Abstract Current treatment of ulcerative colitis (UC) remains challenging, with the mainstay of therapy being 5-aminosalicylic acid-based drugs, which have limited and inconsistent results. Atractylodes macrocephala (AM) is a traditional Chinese medicine commonly used in the clinical treatment of various inflammatory diseases. Herein, we demonstrate that AM-derived extracellular vesicle-like particles (AMEVLP) can effectively modulate the gut microbiota, thereby significantly improving the treatment efficiency of UC. This is achieved by enhancing the alpha diversity of the gut microbiota and re-establishing beneficial types, which in turn alter tryptophan metabolism, leading to an increase in indole derivatives within the gut. This process also protects the gut barrier and exerts anti-inflammatory effects. The mechanism behind these anti-inflammatory effects is closely associated with the Th17 cell differentiation signaling pathway. It is believed that the AMEVLP enable them to efficiently remodel gut microbiota, providing an avenue for the treatment of various inflammatory diseases. Significantly, preliminary clinical trials have shown that AMEVLP can substantially slow the progression of the disease in UC patients. Graphical abstract [44]graphic file with name 12951_2025_3506_Figa_HTML.jpg Supplementary Information The online version contains supplementary material available at 10.1186/s12951-025-03506-8. Keywords: Atractylodes macrocephala, Extracellular vesicle-like particles, Ulcerative colitis, Intestinal microecological, Inflammation Introduction Ulcerative colitis (UC) is a chronic inflammatory bowel disease, distinguished by extensive lesions on the colonic mucosa, which has significantly impaired the quality of life for affected individuals [[45]1]. The clinical manifestations of UC are diverse, encompassing symptoms such as abdominal pain, diarrhea, and the presence of mucopurulent blood in stools. These symptoms are frequently accompanied by systemic manifestations, including lethargy and fatigue [[46]2]. Consequently, the pathogenesis of UC is intricate, involving the interplay of genetic, environmental, and psychological factors that lead to neuroendocrine dysfunction, disruption of the intestinal mucosal barrier, and immune imbalance [[47]3, [48]4]. Currently, the treatment of UC relies heavily on medications, including aminosalicylates, corticosteroids, and immunosuppressants [[49]5- [50]7]. However, these medications have high side effects and are prone to drug dependence and recurring conditions [[51]8, [52]9]. Salicylic acid drugs are prone to allergic reactions, hormonal drugs can adversely affect multiple organ systems with prolonged use, and immunosuppressants are commonly used for maintenance therapy. And increasing evidence suggests that gut flora dysbiosis plays a key role in the pathogenesis of UC [[53]10-[54]14]. Gut microbes can produce a variety of metabolites that can shape the intestinal immune system and affect the integrity of the intestinal barrier [[55]10, [56]15, [57]16]. Therefore, remodeling the microenvironment of intestinal flora to establish a homeostatic equilibrium between beneficial and detrimental bacteria emerges as an effective strategy for both the prevention and treatment of UC. However, existing therapeutic strategies are insufficient for effectively restoring intestinal microecological balance and promoting UC recovery. Extracts of medicinal plants, especially plant derived extracellular vesicle-like particles (PEVLP), may be a potential option for UC treatment. PEVLP is a tiny membrane structure secreted from plant cells to transfer substances and information between plants and their environment. PEVLP is safe and non-toxic, with low immunogenicity, and can be mass-produced. Moreover, PEVLP can inherit nucleic acids, lipids, proteins and other biologically active substances from its source plants [[58]17-[59]21]. Recent studies have shown that these PEVLP can be absorbed by microorganisms in the gut and have an impact on the composition and function of the gut microbiota [[60]12, [61]22]. Moreover, PEVLP may also play an important role in modulating the host’s immune response, mitigating inflammation, and ameliorating metabolic disorders through the interaction with the intestinal flora. Atractylodes macrocephala (AM) is a traditional Chinese medicine with medicinal and food origin, which is widely used treatment of various inflammatory diseases [[62]23, [63]24]. AMEVLP can inherit the anti-inflammatory effect of the traditional Chinese medicine Atractylodis Macrocephalae, and compared with the traditional treatment method, it can accurately target the damaged part of the colon and reduce the generation of side effects. Herein, we demonstrated that Atractylodes macrocephala-derived extracellular vesicles-like particles (AMEVLP) significantly enhanced the recovery of ulcerative colitis by remodeling intestinal microecological balance. We conducted an in-depth exploration of the bioactivity of AMEVLP and its active ingredients, and validated its therapeutic efficacy in UC through both in vitro and in vivo modeling (Scheme [64]1). In conjunction with intestinal flora and multi-omics analysis, it has been revealed that AMEVLP facilitates the enhancement of alpha diversity within gut microbiota and the reestablishment of beneficial types. This, in turn, modifies tryptophan metabolism, resulting in an increase in indole derivatives within the gut. This process also serves to protect the gut barrier and exert anti-inflammatory effects. The mechanism underlying these anti-inflammatory effects is intimately linked with the Th17 cell differentiation signaling pathway. We initially admitted two patients with UC as study subjects to observe the clinical effects of AMEVLP combined with mesalazine in the treatment of UC for the first time from the point of view of herbal vesicles combined with conventional Western medical treatment. Scheme 1. [65]Scheme 1 [66]Open in a new tab Diagram depicting the role of and the AMEVLP mechanism of action for the relief of UC Materials and methods Isolation of AMEVLP Slices of Atractylodis macrocephalae were purchased from Zhixin Pharmaceuticals, originating from Zhejiang Province. The dried rhizomes of AM were identified by the Traditional Chinese Medicine Department of the Third Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine. The herb AM was pulverized with fresh PBS in a juicer for 10 min, then filtered through gauze and centrifuged. Impurities such as dead cells and cellular debris were removed by continuous differential centrifugation (500× g centrifugation for 10 min, 2000× g centrifugation for 20 min, 5000× g centrifugation for 30 min, and 10,000× g centrifugation for 60 min). The supernatant was collected and ultra-centrifuged at 100,000× g for 70 min. The centrifuged precipitate was resuspended with PBS and filtered through a 0.22 μm filter membrane to collect the purified AMEVLP for immediate use or storage at − 80 °C. Cell culture A mouse monocyte macrophage cell line (RAW264.7) was used to mimic an in vitro inflammation model. RAW264.7 cells were inoculated at 1 × 10^6/mL in culture dishes. Cells were cultured with high-sugar DMEM complete medium containing 10% fetal bovine serum, with 1% streptomycin and