Drugs and reagents

All chemical reagents and biological materials were obtained from commercial sources as follows: Dextran sulfate sodium (DSS; molecular weight: 36,000–50,000) was purchased from MP Biomedicals (Aurora, Ohio, USA); Triton 100X, sucrose, and skimmed milk powder were acquired from Biofroxx GmbH; Cell culture reagents including DMEM medium and goat serum were obtained from Shanghai Darthel Biotechnology Co., Ltd. and Beijing Zhongsui Jinqiao Bio-technology Co., Ltd., respectively. Primary antibodies were sourced from multiple suppliers: anti-HKII from Cell Signaling Technology; anti-TNF-α and anti-HIF-1α from Wuhan Three Eagles Biotechnology Co., Ltd.; anti-iNOS, anti-GLUT1, anti-IL-1β, and corresponding secondary antibodies from Sino Biological Inc. Chemical compounds including 5-Amino Salicylic Acid (5-ASA), Baicalein, Palmatine, and Triptonide were purchased from Shanghai Macklin Biochemical Technology Co., Ltd. (White Shark Biotechnology). Experimental kits and solutions, including the crypt blood test kit (Reagan Bio), phosphate buffer solution (PBS), RIPA lysis buffer, sodium citrate buffer, bovine serum albumin, H&E staining kit, DAB staining kit, and EDTA antigen retrieval solution, were obtained from Beijing Solepol Technology Co., Ltd. and Proteintech Group, Inc. Genetic manipulation reagents,including HIF-α overexpression lentiviruses, HIF-α siRNAs, and plasmid DNA, were purchased from Shanghai Genechem Co., Ltd. Chemical solvents (xylene and 75% anhydrous ethanol) were acquired from Tianjin Zhiyuan Chemical Reagent Co., Ltd. Sodium pentobarbital (1%) was provided by the Animal Experiment Center of Yunnan University of Chinese Medicine. The KMSHTF granule preparation, composed of eight medicinal components, was obtained from Kunming Hospital of Traditional Chinese Medicine. The formulation included: Leigongteng (Tripterygium hypoglaucum Hutch), Guanhuangbai (Phellodendron amurense Rupr), huanglian (Coptis chinensis Franch), Qinpi (Fraxinus rhynchophylla Hance), Cangzhu (Atractylodes lancea DC), Baihuasheshecao (Hedyotis diffusa Spreng), Baitouweng (Pulsatilla chinensis Regel), and Wubeizi (Rhus chinensis Mill).

Network pharmacology analysisIdentification and screening of bioactive components and molecular targets in KMSHTF

The active ingredients of each herbal component in KMSHTF were systematically screened using the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP; https://tcmspw.com/tcmsp.php) (Ru et al. 2014). The screening criteria were established based on pharmacokinetic properties and drug-like characteristics, including: (1) oral bioavailability (OB) ≥ 30% (Zhang et al. 2020); (2) drug-likeness (DL) index ≥ 0.18 (Jia et al. 2020). These parameters were selected to ensure the identification of biologically relevant and pharmacologically active compounds within the KMSHTF formulation.

Construction of compound-target interaction network

The bioactive components of KMSHTF, initially retrieved from the TCMSP database, were subjected to target prediction using the SwissTargetPrediction platform (http://www.swisstargetprediction.ch) to identify their corresponding molecular targets, which were considered as potential therapeutic targets of KMSHTF. Concurrently, UC-related genes were systematically retrieved from the GeneCards database (https://www.genecards.org) using "ulcerative colitis" as keyword, establishing the potential disease targets. The intersection between KMSHTF potential targets and UC-related targets was determined to identify common therapeutic targets. These overlapping targets were subsequently visualized using VENNY 2.1 (https://bioinfogp.cnb.csic.es/tools/venny). Finally, the network relationships between the bioactive components of KMSHTF and their corresponding common target genes were analyzed and visualized through the construction of a comprehensive compound-target-disease interaction network using Cytoscape software (version 3.8.2).

Construction and analysis of Protein–Protein Interaction (PPI) network

The protein–protein interaction (PPI) network was constructed using the STRING database (http://string-db.org, version 12.0) with the following parameters: (1) species restriction to Homo sapiens; (2) minimum interaction confidence score threshold set at > 0.9 (Szklarczyk et al. 2023). To enhance network interpretability and visualization, the raw PPI data were imported into Cytoscape software (version 3.8.2) for further analysis and graphical representation. Network topology analysis was performed to identify key hub genes based onmultiple centrality parameters, including Degree, Eigenvector, Local Average Connectivity (LAC), Betweenness, Closeness, and Network. Genes exhibiting values exceeding the median across all topological parameters were identified as potential key regulators. These candidate genes were subsequently ranked according to their Degree values, and the results were visualized using histogram representations to illustrate their relative importance in the network architecture.

