5.1 Materials and reagents

The plant raw material (XSQ) was collected (Enshi Tujia and Miao Autonomous Prefecture, Hubei, China) and identified by the laboratory of Dr. Dingrong Wan, School of Pharmaceutical Sciences, South Central Minzu University. Dextran sulfate sodium salt (DSS, MW: 36–50 kDa) and metaphosphoric acid were purchased from Aladdin Bio-Chem Technology Co., Ltd. (Shanghai, China). The Bicinchoninic Acid (BCA) protein assay kit and myeloperoxidase (MPO) assay kit were purchased from Beyotime Biotechnology (Shanghai, China). Mice tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interferon-γ (IFN-γ) ELISA kits were purchased from NeoBioscience Technology Co., Ltd. (Shenzhen, China). Universal tissue fixative was purchased from Sevier Biotechnology Co., Ltd. (Wuhan, China). RNA extraction and PCR test kits were purchased from Nanjing Vazyme Biotech Co. (Nanjing, China). E-cadherin, Occludin, and Claudin-1 protein, P38 mitogen-activated protein kinase (P38), Phosphorylation (p)- P38, Jun N-terminal kinase (JNK), p-JNK, Extracellular regulated protein kinases (ERK), p- ERK and β-actin were purchased from ABclonal Technology Co., Ltd. (Wuhan, China). The cDNA transcription kit was purchased from ABclonal Technology (Wuhan, China).

5.2 Preparation of XSQ and UPLC-Q-TOF–MS/MS analysis

The crude powder of XSQ was weighed and extracted twice, using a 6-times-volume of and 5-times-volume of pure water for 1 h each time. Then, the extract was filtered to obtain a filtrate. The filtrate was concentrated at relative density and then dried to obtain the extract powder for the sample.

The samples were analyzed using an Agilent ZORBAX RRHD Eclipse XDB-C18 column (2.1 × 100 mm, 1.8 μm) on the Waters H-Class ultra-high performance Liquid Chromatograph (Waters, USA). The mobile phase consisted of A (0.1% aqueous formic acid) and B (methanol) with the following elution gradient: 0–1 min, 5% to 18% B; 1–4 min, 18% to 24% B; 4–7 min, isocratic at 24% B; 7–12 min, 24% to 46% B; 12–15 min, 46% to 64% B; 15–19 min, 64% to 95% B. The column temperature was maintained at 30 °C, with a flow rate of 0.4 mL/min and an injection volume of 5 μL. Detection was performed at a wavelength of 280 nm.

Mass spectrometry was performed on AB Sciex Triple TOF 4600 high-resolution Mass spectrometer (SCIEX), and a heated electrospray ionization source (ESI) was used by negative ion detection mode. The sheath gas flow was set at 60 mL/min, and the auxiliary gas flow at 20 mL/min. The spray voltage was set to 3.5 kV. The capillary and auxiliary heater temperatures were both maintained at 380 °C. The scan mode was set to full M.S./dd-MS2, with full M.S. resolution at 70,000 and dd-MS2 resolution at 17,500. The scan range was m/z 100 to 1200, and the collision energy was 30 eV.

5.3 Animal experiment

All experimental procedures were reviewed, approved, and supervised by the ethical board at South-central Minzu University (License No. SYXK (E) 2021-0089). All procedures were performed by the guidelines in the Guide for the Care and Use of Laboratory Animals [45].

C57BL/6 male mice (6–8 weeks old, 22 ± 2 g) were supplied by Liaoning Provincial Laboratory Animal Resource Center (License No. SCXK (Liao) 2020-0001). All mice underwent acclimatization with adequate food and sterile drinking water for 1 week before randomization.

On the 8th day, 42 mice were randomized into 6 groups (n = 7 each): control group (Ctrl), dextran sulfate sodium (DSS) group, positive group (SASP, 0.02 g/kg), low-dose XSQ group (L-XSQ, 0.04 g/kg), medium-dose XSQ group (M-XSQ, 0.08 g/kg), and high-dose XSQ group (H-XSQ, 0.16 g/kg). Interventions for each group were orally administered, all mice received assigned interventions, and mice from different groups were kept in different cages to avoid cross-contamination.

All groups other than the Ctrl group were provided a 3% DSS solution for 7 days. In addition, all groups simultaneously received allocated interventions for 10 days.

Data on body weight, stool consistency, and stool color were collected daily to calculate the disease activity index (DAI) [46]. After the intervention period, all mice were ether-euthanatized, and their experiment-related tissues were extracted. Immediately following euthanasia, their organs were weighed, and colon length was measured. 0.5 cm of distal colon tissue samples from each mouse were fixed for histological analysis. The remaining colon and other extracted tissue samples were stored at − 80 °C for subsequent analysis.