penicillin, cultured in 5% CO[2] at 37 °C. Fluid was changed every 2 days, with cells passaged when they reached 90% fusion. Macrophages were inoculated in six-well plates at 1 × 10^6/mL and cultured overnight. The potential toxic effects of AMEVLP on RAW264.7 cells were evaluated. Macrophages were randomly divided into a normal control group, a model control group (LPS group), and an AMEVLP administration group. The protein content of AMEVLP was assessed with a BCA kit and found to be 8330 µg/mL. This protein concentration was used for all subsequent studies. Normal control and model control groups were treated with high sugar DMEM complete medium. The AMEVLP group was pretreated with different protein concentrations (0.1 µg/mL, 0.25 µg/mL, and 0.5 µg/mL) of AMEVLP for 24 h. Complete culture solution was aspirated, macrophages were washed with PBS and solution replaced with 1 µg/mL LPS solution (except for the normal control group) and placed in an incubator containing 5% CO[2] at 37° for 24 h. The supernatants of the cells of each group were collected, centrifuged, and stored at − 80 °C. Uptake of AMEVLP by RAW264.7 cells The appropriate amount of DiI dye was added to AMEVLP. The dye was protected from light for 30 min, and the free dye was removed by differential ultracentrifugation. AMEVLP labeled with DiI dye was cultured with RAW264.7 macrophages and incubated under serum-free conditions at working concentrations of 0.25 µg/ mL and 0.5 µg/ mL for 2 h, 4 h and 8 h. The efficiency of cell internalization was observed using flow cytometry, and images were acquired at a working concentration of 0.5 µg/mL using structured light confocal microscopy. Reactive oxygen species (ROS) antioxidant assay The antioxidant effects of AMEVLP were assayed using an ROS Assay Kit. DCFH-DA was diluted with serum-free culture solution (1:1000) to give a final concentration of 10 µmol/L. Cell culture solution was removed and 1 mL of diluted DCFH-DA was added to a six-well plate and incubated for 20 min at 37 °C in a cell culture incubator. Cells were washed three times with serum-free cell culture medium to adequately remove DCFH-DA that had not entered the cells. Samples loaded with probe in situ were directly visualized by laser confocal microscopy. The cells were collected and detected by flow cytometry. Quantitative real time polymerase chain reaction (RT-qPCR) Macrophages after drug intervention or colon tissues after sampling (temporarily stored at − 80 °C), were processed for total RNA by the Trizol method. cDNA was synthesized by reverse transcription kit PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time), and real-time fluorescence quantitative PCR reactions were performed by UltraSYBR Mixture (High ROX). The primer sequences of RT-qPCR related genes are shown in Table [67]S1. UC mouse therapeutic model Seven week-old female C57BL/6J mice, weighing 20 ± 2 g, were obtained from Guangzhou Ruiye Model Animal Center with approval by the Laboratory Animal Ethics Committee of Guangzhou Ruiye Model Animal Biotechnology Co. C57BL/6J mice were placed in an animal room at 20 ± 2 °C with alternating 12 h of light and 12 h of darkness with a humidity of approximately 55–65%. Mice were grouped into five per cage and had free access to food and water. Experiments were started after 1 week of acclimatization. Animals were randomly divided into a normal control group, a 2.5% dextran sodium sulfate (DSS) group, an AMEVLP high-concentration group (2 mg/kg/d), and a low-concentration group (0.5 mg/kg/d), with an average of 6 animals in each group. DSS (MP Biomedals, UK) was dissolved in distilled water to prepare a DSS solution at a concentration of 2.5%. Mice had free access to purified water in the normal control group and 2.5% DSS solution for 5 days in the other UC groups. The high and low concentration AMEVLP drug groups were started on day 6 by gavage for 5 days. The mice were then sacrificed at day 13, the length of the colon of each group was measured and photographed. Feces were collected for microbiological analysis. Colonic tissues, about 1 cm above the rectum were taken for tissue sections and fixed in 4% paraformaldehyde for subsequent patho-histological analysis and immunofluorescence staining. Colonic tissues were taken and ground with a tissue grinder and used for assessment of inflammatory factors in colonic tissues. Other colonic tissues were divided into segments for subsequent histology-related analyses. The specific parameters of the Disease Activity Index are shown in Table [68]S2. Endoscopic assessment of the intestinal tract of mice Colonoscopy was performed using a high-resolution mouse video endoscopy system (SHINOVA, MiniScope 2 V, Shanghai Maiben Medical Science and Technology Co). Mice were fasted for 24 h and treated with N-acetyl-L-cysteine (NAC) prior to examination, which promote intestinal crypt mucus excretion. Mice were anesthetized with 1.5–2.0% isoflurane. Lubricant was rubbed onto the endoscope end. The colon was then inflated with air to visualize the proximal colon for 3 cm. Live animal imaging Labelled extracellular vesicle-like particles were prepared from AM with DiR dye. The protein content of AMEVLP was assessed with a BCA assay kit, and the appropriate amount of DiR dye was added to AMEVLP. The staining was carried out in a metal bath at 37 °C, protected from light for 30 min, and the solution was subjected to ultracentrifugation (10,000× g, 70 min, and 4 °C) to fully bind the DiR dye to AMEVLP. The supernatant was discarded, and PBS was added to remove the free dye by two ultracentrifugations (10,000× g, 70 min, and 4 °C) to finally obtain the DiR dye-labeled AMEVLP. Eighteen 7-week-old female KM mice were selected and treated by gavage. Animals were randomly divided into positive control DiR and DiR-AMEVLP groups, with the different drug groups further divided into different time points of imaging at 6 h, 12 h, and 24 h, with 3 animals in each group. The animals were fed ad libitum for 1 week prior to the experiment. After 1 week, the DiR-AMEVLP group was administered 2 mg/kg per mouse by gavage, and the positive control DiR group was administered 100 µL per mouse by gavage. The mice were sacrificed at 6 h, 12 h, and 24 h. Relevant organs were excised including the five viscera (heart, liver, spleen, lungs, and kidneys) and the six internal organs (small intestine, cecum and colon, stomach, gallbladder, uterus, and bladder). The distribution and specific targeting of AMEVLP in vivo was determined using fluorescence imaging with an animal in vivo imager. Three mice per group per time point were evaluated. Immunofluorescence staining Frozen sections of colon tissue were used to analyze the expression of Occludin, ZO-1, and Claudin. Colon sections were permeabilized with 0.1% Triton X-100 and non-specific proteins were blocked with 1% BSA/PBS for 1 h. The slides were then incubated overnight at 4 °C with primary antibodies including anti-Occludin (#91131, 1:400), anti-ZO-1 (Abcam, ab61357, 1:200), and anti-Claudin (#13255, 1:100), followed by Alexa Fluor 488 and DAPI. Finally, fluorescently-stained colon sections were imaged and photographed using a Leica microscope. 