Extraction of HPW

The crude extract of polysaccharides from Wuguchong was prepared by water extraction and alcohol precipitation according to our previous method [13]. Briefly, dried insect powder was boiled three times in 95% alcohol in a Soxhlet reflux machine to remove excess lipids. A solid–liquid ratio of 20 mg/L was kept slightly boiling at 110 °C, aqueous extraction for 6 h. The concentrated solution was precipitated with 3 times ethanol for 2–3 days, the supernatant was poured, frozen and dried into powder form, and the crude polysaccharide extract was obtained.

After protein removal by the Sevage method, the crude extract was separated and purified by the DEAE-cellulose column chromatography method to obtain homogenous polysaccharides with similar molecular weights and the same polarity. DEAE Sepharose Fast Flow packing was eluted with distilled water to a neutral pH condition, the flow rate was adjusted to 5 mL/min, and this procedure was maintained for 2 h. The crude polysaccharides were dissolved in distilled water, followed by stepwise elution with distilled water and 0.2, 0.5 and 2.0 M NaCl solutions at a flow rate of 15 mL/min. The phenol–sulfuric acid method was used for tracking and detection. A microplate tester was used for detection at 490 nm, and a scatter diagram was drawn (Fig. 1A).

Fig. 1

Purification and identification of HPW. A Elution curves of crude polysaccharides and the position of HPW. The abscissa is the number of elution tubes, and the ordinate is the absorbance value of the tracking sugar levels at 490 nm by the phenol–sulfuric acid method. B Ion chromatogram of the monosaccharide composition of HPW samples. The abscissa is the retention time (Time, min), and the ordinate is the response value of the ion detection (Response, nC). C Aggregated table of properties and monosaccharide composition of HPW

According to the peak shape, each component was collected, concentrated, dialysed in a 3.5 kDa molecular weight cut-off membrane, and freeze-dried. The fractions that eluted with 0.2 M NaCl were further purified on a Sephacryl S-200 column (1.6 × 80 cm) and were eluted with potassium phosphate buffer (PBS, 0.1 M, pH 7.2) at a flow rate of 0.5 mL/min [15].

Finally, a component with a relatively high concentration was obtained, which was named HPW. The extraction rate was 1.2%, and the purity was 87% by the phenol–sulfuric acid method.

Characterization and monosaccharide composition of HPW

The molecular weight and purity of HPW were assayed with high-performance gel permeation chromatography (HPGPC) (Shimadzu, Japan) using dextrans (MW: 5 kDa, 12 kDa, 25 kDa, 50 kDa, 80 kDa, 150 kDa, 270 kDa, Sigma–Aldrich, USA) as standard samples in a TSK-gel GMPWXL (7.8 mm × 300 mm, 5 μm) [16]. The data showed that the average molecular weight of HPW was approximately 616 kDa, and the purity was 87% (Fig. 1C).

High-performance anion-exchange chromatography (HPAEC) was used to identify the monosaccharide composition of HPW. The chromatographic system used a Thermo ICS5000 ion chromatography system (Thermo Fisher Scientific, USA), and an electrochemical detector was used to analyse the monosaccharide components with the following parameters: flow rate, 0.5 mL/min; injection volume, 5 μL; solvent system, 0.1 M NaOH: (0.1 M NaOH, 0.2 M NaAc); gradient program, 95:5 V/V at 0 min, 80:20 V/V at 30 min, 60:40 V/V at 30.1 min, 60:40 V/V at 45 min, 95:5 V/V at 45.1 min, 95:5 V/V at 60 min.

The data showed that HPW was mainly composed of galactose (21.55%), glucose (20.77%), rhamnose (7.05%), mannose (7%), arabinose (5.02%) and xylose (3.21%). An ion chromatogram of the samples is shown in Fig. 1B, and the monosaccharide composition is detailed in Fig. 1C.

Animals

SPF-grade C57BL/6 male mice weighing 18–22 g and aged 6–8 weeks were purchased from the Experimental Animal Centre of Dalian Medical University, China. All mice were adaptively fed for 7 days for subsequent experiments. The ambient temperature was 20–25 °C, and the relative humidity was 40–60%. Mice were maintained on a 12-h light–dark cycle and were randomly given chow and drinking water. The procedures for animal experiments were performed strictly in accordance with the standard guidelines for laboratory animals and approved by the Ethics Committee of Dalian Medical University (Ethical Approval Number: AEE19074).

Experimental procedure

Forty C57BL/6 male mice were randomly allocated into 5 groups with 8 mice in each group. The groupings were as follows:

Group 1: Water + saline.

Group 2: HPW (100 mg/kg BW) + saline.

Group 3: Water + 5-FU (50 mg/kg BW).

Group 4: HPW (100 mg/kg BW) + 5-FU (50 mg/kg BW).

Group 5: Mesalazine (10 mg/kg BW) + 5-FU (50 mg/kg BW).

To obtain a stable experimental animal model of IM, we used a previously reported protocol [17]: normal saline and 5-FU (50 mg/kg) were injected intraperitoneally during the first three days. Water, HPW or mesalazine was administered orally 1 h before 5-FU administration for one week.

Physical manifestations and tissue collection

The body weight and diarrhoea score of each mouse and the food intake of each group were recorded every day during the experiment. Diarrhoea severity was scored daily by an uninformed researcher based on criteria in previous studies [18]. The scoring criteria for diarrhoea severity were as follows: 0: normal stool; 1: slight (wettish and soft stool); 2: moderate (unformed stool, wet crissum and stained coat); and 3: severe (watery stool).