Functional annotation and pathway enrichment analysis

Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to elucidate the biological functions and signaling pathways associated with the potential therapeutic targets of KMSHTF in UC treatment. The analyses were conducted using the Database for Annotation, Visualization and Integrated Discovery (DAVID, version 6.8) (Sherman et al., 2022) with the following parameters: (1)species restriction to Homo sapiens; (2) significance threshold set at p < 0.05 (Gan et al., 2021). The enrichment results were subsequently visualized and analyzed using the bioinformatics online platform (http://www.bioinformatics.com.cn) to facilitate data interpretation and presentation.

Molecular docking analysis

HIF-1α and HKII were selected as key targets within the HIF-1α signaling pathway for dual-ligand molecular docking studies. The three-dimensional crystal structures of HIF-1α and HKII in PDB format were retrieved from the RCSB Protein Data Bank (https://www.rcsb.org/) to serve as receptor proteins. Thetwo-dimensional molecular structures of KMSHT's bioactive components were obtained from PubChem database (https://pubchem.ncbi.nlm.nih.gov/) and subsequently converted to mol2 format using Open Babel software (version 3.1.1). The molecular docking simulations were performed using PyMOL (version 2.4) forstructural preparation and AutoDock Vina for docking calculations. The binding affinity, expressed as binding energy (typically ≤ −5 kcal/mol), was used to evaluate the interaction potential between target proteinsand KMSHT's bioactive components. The docking conformation exhibiting the most favorable binding energy was selected as the optimal binding mode and visualized using PyMOL 2.4 for structural analysis and interpretation.

AnimalsEstablishment of DSS-Induced Colitis Rat Model

Fifty specific pathogen-free (SPF) Sprague–Dawley (SD) rats (weight range: 160–180 g) were obtained from Beijing Belgi Company. The animals were maintained in the Animal Experimentation Center of Yunnan University of Chinese Medicine under controlled environmental conditions: temperature 22–25 °C, relative humidity 40–70% and a 12-h light–dark cycle. All rats were provided with standard laboratory diet and allowed one week of acclimatization prior to experimentation. The experimental protocol was approved by the Institutional Animal Care and Use Committee of Yunnan University of Chinese Medicine (Approval No.: [2022]XWB(145)).Following the acclimatization period, rats were randomly allocated into five experimental groups (n = 10 per group): (1) control group (normal saline), (2) model group (DSS-induced colitis), (3) KMSHTF treatment group (1.26 g/kg/day), (4) 5-Amino Salicylic Acid (5-ASA) treatment group (0.1 g/kg/day), and (5) combination treatment group (KMSHTF 1.26 g/kg/day + 5-ASA 0.1 g/kg/day). The dosage of KMSHTF conversion between human and rat equivalents was calculated based on body surface area ratio, following the standard conversion formula (rat dose [mg/kg] = human dose [mg/kg] × 6.3). Colitis was induced by administering 4% dextran sulfate sodium (DSS) in drinking water ad libitum for 14 days to all experimental groups except the control group, which received regular drinking water. Treatment groups received daily oral gavage of their respective therapeutic agents for 14 consecutive days, while control and model groups received equivalent volumes of distilled water. The administration volume and duration were standardized across all groups to ensure experimental consistency.

Assessment of Disease Activity Index (DAI)

Throughout the experimental period, daily monitoring of parameters was conducted, including body weight measurement, stool consistency evaluation, and fecal occult blood detection. The Disease Activity Index (DAI) was calculated according to the established scoring system (Yu et al., 2023) based on three parameters: (1) Weight loss assessment: (0: No weight loss; 1: 1–5% weight reduction; 2: 5–10% weight reduction; 3:10–20% weight reduction; 4: > 20% weight reduction). (2) Stool consistency evaluation: (0: Normal stool; 2: Loose stool; 3: Watery diarrhea). (3) Fecal blood detection: (0: No blood in stool; 2: Minor bleeding; 3, major bleeding). The DAI score was calculated by summing the individual scores from these three parameters and dividing by 3. The severity of ulcerative colitis was quantitatively assessed based on the final DAI score, with higher scores indicating more severe disease progression.

