVs intervention, however, the activities of these enzymes were restored to levels comparable to those of the CTRL group, indicating that KGEVs effectively restore the endogenous antioxidant defence system.

To further validate this effect at the cellular level, an oxidative stress model in RAW264.7 cells using the oxidant ROSUP was developed. Fluorescence microscopy (Fig. 5C) shows that KGEV treatment significantly attenuated intracellular ROS levels, with the most pronounced effect at 50 ng/mL.

Furthermore, the DPPH assay confirmed the intrinsic radical-scavenging capacity of KGEVs, demonstrating significant in vitro antioxidant activity in a dose-dependent manner (Fig. 5G).

Taken together, these in vivo and in vitro findings consistently demonstrate that KGEVs exert potent antioxidant effects.

KGEVs induced a reconfiguration of the gut Microbiome composition

An increasing body of evidence suggests that the gut microbiota is closely associated with the pathogenesis of IBD, such as UC. Therefore, 16 S rRNA gene sequencing analysis of fecal samples from different groups of mice was undertaken to investigate whether KGEVs could modulate gut microbiota homeostasis and to perform correlation analyses between intestinal flora and UC phenotypes. Alpha diversity was assessed using the ACE and Shannon indices, and the results are presented in Fig. 6AB. KGEVs yielded an alpha-diversity profile that closely resembled that of the CTRL group. Principal Coordinate Analysis 2 (PCoA2), depicted in Fig. 6C, shows a tendency for samples within each group to cluster, indicating high similarity among them. In contrast, a greater distance between groups suggests significant differences among the communities. Figure 6D and E illustrate differences in the relative abundance of gut microbiota at the phylum and genus levels, respectively. Overall, the KGEVs group show intestinal microbial composition, community richness, and species diversity more akin to the CTRL group than to the DSS group.

Fig. 6.

KGEVs induced a reconfiguration of the gut microbiome composition. (A) Abundance-based Coverage Estimator index (ACE) (n = 7–8). (B) Shannon Wiener Diversity Index (n = 7–8). (C) Principal Coordinate Analysis 2 (PCoA2) (n = 7–8). (D) Relative abundance of gut microbiome on phylum level (n = 7–8). (E) Relative abundance of gut microbiome on genus level (n = 7–8). (F) Taxonomic cladogram (n = 7–8). (G) Least discriminant analysis (LDA) (n = 7–8). (H) Differentially abundant taxa at various taxonomic levels (phylum, order, class, family, genus). (I) Correlation analysis between the intestinal flora and UC phenotypes (n = 7–8). Statistical comparisons were performed using the Kruskal-Wallis test combined with the pairwise Wilcoxon test. *p < 0.05, **p < 0.01, and ***p < 0.001

At the phylum level, 16 S rRNA sequencing identified Bacteroidetes and Firmicutes as two pivotal phyla in the gut ecosystem. These taxa are crucial for host health, influencing key physiological processes such as digestion, nutrient absorption, immune regulation, and gut barrier integrity [37, 38]. As shown in Fig. 6DH, the relative proportions of Bacteroidetes and Firmicutes differed significantly between the KGEVs and DSS groups.

The trends observed at the Order (Bacteroidales) and Class (Bacteroidia) levels are consistent with those at the Phylum level. Similarly, the trends for Muribaculaceae and Butyricicoccaceae at the family level align with those at the genus level, with significant differences observed between the MOD and KGEVs groups. Notably, Muribaculaceae, which exhibited higher abundance, has been identified as an anti-inflammatory taxon. At the genus level, Muribaculaceae was identified as an anti-inflammatory genus. This genus can inhibit the activation of CD8 + T lymphocytes, promoting tolerance to immune stimulation and being negatively associated with inflammatory states [38]. Additionally, Muribaculaceae can produce metabolites, such as butyrate, which have been shown to reduce the severity of DSS-induced UC [39, 40]. As shown in Fig. 6H, the relative abundance of Muribaculaceae in the level of Family and Genus was significantly reduced in the DSS group compared to other experimental conditions, and the application of KGEVs resulted in a substantial increase in the abundance of Muribaculaceae.

Analysis of the Cladogram and the LDA score distribution histogram for significantly different species (Fig. 5F, G) revealed that f-Muribaculaceae, g-Muribaculaceae, o-Bacteroidales, p-Bacteroidetes, c-Bacteroidia, and f-Erysipelotrichaceae were significantly enriched in the KGEVs group. Among these, f-Muribaculaceae showed significant differences between groups and the highest abundance among the significantly enriched taxa, and its LDA score was higher than that of other taxonomic units, indicating the greatest impact on intergroup differences. f-Butyricicoccaceae, g-Butyricicoccus, c-Clostridia, and p-Firmicutes were enriched in the DSS group, with two unclassified genera observed. Correlation analysis results indicated that f-Muribaculaceae and g-Muribaculaceae were closely associated with the CAT, body weight changes, colon length, and the DAI index, while c-Bacteroidia, p-Bacteroidetes, o-Bacteroidales, p-Firmicutes, and c-Clostridia were associated with the CAT and DAI, g-Butyricicoccus was related to colon length, and f-Erysipelatoclostridiaceae was related to GSH, colon length, DAI. The correlation results suggest a close relationship between the gut microbiota and UC phenotypes, indicating that KGEVs can improve or adjust the gut microbiota, exerting a therapeutic effect on UC.