16 S rDNA amplicon sequencing Mouse fecal samples were shipped on dry ice to Beijing Novozymes Technology Co. for 16 S rDNA Amplicon Sequencing. Genomic DNA was extracted from mouse fecal samples using the magnetic bead method and fecal genomic DNA extraction kit (TianGen). We then tested the extracted genomic DNA for DNA purity and concentration by 1% agarose gel electrophoresis. PCR products were obtained and assessed by electrophoresis using an agarose gel. The PCR products were purified with magnetic beads, quantified by UV spectrophotometry, mixed in equal amounts according to the concentration of PCR products, and the PCR products detected by agarose gel electrophoresis using 2% agarose after sufficient mixing. Products were recovered by using a pass-through DNA Purification and Recovery Kit (TianGen) for the target bands. Library construction was performed using the NEB Next⑥ Ultra™ II FS DNA PCR-free Library Prep Kit (New England Biolabs). The constructed library was quantified by Qubit and RT-qPCR, and after the library was qualified, the PE 250 was up-sequenced using NovaSeq 6000. Finally, bioinformatics analysis was performed using QIIME2 software. Intestinal metabolomics analysis Metabolites were extracted from feces (100 mg per sample). Non-targeted LCMS/MS analysis as well as data preprocessing and annotation were performed using NOHZYUAN Technology LC-MS/MS and a Vanquish UHPLC (Thermo Fisher, Germany) with a Hypesil Gold column (1.9 μm 2.1*100 mm, Thermo Fisher, USA) coupled to a Q Exactive™ HF (Thermo Fisher, Germany). MS/MS spectra were acquired by data-dependent scans using a Q Exactive™ HF mass spectrometer. All MS raw data files were processed by Compound Discoverer 3.3 software for spectral processing and database searching, and each metabolite was screened for retention time (RT), mass-to-charge ratio (m/z) value, and other parameters to obtain qualitative and quantitative metabolite results. Then metaX software was used to perform quality control on the data to ensure accuracy and reliability. The KEGG database, HMDB database, and LIPIDMaps database were applied for metabolite identification. Next, the metabolites were subjected to multivariate statistical analysis, and the metabolomics data processing software metaX was used to convert the data to Principal Component Analysis (PCA) and Partial Least Squares Discriminant Analysis (PLS-DA), which led to the VIP value of each metabolite, revealing the differences in metabolic patterns among the different groups. A volcano map was plotted with the R package ggplot2, which can combine the three parameters of metabolite VIP value, log2 (Fold Change) and -log10 (P-value) to filter metabolites of interest. Clustered heat maps, plotted with the R package Pheatmap, were normalized to the metabolite data using z-score. Correlation analysis (Pearson’s correlation coefficient) between the different metabolites was performed using the R language cor (), with statistical significance achieved by cor.mtest() in R. A P value of < 0.05 was considered to be statistically significant, and correlation graphs were plotted using the corrplot package in R. Bubble plots were performed with the R package ggplot2, and the KEGG database was used to investigate metabolite functions and metabolic pathways, considered to be enriched when x/n > y/n, and significantly enriched when the P-value of the metabolic pathway was < 0.05. Immunohistochemistry To determine levels of IL-1β ([69]AB234437, 1:50), IL-10 ([70]AB189382, 1:100), IL-21 (AB5978, 1:800), and TNF-α (AB1793, 1:100) in the colon, we performed immunohistochemical analyses of colon tissue. After deparaffinization, rehydration, and antigen repair of the colon tissue, the colon sections were treated with 3% H[2]O[2] to block endogenous peroxidase and washed with PBS for 3–5 min. The colon sections were then incubated with the primary antibody at 4 °C for 12 h. After washing with PBS, the slides were immersed in the corresponding secondary antibodies at 37 °C for 30 min. The color was developed with diaminobimane (DAB) and restained with hematoxylin, and finally differentiated using hydrochloric acid alcohol. Quantification of DAB-stained positive areas was performed using ImageJ software. AMEVLP omics analysis Lipid and metabolite analysis, as well as data preprocessing for untargeted metabolomics of AMEVLP were performed by Shanghai Lumine Biotechnology Co. Sample pretreatment, metabolite extraction, LC-MS full-scan detection, data preprocessing, and statistical analysis were performed. Non-targeted metabolomics based on ultra-high performance liquid tandem high-resolution mass spectrometry (UPLC-HRMS) was combined with the metabolomics data processing software, Progenesis QI v2.3, to perform qualitative and relative quantitative analyses and standardized pre-processing of the raw data. Comparative analysis of metabolic differences was performed by multivariate and univariate statistical analyses, screening for differential metabolites, followed by correlation analysis and pathway enrichment analysis. The small RNA-seq of AMEVLP was analyzed by Xiamen Life Interconnect. The PE150 sequencing protocol was used for the small RNA sequencing libraries, and the quality values of the sequenced libraries were assessed using fastqc. For non-model species, the miRbase library did not record their small RNA sequences, so small RNAs from all plants in the miRbase library were utilized as a reference to quantify possible small RNAs in the samples. Literature search was also conducted, and if small RNA sequences for the species were provided in the literature, they were combined with plant small RNAs for analysis. Multi-omics correlation analysis The joint multi-omics analysis of AMEVLP was performed by Genesky Biotechnologies Inc. After the raw multi-omics data were normalized, the database was searched to screen for metabolites shared between AMEVLP and the gut, and then the data were quality controlled to ensure the accuracy and reliability of the data results. Differential metabolites shared by AMEVLP and the gut were analyzed in a two-by-two joint analysis with the gut flora, the gut transcriptome, and the vesicle transcriptome to analyze the metabolites shared by each omic and analyzed differently, with Pearson correlation analyses used for the metabolites with significant differences. Finally, based on the differential metabolites obtained from the screening, and the KEGG annotation ID, the differentially expressed genes were analyzed for KEGG enrichment of metabolites. Modeling of antibiotics Seven week-old female C57BL/6J mice weighing 20 ± 2 g were obtained from Guangzhou Ruiye Model Animal Center, as approved by the Laboratory Animal Ethics Committee of Guangzhou Ruiye Model Animal Biotechnology Co. C57BL/6J mice were placed in an animal room at 20 ± 2 °C with alternating 12 h of light and 12 h of darkness and a humidity of approximately 55–65%. Mice were placed five per cage and had free access to food and water. The groups were randomized into normal control (NC), antibiotic alone (Antibiotic + DSS), and AMEVLP (2 mg/kg/d) high concentration group (Antibiotic + DSS + AMEVLP), which will be referred to later as the Ab group versus the Ab + AMEVLP 2.0 mg group. Each group averaged eight mice. DSS was dissolved in distilled water to prepare a solution of DSS at a concentration of 2.5%. Experiments were started after 1 week