Histopathological Analysis by Hematoxylin and Eosin (H&E) Staining

Following anesthesia induction with sodium pentobarbital, rats were euthanized via abdominal aorta exsanguination. The entire colon was carefully dissected, and its length was measured and documented photographically for morphological assessment. Colon tissue samples were immediately fixed in 4% paraformaldehyde solution for 24 h at 4 °C to preserve tissue architecture. The fixed tissues underwent standardhistological processing, including paraffin embedding, sectioning at 4–5 μm thickness, deparaffinization withxylene, and rehydration through a graded ethanol series. Tissue sections were then stained with hematoxylinand eosin (H&E) according to established protocols. Histopathological evaluation was performed using light microscopy (Nikon Eclipse E100, Japan) to assess tissue morphology, inflammatory cell infiltration,andepithelial damage. Representative images were captured at 100 × and 400 × magnifications for comparative analysis.

Western Blot (WB) Analysis

Total proteins were extracted from colon tissues or co-cultured cells using RIPA lysis buffer supplemented with protease inhibitors. The tissues were homogenized or cells were lysed in the buffer and incubated for 30 min at 4 °C. Following incubation, the lysates were centrifuged at 12,000 × g for 10 min at 4 °C to remove insoluble debris. The supernatant was collected, and protein concentration was determined using a BCA assay kit. Subsequently, equal amounts of protein were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a Bio-Rad system. The separated proteins were then transferred onto a polyvinylidene difluoride (PVDF) membrane. To block non-specific binding sites, the membrane was incubated with 5% skim milk powder in Tris-buffered saline with Tween-20 (TBST)for 1 h at room temperature. The membrane was then probed with specific primary antibodies agains β-catenin, IL-6, TNF-α, IL-4, IL-1β, HIF-1α, HKII, GLUT1, and iNOS overnight at 4 °C. After washing, the membrane was incubated with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies for 2 h at room temperature. Protein bands were visualized using an enhanced chemiluminescence (ECL) detection system. The optical density of the bands was quantified using Image Pro Plus 6.0 software, and the protein expression levels were normalized to the β-actin loading control.

Immunohistochemistry (IHC) Analysis

Paraffin-embedded tissue sections were deparaffinized and rehydrated through a graded alcohol series. Endogenous peroxidase activity was quenched by incubating the sections with 3% H2O2 for 10 min at room temperature. Antigen retrieval was performed by heating the sections in 10 mM citrate buffer (pH6.0)at 95 °C for 30 min. After cooling to room temperature, non-specific binding sites were blocked by incubating the sections with 5% goat serum for 1 h. The sections were then incubated overnight at 4 °C with primary antibodies against HIF-1α, TNF-α, HKII, and IL-6. Following primary antibody incubation, the sections were washed and incubated with an appropriate horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature. After washing, the immunoreactivity was visualized using diaminobenzidine (DAB, Solarbio) as the chromogen. The nuclei were Counter stained with hematoxylin (Solarbio), and the sections were dehydrated and mounted with neutral resin. Images were captured using a light microscope, and antibody expression was quantitatively analyzed using the ImageJ® software with the Color Deconvolution plugin. The grayscale values of positively stained areas were measured to determine the degree of immunoreactivity.

Cell culture

Caco-2 cells were obtained from the Cell Bank of the Typical Culture Preservation Committee of theChinese Academy of Sciences, while THP-1 cells were acquired from the same repository. Both cell lineswere maintained in a humidified cell culture incubator at 37 °C with 5% CO2. The culture medium was supplemented with 12% heat-inactivated fetal bovine serum (F8318; Sigma, St. Louis, MO, USA) and 1%penicillin/streptomycin (V900929; Sigma) to support cell growth and prevent contamination.

THP-1 Differentiation and Co-culture

Caco-2 cells were seeded and cultured in Dulbecco's Modified Eagle Medium (DMEM) in the apicalcompartment of transwell plates. The medium in the basolateral compartment was replaced with Roswell ParkMemorial Institute (RPMI)-based THP-1 medium (51536C; Sigma) supplemented for THP-1 cell culture, excluding β-mercaptoethanol.THP-1 cells were initially maintained in 25-cm2 flasks at a density of 3 × 106cells cells and differentiated into macrophage-like cells by treatment with phorbol 12-myristate13-acetate(PMA, 200 ng/mL) for 24 h. Following differentiation, the cells were detached using a cell dissociationreagent (T4049; Sigma) and seeded at a density of 1.8 × 105 cells/well in the basolateral compartment of6-well transwell plates. The cells were allowed to reattach for 1.5 h under standard culture conditions. To establish the co-culture system, transwell inserts containing confluent Caco-2 monolayers were transferred to the wells containing differentiated THP-1 cells. The co-culture was maintained for 24 h withoutfurther intervention. The experimental groups for co-culture Were designed as follows: Control group Co-culture of Caco-2 and THP-1 cells without treatment; Model group: Co-culture of Caco-2 and THP-1 cells treated with lipopolysaccharide (LPS); treatment groups: Co-culture of Caco-2 and THP-1 cells treated with LPS and KMSHTF core effective compounds at low (40 µM), medium (80 µM), and high (100 µM) concentrations.

