The herb Amomum tsaoko donated by Dr. Yanqiong Yang. According to the quality control criteria specified in the Chinese Pharmacopoeia (2020), a clinical dose of 6 g per day for humans was utilized, alongside the human-to-animal surface area ratio, to determine the equivalent dose (He et al., 2024). As a result, the dosage of CG employed in this study was calculated to be 0.91 g/kg. Furthermore, to determine a more appropriate dosage, a high concentration—defined as twice the standard dose—was established at 1.82 g/kg. For the in vivo experiments, the CG concentration was diluted to 182/91 mg/mL with water, and a dose of 200 μL was administered per mouse. The administration frequency of CG is once a day.

The CG herb was initially steeped in distilled water at a tenfold volume for 2 hours, followed by a boiling period of 40 minutes. A subsequent decoction was prepared using an eightfold volume of distilled water, following the same procedure. The final extract was produced by combining the two decoctions, which were then filtered through gauze and distilled. After a 48-hour drying period under vacuum conditions, the CG was freeze-dried to obtain a powdered form. The extraction yield was determined to be 13.3%.

The chemical analysis, prototype, and metabolite components of CG were performed utilizing a Waters H-Class UPLC System (Waters Technology Co., Ltd, Milford, USA) in combination with Sciex Triple TOF® 4600 high-resolution mass spectrometry (AB Sciex, Darmstadt, Germany). The chromatographic separation was achieved using a Waters ACQUITY UPLC CSH C18 column (2.1 × 100 mm, 1.7 µm). The mobile phase consisted of water with acetonitrile (solvent A) and 0.1% formic acid (solvent B). The gradient elution was programmed as follows: 0–3 min, 3% A and 97% B; 5–10 min, 3%–10% A and 97%–90% B; 7–20 min, 10%–20% A and 90%–80% B; 20–30 min, 20%–50% A and 80%–50% B; 30–40 min, 50%–90% A and 50%–10% B; 40–44 min, 90% A and 10% B; 44–44.1 min, 90%–3% A and 10%–97% B; 44.1–48 min, 3% A and 97% B. The column oven temperature was maintained at 30 °C, with a flow rate set at 0.3 mL/min. Mass spectrometric detection was conducted in both negative and positive ion modes using an electrospray ionization source.

In this study, data collection was facilitated by the use of Analyst TF 1.7.1 software, while data analysis was conducted using Peakview 1.2. During the identification process, mass spectrometry data were initially compared against the Natural Products HR-MS/MS Spectral Library 1.0 database. Subsequently, the compounds underwent a preliminary evaluation based on the score data associated with each chromatographic peak. Further verification was performed by examining both the primary and secondary data corresponding to each chromatographic peak. The Natural Products HR-MS/MS Spectral Library 1.0 database includes multistage mass spectrometry data derived from our standard products as well as other standard products. This encompasses various collection modes, admixtures, collision energies, and additional parameters, resulting in a comprehensive repository of compound information. The database exclusively contains authentic maps of the collected standard products, devoid of any simulated speculation, thereby ensuring highly accurate matching results. Compounds not present in the database were identified through literature reports and adherence to established mass spectrum fragmentation rules.

High-throughput genomic data from the blood samples of patients diagnosed with clinical ulcerative colitis were systematically retrieved and analyzed using the GEO database. Following a rigorous screening process, GSE193677 and GSE186507 were identified and selected for further study. Each dataset comprised samples from both healthy volunteers and UC patients, with a minimum of three samples per group. Subsequent to data collection, gene expression data were exported, and Gene Symbol conversion was conducted utilizing the DAVID platform. Our aim was to identify differentially expressed genes and enriched pathways associated with the systemic inflammatory state of UC, thereby providing insights and directions for subsequent investigations into tissue-specific mechanisms using animal models.

For the RNA-seq datasets (GSE193677 and GSE186507), the data acquisition involved downloading the original gene count matrix from the GEO database. In the preprocessing stage, we employed the ggplot2 package in R to generate box plots for visual inspection, and utilized the normalizeBetweenArrays function from the limma package for normalization. Differential expression analysis was subsequently conducted on this standardized data. Differentially expressed genes between the subject and control groups in each dataset were identified using the limma package, applying a filtering threshold of LogFC >1 and P-value <0.05. We then applied the RobustRankAggreg (RRA) method to integrate genes that were consistently upregulated or downregulated across both datasets.

ClusterProfiler was used to study gene distribution in key modules via GO and KEGG analyses. After identifying the target module, intersections of all key genes in the module with CG (Supplementary Table S2) and necroptosis-related genes (Supplementary Table S3) were determined. Protein interaction network analysis was performed using PPI through the STRING database, leading to the identification of potential targets for treating ulcerative colitis by adding CG to inhibit necroptosis.

