Chemicals and reagents

Notoginsenoside R1 (NGR1, BP1010, C47H80O18, purity ≥98%, CAS No 80418-24-2, MW: 933.13 Da) was purchased from Chengdu Purifa Technology Development Co. Ltd (Chengdu, China). Dextran sulfate sodium salt (DSS, 0216011010, MW: 36 kDa–50 kDa) was purchased from MP Biomedicals (Shanghai, China). Salicylazosulfapyridine (SASP, S0883, C18H14N4O5S, CAS No 599-79-1, MW: 398.39 Da) and FITC-dextran (FD4, CAS No 60842-46-8) was purchased from Sigma-Aldrich (Darmstadt, Germany). ICG-001 (T6113, C33H32N4O4, purity ≥ 98%, CAS No 780757-88-2, MW: 548.64 Da) was acquired from TOPSCIENCE (Shanghai, China). Water-DEPC treated (693520) and DMSO (D8418) were obtained from MilliporeSigma (Burlington, MA, USA).

Cell culture

NCM460 human intestinal epithelial cells and CT26 murine colon carcinoma cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). NCM460 and CT26 cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 culture medium (11875085, Gibco, NY, USA) supplemented with 10% fetal bovine serum (10099158, Gibco, NY, USA). The culture conditions included a humidified atmosphere containing 5% CO2, with a constant temperature maintained at 37 °C.

Animals

The Laboratory Animal Center of Shanghai University of Traditional Chinese Medicine provided female C57BL/6 mice weighing 20 ± 2 g. These mice were housed in a specific pathogen-free facility under meticulously controlled conditions, including a temperature range of 23–25 °C, humidity maintained at 60%–70%, and a well-regulated 12-h light-dark cycle. The Animal Experimentation Ethics Committee of Shanghai University of Traditional Chinese Medicine granted approval (PZSHUTCM2307310004) for experimental procedures conducted on the animals. All experiments were conducted in accordance with institutional animal care guidelines and protocols approved by the committee.

Establishment of acute colitis mouse model

According to the method reported by Yue [26], we established the acute colitis mouse model. Briefly, female C57BL/6 mice were divided randomly into four groups: Control, DSS, DSS + SASP, and DSS + NGR1. Acute colitis was induced by administering 3% DSS in the drinking water of mice for a period of 8 days. Mice in the DSS + SASP group were treated orally with SASP (260 mg/kg) once per day for the same duration. The DSS + NGR1 group received NGR1 (25, 50, 125 mg/kg) by oral gavage once per day for 10 days. Mice in the Control and DSS groups were administered the same volume of Control. Daily monitoring of body weight and rectal bleeding was conducted throughout the 10-day period. At the end of the experiment, mice were euthanized, and the colon was collected for further analysis.

Female C57BL/6 mice were randomly divided into four groups: DSS, DSS + ICG-001, DSS + NGR1 and DSS + ICG-001 + NGR1. To establish an acute enteritis model, mice were subjected to the protocol described above. Mice in the DSS + NGR1 and DSS + ICG-001 + NGR1 group were given NGR1 (25 mg/kg) orally once daily for 10 consecutive days. Meanwhile, mice in the DSS + ICG-001 and DSS + ICG-001 + NGR1 groups were given ICG-001 (20 mg/kg) via intraperitoneal injection three times per week. The DSS and DSS + NGR1 groups received the same volume of Control.

Xenograft tumor transplantation model

Male BALB/c mice were acclimated for 1 week in a specific pathogen-free environment. Subsequently, CT26 cells (2 × 105 cells/mouse) were subcutaneously transplanted into the left axillary region of each mouse. Once the tumor size reached 200 mm3, the mice were randomly assigned to the vehicle group or the NGR1 group based on tumor size. Throughout the 18-day experiment, mice in the vehicle group received 0.5% CMC-Na, while those in the NGR1 group were administered 25 mg/kg NGR1. Tumor volume = 0.5 × length (mm) × width (mm)2.

Measurement of intestinal permeability

C57BL/6 mice were fasted for 4 h before execution. Mice were then orally administered 60 mg/100 g body weight of FITC-dextran in 200 µL of sterile saline. After 4 h, blood samples were collected via retro-orbital bleeding, and serum was separated by centrifugation. The serum FITC-dextran levels were measured at an excitation wavelength of 485 nm and an emission wavelength of 528 nm using a fluorometer (VARIOSKAN FLASH, Thermo Fisher, MA, USA).

H&E staining

Colonic tissues were collected from mice and fixed in 4% paraformaldehyde. Tissues were then dehydrated, embedded in paraffin, and sectioned into 4 μm thick slices. The sections were then stained with hematoxylin and eosin (H&E) using standard protocols. Stained sections were analyzed under a light microscope (BX61VS, Olympus, Tokyo, Japan), and images were captured for further analysis.