HIF-1α Knockdown in THP-1 Cells

After determining the optimal siRNA concentration targeting HIF-1α, THP-1 cells were treated with 200 ng/mL phorbol 12-myristate 13-acetate (PMA) to induce differentiation into M0 macrophages. The differentiated macrophages were then stimulated with 2 µg/mL lipopolysaccharide (LPS) to promote polarization into the M1 phenotype. Following polarization, the M1 macrophages were transfected with siRNA using a prepared siRNA storage solution and cultured for 48 h to achieve HIF-1α knockdown. To establish the co-culture system, transwell inserts containing confluent Caco-2 cell monolayers were transferred to the wells containing siRNA-transfected THP-1 cells. The co-culture was maintained for 24 h without further intervention. The experimental groups for the knockdown study were designed as follows: negativecontrol(THP-1 cells transfected with scrambled siRNA);HIF-1α knockdown group(THP-1 cells transfected with siRNA targeting HIF-1α); Negative control + drug intervention group (THP-1 cells transfected with scrambled siRNA + treated with the optimal concentration of the effective compounds);HIF-1α knockdown + drug intervention group (THP-1 cells transfected with siRNA targeting HIF-1α + treated with the optimal concentration of the effective compounds).

Lentivirus Transfection in THP-1 Cells

The HIF-1α overexpression lentiviral particles were produced using 293 T cells, which were obtained from the Cell Bank of the Typical Culture Preservation Committee of the Chinese Academy of Sciences.THP-1cells were treated with 200 ng/mL phorbol 12-myristate 13-acetate (PMA) to induce differentiation into M0 macrophages. The differentiated macrophages were then stimulated with 2 µg/mL lipopolysaccharide (LPS)to promote polarization into the M1 phenotype. Subsequently, the M1 macrophages were infectedwith the HIF-1α overexpression lentiviral particles and cultured for 48 h to achieve stable overexpression of HIF-1α.To establish the co-culture system, transwell inserts containing confluent Caco-2 cell monolayers were transferred to the wells containing lentivirus-infected THP-1 cells. The co-culture was maintained for 24 h without further intervention. The experimental groups for the overexpression study were designed as follows: Negative Control group(M1 macrophages infected with lentiviral particles carrying a negative control plasmid, co-cultured with Caco-2 cells); HIF-1α overexpression group(M1 macrophages infected with lentiviral particles carrying the HIF-1α overexpression plasmid, co-cultured with Caco-2 cells).Negative control + drug intervention group(Negative control treated with the optimal concentration of the effective compounds); HIF-1α overexpression + drug intervention group(HIF-1α overexpression group treated with the optimal concentration of the effective compounds).

Statistical analysis

All experimental data were analyzed using SPSS 22.0 statistical software (IBM Corp., Armonk, NY, USA), and graphical representations were generated using GraphPad Prism 6.0 software (GraphPad Software Inc., San Diego, CA, USA). Data were derived from at least three independent experiments, each performed in triplicate, and are presented as the mean ± standard deviation (SD). For comparisons among multiple groups, one-way analysis of variance (ANOVA) was performed, followed by the least significant difference (LSD) post hoc test. A P-value of less than 0.05 (P < 0.05) was considered statistically significant.

Results of network pharmacology analysis.

KMSHTF effective compounds from TCMSP

A comprehensive search of the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database was conducted to identify the active ingredients of each herbal component in KMSHTF. This search yielded a total of 126 active compounds. The distribution of these compounds across the individual herbs was as follows: 51 compounds from Leigongteng, 30 from Guanhuangbai, 14 from huanglian, 3 from Qinpi, 9 from Cangzhu, 7 from Baihuasheshecao, 11 from Baitouweng, and 1 from Wubeizi. Detailed information on these compounds is provided in Supplementary Data Table 1.

Identification of Potential Targets of KMSHTF for UC Treatment

Potential targets of KMSHTF were predicted using the SwissTargetPrediction database, resulting in the identification of 744 target genes. Additionally, a search of the GeneCards database yielded 5,680 targets associated with ulcerative colitis (UC). By intersecting the KMSHTF potential targets with UC-related targets, 365 common target genes were identified (Fig. 1). These overlapping targets represent potential therapeutic candidates for UC treatment mediated by KMSHTF.