of acclimatization feeding. On days 1, 2, 6, and 7, mixed antibiotics were given at a dose of 1000 mg/kg/d for gut flora removal, which consisted of a mixture of vancomycin, ampicillin, metronidazole, and neomycin sulfate in a ratio of 2:4:4:4 by mass, and prepared as an aqueous solution with distilled water for gavage. On days 3–5, the mice were routinely given drinking water, and on days 8–9, all were given a 2.5% DSS solution. On days 3–11, mice in the dosing group were administered 200 µL of AMEVLP at a concentration of 2 mg/kg/d by gavage, and an equal amount of PBS was given to mice in the Ab group. Finally, the mice were sacrificed at day 12. The length of the colon for each group of mice was measured and photographed. Colon tissue about 1 cm above the rectum was taken for tissue sectioning and fixed in 4% paraformaldehyde for subsequent patho-histologic analysis. Mouse body weight, fecal character, and blood stools were recorded daily as well as the calculation of disease activity index scores. Clinical data The clinical data of this study were collected from September 2023 to September 2024 in two patients with ulcerative colitis admitted to the Department of Gastroenterology of the Third Affiliated Hospital of Guangzhou University of Chinese Medicine for treatment. Patients were divided into mesalazine and AMEVLP groups according to the randomized numerical table method and treated for 2 weeks. Patients in the mesalazine group were treated with oral mesalazine alone, 0.5 g/dose, 3 times/day. The AMEVLP group was treated with AMEVLP in combination with mesalazine group, 5 g/dose, 2 times/day. Inclusion criteria: 18 years ≤ age ≤ 65 years; no history of antimicrobial drugs or related systemic therapy in the last month; all were primary or chronic relapsing; good compliance, patients and their families were clear about the treatment and the study process, and signed informed consent. Exclusion criteria: patients with contraindications or allergies to the study drug; patients with serious complications such as intestinal obstruction or perforation; patients with a history of previous intestinal surgery; patients with malignant tumors, acute or chronic infections, other immune system diseases, severe cardiac, hepatic, and renal disorders; pregnant or breastfeeding females; patients with severe fulminant ulcerative colitis; patients who do not take the medication according to the requirements of the study or who self-adopt other treatment regimens; those who fail to provide feedback on their therapeutic efficacy. The study was approved by the Ethics Committee of the Third Affiliated Hospital of Guangzhou University of Chinese Medicine under Grant No. PJ-XS-20230919-001, and the patients all signed the informed consent form for this study. Data statistics Significance levels were determined by appropriate statistical analysis based on whether the data were normally distributed and the number of test groups used for comparison. Comparisons between the two groups were made using an unpaired t-test (Student’s t-test). All statistical analyses were performed using GraphPad Prism version 9.0 (GraphPad Software, USA). Results are expressed as the means ± standard deviation. Differences were considered significant if the P value was less than 0.05. ***P < 0.001; **P < 0.01; *P < 0.05. Results Isolation and characterization of AMEVLP AM was isolated from AMEVLP by juicing, differential ultracentrifugation, and filtration (Fig. [71]S1A). TEM analysis showed that the AMEVLP were uniform in size and had a round- or cup-shaped morphology (Fig. [72]1A). The mean diameter of AMEVLP was 90.61 ± 19.66 nm and the concentration was 6.27 × 10^11 particles/mL (Fig. [73]1B). The BCA method measured a protein concentration of 8330 µg/mL, and subsequent uniform administration was expressed as protein concentration. After incubation and lysis using a Triton X-100, we utilized a nanoflow detector to analyze the percentage of particles with membrane structures in the AMEVLP samples. TritonX-100 membrane-breaking experiments demonstrated that the purity of the isolated AMEVLP reached nearly 80% (Fig. [74]1C). The gastrointestinal stability test of AMEVLP showed that AMEVLP had a particle size of 182.2 nm, a particle size of 215.7 nm in gastric fluid, and a particle size of 161.2 nm in gastric fluid and small intestine fluid. These results indicate that AMEVLP achieve a steady state in both particle size and potential in gastric and small intestinal fluids (Fig. [75]1D). In addition, AMEVLP is rich in bioactive components such as nucleic acids (Fig. [76]1E), lipids, and metabolites (Fig. [77]1F and Fig. [78]S1B). The metabolites detected in the positive ion mode at the time of LC-MS data acquisition were Atractylenolide, Ginsenoside F2, and Dendrolasin, while Resveratrol 3-sulfate, and Artemisinin detected in the negative ion mode (Fig. [79]S2A). The metabolites specifically enriched in AMEVLP include monoterpenes and terpene lactones, steroids, steroid derivatives, as well as indoles and their derivatives (Fig. [80]S2B-D). These results demonstrate that high-purity and high-quality AMEVLP were prepared. These data support the subsequent use of gavage administration and lay the foundation for the subsequent study of the biological activity and mechanism of action of AMEVLP. Fig. 1. [81]Fig. 1 [82]Open in a new tab Identification of AMEVLP. A. TEM images of AMEVLP. Scale bar indicates 200 nm and 50 nm. B. Nano FCM for detection of particle size distribution of AMEVLP. C. Triton X-100 detection of lipid membrane structure of AMEVLP. ***P < 0.001. D. Stability of AMEVLP in simulated gastric and small intestinal fluids. E. Agarose gel electrophoresis for identification of nucleic acids of AMEVLP. F. Lipid composition and metabolite composition mapping of AMEVLP Anti-oxidant and anti-inflammatory effects of AMEVLP in vitro RAW264.7 cells are highly phagocytic and are commonly used as inflammatory cell models. Therefore, we chose different concentrations of AMEVLP to act on RAW264.7 cells to observe the ability of RAW264.7 cells to uptake AMEVLP. The anti-inflammatory and anti-oxidant effects of AMEVLP on RAW264.7 were investigated as well. The efficiency of cellular uptake of AMEVLP determines the extent to which AMEVLP affects cell function and viability. Therefore, through pre-experimental screening we finally chose 0.25 and 0.5 µg/mL protein concentrations of AMEVLP for uptake experiments. Flow cytometry results showed that the proportion of cells containing fluorescent signals in RAW264.7 macrophages increased with increasing concentrations of AMEVLP protein and with prolonged incubation time. When the concentration of AMEVLP was 0.5 µg/mL and the incubation time was 8 h, the proportion of cells containing AMEVLP reached 99.12% (Fig. [83]2A, Fig. [84]S3A). The images taken by confocal microscopy showed that RAW264.7 effectively internalized and absorbed AMEVLP and achieved nearly 100% internalization after 8 h (Fig. [85]2B, Fig. [86]S3B). These results suggest that AMEVLP can be taken up by AMEVLP macrophages, possibly regulating biological processes such as cell metabolism and proliferation. Subsequently, we induced an oxidative stress model in RAW264.7 cells with 0.5 mM H[2]O[2], and by both flow cytometry and confocal imaging