KGEVs exhibit an excellent biocompatibility and safety profile

To support the clinical translation of KGEVs, their biosafety was comprehensively evaluated both in vitro and in vivo. For the in vitro assessment, the biocompatibility of KGEVs was first tested in RAW 264.7 macrophages. CCK-8 assay revealed no cytotoxicity even at a high concentration of 1000 µg/mL after 24 or 48 h of incubation (Fig. 7A, B). Additionally, at a functional working concentration (12.5 ng/mL), KGEVs treatment did not induce M1 macrophage polarisation (Fig. 4I) or upregulate pro-inflammatory cytokine expression compared to the control group (Fig. 4K).

Fig. 7.

Evaluating the biosafety of KGEVs from cells to animal models. (AB) Cell viability of RAW 264.7 cells treated with KGEVs (5-1000 µg/mL) for 24 h and 48 h, measured by CCK-8 assay. (C) Body weight change expressed as the percentage of the day-zero weight (n = 6) (D-G). Analysis of serum AST, ALT, Cre, and Bun levels (n = 6). (H) HE staining of major organs (heart, liver, spleen, lung, and kidney). Statistical analysis was conducted using a two-tailed, unpaired Student’s t-test to compare the differences between the groups, *p < 0.05, **p < 0.01, ***p < 0.001

For the in vivo evaluation, mice were orally administered KGEVs at a dose of 100 mg/kg daily for nine consecutive days. The KGEVs-treated mice showed a body weight trajectory similar to that of the control group, with no statistically significant difference between the groups (Fig. 7C). To assess potential hepatorenal toxicity, serum biomarkers including ALT, AST, Cr, and BUN were measured. The results indicate no significant differences in these biochemical parameters between the KGEVs and control groups (Fig. 7D-G). Finally, histopathological examination of major organs (heart, liver, spleen, lung, and kidney) via HE staining revealed no abnormalities, including cellular edema, epithelial damage, or inflammatory cell infiltration, in either group (Fig. 7H).

Collectively, these findings demonstrate the excellent biosafety profile of KGEVs, providing strong support for their preclinical development and future clinical translation.

Discussion

This study demonstrates that KGEVs exhibit significant anti-inflammatory, antioxidative, intestinal mucosal protective, gut microbiota regulatory, and inhibitory properties against macrophage M1 polarisation. Notably, they demonstrate targeted tropism and accumulation at damaged colonic sites. These characteristics enable KGEVs to treat UC via oral administration effectively. This research expands the scope of UC drug therapy and validates the pharmaceutical potential of KGEVs.

The multi-omics analysis reveals that KGEVs are enriched in several bioactive molecules with known therapeutic potential in inflammatory diseases. For instance, the lipidomic profiling identified a significant proportion of sphingosine (SPH) and its precursor N-acylethanolamine (AEA). SPH and its metabolites are critical regulators of the immune system, and the therapeutic potential of targeting their pathways is highlighted by the clinical investigation of drugs such as CBP-307 for ulcerative colitis. Similarly [41], the metabolomic analysis detected high levels of dehydroepiandrosterone (DHEA), a steroid known to mitigate inflammatory responses in IBD by activating the GPR30-Nrf2 pathway and inhibiting the NLRP3 inflammasome [42]. The presence of these components suggests that KGEVs may exert their therapeutic effects, at least in part, through modulating these established anti-inflammatory and barrier-protective pathways.

In UC, the the intestinal mucosal barrier is impaired, leading to increased permeability, dysregulated immune responses, and exacerbated inflammation. Protecting and repairing the intestinal mucosal barrier is a key strategy in the treatment of UC. The data derived in this study indicate that KGEVs significantly reduce intestinal permeability. Furthermore, results from both immunohistochemistry and immunofluorescence confirmed that KGEVs contribute to the maintenance of barrier integrity.

Oxidative stress plays a pivotal role in the onset and progression of UC, exacerbating the damage to the intestinal mucosal barrier, promoting inflammatory responses, impairing cellular function, and disrupting the balance of the intestinal microbiota, thus creating a vicious cycle. Previous studies have found that kaempferol possesses antioxidant properties [43]. Consistent with this, this study also confirmed that KGEVs can modulate oxidative stress-related indices in UC mice, validating their effective clearance of colonic and RAW264.7 ROS. Treatment with KGEVs significantly increased the activities of CAT, SOD, and GSH in mice compared to the DSS group. Additionally, in vitro DPPH assays demonstrate that KGEVs exhibit significant antioxidant capacity and display a dose-dependent response.