Fig. 1

Identification of Potential Targets of KMSHTF for UC Treatment. The Venn diagram illustrated the overlap between KMSHTF target genes and UC-related target gene (a). The intersection of KMSHTF potential targets and UC targets yielded 365 common target genes.Detailed information on these 365 common target genes is provided in Supplementary Data Table 2

Protein–Protein Interaction (PPI) Analysis and Identification of Key Target Genes for KMSHTF in UC Treatment

The 365 common target genes were analyzed using the STRING 11.5 database to construct a protein–protein interaction (PPI) network. The resulting network comprised 365 nodes and 1,579 edges (Fig. 2a),where nodes represent proteins and edges indicate PPI relationships, with an average node degree of 8.65.The PPI data were downloaded in TSV format and subsequently visualized using Cytoscape 3.8.2 (Fig. 2b).To identify key genes, topological parameters including Degree, Eigenvector, Local Average Connectivity (LAC), Betweenness, Closeness, and Network values were calculated. Based on these analyses, 34 genes were identified as central to the network, one of which was HIF-1α. The relevance of these key genes was further illustrated by constructing a histogram based on their degree values (Fig. 2c).

Fig. 2

PPI Analysis and Key Target Genes of KMSHTF in UC Treatment. The protein–protein interaction (PPI) network was constructed with a minimum interaction score threshold of 0.9 (a). The PPI results were visualized using Cytoscape 3.8.2 (b), where larger nodes represent genes with a higher degree (number of connected genes), and thicker edges indicate a higher combined interaction score. A histogram of the top 34 key target genes, ranked by their degree values, isshown in c. The x-axis represents individual genes, while the y-axis represents the degree value (number of connections) for each gene

GO and KEGG Enrichment Analysis of KMSHTF in UC Treatment and Exploration of Potential Therapeutic Mechanisms

Gene Ontology (GO) enrichment analysis of the 365 common target genes identified a total of 1,502 significantly enriched terms (P < 0.05), including 1,165 biological processes (BP), 120 cellular components (CC), and 242 molecular functions (MF). The BP terms were significantly enriched in inflammatory response, protein phosphorylation, signal transduction, negative regulation of apoptotic process, positive regulation of RNA polymerase II promoter transcription, and positive regulation of cell proliferation. The CC terms were significantly enriched in cytosol, plasma membrane, cytoplasm, cytoskeleton, and nucleus. The MF terms were significantly enriched in ATP binding, protein binding, and identical protein binding. The top 10 significantly enriched terms in each category (BP, CC, and MF) were visualized in a bar graph (Fig. 3a). Subsequently, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysiswas performed on the 365 common target genes, revealing 172 significantly enriched pathways (P < 0.05). The top 20 enriched pathways were selected and visualized (Fig. 3b). Notably, the HIF-1α signaling pathway ranked 18th among all enriched pathways, suggesting that KMSHTF may exert its anti-inflammatory effects in UC by modulating the HIF-1α pathway. To further elucidate the potential mechanisms of KMSHTF in UC treatment, a comprehensive disease-pathway-target-component-drug network was constructedusing Cytoscape software (Fig. 3c). This network integrated the identified targets, pathways, and active components of KMSHTF, providing a holistic view of its potential therapeutic mechanisms.

Fig. 3

GO and KEGG Enrichment Analysis of KMSHTF in UC Treatment and Exploration of Potential Therapeutic Mechanisms. The top 10 significantly enriched terms in the categories of biological processes (BP), cellular components (CC), and molecular functions (MF) are visualized in a bar graph (a). KEGG pathway enrichment analysis of the 365 common target genes identified 172 significantly enriched pathways (P < 0.05). The top 20 enriched pathways are displayed in b, with the HIF-1α signaling pathway ranking 18th.This finding supported the hypothesis that KMSHTF may alleviate UC inflammation by modulating the HIF-1α pathway. To further elucidate the potential mechanisms of KMSHTF in UC treatment, a comprehensive disease-pathway-target-component-drug network was constructed using Cytoscape software (c). c, the oval shapes in the upper right represented the active compounds of each herb in KMSHTF: yellow for Jiuhuanglian, orange for Kunmingshanhaitang, dark blue for Baitouwen, gray for Wubeizi, green for Guanhuangbai, dark green for Baihuasheshecao, purple for Cangzhu, pink for Qinpi, and red for compounds shared by multiple herbs. The blue rectangles in the lower section represented the overlapping target genes for KMSHTF in UC treatment, while the red triangles denoted hub genes, as detailed in Supplementary Data Table 2. The rhombus shapes in the upper left represented the top 20 enriched pathways,with the red rhombus (hsa04660) highlighting the HIF-1α pathway. Additional pathway information corresponding to the numbering is provided in Supplementary Data Table 3