showed that AMEVLP significantly reduced ROS expression, achieving anti-oxidant effects (Fig. [87]2C-D, Fig. [88]S3C-D). RT-qPCR results in the LPS-induced inflammation model of RAW264.7 cells showed that AMEVLP up-regulated the expression of the inflammation-suppressing factor IL-10 (Fig. [89]S3E) and inhibited the expression of the pro-inflammatory factors IL-1β, IL-6, IL-12, and TNF-α (Fig. [90]S3F). These results suggest a promising in vitro anti-inflammatory effect, and a concentration-dependent effect. Based on these results, we concluded that AMEVLP has good anti-oxidant and anti-inflammatory effects in vitro. Fig. 2. [91]Fig. 2 [92]Open in a new tab In vitro anti-inflammatory and anti-oxidant effects of AMEVLP. A. Fluorescence signal detection of RAW264.7 cells after internalized uptake of Dil-labeled AMEVLP (0.25 µg/mL and 0.5 µg/mL) at different time points (2, 4, 8 h). B. Incubation of RAW264.7 cells with DiI-labeled AMEVLP (0.5 µg/mL). Fluorescence images for 2, 4, and 8 h. Scale bar indicates 20 μm. C-D. Fluorescence signal distribution and representative fluorescence images of DCF in RAW264.7 cells induced by H[2]O[2] and treated with different protein concentrations of AMEVLP for 6 h. Scale bar indicates 100 μm AMEVLP is safe, nontoxic, and entero-targeted in vitro and in vivo To verify the biocompatibility of AMEVLP in vivo, we examined the effect of different concentrations of AMEVLP on the growth activity of RAW264.7 macrophages at 24 h and 48 h by MTT assay. The results showed that AMEVLP at 1–20 µg/mL had no significant effect on the viability of RAW264.7 macrophages and no potential toxicity (Fig. [93]S4A and B). Hemolysis experiments revealed that different concentrations of AMEVLP did not cause rupture of erythrocytes (Fig. [94]S4C). AMEVLP had no significant effect on body weight in mice (Fig. [95]S4D). AMEVLP had no effect on apoptosis of RAW264.7 macrophages as detected by flow cytometry (Fig. [96]S4E). The results of H&E staining showed that the pathological changes of various tissue organs (heart, liver, spleen, lungs, kidneys, and colon) by different concentrations of AMEVLP were not significantly different from those of the NC group, indicating that AMEVLP did not have a significant effect on organs of the body. Results of the blood and biochemical assays showed that AMEVLP had no effect on indices related to blood, liver, or renal function (Fig. [97]S4F and G). These results show that AMEVLP have good biocompatibility and can be used for subsequent biological activity studies. Targeted distribution of AMEVLP in vivo was explored using a small animal in vivo imager. The results showed that at 6 h, 12 h, and 24 h, the fluorescence signals of the colon tissue of DiR-AMEVLP were significantly higher than those of the control DiR. These results indicate that AMEVLP can be targeted and transported to the colon, where they may be enriched for precise therapeutic effects (Fig. [98]S5). AMEVLP treats DSS-induced UC of mice To validate the biological activity of AMEVLP, we induced a UC model in mice with DSS (Fig. [99]3A). Changes in UC induced body weight were reversed in the AMEVLP-administered group compared to the DSS-modeled group (Fig. [100]3B). Disease activity index scores showed improvement in the administered group (Fig. [101]3C). Colon length was significantly shorter in the DSS group and significantly improved in the AMEVLP high-concentration administration group (Fig. [102]3D-E). RT-qPCR results showed that AMEVLP up-regulated the mRNA expression of the inflammation-suppressing factor IL-10 (Fig. [103]3F) and inhibited the mRNA expression of the pro-inflammatory factors IL-1β, IL-6, IL-12, and TNF-α (Fig. [104]3G), suggesting an in vivo anti-inflammatory effect. The endoscopic results showed that there were obvious white ulcers in the DSS group, with the therapeutic effect significant in the AMEVLP high-concentration administration group, with the mucosal status similar to normal mice (Fig. [105]3H), indicating that AMEVLP improve the mucosal status of the intestinal tract. H&E staining showed significant crypt gland destruction and atrophy, disorganized arrangement, altered cup cell morphology, undulating and uneven mucosal surface, as well as large inflammatory cell infiltration in the 2.5% DSS group compared to the NC group. After treatment with high and low concentrations of AMEVLP, inflammatory cell infiltration was still seen in the colon mucosa, and the glandular structure was incomplete, but it was significantly less than that the DSS group The cupular cell morphology was also significantly better in the AMEVLP group (Fig. [106]3I). These results show that AMEVLP can treat DSS-induced UC in mice, and that the treatment effect was better in the group with the higher concentration of the drug. To explore the restorative effects of AMEVLP on colonic barrier function, we stained the colonic tissues of mice for tight junction proteins and with Alcian blue. The results of tight junction protein staining showed Occludin red fluorescence, ZO-1 green fluorescence, and Claudin green fluorescence were significantly reduced in the DSS group compared with the NC group, while Occludin red fluorescence, ZO-1 green fluorescence, and Claudin green fluorescence were significantly increased in the 2.0 mg/kg AMEVLP group compared to the DSS group. All of the above indicate that AMEVLP up-regulates the protein expression of Occludin, ZO-1, and Claudin in the colon tissues of DSS-treated mice, and that DSS-induced inflammation in the colon was reduced and that impaired colonic barrier function was repaired (Fig. [107]3J). The microscopic results of Alcian blue staining showed that the cup cells in the DSS group were significantly impaired compared with the NC group, and the blue mucus area was significantly reduced. The cup cell structure in the 2.0 mg/kg AMEVLP group was significantly restored compared with that in the DSS group, and was similar to that in the NC group, with a significant increase in the blue mucus area. All of the above indicate that the intestinal glands were significantly damaged in mice induced by DSS, and AMEVLP administration restored the damaged glands (Fig. [108]3K). Fig. 3. [109]Fig. 3 [110]Open in a new tab AMEVLP alleviates DSS-UC and repairs colonic barrier function in mice. A. Experimental design of the mouse model of DSS-induced UC treated with AMEVLP. B. Body weight changes of each group were monitored daily and expressed as a percentage of initial body weight. C. Disease Activity Index (DAI) scores. n = 6. D. Morphological observation of the colons of representative mice in each group. E. Colonic lengths. n = 5. F. RT-qPCR showed that the administration of AMEVLP up-regulated the IL- 10 mRNA expression level. G. RT-qPCR showing inhibition of IL-1β, IL-6, IL-12. and TNF-α mRNA expression after AMEVLP administration. Data are shown as means ± standard deviation, n = 3. **P < 0.01, *** P < 0.001. H. Colonic endoscopic images of colonic inflammation in mice. I. H&E-stained colonic tissue sections. Scale bar indicates 200 μm. J. Images of immunofluorescence staining for the expression of colonic tight junction proteins (Occludin, ZO-1, and Claudin). Scale bar indicates 100 μm. K. Alcian blue stained images. Scale bars indicate 50 μm and 20 μm AMEVLP prevent