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 × 106/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% CO2 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 × 106/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% CO2 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 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 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 (AB234437, 1:50), IL-10 (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% H2O2 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.