5.1 Materials

Dextran sulfate sodium (DSS; MW: 40,000) and metaphosphoric acid were purchased from Aladdin Technology (Shanghai, China), providing a diverse range of materials for our study. The myeloperoxidase (MPO) detection kit was purchased from Jiancheng Biotechnology Co., Ltd. (Nanjing, China). Mouse ELISA kits of interleukin (IL)-10, tumor necrosis factor (TNF)-α, IL-6 and IL-1β were obtained from Neobioscience (Shenzhen, China). Neutral-buffered paraformaldehyde was purchased from Wuhan Service Bio (Wuhan, China). An enhanced bicinchoninic acid (BCA) protein assay kit, NP-40 lysis buffer, protease and phosphatase inhibitor cocktails for mammalian cells and tissue extracts, and assay kits for superoxide dismutase (SOD), water-soluble tetrazolium-8, glutathione (GSH), and malondialdehyde (MDA) were obtained from Beyotime (Shanghai, China). Occludin-1, claudin-1, Zonula Occludens (ZO-1), signal transducer and activator of transcription 3 (STAT3), p-STAT3, nuclear factor erythroid 2-related Factor 2 (Nrf2), heme oxygenase (HO-1), Janus kinase 2 (JAK2), p-JAK2, GPR41/43 and β-actin were purchased from ABclonal Technology Co., Ltd. (Wuhan, China). A microplate reader (Spark 20 M Sunrise) was purchased from Tecan Trading (Shanghai, China). The electrophoresis apparatus, mini-transfer tank, and electrophoresis power supply were procured from Liuyi Biotechnology (Beijing, China).

Huangqin (Radix Scutellariae, the dried root of Scutellaria baicalensis Georgi) voucher specimens (No. SC-2019028), Shiliupi (Garden Burnet Root, dried root of Sanguisorba officinalis L.) voucher specimens (No. SC-2019016) and diyu (Pomegranate Rind, the dried rind of Punica granatum L.) voucher specimens (No. SC-2019020) were deposited in the Herbarium of Medical Plants, College of Pharmacy, South-Central Minzu University. XYKJP (pharmaceutical batch number: Z20180126) was provided by Renmin Hospital of Wuhan University. We used Sulfasalazine (SASP, Shanghai Sine Tianping Pharmaceutical Co., Ltd. Shanghai, China) enteric-coated tablets as positive controls, a choice that underscores the robustness of our study's methodology.

For the animal experiment, the XYKJP sample solution was prepared as follows: 0.3, 0.6, and 1.2 g/kg of XYKJP powder were dissolved in 20 mL of saline.

5.2 Preparation of XYKJP

XYKJP is composed of Huangqin, Shiliupi, and Diyu. Huangqin, Shiliupi, and Diyu were weighed in a ratio of 3:2:2 and were extracted twice with six volumes of 60% ethanol for 1 h. The filtrate was concentrated, and the extract powder was obtained by spray drying. The dry extract powder was evenly mixed with crosslinked povidone, sodium carboxymethyl starch, and microcrystalline cellulose, and then a suitable amount of 60% ethanol solution was applied to make particles and dried at 50 °C, then pressed into tablets with a final drug dose of 30 mg/100 mg.

XYKJP is composed of Huangqin, Shiliupi, and Diyu. These ingredients were weighed in a ratio of 3:2:2 and were subjected to a thorough extraction process. They were extracted twice with six volumes of 60% ethanol for 1 h, ensuring the maximum extraction of beneficial compounds. The filtrate was concentrated, and the extract powder was obtained by spray drying. The dry extract powder was then mixed with crosslinked povidone, sodium carboxymethyl starch, and microcrystalline cellulose, and a suitable amount of 60% ethanol solution was applied to make particles and dried at 50 °C, then pressed into tablets with a final drug dose of 30 mg/100 mg. This thorough extraction process guarantees the efficacy of the product.