Necrostain-1 (Nec-1) (S8037) was acquired from Selleck. Calcein-AM/PI Cell Viability/Cytotoxicity Assay Kit was bought from Beyotime (C2015M). CellTiter-Glo Luminescent Assay was bought from Beyotime (C0065L). Human-TNF-alpha (C008) was purchased from Novoprotein. SM-164 Hydrochloride (HY-15989A) was acquired from Med Chem Express. Z-VAD-fmk (T6013) was bought from TargetMol. Anti-RIPK1 (3493S) and human-specific anti-phospho-RIPK1 (65746S) was bought from Cell Signaling Technology. The BCA protein assay kit (CW0014S) was acquired from CWBIO. The RIPK3 polyclonal antibody (17563-1-A) and Anti-MLKL (66675-1-Ig) were from Proteintech. Anti-RIPK3/p-RIPK3 (ab209384), anti-MLKL (ab184718), human-specific anti-phospho-MLKL (ab187091) and mouse-specific anti-phospho-RIPK3 (ab195117) were bought from Abcam. The mouse-specific anti-phospho-MLKL (bsm-54104R) came from Bioss (Beijing, China). The mouse-specific anti-phospho-RIPK1 (BX60008) was bought from Biolynx (Hangzhou, China). The Occludin (GB111401), p-STAT3 (GB150001) and ZO-1 (GB111402) antibodies were supplied by Servicebio (Wuhan, China). Anti-STAT3 came from Selleck (F0200). Dextran sulfate sodium (DSS) (MW: 36,000–50,000, cat#CD4421) was bought from Coolaber (Beijing, China). The Anti-GAPDH (ab181602) was purchased from Abcam. The TUNEL kit (G1502) was supplied by Servicebio. The HRP Goat anti-rabbit IgG (abs20040) was bought from Absin. The high-intensity ECL Western blotting substrate was from Tanon.

The HT-29 cell line (Procell, CL-0118) was obtained from Procell Life Science & Technology Co., Ltd. The cells were cultured at 37 °C with 5% CO₂ in growth medium (Procell, CM-0118) supplemented with 10% fetal bovine serum (BI, C04001-500) and 100 U/mL penicillin/streptomycin (Gibco, 15140-122).

HT-29 cells were seeded in 96-well plates at 10,000 cells per well, with triplicates for each group. After 12 h, freeze-dried CG powder was diluted to final concentrations ranging from 100–1.5 mg/mL with pre-treated with 10 nM SM-164 hydrochloride and 20 μM caspase inhibitor z-VAD-fmk for 30 min. Then the cells were stimulated with 20 ng/mL h-TNF-α for another 12 h (TSZ, a combination of h-TNF-α, SM-164, and Z-VAD-FMK). Cell viability was measured with the CellTiter-Glo Luminescent Assay and luminescence was recorded using a SpectraMax M5 (Molecular Devices).

HT-29 cells were collected in accordance with previously established protocols, and 100 μL of a Calcein-AM/PI working solution (dilution 1:1000) was subsequently introduced to each well after a 12-h incubation period. The plates were then incubated for an additional 30 min at 37 °C, protected from light. Cellular viability within each group was evaluated using a microscope (Nikon, Tokyo, Japan), with red fluorescence signifying non-viable cells and green fluorescence signifying viable cells.

Forty male C57BL/6J mice (6–7 weeks old, 21–23 g) were sourced from Liaoning Changsheng Biotechnology Co., Ltd. They were kept at 23 °C–25 °C, 50%–60% humidity, with a 12-h light/dark cycle, and given unlimited access to standard rodent chow and tap water. The Animal Care and Use Committee of Henan University of Chinese Medicine approved all procedures (approval no. DWLLSZR202508058; Henan, China).

Male C57BL/6J mice were allocated into five distinct experimental groups: the control group (Con), the DSS-treated group (DSS), the low-dose CG group (CG-L 0.91 g/kg), the high-dose CG group (CG-H 1.82 g/kg), and the Nec-1 group (5 mg/kg), with each group comprising eight mice. Based our approach on both our previous research and existing literature. The final dosage of Nec-1 is set at 5 mg/kg, with a solvent composition of 5% DMSO and 95% physiological saline. The method of administration is intraperitoneal injection, with a frequency of once per day. The control group was maintained on a standard diet and provided with water, whereas the remaining groups received 2.5% DSS for a duration of 7 days to induce UC. The administration period for DSS spans from day 0 to day 7, after which the administration commences. The administration of CG and Nec-1 was from the 8th day to the 13th day. On the 13th day of the study, all animals were humanely euthanized by certified personnel via the administration of an overdose of sodium pentobarbital (150 mg/kg, administered intraperitoneally). Following the induction of unconsciousness, cervical dislocation was performed as a secondary measure to ensure the complete cessation of cerebral activity. The confirmation of death for each animal was established by the absence of detectable cardiac activity and the loss of corneal reflexes. All animal procedures were conducted at the Animal Experimental Center of Henan University of Chinese Medicine. Post-mortem, colonic tissue samples were collected for further analysis.

The DAI score is derived from the aggregation of three distinct parameters: body weight loss, presence of blood in stool, and severity of diarrhea. Body weight loss is evaluated on a scale ranging from 0, indicating no loss, to 4, representing a loss exceeding 20%. The presence of blood in stool is similarly assessed, with a score of 0 denoting no blood and a score of 4 indicating gross bleeding. Diarrhea severity is categorized as follows: 0 for normal consistency, 1 for soft stools, 2 for soft and pasty stools, 3 for stools that are soft and pasty and adhere to the anus, and 4 for liquid stools. The cumulative DAI score is calculated by summing the scores from these individual parameters.