Identification of Baicalein, Palmatine, and Triptonide as Potential Key Compounds in KMSHTF for UC Treatment

In this study, molecular docking was employed to identify the core active ingredients of KMSHTF with potential therapeutic effects against ulcerative colitis (UC). Based on the HIF-1α signaling pathway, which was highlighted as a key therapeutic pathway in this study, the critical target genes HIF-1α and HKII were selected for molecular docking with the active compounds of KMSHTF. According to the receptor-ligand docking theory, the docking energy is inversely correlated with binding affinity; thus, more negative docking energy values indicate stronger binding affinity between the protein and ligand. Detailed docking results are provided in Supplementary Data Table 4. Among the tested compounds, Baicalein, Palmatine, and Triptonide (Fig. 4, Table 1) exhibited strong binding affinities to both HIF-1α and HKII, as well as significant relevance to the HIF-1α signaling pathway. Consequently, these three compounds were selected for further in vitro experimental validation Tables 2, 3 and 4.

Fig. 4

Baicalein, Palmatine, and Triptonide as Potential Key Compounds in KMSHTF for UC Treatment. Molecular docking results revealed the binding interactions of the key compounds: Baicalein forms hydrogen bonds with residues ASN-448 and ASP-238 of HIF-1α (a) and with residues ARG-462, ARG-769, and GLY-765 of HKII (b). Triptonide formed hydrogen bonds with residue ASP-249 of HIF-1α (c) and with residue LYS-638 of HKII (d). Palmatine formed hydrogen bonds with residue ARG-440 of HIF-1α (e) and with residues ASN-223 and ARG-407 of HKII (f)

Table 1 The total available compounds of KMSHTFTable 2 The common target genes of KUSHTF and UCTable 4 Molecular docking resultResults of In Vivo and In Vitro Biological ExperimentsKMSHTF Alleviated Disease Severity and Improved Colonic Pathology in UC Rats

Compared to the control group, the disease activity index (DAI) scores in the model group increase dpogressively, peaking on day 10. All rats in the model group exhibited diarrhea and bloody stools, alongwith severe perianal lesions. Treatment with KMSHTF and 5-aminosalicylic acid (5-ASA) significantly improved these symptoms (Fig. 5a).Specifically, compared to the model group, the KMSHTF, 5-ASA, and KMSHTF + 5-ASA groups showed reduced fecal occult blood (Fig. 5c) and significantly lower DAI scores(Fig. 5e).Additionally, the model group exhibited a statistically significant shortening of colon length compared to the control group (p < 0.05).This pathological change was reversed in the KMSHTF, 5-ASA, and KMSHTF + 5-ASA groups (Fig. 5d and f). Histological analysis revealed that the colonic mucosa of rat sin the model group displayed epithelial loss, irregular glandular arrangement, extensive inflammatory cell infiltration, and a reduction in goblet cells. In contrast, treatment with KMSHTF promoted epithelial cell proliferation and repair, restored glandular organization, significantly reduced inflammatory cell infiltration, increased goblet cell numbers, and minimized hemorrhagic spots. Similar improvements were observedin the 5-ASA and KMSHTF + 5-ASA groups (Fig. 5g). These results demonstrate that KMSHTF significantly alleviated disease severity and mitigated colonic injury in UC rats. Moreover, the combination of KMSHTF and 5-ASA exhibited the most pronounced therapeutic efficacy.

