Hedyotis diffusa Suppresses Colitis-Associated Colorectal Cancer via Inhibition of the IL-17A-IL-17RA Axis and NF-κB Signaling

TL;DR

Hedyotis diffusa and its component ferulic acid reduce inflammation and tumor growth in colorectal cancer by targeting immune pathways.

Contribution

The study reveals a novel mechanism by which Hedyotis diffusa and ferulic acid suppress CRC through inhibition of the IL-17A-IL-17RA axis and NF-κB signaling.

Findings

HD and FA reduced tumor number, size, and colitis in a CRC mouse model.

Transcriptomic analysis showed suppressed IL-17A and NF-κB signaling.

FA binds to IL-17A, potentially disrupting its interaction with IL-17RA.

Abstract

Chronic inflammation-driven colorectal cancer (CRC) is critically mediated by interleukin-17A (IL-17A)-dependent immune responses and nuclear factor-κB (NF-κB) signaling, which promote immune cell infiltration and tumor progression. In this study, the anti-tumor efficacy and molecular mechanisms of a standardized extract of Hedyotis diffusa Willd. (HD) and its constituent, ferulic acid (FA), were investigated using an azoxymethane/dextran sulfate sodium (AOM/DSS)-induced colitis-associated CRC mouse model. HD and FA treatment markedly alleviated colitis, reduced tumor number and size, improved survival, and attenuated histopathological damage. Transcriptomic analysis revealed significant modulation of immune-related pathways, with prominent suppression of IL-17A and NF-κB signaling. Molecular docking demonstrated binding of FA to IL-17A at Pro59 and Arg101, suggesting potential disruption of the IL-17A/IL-17RA interaction. Consistently, both HD and FA reduced immune cell infiltration, downregulated IL-17A production, and inhibited NF-κB activation in colonic tissues. Collectively, these findings demonstrated that HD exerted protective effects against inflammation-associated CRC through targeting the IL-17A/IL-17RA axis and downstream NF-κB signaling, providing mechanistic insight into IL-17A-centered immunomodulation in colorectal tumorigenesis.

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Full text

1. Introduction

Colorectal cancer (CRC) is one of the leading causes of cancer-related morbidity and mortality worldwide, accounting for approximately 10% of all cancer cases and ranking as the second most common cause of cancer death globally [1,2]. Despite advances in screening and treatment, the global burden of CRC continues to increase, driven by population aging, dietary patterns, and lifestyle changes, with incidence and mortality projected to rise substantially by 2040 [3]. These trends underscore the urgent need to identify novel preventive and therapeutic strategies targeting key pathogenic mechanisms of CRC.

Chronic inflammation is a central driver of colorectal carcinogenesis, particularly in colitis-associated CRC [4]. Patients with inflammatory bowel disease (IBD) and individuals exposed to proinflammatory dietary and environmental factors exhibit a markedly increased risk of CRC [5,6]. In the inflamed colon, cytokines such as interleukin (IL)-1β, IL-6, and IL-17A mediate crosstalk between immune and epithelial cells, thereby sustaining a tumor-promoting microenvironment. Among these mediators, IL-17A has emerged as a critical regulator of inflammation-driven tumorigenesis by enhancing epithelial proliferation, recruiting immune cells, and activating downstream signaling pathways, including nuclear factor-κB (NF-κB) [7]. Consequently, interventions capable of modulating IL-17A-dependent inflammatory signaling represent promising approaches for CRC prevention and treatment [8].

Hedyotis diffusa Willd. (HD), also known as Oldenlandia diffusa or Bai Hua She She Cao, is a medicinal herb widely used in East Asia and has been extensively investigated for its anti-inflammatory and anti-cancer properties [9]. Clinical prescription analyses indicate that HD is among the most frequently used single herbs for CRC treatment, particularly in Taiwan [10,11]. Experimental studies have demonstrated that HD suppresses tumor growth and metastasis through regulation of multiple oncogenic pathways, including Akt, extracellular signal-regulated kinases (ERK), signal transducer and activator of transcription 3 (STAT3), NF-κB, and Wnt/β-catenin, primarily in cancer cell lines and xenograft models [12,13]. In addition, HD-derived polysaccharides have been shown to enhance T-cell activation and improve immune checkpoint inhibitor efficacy in CRC xenografts [14]. However, these models do not fully recapitulate the complex inflammatory microenvironment characteristic of colitis-associated CRC.