5.4 Histological analysis

The colon tissues were fixed with 4% paraformaldehyde for at least 24 h. First, an appropriate amount of colonic tissue was taken, dehydrated, deparaffinized, and performed microtomy (4 µm). Next, the samples were then stained for H&E [47]. The pathological morphology of the colon tissues was examined and photographed with an optical microscope (Table 1).

Table 1 Anti-body used for western blot experiment

The pathology score was calculated with the following formula: Pathological score = (Tissue structural integrity + Epithelial cell loss + Inflammatory cell infiltration + Degree of edema) / 4. The specific criteria of the scoring standards are provided in Table 2.

Table 2 Pathological scoring criteria5.5 Myeloperoxidase (MPO) activity assay

First, prepare a 5% colon tissue homogenate with reagent 2. Then, 40 µL of reagent 3 was added to 360 µL of homogenate, mixed, and incubated in a 37 °C water bath for 15 min. 40 µL of Reagent 4 and 600 µL of dye were added to a new tube, followed by 40 µL of the above solution. Afterward, the reagents are mixed well and incubated in a 37 °C water bath for 30 min. After incubation, 10 µL Reagent 7 was added, mixed well, and incubated in a 60 °C water bath for 30 min. Finally, the O.D. value was measured at the excitation wavelength of 460 nm, and MPO activity was calculated with the following formula:

$$}\left( }/}} \right) \, = \, \left( }_}}} }_}}} } \right) \, /. \times }\left( } \right).$$

5.6 Cytokine assay

First, 10% colon tissue homogenates were prepared with phosphate buffer solution (PBS) and centrifuged with 12,000 g at 4 °C for 10 min; the supernatant was collected and stored in − 80 °C storage for subsequent analysis. Testing was completed according to the instructions provided by the kit vendor.

5.7 16s rRNA sequencing

The test kits and sequencing platform are purchased from Beijing Bemac Biotechnology Co., Ltd. (Beijing, China) in this experiment segment. First, total DNA was extracted with the TGuide S96 Magnetic Soil/Stool DNA Kit. Next, 50 ng of DNA sample was prepared for V3-V4 region amplification; the primers used are 338F (5′-ACTCCTACGGGAGGCAGCA-3′) and 806 R (5′-GGACTACHVGGGTWTCTAAT-3′). Samples of the PCR amplification experiment are purified with an OMEGA DNA kit. Finally, the samples were sequenced on the Illumina Novaseq 6000 platform.

The raw sequencing output (stitch, quality control, remove chimera) was cleaned to obtain the high-quality tags sequence. Then, the USEARCH tool (version 10.0, https://www.drive5.com/usearch) was used to cluster tag sequences with 97% or more agreement, and a 0.005% threshold was used to filter OTUs. Next, QIIME 2™ (National Institutes of Health, U.S.) assessed α and β diversity. Finally, the Metastats® tool (Center of Bioinformatics and Computational Biology, University of Maryland) was used to analyze species abundance [48, 49].

5.8 Detection of SCFAs in cecum contents

First, 100 mg cecum contents were suspended in 100 µL methanol, vortexed, and centrifuged with 12000 g at 4 °C for 10 min. 80 µL supernatant was collected and mixed well with 16 µL 25% metaphosphoric acid and let set overnight at 4 °C. Then, the supernatant was centrifuged with 12000 g at 4 °C for 10 min and collected for subsequent gas chromatography.

Use TRACE 1300 GC and TR-FAME GC columns to perform gas chromatography; the specification of the columns was 60 m × 0.25 mm ID × 0.25 µm. The temperature program we designed was: start at 75 °C, increase steadily by 2 °C/min until 180 °C, and maintain at this level for 2 min [50, 51].

5.9 Western blotting (W.B.) protein assay

First, the enzymatic digestion method was used to extract protein, and protein concentration was measured with a BCA test kit [52]. Next, proteins were separated using the SDS-PAGE method and transferred to the PVDF membrane [53]. Blocked with 5% nonfat dry milk diluted in TBST buffer, antibody 1 was incubated at 4 °C overnight. The next day, the membrane was washed with TBST buffer, and the appropriate amount of diluted antibody 2 was incubated for 1 h at room temperature. Afterward, The membranes were washed 3 more times with TBST buffer and then blotted by ECL [54]. Finally, the results were analyzed using the Image J® software (National Institutes of Health, U.S.).

5.10 Statistical analysis

We used ANOVA to compare the difference between trial groups on the GraphPad Prism software (version 8.0.2, La Jolla, CA, USA). The test significance threshold was set to *P = 0.05 and **P = 0.01. All data was reported as mean ± standard error of the mean (SEM).