Fig. 5

KMSHTF Alleviated Disease Severity and Improved Colonic Pathology in UC Rats. Compared to the control group, the disease activity index (DAI) scores in the model group increasedprogressively, peaking on day 10.All rats in the model group exhibited diarrhea, bloody stools, and severeperianal lesions. Treatment with KMSHTF and 5-aminosalicylic acid (5-ASA) significantly improved thesesymptoms (a). Specifically, compared to the model group, the KMSHTF, 5-ASA, and KMSHTF + 5-ASA groups showed KMSHTF significantly reduced fecal occult blood (c) and lowed DAI scores (e). Additionally, the model group exhibited a statistically significant shortening of colon length compared to the control group (p < 0.05). This pathological change was reversed in the KMSHTF, 5-ASA, and KMSHTF + 5-ASA groups (d and f). Histological analysis revealed that the colonic mucosa ofrats in the model group displayed epithelial loss, irregular glandular arrangement, extensive inflammatory cell infiltration, and a reduction in goblet cells. In contrast, treatment with KMSHTF promoted epithelial cell proliferation and repair, restored glandular organization, significantly reduced inflammatory cell infiltration, increased goblet cell numbers, and minimized hemorrhagic spots. Similar improvements were observed in the 5-ASA and KMSHTF + 5-ASA groups (g). These results demonstrated that KMSHTF significantly alleviated disease severity and mitigated colonic injury in UC rats, with effects comparable to those of 5-ASA. Moreover, the combination of KMSHTF and 5-ASA exhibited the most pronounced therapeutic efficacy. * indicates Statistical significance of the model group compared to the control group (*: p < 0.05, **: p < 0.01, ***: p < 0.001); # indicates statistical significance of each treatment group compared to the model group (#: p < 0.05, ##: p < 0.01, ###: p < 0.001, ####: p < 0.0001)

KMSHTF Reduced Expression Levels of Inflammatory Factors in Colon Tissue of UC Rats

As shown in Fig. 6a and b, the protein expression levels of TNF-α and IL-6 were significantly elevated in the model group compared to the control group. In contrast, treatment with KMSHTF significantly reduced the expression of these inflammatory factors, with the most pronounced reduction in the combined group (KMSHTF + 5-ASA) (Fig. 6a and b). Immunohistochemical analysis further confirmed these findings. The expression of TNF-α and IL-6 was markedly increased in the model group compared to the control group. However, KMSHTF treatment significantly downregulated the expression of these cytokines, and the combined treatment group exhibited the most substantial reduction in TNF-α and IL-6 levels (Fig. 6c and d).

Fig. 6

KMSHTF Reduced Expression Levels of Inflammatory Factors in Colon Tissue of UC Rats. As shown in a and b, the protein expression levels of TNF-α and IL-6 were significantly elevated in the model group compared to the control group. In contrast, treatment with KMSHTF significantly reduced the expression of these inflammatory factors, with the most pronounced reduction observed in the combined treatment group (KMSHTF + 5-ASA) (a, b).Immunohistochemical analysis further confirmed these findings.The expression of TNF-α and IL-6 was markedly increased in the model group compared to the control group. However, KMSHTF treatment significantly downregulated the expression of these cytokines, and the combined treatment group exhibited the most substantial reduction in TNF-α and IL-6 levels (c, d). * indicates statistical significance of the model group compared to the control group (*: p < 0.05, **: p < 0.01, ***: p < 0.001); # indicates statistical significance of each treatment group compared to the model group (#: p < 0.05, ##: p < 0.01, ###: p < 0.001)

KMSHTF Promoted Macrophage Polarization by Upregulating IL-4, an M2-Type Macrophage Polarization Factor

Previous results demonstrated that KMSHTF significantly alleviated disease severity and colonic injury in UC rats, while it reduced the protein expression levels of TNF-α and IL-6,which are specific markers of M1-type macrophages (Kałużna et al. 2022).Based on these findings, we hypothesized that KMSHTFmight facilitate ulcerative colitis healing by modulating macrophage polarization. To test this hypothesis, our team investigated the expression levels of macrophage polarization-related proteins. Compared to the control group, the model group exhibited a significant increase in the expression of IL-1β, an M1-type macrophage polarization factor, and a significant decrease in the expression of IL-4, an M2-type macrophage polarization factor. Treatment with KMSHTF reversed these changes, reducing IL-1β expression and increasing IL-4 expression. These effects were further enhanced in the combination treatment group (KMSHTF + 5-ASA) (Fig. 7).

Fig. 7

KMSHTF Promoted Macrophage Polarization by Upregulating IL-4, an M2-Type Macrophage Polarization Factor. Compared to the control group, the expression of the M1-type macrophage polarization factor IL-1β was significantly increased in the model group, while the expression of the M2-type macrophage polarization factor IL-4 was significantly decreased. Treatment with KMSHTF reversed these changes, reducing IL-1β expression and increasing IL-4 expression. These effects were further enhanced in the combination treatment group (KMSHTF + 5-ASA) (Fig. 7). * indicates statistical significance of the model group compared to the control group (*: p < 0.05, **: p < 0.01, ***: p < 0.001); # indicates statistical significance of each treatment group compared to the model group (#: p < 0.05, ##: p < 0.01, ###: p < 0.001)