At the end of the experiment (7 days after treatment), fresh faeces were collected and quickly placed into liquid nitrogen for preservation and used for SCFA analysis. Mice were deprived of water and food for 12 h. Blood was collected after pentobarbital anaesthesia for enzyme-linked immunosorbent assay (ELISA) analysis. The entire small intestine was rapidly dissected. A 2-cm intestinal segment of the jejunum was taken 15 cm behind the pylorus and fixed with 4% paraformaldehyde for haematoxylin–eosin (HE) staining and periodic acid-Schiff (PAS) staining. The rest of the small intestine was used for other molecular biological experiments.

Histological analysis

The 2-cm jejunum segment was divided into two portions and placed in 4% paraformaldehyde fixative and Karnovsky fixative. After paraformaldehyde fixation for 12–24 h, the tissues were subjected to procedures such as dehydration and paraffin embedding. Slices with a thickness of 5 μm were cut and rehydrated with graded ethanol after being dewaxed with xylene. HE staining was then performed, the dye was washed off with water, and the slices were dehydrated and sealed. Villus length and crypt depth, which are specific indicators of intestinal barrier function and absorption, were measured under a microscope (Olympus BX-40, Japan) using Image-Pro Plus 6.0 software.

The other part of the jejunal tissue was fixed in Karnovsky buffer and then similarly processed according to dehydration, embedding and sectioning procedures. The sections were stained with Schiff's reagent for 20 min, followed by haematoxylin for 20 min. The staining solution was washed away, and the slices were subsequently dehydrated and sealed. The number of goblet cells on each villus was counted by Image-Pro Plus 6.0 software. Goblet cells appear purplish red under a microscope and are an important indicator of the small intestinal mucosal barrier. Reagents were provided by Wuhan Servicebio Biotechnology Co., Ltd.

Reagents and antibodies

A Diamine oxidase activity (DAO) detection kit was purchased from Beijing Solarbio Technology Co., Ltd. A total antioxidant capacity (T-AOC) test kit was obtained from Nanjing Jiancheng Biological Engineering Research Institute Co., Ltd. ELISA kits (Shanghai Langton Biotechnology Co., Ltd.)were used to measure D-lactate, a mechanical barrier marker, and SIgA, a mucosal barrier marker. Claudin-1, proliferating cell nuclear antigen (PCNA), TNF-α and GAPDH antibodies were provided by Wuhan Proteintech Biotechnology Co., Ltd. Occludin, NF-κB (P65) and COX-2 antibodies were purchased from Abcam (Cambridge Science Park in Cambridge, UK). Mouse IL-10 and IL-1β ELISA kits was purchased from Beijing Solarbio Technology Co., Ltd.

Western blot analysis

The expression of Claudin-1, Occludin, PCNA, TNF-α, NF-κB (P65) and COX-2 in jejunum and ileum tissue samples was measured by Western blotting. Tissue samples were lysed with RIPA buffer containing protease and phosphatase inhibitors to obtain total proteins, followed by BCA protein quantification. Related reagents were purchased from Sevenbio (Beijing) Co., Ltd. The lysed supernatant (approximately 80 μg of protein) was transferred to a PVDF membrane (Millipore, USA) by 10% SDS–PAGE. After being blocked for two hours at room temperature in 5% nonfat milk, the membranes were incubated with the following primary antibodies overnight at 4 °C: Claudin-1 (1:1000), Occludin (1:2000), PCNA (1:5000), TNF-α (1:1000), NF-κB (p65) (1:1000), COX-2 (1:1000), and GAPDH (1:2000). RP-conjugated rabbit anti-mouse IgG and goat anti-rabbit IgG secondary antibodies were added and incubated at room temperature for 1 h, and the protein bands were visualized with an ECL detection system (Syngene, UK). Image-Pro Plus 6.0 software was used to analyse the grey values.

Measurement of SCFAs

The SCFA levels in mouse faecal samples were measured according to a previously reported method [19]. An appropriate amount of the sample was added to 0.3 mL of water, 100 μL of 50% sulfuric acid, 25 μL of 500 mg/L internal standard (cyclohexanone) solution and 0.5 mL of ether, after which the mixed solution were homogenated for 1 min and centrifuged at 12,000 rpm at 4 °C for 10 min. The supernatant was placed on the instrument for testing (Gas Chromatograph-Mass Spectrometer, Shimadzu GCMS QP2010-Ultra, Japan). The chromatographic system was as follows: Agilent DB-WAX capillary column (30 m × 0.25 mm × 0.25 μm). The carrier gas was high purity helium (≥ 99.9%), and the flow rate was 1.0 mL/min. The inlet temperature was 220 °C, the injection volume was 1 μL, and the solvent delay time was 2.5 min for splitless injection. For the mass spectrometry system, an electron bombardment ion source (EI) was used, the ion source temperature was 230 °C, and the interface temperature was 220 °C. The chromatograph was connected to a microcomputer with a detector for collecting the results of the chromatographic analysis with the GC Solution program (Shimadzu, Japan).

Statistical analysis

The data are presented as the means ± SEM and were analysed using one-way ANOVA with GraphPad Prism 8.0 followed by Dunnett’s test. We considered the data significant when p < 0.05.