In the pathological state of IBD such as UC, the equilibrium between M1 and M2 macrophages is altered, typically leading to a dominance of pro-inflammatory M1 macrophages. In contrast, the anti-inflammatory and tissue-repair functions of M2 macrophages are repressed. The current study demonstrated the inhibitory impact of KGEVs on the polarisation of M1 macrophages at both the animal and cellular levels, thereby diminishing pro-inflammatory effects and enhancing therapeutic outcomes for UC.

The gut microbiota plays a multifaceted role in UC, being not only associated with the onset of the disease but also closely linked to its persistence and progression. This study demonstrates that KGEVs can ameliorate gut microbiota dysbiosis, with the microbial community post-administration more closely resembling the control group and alleviating UC symptoms.

Furthermore, in an era of probiotic and biologic therapies for UC, 5-ASA remains a first-line treatment for mild to moderate UC and is widely utilised. The effects of 5-ASA in improving certain colitis indicators in our study were not as pronounced as expected. A primary reason for the modest efficacy of 5-ASA likely stems from its inherent pharmacokinetic limitations. The poor water solubility of 5-ASA leads to its extensive absorption in the stomach and small intestine, resulting in insufficient drug concentration reaching the colon and a compromised therapeutic efficacy [44].

Previous studies have confirmed that the chemical constituents in KG, such as kaempferol, possess therapeutic effects on UC. However, the role of EVs from KG in the treatment of UC has not been previously reported. The current study not only confirms the significant therapeutic effects of KGEVs on UC but also optimises the extraction method, substantially improving the efficiency of plant-derived EVs. In contrast to the single compounds typically used in contemporary Western and modern Chinese herbal research, EVs consist mainly of lipids, proteins, and nucleic acids, offering a new avenue for exploring the material basis of traditional Chinese medicine’s efficacy.

Conclusion

This study elucidates that KGEVs possess notable anti-inflammatory, antioxidative, mucosal-protective, gut microbiota-regulating, and macrophage M1 polarisation-inhibitory properties. Oral administration of KGEVs can effectively target and treat UC, thereby opening new therapeutic avenues for UC pharmacotherapy. The results not only expand the scope of drug treatment for UC but also validate the pharmaceutical potential of KGEVs.

Abbreviations

UC

Ulcerative colitis

CD

Crohn’s disease

IBD

Inflammatory bowel disease

KGEVs

Kaempferia galanga L.-derived extracellular vesicles

KG

Kaempferia galanga L.

IL-6

Interleukin-6

IL-1β

Interleukin-1β

TNF-α

Tumour necrosis factor-alpha

IL-10

Interleukin-10

PBS

Phosphate-buffered saline

DSS

Dextran sulphate sodium salt

LPS

Lipopolysaccharide

DiD

1,1’-Dioctadecyl-3,3,3’,3’-Tetramethylindocarbocyanine Perchlorate

FITC

Fluorescein isothiocyanate

BCA

Bicinchoninic acid assay

NTA

Nanoparticle tracking analysis

TEM

Transmission electron microscopy

DMEM

Dulbecco’s modified Eagle’s medium

ELISA

Enzyme-linked immunosorbent assay

CAT

Catalase

SOD

Superoxide dismutase

GSH

Glutathione

H&E

Hematoxylin and eosin staining

AB-PAS

Alcian blue-periodic acid-schiff stain

IHC

Immunohistochemical

HRP

Horseradish peroxidase

DAB

3,3’-Diaminobenzidine tetrahydrochloride

DAI

Disease Activity Index

5-ASA

5-Aminosalicylic acid

ACE

Abundance-based coverage estimator index

Author contributions

L.Z.L., X.H.Z., and B.Z.P. were responsible for designing the entire research protocol, completing the primary experiments, and drafting the original manuscript. J.M., FW., C.H.W. and Z.H.J. conducted the animal experiments. Z.E.H. and Z.P.Z. contributed to the cell experiments. S.Y.L., Y.C., L.N.Z., C.Y.S. assisted with the experiments. S.H.G. and X.S.Z. offered suggestions for the experimental design and guided the experimental process. C.P.J. and H.Y.K. revised the manuscript. All authors have read and approved the final manuscript.

Funding

This research was supported by the Noncommunicable Chronic Diseases-National Science and Technology Major Project (2025ZD0550800), The Joint Funds of National Natural Science Foundation of China (U22A20365), National Natural Science Foundation of China (82405279), GuangDong Basic and Applied Basic Research Foundation (2023A1515110757), Dongguan social development technology program (High level hospital construction project: 20231800913372), Natural Science Foundation of Guangdong Province (2023A1515010720, Guangdong Traditional Chinese Medicine Special Fund (20221260, 20261249), The Major scientific and technological project of Guangzhou Municipal Health Commission (20252D003).

Data availability

The datasets used for and/or subjected to detailed analysis in the context of this scholarly inquiry can be accessed from the corresponding author upon the submission of a formal request, provided that ethical and confidentiality protocols are adhered to.

Declarations

Ethics approval and consent to participate

All animal experiments adhered to the guidelines outlined by the China Council on Animal Care and Use and received approval from the Animal Ethical Committee of Southern Medical University (Approval No. SMUL202403012).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.