To date, the therapeutic efficacy and molecular mechanisms of HD have not been evaluated in an inflammation-driven CRC model. Therefore, the present study investigated the effects of HD in an azoxymethane/dextran sulfate sodium (AOM/DSS)-induced murine CRC model, which closely mimics the pathogenesis of human colitis-associated CRC [15]. By integrating ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), RNA sequencing, molecular docking, and immunohistochemistry (IHC), we aimed to elucidate the molecular mechanisms and bioactive constituents underlying HD activity. Our findings demonstrated that HD attenuated inflammation-associated colorectal tumorigenesis by modulating IL-17A-mediated immune responses and NF-κB signaling, and identified ferulic acid (FA) as a key contributor to these effects.

2. Results

2.1. HD Ameliorated DSS-Induced Colitis in Mice

The NF-κB-luciferase transgenic mouse model, previously established and validated for bioluminescent correlation with NF-κB activation [16], was used to evaluate the anti-inflammatory effects of HD. Mice were treated with 3% DSS to induce colitis and co-administered various dosages of HD. Bioluminescent imaging was performed on Day 7. As shown in Figure 1, a minimal luminescent signal was detected in the mock group. In contrast, DSS treatment markedly increased luminescence in the colon, with a 170-fold increase in photon flux (photon/sec/cm^2^/sr) compared to the mock group. HD treatment significantly attenuated DSS-induced luminescence in a dose-dependent manner, indicating suppression of NF-κB activity. These results suggested that HD effectively ameliorated DSS-induced colitis in vivo.

2.2. HD Ameliorated AOM/DSS-Induced CRC in Mice

Given the protective effects of HD observed in the DSS-induced colitis model, we next evaluated whether HD could attenuate tumor development in a colitis-associated CRC model. BALB/c mice were administered AOM followed by cycles of DSS to induce CRC, and HD was administered orally at 200 mg/kg, a dose selected based on a previous experiment. The experiment spanned 91 days, during which clinical signs, particularly rectal bleeding (a hallmark symptom of CRC), were closely monitored. All mice in the mock group remained free of rectal bleeding throughout the study, while 100% of mice in the CRC group exhibited this symptom. Notably, HD treatment significantly reduced the incidence of rectal bleeding. At the experimental endpoint, the survival rate was 100% in both the mock and HD-treated groups, compared to 83.3% in the CRC group. These findings suggested that HD mitigated CRC-associated clinical symptoms and improved survival in the AOM/DSS-induced CRC mouse model.

On day 91, mice were sacrificed, and colons were excised and longitudinally opened for macroscopic tumor assessment. As shown in Figure 2a, colons from the mock group appeared smooth and tumor-free, while those from the CRC group showed numerous tumor nodules of varying sizes. Although some tumors were also observed in the HD-treated group, their numbers were markedly reduced. Tumor nodules were categorized by diameter into three groups: <2 mm, 2–4 mm, and >4 mm. Quantitative analysis revealed that the CRC group developed an average of 26.11 ± 5.23 tumors per mouse, including 4.44 ± 2.46 large tumors (>4 mm) (Figure 2b). Tumor size distribution was approximately 31% (<2 mm), 52% (2–4 mm), and 17% (>4 mm). In contrast, the HD group had significantly fewer tumors, averaging 8.00 ± 4.21 per mouse, a 69.03 ± 16.12% reduction compared to the CRC group. The number of large tumors (>4 mm) was also significantly lower at 1.13 ± 0.83 per mouse, corresponding to a 74.66 ± 18.79% reduction (vs. CRC group).

Histopathological analysis (Figure 2c) showed normal colonic architecture in the mock group, with intact surface epithelium and organized submucosa. In the CRC group, epithelial dysplasia, inflammatory infiltration, and features of tubular adenocarcinoma were observed. In contrast, colonic tissues from the HD-treated group exhibited preserved structures and improved histological features. Collectively, these findings demonstrated that HD alleviated clinical symptoms, reduced tumor burden, and ameliorated histopathological damage in the AOM/DSS-induced CRC mouse model.