Enzyme-linked immunosorbent assay (ELISA)

The concentrations of DAO (CSB-E10090m) and LPS (CSB-E13066m) in mouse serum samples were determined using the respective ELISA kit (Wuhan Huamei Biological Engineering Co., Ltd, Wuhan, China). Specifically, serum samples were added to a 96-well plate coated with DAO or LPS-specific antibodies, followed by incubation with detection reagents and substrate solution. Absorbance was measured at 450 nm, and concentrations were calculated using standard curves.

Immunofluorescent staining

Colonic tissues were fixed in 4% paraformaldehyde, embedded in OCT compound, and sectioned into 5-μm slices. After permeabilization and blocking, sections were incubated with primary antibodies against ZO-1 (#13663, Cell Signaling Technology, CST, MA, USA) and Occludin (#91131, CST, MA, USA), followed by secondary antibodies conjugated to fluorophores (9300039001, ABclonal, Wuhan, China). Nuclei were counterstained with DAPI (#4083, CST, MA, USA), and images were obtained using a fluorescence microscope (BX61VS, Olympus, Tokyo, Japan). Quantification of ZO-1 and Occludin expression was performed using ImageJ software (NIH, Bethesda, MD, USA).

Alcian blue staining

Colonic tissue samples were obtained from mice, fixed, dehydrated, embedded in paraffin blocks, sectioned, and stained with Alcian blue using a commercial kit. Under a light microscope (BX61VS, Olympus, Tokyo, Japan), the stained sections were examined and images were captured for subsequent analysis.

Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR)

RNA was extracted using the TRIzol method, and RNA quantity and purity were measured by NanoDrop spectrophotometer (Thermo Fisher Scientific). The RNA was then reverse-transcribed using an Evo M-MLV RT Premix for qPCR kit (AG11706, Accurate Biotechnology Co., Ltd., Chengdu, China), and qPCR was performed using a SYBR® Green Premix × Pro Taq HS qPCR Kit (AG11718, Accurate Biotechnology Co., Ltd., Chengdu, China) (Table 1). The amplification was carried out using an ABI Prism 7900HT Sequence Detection System (Life Technologies, CA, USA), and data were analyzed using the 2−ΔΔCt method.

Table 1 Genes and associated primer sequences used for RT-qPCR analysis.Western blot

Colonic tissues were extracted and homogenized, and protein was obtained using RIPA lysis buffer with phosphatase and protease inhibitors. Protein concentration was measured using a BCA assay kit (20201ES76, Yeasen Biotech Co., Ltd, Shanghai, China). Equal amounts of protein were loaded onto SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) gels and separated by electrophoresis. Subsequently, the separated proteins were transferred onto PVDF membranes (000025736, Milipore, MA, USA). The membrane was then blocked with 5% BSA solution for 2 h. After blocking, the membrane was incubated with primary antibodies overnight at 4 °C, followed by incubation with HRP-conjugated secondary antibodies for 1 h at room temperature. Protein bands were visualized using ECL reagents (WBKLS0500, Millipore) and imaged with a GS-700 imaging densitometer (Bio-Rad, CA, USA). Protein expression levels were quantified using ImageJ software (NIH, Bethesda, MD, USA). The following primary antibodies were used: rabbit anti-β-Catenin (1:1000, #8480, CST, MA, USA), rabbit anti-p-GSK-3β (1:1000, #5558, CST, MA, USA), rabbit anti-GSK-3β (1:1000, #12456, CST, MA, USA), rabbit anti-Cyclin D1 (1:1000, #2922, CST, MA, USA), rabbit anti-c-Myc (1:1000, #5605, CST, MA, USA) and rabbit anti-β-actin (1:1000, #4970, CST, MA, USA).

Immunohistochemistry (IHC)

Colonic tissue sections were fixed in 4% paraformaldehyde, embedded in paraffin, and sliced into 5 μm thick sections. Antigen retrieval was performed using citrate buffer solution (pH = 6.0) and heating in a microwave oven. Non-specific binding was blocked with 5% goat serum for 30 min. Sections were incubated overnight at 4 °C with primary antibodies, followed by incubation with a secondary antibody and staining with DAB (3,3′-diaminobenzidine). Hematoxylin was used for counterstaining before the sections were examined microscopically and images were captured.

RNA-sequencing

Total RNA was extracted from mouse intestinal tissues using TRIzol reagent according to the manufacturer’s instructions. The extracted RNA was evaluated for quality using a NanoDrop spectrophotometer (Thermo Fisher). RNA sequencing libraries were then constructed with the NEBNext Ultra RNA Library Prep Kit for Illumina, and sequencing was performed on an Illumina HiSeq platform. The differential gene was carried out on the cloud platform of majorbio (https://www.majorbio.com/).