UC induced by DSS in mice After verifying that AMEVLP could treat DSS-induced UC, we wanted to verify whether AMEVLP could prevent DSS-induced UC (Fig. [111]S6A). Changes in body weight of mice were found to be restored in the AMEVLP-administered group compared to the DSS-modeled group (Fig. [112]S6B). Disease activity index scores showed improvement in the administered group (Fig. [113]S6 C). Colon length was significantly shorter in the DSS group with successful modeling and significantly better in the AMEVLP high-concentration administration group (Fig. [114]S6D and E). RT-qPCR results showed that AMEVLP up-regulated the mRNA expression of the inflammation-suppressing factor IL-10 (Fig. [115]S6F) and inhibited the mRNA expression of the pro-inflammatory factors IL-1β, IL-6, IL-12, and TNF-α (Fig. [116]S6G), suggesting an in vivo anti-inflammatory effect. Endoscopic results showed obvious white ulcers in the DSS group, and the preventive effect was significant in the AMEVLP high-concentration administration group, with the mucosal status similar to normal mice (Fig. [117]S6H). H&E staining showed significant crypt gland destruction and atrophy in the DSS group, which was significantly improved in the administered group (Fig. [118]S6I). These results show that AMEVLP prevents DSS-induced UC in mice, and the treatment effect was better in the group with the higher concentration of the drug. AMEVLP regulates intestinal flora and metabolite composition in mice with UC To elucidate how AMEVLP regulate the composition of the intestinal flora in mice with UC, we analyzed the feces of mice for intestinal flora. The richness and diversity of microbial communities within the samples were reflected by diversity analysis (Alpha diversity) with the results of the recovery of Shannon’s index in the AMEVLP group (vs. DSS) reflects restored community evenness, while the increase in Observed species and Chao1 indicates enhanced species richness. (Fig. [119]4A-C). The PCA results showed that the first principal component contributed 15.72% and the second principal component contributed 12.34% to the sample differences, and the results showed that the community composition of the samples was similar between the groups (Fig. [120]4D). Subsequently, we compared the intestinal flora at the phylum level and at the genus level, and found that the results of the intergroup comparison at the phylum level showed that Bacteroidota and Firmicutes were significantly decreased in the DSS group compared to the NC group, and significantly increased after AMEVLP administration compared to the DSS group. Proteobacteria were significantly increased in the DSS group compared to the NC group and significantly decreased after AMEVLP administration compared to the DSS group (Fig. [121]4E). There was a significant decrease in Escherichia-Shigella and a significant increase in beneficial bacteria at the genus level (such as Bacteroides) after administration of the drug (Fig. [122]4F). The above results indicate that AMEVLP can decrease the abundance of harmful bacteria and increase the abundance of beneficial bacteria at the phylum level and genus level. In the differential species evolutionary branching diagram, the expression of species with significant differences at different taxonomic levels can be seen, and the microbial taxa that play an important role in the AMEVLP group are f_Bacteroidaceae and f_Rikennellaceae (Fig. [123]4G). Based on the species annotation results at different taxonomic levels, we selected the species with the highest ranked and statistically different maximum relative abundance at the genus level for each group of NC, DSS, and AMEVLP, and plotted the relative abundance bar charts at the genus level for the corresponding species annotation results for each group. The results of the relative abundance plot showed that AMEVLP at the genus level showed significant differences in abundance among the groups Muribaculaceae, Rikenellaceae_RC9_gut_group, Lachnospiraceae_NK4A136_group, Alistipes, Bacteroides, and Escherichia-Shigella. DSS decreased the abundance of the beneficial bacterial flora in the feces of mice in the phylum Bacteroidetes and the phylum Thick-walled Bacteria, whereas AMEVLP reversed the trend, with an increase in the abundance of the bacterial flora after the administration of the drug (Fig. [124]4H). DSS increased the abundance of harmful bacteria in the feces of mice (Fig. [125]4I). We enriched KEGG pathways by untargeted metabolomics techniques for differential metabolites of mouse feces (Fig. [126]4J) and found that the top two rankings were the tryptophan metabolism pathway as well as the steroid hormone biosynthesis pathway, respectively. The results of the tryptophan metabolic pathway heat map showed that 6-Hydroxymelatonin, L-Kynurenine, Melatonin, and Indole-3-acetamide were significantly increased after AMEVLP administration compared to the DSS group (Fig. [127]4K). The results of the steroid hormone biosynthesis pathway heat map showed a significant increase in hydrocortisone. These results show that AMEVLP can alter tryptophan metabolism by modulating changes in the intestinal flora, which in turn increase indole derivatives in the intestinal tract allowing for protective and anti-inflammatory effects on the intestinal barrier following AMEVLP administration (Fig. [128]4L). Fig. 4. [129]Fig. 4 [130]Open in a new tab AMEVLP regulation of gut microbial and metabolite composition. A. Observed species index (Alpha diversity). B. Chao1 index (Alpha diversity). C. Shannon index (Alpha diversity). D. Principal component analysis (PCA). D. Intergroup comparison of relative abundance of gut microbiota at the gate level. E. Heat map of relative abundance of taxa at the genus level of each group abundance. F. Heat map of categorical relative abundance of groups at the genus level. G. Evolutionary dendrograms of species with significant differences. H. Relative abundance of beneficial flora with significant differences at the genus level. I. Relative abundance of harmful flora with significant differences at the genus level. Data are shown as means ± standard deviation, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001. J. KEGG pathway enrichment bubble plots showing differential metabolite expression. K. Tryptophan metabolism pathway heat map. L. Steroid hormone biosynthesis pathway heat map AMEVLP treats UC mice in a microbiota-dependent manner To further confirm whether the effect of AMEVLP on DSS-induced colitis in mice was microbiota-dependent, we pretreated C57BL/6 mice with a mixture of broad-spectrum oral antibiotics (Ab) to disrupt the intestinal microbiome following administration of 2.5% DSS (Fig. [131]S7A). The AMEVLP untreated group was treated with an antibiotic mixture alone (group Ab) as a control. Body weight changes and disease activity index scores of mice were recorded daily. Compared with the Ab group, the Ab + AMEVLP group did not significantly improve the symptoms of DSS-induced colitis, and the results show similar changes in body weight (Fig. [132]S7B). Disease activity index scores (Fig. [133]S7C) and colon length (Fig. [134]S7D) were not statistically different. These results suggest that AMEVLP can alleviate DSS-induced colitis in mice by modulating the gut