2.3. HD Modulated Immune-Related Gene Expression Pathways in Rectal Tissues of CRC Mice

To elucidate the anti-cancer mechanisms of HD, total RNA was extracted from rectum tissues and subjected to RNA-sequencing (RNA-Seq) analysis. Differentially expressed genes (DEGs) were identified using a threshold of fold change ≥ 2 or ≤ −2 and adjusted p ≤ 0.05. Compared with the mock group, the CRC group exhibited 3909 DEGs, including 2604 upregulated and 1305 downregulated genes. In comparison to the CRC group, HD treatment resulted in 3768 DEGs, including 1852 upregulated and 1916 downregulated genes. Functional enrichment of DEGs was performed using Gene Ontology (GO) biological process and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. GO analysis revealed that HD significantly affected 1529 biological processes, with the top 20 shown in Figure 3a. These included processes related to immune cell locomotion (e.g., leukocyte chemotaxis and migration) and cell adhesion (e.g., leukocyte cell–cell adhesion) (Figure 3b). KEGG pathway analysis showed that HD significantly affected 70 pathways, with the top 20 listed in Table 1. Notably, 11 of these were immune-related pathways. In addition, two signal transduction pathways, including cytokine-cytokine receptor interaction and NF-κB signaling pathways, were also implicated in immune response. Of particular interest, two were directly related to the IL-17 signaling axis: Th17 cell differentiation and the IL-17 signaling pathway. These results suggested that HD exerted anti-CRC effects by regulating genes involved in immune response, with IL-17-related mechanisms serving as potential therapeutic targets of HD.

2.4. Identification of FA from Standardized Concentrated Hedyotidis Diffusae Herba as a Potential Anti-CRC Compound in HD via UHPLC-MS/MS and IL-17A Docking Analysis

To identify bioactive constituents responsible for the anti-CRC effects of HD, the chemical composition of the HD extract prepared from standardized concentrated Hedyotidis Diffusae Herba was analyzed using UHPLC-MS/MS. Compounds were tentatively identified by comparing retention time and mass-to-charge ratios (m/z) with data from spectral libraries and literature. A total of 30 compounds were identified, nearly half of which were phenolic compounds (Figure 4 and Table 2). The five most abundant compounds identified in the HD extract were quercetin (3.094%), sinapinic acid (2.955%), kaempferol (2.334%), FA (2.212%), and 4-coumaric acid (0.736%). The identity of FA was confirmed by comparison of its UHPLC retention time (25.544 min) with that of the FA standard (Figure 4).

Based on RNA-Seq results indicating significant modulation of the IL-17 signaling and Th17 cell differentiation pathways, the five most abundant compounds identified in the HD extract, including quercetin, sinapinic acid, kaempferol, FA, and 4-coumaric acid, were subjected to molecular docking analysis using the IL-17A/IL-17RA complex (PDB ID: 4HSA) as the target. For each compound, nine independent docking runs were performed, and both the predicted binding free energy (ΔG) and binding site distribution were evaluated.

The comparative docking results are summarized in Table 3. Region i corresponds to the IL-17A/IL-17RA interaction interface previously reported to be critical for receptor–ligand signaling [17]. Among the tested compounds, FA exhibited the highest binding frequency at the IL-17A/IL-17RA interface region i, with 6 out of 9 docking poses localized to this region and a favorable binding free energy (ΔG = −7.2 kcal/mol). In contrast, 4-coumaric acid and sinapinic acid showed comparable binding frequencies at region i (5/9 poses each), but with weaker binding energies (−5.9 and −6.8 kcal/mol, respectively). Although quercetin displayed a lower minimum ΔG value (−9.9 kcal/mol), its binding to region i occurred less frequently (3/9 poses), indicating lower site-specific binding consistency. Kaempferol did not exhibit any docking pose localized to region i across the nine simulations. The docking poses localized to region i are shown in Figure S1.

Consistent with these comparative results, FA was selected for detailed interaction analysis. As shown in Figure 5, FA bound to region i of the IL-17A/IL-17RA interface, interacting primarily with residues Pro59 and Arg101 on the IL-17A chain B. FA formed hydrogen bonds with Pro59 (3.4 Å) and Arg101 (2.4 and 2.5 Å), residues known to be critical for IL-17A-IL-17RA recognition [17]. Notably, Arg101 of IL-17A and Trp31 of IL-17RA form conserved contacts essential for receptor signaling [17]. Binding of FA at this interface was therefore predicted to interfere with IL-17A/IL-17RA complex formation, potentially attenuating downstream IL-17-mediated inflammatory signaling. Together with transcriptomic evidence, these results supported FA as a bioactive constituent of HD contributing to its anti-CRC effects, at least in part through modulation of IL-17-driven inflammatory responses.