Transepithelial electrical resistance (TEER)

Caco-2 cells were seeded in Millicell inserts of 24-well plates at a density of 5 × 104 cells/400 μL per well. The outer chamber was filled with 600 μL DMEM medium (2323012, Gibco, NY, USA) and replaced every other day. TEER values were measured using a MERS00002 volt-ohm meter system (Milipore), and the electrode was sterilized with 70% ethanol and rinsed with sterile phosphate-buffered saline (PBS) before each measurement. Monolayer formation was assumed at TEER values of 400 Ω/cm2. Measurements were taken at regular intervals using the same electrode and recorded.

Organoid extraction, culture and treatment

The intestinal crypts were isolated from the small intestine of C57BL/6 mice (6- to 8-week-old). The small intestine was removed and flushed with ice-cold PBS. The intestine was opened longitudinally and cut into 2- to 3-mm pieces. The pieces were then washed with ice-cold PBS and incubated in 3 mM EDTA solution at 4 °C for 20 min with gentle shaking. After incubation, the crypts were released by vigorously shaking the tubes. The supernatant containing the crypts was collected and filtered through a 70-μm cell strainer. The crypts were then centrifuged at 1200 r/min for 5 min and resuspended in Matrigel (Corning, NA, USA). The crypt-Matrigel mixture was plated in 24-well plates and incubated at 37 °C for 30 min to allow the Matrigel to solidify. The IntestCultTM OGM Mouse Basal Medium (#06005, STEMCELL, Vancouver, Canada) was then added to the wells and changed every other day.

After cultured 2 days in a 24 well plate, the intestinal crypts were randomly divided into control, DSS model group and DSS + NGR1 group. Then, the organoids were administered DSS (20 μg/mL), DSS (20 μg/mL) plus NGR1 (100 μM) for 4 days. The organoid growth conditions were recorded by the microscope (Olympus CKX4, Tokyo, Japan). IHC assay was conducted to examine the fluorescent protein expression of Lgr5 and β-Catenin (refer to the above method of IHC).

Molecular docking

The molecular docking was performed using AutoDock Vina software. The 3D crystal structure of β-Catenin protein (PDB: 1JDH) was obtained from the Protein Data Bank (PDB) database. The structure of NGR1 was drawn and optimized using ChemDraw software and converted to a PDB file using Open Babel software. The protein and ligand files were prepared using AutoDock Tools. Docking simulations were performed and the conformation with the lowest binding energy was selected as the final docking result. The docking results were analyzed using PyMOL software.

TOPFlash

The TOPFlash assay was performed as previously described with slight modifications [27]. HEK293T cells were seeded in 24-well plates and cultured overnight. The cells were transfected with the 500 ng TOPFlash luciferase reporter plasmids (Beyotime Biotechnology, Shanghai, China) and 50 ng Renilla luciferase (Promega GmbH, Mannheim, Germany) using Lipofectamine 3000 (Thermo Fisher). After 24 h, the cells were treated with NGR1 (50 μM) and BIO (0.5 μM) for 24 h, separately. Subsequently, cells were lysed in 150 μL/well passive phenylbenzothiazole (PPBT) buffer, and the luciferase activity was measured using a Dual-LuciferaseTM Reporter Assay System (Promega Corporation, WI, USA). The firefly luciferase activity was normalized to Renilla luciferase activity.

Wound healing assay

A scratch wound was created using a plastic pipette (10 μL) tip. NCM460 cells were then washed with PBS to remove any debris and treated with either DSS (20 μg/mL) or DSS (20 μg/mL) + NGR1 (100 μM) for 24 h. The width of the scratch was measured using microscopy at 0 and 24 h post-dosing, and the percentage of wound closure was calculated by comparing the scratch width at 24 h to the initial scratch width.

Flow cytometry

NCM460 cells were treated with either DSS (20 μg/mL) or DSS (20 μg/mL) + NGR1 (100 μM) for 24 h. Then, NCM460 cells were harvested and washed with PBS after experimental treatment. Cells were then suspended in a binding buffer containing Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI), and incubated in the dark at room temperature for 15 min. Flow cytometry analysis was performed to detect apoptotic cells. The data were analyzed using Guava software, and the percentage of apoptotic cells was expressed.

Statistical analysis

Statistical analysis was performed using GraphPad Prism 9.0 software. Data were presented as mean ± standard deviation (SD). Differences between groups were analyzed using one-way analysis of variance (ANOVA). P < 0.05 was considered statistically significant. All experiments were repeated at least three times.