5.3 LC–MS analysis of XYKJP components

The UHPLC system (Ultimate 3000 UPLC, Dionex) connected to a Q Exactive /MS (Thermo Scientific, Bremen, Germany) was selected to analyze the compound composition of XYKJP as the LC–MS platform. The main active ingredients were separated by using an Accucore™ aQ C18column (100 × 2.1 mm, 2.6 μm) with a constant flow of 0.2 mL/min. The column temperature was 25 °C. The injection volume was 5 μL. The mobile phases were solvent (A): Acetonitrile, B: 0.1% aqueous solution of formic acid. The gradient elution process was meticulously designed and executed: 95–90% (0–3 min), 90–83% A (3–9 min), 83–80% A (9–11 min), 80–72% A (11-14 min), 72–70% A (14–16 min), 70–65% A (16–19 min), 65–63% A (19–21 min), 63–45% (21–29 min), 45–37% (29–33 min), 37–5% (33–43 min). The samples were analyzed by a Q-Exactive mass spectrometer in an ESI ± ms ion source. The capillary temperature was 300 °C, the sheath gas was 30 Arb, the auxiliary gas was 10 Arb, the max spray current was 100.00 µA, and the probe heater temp was 320 °C.

5.4 Animal experiments

All ethical procedures complied with the requirements of the Ethics Committee of South-Central Minzu University and followed the Guide for the Care and Use of Experimental Animals (Licence No. SYXK (Hubei) 2016–0089). C57BL/6 mice (SPF, 6–8 w), a commonly used model for this type of research due to their genetic stability and susceptibility to certain diseases, were supplied by the Liaoning Provincial Laboratory Animal Resource Center (Licence No: SCXK (Liaoning) 2020-01) and fed in an SPF animal laboratory.

The 60 mice were meticulously and randomly divided into 6 groups (n = 10), each serving a specific purpose: Ctrl group, DSS group (DSS), DSS + 0.3 g/kg XYKJP intervention group (L-XYKJP), DSS + 0.6 g/kg XYKJP intervention group (M-XYKJP), DSS + 1.2 g/kg XYKJP intervention group (H-XYKJP), and DSS + 0.2 g/kg SASP group (SASP). In the JAK2-STAT3 pathway inhibitor experiments, 50 mice were similarly and randomly divided into five groups (n = 10): Ctrl, DSS, DSS + H-XYKJP, DSS + AG490 (5 mg/kg, intraperitoneal), DSS + H-XYKJP + AG490 (5 mg/kg).

All mice were carefully acclimatized and fed for 7 days before the experiments, ensuring their comfort and well-being. On day 8, all mice, except the control group, were fed 3.0% DSS in drinking water for 1 week. XYKJP and SASP dissolved in saline were orally administered to mice in the corresponding groups for 10 consecutive days. The control and DSS groups were treated with saline (0.1 mL/10 g).

5.5 Disease activity index (DAI) score and spleen index

After modeling and intervention in each group, the mice were observed and the DAI (including stool bleeding, stool consistency, and weight loss) was evaluated (Table 1). All mice were euthanized on day 18 with the utmost care and respect for ethical considerations, and the entire colon was extracted and measured for length. The spleen was collected and weighed to calculate the spleen index. All other samples were stored at −80 °C for subsequent evaluation.

Table 1 Evaluation of DAI scores5.6 Histopathological assay

The isolated colon tissues were fixed with 4% paraformaldehyde and then placed in paraffin and cut into 4-μm-thick slices, then stained with hematoxylin and eosin (H&E). Colonic histopathologic changes were scored according to previous studies [35].

5.7 Reactive oxygen species (ROS) measurement

ROS levels were detected by immunofluorescence analysis in a thorough and systematic manner. The process began with staining colon tissue sections using a ROS kit, followed by the incubation of the probes (dichlorodihydrofluorescein diacetate, DCFH-DA, 10 μM) in the dark at 37 °C for 30 min. This was then followed by three washes with high-dextrose dulbecco's modified eagle's medium (DMEM). The results, a product of our meticulous protocol, were observed using a fluorescence microscope (ECLIPSE Ti2, Nikon, Japan), and the fluorescence intensity (MFI) of ROS was assessed using ImageJ [36].