KMSHTF Modulated Macrophage Metabolism via the HIF-1α Pathway to Promote M2-Type Macrophage Polarization

The above results indicated that KMSHTF may alleviate UC inflammation by promoting M2-type macrophage polarization in the intestinal wall. Recent studies have demonstrated that HIF-1α can drive M2-type macrophage polarization by regulating macrophage metabolism, thereby ameliorating ulcerative colitis symptoms (Wu et al. 2021; Zhuang et al. 2021). This aligns with our earlier network pharmacology prediction that KMSHTF may exert its therapeutic effects in UC by modulating the HIF-1α pathway. To validate whether KMSHTF mitigates UC inflammation through the HIF-1α pathway to promote M2-type macrophage polarization, we conducted the following experiments. Western blot analysis revealed that the protein expression levels of HIF-1α, HKII, GLUT1, and iNOS were significantly elevated in the model group compared to the control group. However, these levels were markedly reduced in the KMSHTF, with the most pronounced reduction observed in the KMSHTF + 5-ASA combination group (Fig. 8a and b). Immunohistochemical results further confirmed these findings: the expression of HIF-1α, HKII, and GLUT1 was significantly higher in the model group compared to the control group. Treatment with KMSHTF or 5-ASA significantly reduced the expression of these proteins, with the greatest reduction observed in the KMSHTF + 5-ASA group (Fig. 8c and d). The green arrows in Fig. 8c highlight the changes in protein expression.

Fig. 8

KMSHTF Modulated Macrophage Metabolism via the HIF-1α Pathway to Promote M2-Type Macrophage Polarization. The protein expression levels of HIF-1α, HKII, GLUT1, and iNOS were significantly elevated in the model group compared to the control group. However, these levels were markedly reduced in the KMSHTF and 5-ASA treatment groups, with the most pronounced reduction observed in the KMSHTF + 5-ASAcombination group (a, b). Immunohistochemical results further confirmed these findings. The expression of HIF-1α, HKII, and GLUT1 was significantly higher in the model group compared to the control group. Treatment with KMSHTF or 5-ASA significantly reduced the expression of these proteins, with thegreatest reduction observed in the KMSHTF + 5-ASA group (c, d). The green arrows highlighted the changes in protein expression. * indicates statistical significance of the model group compared to the control group (*: p < 0.05, **: p < 0.01, ***: p < 0.001); # indicates statistical significance of each treatment group compared to the model group (#: p < 0.05, ##: p < 0.01, ###: p < 0.001)

Baicalein, Palmatine, and Triptonide, the Key Compounds of KMSHTF, Significantly Reduced the Expression Levels of HIF-1α, HKII, GLUT1, and IL-6 In Vitro

Based on the results of previous animal experiments, we hypothesized that KMSHTF might promote M2 macrophage polarization through the HIF-1α pathway. To further validate this mechanism, we conducted cellular experiments using the three compounds: Baicalein, Palmatine and Triptonide—identified through molecular docking as having the strongest binding affinity to HIF-1α and HKII. The results demonstrated.

that the protein expression levels of HIF-1α, HKII, GLUT1 and IL-6 were significantly elevated in the Model group compared to the control group. However, treatment with Baicalein, Palmatine and Triptonide significantly reduced the expression of these proteins in a concentration-dependent manner. Among the three compounds, Baicalein exhibited the most pronounced effect (Fig. 9).

Fig. 9

Baicalein, Palmatine, and Triptonide, the Key Compounds of KMSHTF, Significantly Reduced the Expression Levels of HIF-1α, HKII, GLUT1, and IL-6. The active compounds of KMSHTF—Baicalein, Palmatine, and Triptonide—significantly decreased the expression of HIF-1α, HKII, GLUT1, and IL-6 in a concentration-dependent manner. Among the three compounds, Baicalein exhibited the most pronounced effect (a-o). * indicates statistical significance of the model group compared to the control group (*: p < 0.05, **: p < 0.01, ***: p < 0.001); # indicates statistical significance of each treatment group compared to the model group (#: p < 0.05, ##: p < 0.01, ###: p < 0.001)

Combined Treatment with Baicalein, Palmatine, and Triptonide More Effectively Reduced the Expression of HIF-1α, HKII, GLUT1, and IL-6 Compared to each Compound alone In Vitro

Furthermore, when Baicalein, Palmatine and Triptonide were administered in combination, the reduction in protein expression levels of HIF-1α, HKII, GLUT1 and IL-6 was significantly more pronounced compared to treatment with each compound alone (Fig. 10).

Fig. 10