microbiome. Combined multi-omics analysis of signaling pathways involved in AMEVLP induced effects To explore the signaling pathways that underlie the specific actions of AMEVLP in mice, we combined multiple omics for analysis. We first found the common metabolites of the vesicles and the intestine (Fig. [135]S8) and analyzed these for differences. The results revealed that L-Tyrosine, 1-Methyladenosine, Cinchophen, Octopine, Valylproline, Thymidine, and Leucylproline were differentially expressed in the DSS and AMEVLP groups, and were significantly greater in the AMEVLP group (Fig. [136]5A). The common differential metabolites of the vesicle and intestine may indicate that the metabolites in the vesicle enter the intestine and thus play a regulatory role. We correlated shared differential metabolites with intestinal flora and found that Alistipes, Bacteroides, Rikenellaceae_RC9_gut_group, and UCG-010 were significantly correlated with shared differential metabolites; whereas metabolites differentially expressed in the AMEVLP group down-regulated Escherichia- Shigella, and Ralstonia colony expression. Vesicles modulate changes in intestinal flora through intra-vesicular metabolites, decreasing the expression of harmful flora and increasing the abundance of beneficial flora (Fig. [137]5B). We then evaluated the gut transcriptome (Fig. [138]S9) and genes associated with shared differential metabolites (Fig. [139]5C), genes associated with the gut transcriptome and intestinal flora (Fig. [140]5D), and genes associated with vesicular miRNAs and transcriptome for pathway enrichment analysis (Fig. [141]5E). We explored the correlation between shared differential metabolites and the transcriptome, between gut flora and the gut transcriptome, and between the vesicular transcriptome and the gut transcriptome. Relevant pathways were found by pathway analysis, and most were found to be related to immunity. KEGG analysis found co-enrichment of the Th17 cell differentiation signaling pathway. To confirm that AMEVLP is functioning through this signaling pathway, we used immunohistochemistry to validate the expression of cytokine proteins associated with the Th17 cell differentiation signaling pathway. Expression of pro-inflammatory factors IL-1β, IL-21, and TNF-α was significantly lower in the AMEVLP-administered group than in the DSS group, and the expression of the inflammation-suppressing factor protein IL-10 was greater than that in the DSS group (Fig. [142]5F, Fig. [143]S10). These results demonstrate that AMEVLP treat UC of mice through the Th17 cell differentiation signaling pathway. Fig. 5. [144]Fig. 5 [145]Open in a new tab AMEVLP alleviates UC in mice by regulating the Th17 cell differentiation signaling pathway. A. Heat map of AMEVLP and intestinal common differential metabolites. B. Correlation analysis of common differential metabolites and intestinal flora. C. KEGG pathway enrichment analysis of common differential metabolites and colon tissue transcriptome. D. KEGG pathway enrichment analysis of colon tissue transcriptome and intestinal flora. E. Immunohistochemical enrichment analysis of AMEVLP transcriptome and colon tissue transcriptome. F. Quantitative plot of immunohistochemical staining for cytokines associated with the Th17 cell differentiation signaling pathway, n = 3, **P < 0.01 AMEVLP prevents ulcerative colitis induced by TNBS in mice To fully assess the efficacy of AMEVLP, we performed additional experiments using the TNBS-induced colitis model. (Fig. [146]S11A). Changes in body weight of mice revealed a rebound in the AMEVLP-administered group compared to the TNBS modeling group (Fig. [147]S11B). Disease activity index scores showed improvement in the dosed group (Fig. [148]S11C). Colon length was significantly shorter in the DSS group with successful modeling and significantly better in the AMEVLP high concentration administration group (Fig. [149]S11D). Endoscopic results showed obvious white ulcers in the TNBS group, and the preventive effect was obvious in the AMEVLP high-concentration administration group, and the mucosal status was close to the level of normal mice (Fig. [150]S11E). HE staining showed obvious crypt gland destruction and atrophy in the TNBS group, and it was obviously improved in the drug administration group (Fig. [151]S11F). Flow cytometry was used to detect changes in Th17 and Treg cells in the spleens of colitis mice, and the proportion of proinflammatory Th17 cells (CD4⁺IL-17 A⁺) in the AMEVLP-treated group was significantly reduced by 7.88% compared with that of the colitis control group (Fig. [152]S11G), although a significant reduction in the proportion of regulatory T cells (CD4 ⁺FoxP3⁺) numbers showed a 1.83% decreasing trend (Fig. [153]S11H), but did not reach statistical significance (Fig. [154]S11I). All these results showed that AMEVLP prevented TNBS-induced ulcerative colitis in mice, and the treatment effect was better in the group with high concentration of the drug, which was superior to that of the positive drug mesalazine. Clinical efficacy The colonoscopic results of the two patients currently admitted to the AMEVLP group after oral administration of AMEVLP showed. Patient A had fewer foci of rectal mucosal erosion and purulent discharge than before treatment. Patient B had significantly better rectal mucosal erosions and ulcers than before treatment (Fig. [155]6A). Pre-treatment HE staining of Patient A showed cryptitis, crypt abscess, chronic active inflammation of the mucosa with erosions, lymphoid tissue hyperplasia and lymphoid follicle formation. Post-treatment results showed a slight decrease in crypt abscess and lymphocytic infiltration compared to the previous period, with cryptitis and chronic active inflammation of the mucosa still present. HE staining of Patient B before treatment showed crypt abscess and crypt atrophy, partial distortion of mucosal crypts, and more lymphocytic and neutrophilic infiltration in the interstitium. The results after treatment did not show any significant crypt abscess, crypt atrophy and lymphocytic infiltration were slightly better than before, and some crypts were still distorted (Fig. [156]6B). Fig. 6. [157]Fig. 6 [158]Open in a new tab Clinical efficacy of AMEVLP. A. Comparison of colonoscopic findings after oral administration of AMEVLP in Patient A and Patient B. B. H&E-stained rectum tissue sections. Scale bars indicate 200 μm Discussion In this study, we combined the pharmacological activity of the traditional Chinese medicine AM with the biological function of PEVLP, creatively extracted an AMEVLP that can regulate intestinal microecology and has anti-inflammatory activity, and explored its specific mechanism of action in the treatment of UC. The current treatment of UC remains challenging. Currently, 5-aminosalicylate, corticosteroids, intestinal probiotics and immunosuppressants are commonly used therapeutic drugs in Western clinical practice [[159]1]. Salicylic acid drugs are prone to allergic reactions, hormonal drugs can cause adverse effects on multiple organ systems with long-term use, and immunosuppressive drugs are commonly used in maintenance therapy, all of which work by regulating the body’s immune response, but have significant side effects and adverse reactions [[160]25-[161]27]. Supplementation