5.8 Measurement of myeloperoxidase (MPO) activity

An MPO kit was used to determine MPO activity in the colon tissue. Briefly, appropriate colonic tissue was weighed and added to the prepared reagent at a ratio of 1:19. A 5% tissue homogenate was harvested, and the MPO activity was quantified. The OD density was measured at 460 nm. MPO activity was calculated using the following equation:

MPO (U/g colon tissues) = (OD value of the sample–OD value of the control)/11.3 × sample volume (g).

5.9 Quantification of superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione (GSH) levels

Colon tissue was homogenized on ice with 5 mL of 5% trichloroacetic acid (TCA) per gram. The homogenate was then centrifuged at 4 °C for 15 min at 1000 g, and the supernatant was collected for a comprehensive biochemical analysis.The levels of SOD, MDA and GSH were meticulously determined according to the manufacturer's instructions, leaving no stone unturned in the pursuit of accurate results.

5.10 Cytokine measurements in colon tissue

To explore the effects of XYKJP's anti-inflammatory properties, the contents of tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), interferon-γ (IFN-γ), interleukin 10 (IL-10), and interleukin 1 beta (IL-1β) were measured by enzyme-linked immunosorbent assay (ELISA) kits respectively.

5.11 Quantitative real-time PCR (qRT-PCR) analysis

The gene expression of inflammatory factors IFN-γ, IL-6, interleukin 22 (IL-22), IL-1β, and TNF-α was analyzed using qRT-PCR. Total RNA was extracted using the TRIzol reagent, followed by cDNA amplification. The primers used for the target genes are listed in Table 2. Finally, qRT-PCR (Applied Biosystems, Singapore) was performed, with the highly reliable β-actin serving as the reference gene, based on the 2(-ΔΔCt) method [37].

Table 2 List of primer sequences for RT-qPCR5.12 Western blotting analysis

Proteins were enzymatically extracted using NP-40 lysis buffer containing a cocktail of protease and phosphatase inhibitors (50:1). The proteins were separated by 10% SDS-PAGE, a thorough process, and then transferred into a polyvinylidene difluoride (PVDF) membrane for 1.5 h. The membranes were then blocked with 5% skim milk and incubated overnight at 4 °C with primary antibodies (Table 3). Subsequently, the PVDF membranes were incubated with a secondary antibody for 1 h. The target proteins were identified using ECL reagents and analyzed using the ImageJ software (NIH Image, USA).

Table 3 Anti-body used for western blot experiment5.13 16S rRNA sequencing

To assess the regulatory effects of XYKJP on the intestinal flora, we performed 16S rRNA gene sequencing. The bacterial DNA in the colon contents was extracted using a DNA Kit (MN NucleoSpin 96 Soi), which was used to amplify the V4 region.16S rRNA gene V4 sequencing was conducted using an Illumina NovaSeq 6000 platform (Beijing Baimaike Biotechnology Co., Ltd, Beijing, China). We then provided detailed information on library construction, bacterial abundance analysis, and most importantly, the functional prediction of fecal samples, which holds the key to our significant findings, in the Supplementary Methods.

5.14 Determination of short-chain fatty acids (SCFAs)

To detect the levels of SCFAs, we homogenized 50 mg of fecal samples with 50 ml of 1% (V/V) aqueous methanol solution (containing 25.0% HPO3), shaken on a vortex shaker for 10 min, then centrifuged at 1.2 × 104 g at 4 °C for 10 min, then collected supernatant for subsequent analysis. SCFA content was meticulously analyzed using gas chromatography (GC). These conditions are described in the Supplementary Methods.

5.15 Statistical analysis

All the data were expressed as the mean ± SEM. The results were evaluated using Student's t-test and one-way analysis of variance (ANOVA). ImageJ software was used to analyze the histological and fluorescent images. Bar graphs were generated using the Prism 8.0.2 software (GraphPad, La Jolla, CA, USA).