Mouse colon tissues underwent fixation, dehydration, and embedding into sections, followed by deparaffinization in water. Subsequently, the sections were stained with hematoxylin for 3–5 min at room temperature, rinsed, and blued. Thereafter, the sections were dehydrated in 95% alcohol for 1 min and counterstained with eosin for 15 s at room temperature. Finally, the sections were further dehydrated, mounted, and cover slipped in preparation for imaging and analysis.

Mouse colon tissues underwent fixation, dehydration, and embedding into sections, followed by deparaffinization in water. Subsequently, the slides were sequentially stained with AB-PAS staining solutions C, B, and A, followed by sealing with neutral gum. Micro-microscopic examination, image acquisition and analysis.

Paraffin-embedded sections of mouse colon tissue underwent deparaffinization to water, followed by a 20-minute incubation at 37 °C with a proteinase K working solution to facilitate antigen retrieval. The sections were then subjected to three washes with phosphate-buffered saline (PBS) for 5 min each at room temperature. A membrane-permeabilization solution was applied to cover the tissue, followed by a 20-minute incubation at room temperature, and another PBS wash. Subsequently, the sections were incubated in the TUNEL reaction solution for 1 h at 37 °C. The quantity of terminal deoxynucleotidyl transferase (TDT) enzyme from the TUNEL kit (catalog number G1502; Wuhan Servicebio Technology Co., Ltd.) was adjusted according to the number of slides and tissue size and mixed with dUTP and buffer in a 1:5:50 ratio. After thorough washing with PBS, the sections were stained with DAPI for 10 min at room temperature, protected from light. A final PBS wash was performed, and the sections were mounted using an anti-fluorescence quenching mounting medium. Images were subsequently captured using a fluorescence microscope.

Mouse colon tissues were subjected to fixation, dehydration, and embedding into sections, followed by deparaffinization in water. Subsequently, antigen retrieval was performed, followed by the inhibition of endogenous peroxidase activity and serum blocking. The sections were then incubated with a primary antibody, followed by a secondary antibody. DAB staining was applied, and the cell nuclei were counterstained. Finally, the sections underwent dehydration and sealing. The results were interpreted using a white light microscope.

Paraffin-embedded intestinal tissue sections were cut into slices. After removing the paraffin, the epithelial monolayer was immunostained. Slides were incubated overnight with antibodies at 4 °C, rinsed with PBS four times, and then incubated with a secondary antibody for 1 h in the dark. They were stained with DAPI (G1012, Servicebio, China) for 10 min at room temperature, rinsed with PBS, and treated with an anti-fluorescence quencher. Following the primary antibodies, Affini Pure goat anti-rat IgG (1:200) (GB21303, Servicebio, China) and goat anti-mouse IgG (1:200) (GB22301, Servicebio, China) were used as secondary antibodies. The fluorescent images of samples were captured using confocal microscopy.

HT-29 cells were cultured in 6-well plates at a density of 100,000 cells per well and subjected to treatment with varying concentrations of CG or stimulation with TSZ for different time intervals to evaluate their anti-necroptosis activity. Post-treatment, cell lysis was performed using RIPA Lysis Buffer (CWBIO, Jiangsu, China), and protein concentrations were quantified utilizing a BCA protein assay kit (CWBIO, Jiangsu, China). The protein extracts were then resolved by 10% SDS-PAGE and transferred onto PVDF membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5% BSA for 2 h and subsequently incubated overnight at 4 °C with various primary antibodies at a 1:1000 dilution. Following a 1-hour incubation with HRP-conjugated secondary antibodies, the membranes were analyzed using an imaging system (Bio-Rad, Hercules, USA), and band optical densities were quantified using ImageJ software.

To investigate the potential active role of STAT3, the known STAT3 activator Colivelin was employed. HT-29 cells were treated with 50 mg/mL of CG, both with and without the presence of Colivelin (0.4 μM), within a TSZ-induced necroptosis model. Cell viability was assessed using the CellTiter-Glo Luminescent Assay, and luminescence was recorded utilizing a SpectraMax M5 (Molecular Devices).

For longitudinal data collected at multiple time points, such as daily weight or disease activity index, repeated measures analysis of variance (ANOVA) is employed for statistical evaluation. Initially, the Mauchly’s test of sphericity is performed. If the assumption of sphericity is violated (p < 0.05), the Greenhouse-Geisser correction is applied to adjust the degrees of freedom. In the analysis model, “treatment” is considered as the between-subjects factor and “time” as the within-subjects factor, with the interaction effect of “treatment × time” being assessed. Upon finding a significant interaction effect, simple effect comparisons are conducted between groups at each time point, with multiple comparisons corrected using the Bonferroni method. For data measured at a single time point, such as colon length or histological score, a one-way ANOVA followed by Tukey’s post-hoc test is utilized. A p-value of less than 0.05 was considered to indicate statistical significance.