with gut probiotics is also a complementary combination therapy, but it is not as effective [[162]28]. With the increasing research on the pathogenesis of ulcerative colitis, more and more evidence suggests that the development of UC is closely related to intestinal flora dysbiosis. Imbalance of microbial homeostasis leads to colonization and invasion of opportunistic pathogenic bacteria in the gut, increasing the risk of host immune response and promoting the development of UC. Therefore, remodeling the microenvironment of intestinal flora to establish a homeostatic balance of intestinal microorganisms has become an important strategy for the prevention and treatment of UC. At present, few studies have been conducted on UC therapeutic drugs that regulate intestinal microecology, and there is an urgent need to find new drugs that regulate intestinal microecology and have anti-inflammatory effects, are safe and reliable, and can be taken for a long period of time. It has been shown that PEVLP extracted from various fresh herbs (e.g., ginseng, turmeric, etc.) is widely used in a wide range of diseases and is a completely new medicinal substance [[163]29-[164]31]. However, the therapeutic mechanisms of UC in regulating intestinal microecology have been little studied. PEVLP is biocompatible and biologically active and has a unique targeting property that allows for targeted delivery of drugs to sites of inflammation [[165]32, [166]33]. Several studies have shown that PEVLP not only concentrates the active ingredients of traditional Chinese medicine, but also improves bioavailability and achieves precise therapeutic effects [[167]34-[168]41]. PEVLP is a good choice for novel pharmacodynamic substances that can inherit the bioactive components of source Chinese medicines. Atractylodes macrocephala, a traditional Chinese medicine that is homogeneous with food and medicine, affordable and rich in efficacy, has been widely used in the clinical treatment of UC [[169]42]. However, most studies on AM drugs have focused on single extracts (e.g., atractylenolide, atractylenolide polysaccharide, etc.), with insufficient bioavailability of the active ingredients and unclear target distribution sites [[170]43, [171]44]. In this study, we hypothesized that AMEVLP inherited the bioactive components of AM for the treatment of UC. Multiple characterization and identification methods proved that we isolated high-purity and high-quality AMEVLP, verified the gastrointestinal stability of AMEVLP, and identified its main contents, laying a foundation for the subsequent study of the biological activity and mechanism of action of AMEVLP. Cellular level studies showed that AMEVLP could be effectively taken up by RAW264.7 macrophages with better drug uptake characteristics. Moreover, it demonstrated significant anti-inflammatory activity by up-regulating the gene expression of anti-inflammatory factors and inhibiting pro-inflammatory factors. In addition, AMEVLP demonstrated the ability to possess antioxidant properties and reduce the expression of ROS in macrophages, proving that AMEVLP has biological activity as a therapeutic agent for UC. In vivo studies further confirmed the potential of AMEVLP in the treatment of UC. AMEVLP not only showed good biocompatibility and targeting in vivo, but also effectively improved the colonic barrier function and restored the expression of tight junction proteins and glandular structure in UC mice. It has been reported that the occurrence of UC is closely related to intestinal flora dysbiosis, which plays an important role in the regulation of intestinal metabolites. In our study, gut flora analysis showed that AMEVLP slowed disease progression by modulating the structure of the intestinal microbial community, increasing the abundance of beneficial bacteria, inhibiting the growth of harmful bacteria, and reshaping the intestinal microecological balance. Interestingly, AMEVLP improved UC mice in a microbiota-dependent manner. Metabolomics and transcriptomics analyses further revealed that AMEVLP repaired the colonic epithelial barrier and attenuated inflammation by modulating tryptophan metabolism, increasing indole derivatives, and its mechanism of action was closely related to the Th17 cell differentiation signaling pathway, which provided a new strategy for suppressing UC immune imbalance. The results from colonoscopies and HE staining of clinical patients further confirm that AMEVLP can effectively repair intestinal ulcers and damaged intestinal mucosa, demonstrating notable clinical efficacy. Immunohistochemistry and TNBS-induced colitis model demonstrated that AMEVLP could regulate Th17 cells, and the results suggested that AMEVLP could form a synergistic regulatory network through the “metabolite-flora-immunity axis”. We suggest that AMEVLP may preferentially regulate changes in the intestinal flora via intravesicular metabolites, thereby restoring intestinal flora homeostasis and ultimately inhibiting the Th17 pathway, thereby alleviating colonic inflammation. Although this study provides strong evidence for the use of AMEVLP in the treatment of UC, limitations remain. Batch-to-batch variability in the extraction process and under-explored bioactive molecules such as proteins in AMEVLP. It is suggested that future studies need to establish a more comprehensive quality control system and explore the specific composition of vesicles in depth to optimize therapeutic efficacy and biosafety. In summary, AMEVLP, as the concentrated essence of the active ingredients of AM, not only inherited the properties of its source Chinese medicine, but also demonstrated significant anti-inflammatory, antioxidant, and intestinal barrier repairing abilities in ex vivo and in vivo experiments. By regulating intestinal flora, metabolites, and immune responses, AMEVLP provides a new pathway and mechanistic understanding of UC treatment and opens up a broad prospect for the application of herbal vesicles in disease treatment. Initial clinical trials have limitations as early exploratory trials, and further validation of the efficacy of AMEVLP through larger samples, multicenter designs, and long-term follow-up is needed. However, to a certain extent, it shows that AMEVLP has a great clinical application in the treatment of UC. Conclusions In summary, AMEVLP, as the concentrated essence of the active ingredients of AM, not only inherited the properties of its source Chinese medicine, but also demonstrated significant anti-inflammatory, antioxidant, and intestinal barrier repairing abilities in ex vivo and in vivo experiments. By regulating intestinal flora, metabolites, and immune responses, AMEVLP provides a new pathway and mechanistic understanding of UC treatment. More importantly, the mechanism of action of AMEVLP is to exert therapeutic effects through microbial dependence. This study contributes to the development of a novel natural UC therapeutic drug with dual efficacy of regulating intestinal microecology and anti-inflammation, which has great potential for clinical translation and lays the foundation for the clinical application of herbal vesicles. Electronic supplementary material Below is the link to the electronic supplementary material. [172]Supplementary Material 1^ (10.8MB, docx) Acknowledgements