2.5. FA Ameliorated AOM/DSS-Induced CRC in Mice

We further analyzed whether FA ameliorated tumor development in the AOM/DSS CRC model. FA (100 mg/kg) was orally administered for 91 consecutive days. On day 91, mice were sacrificed, and colons were excised and longitudinally opened for macroscopic tumor assessment. Figure 6a shows that colons from the CRC group showed numerous tumor nodules of varying sizes. Some tumors were also observed in the FA-treated group; however, their numbers were markedly reduced. Quantitative analysis revealed that the CRC group developed an average of 23.83 ± 7.19 tumors per mouse, including 5.17 ± 2.71 large tumors (>4 mm) (Figure 6b). Tumor size distribution was approximately 36% (<2 mm), 42% (2–4 mm), and 22% (>4 mm). In contrast, the FA group had significantly fewer tumors, averaging 7.00 ± 3.63 per mouse, a 70.63 ± 15.25% reduction compared to the CRC group. The number of large tumors (>4 mm) was also significantly lower at 0.67 ± 0.82 per mouse, corresponding to an 87.11 ± 15.79% reduction. These data suggested that FA reduced tumor burden in the AOM/DSS-induced CRC mouse model.

2.6. HD and FA Suppressed NF-κB Activation, IL-17A Production, and Granulocyte Infiltration in Mice with AOM/DSS-Induced CRC

Based on RNA-Seq findings and molecular docking analysis, HD was shown to modulate genes involved in the NF-κB and IL-17A signaling pathways in rectal tissue of mice with CRC. To validate these findings in vivo, we evaluated whether HD and FA could inhibit the activation of NF-κB (p65), IL-17A production, and granulocytes (CD11b) infiltration in colon tissues using IHC staining. As shown in Figure 7, the CRC group exhibited a marked increase in p65- and CD11b-positive cells, as well as an elevated IL-17A-stained area, compared with the mock group. Treatment with HD or FA significantly reduced the percentages of p65- and CD11b-positive cells and decreased IL-17A expression in the colon. These findings suggested that both HD and FA suppressed immune cell infiltration by downregulating IL-17A production and NF-κB signaling, thereby attenuating inflammation and tumorigenesis in the AOM/DSS-induced colitis-associated CRC model. Notably, these results supported a mechanistic link between the anti-cancer activity of FA and its suppression of IL-17A-mediated immune signaling.

3. Discussion

A wide range of murine models has been established to investigate CRC, including genetically engineered models, xenografts, and chemically induced systems [18,19]. Among these, the AOM/DSS-induced colitis-associated cancer model is widely recognized for recapitulating the stepwise progression from chronic intestinal inflammation to tumorigenesis observed in human CRC. This model closely mirrors key histopathological and molecular features of inflammation-driven CRC while offering high reproducibility within a relatively short experimental timeframe [15,20]. Accordingly, it represents a robust in vivo platform for mechanistic studies targeting inflammation-associated tumorigenesis.

HD has been extensively reported to exhibit anti-inflammatory and anti-cancer properties; however, most previous investigations were limited to in vitro systems or xenograft models, which do not adequately reflect the complex inflammatory microenvironment characteristic of colitis-associated CRC [12,13]. In the present study, we demonstrated for the first time that long-term oral administration of HD significantly improved survival, reduced tumor incidence and burden, and alleviated disease-associated symptoms in the AOM/DSS-induced CRC model. These findings established HD as an effective intervention in a physiologically relevant inflammation-driven CRC model and provided a foundation for mechanistic dissection at the molecular level.

Previous studies have shown that HD suppresses CRC cell proliferation and induces apoptosis through mitochondrial dysfunction, cell cycle arrest, and inhibition of oncogenic signaling pathways, including STAT3, ERK, and Akt, primarily in human colon cancer cell lines [21,22,23]. Antitumor activity via STAT3 inhibition has also been reported in CRC xenograft models [24]. In addition, network pharmacology analyses suggest that HD targets multiple cancer-related biological processes, encompassing inflammation, immune regulation, and intercellular signaling [25,26]. Nonetheless, the molecular mechanisms by which HD modulates inflammation-driven CRC in vivo remained unclear.

To address this gap, we employed RNA sequencing to profile transcriptional changes in rectal tissues from AOM/DSS-treated mice. HD administration markedly altered inflammation-associated gene networks, with significant enrichment of pathways related to B cell receptor signaling, Wnt signaling, NF-κB signaling, IL-17 signaling, mitogen-activated protein kinase pathways, and chemokine signaling. Importantly, genes implicated in IBD were also significantly modulated, reinforcing the mechanistic link between chronic inflammation and CRC pathogenesis. Among the dysregulated cytokines, IL-17A emerged as a key node, consistent with its established role in promoting epithelial proliferation and sustaining a pro-tumorigenic inflammatory microenvironment [7]. Immunohistochemical analyses further confirmed that HD significantly reduced IL-17A expression in colonic tissues, suggesting that attenuation of IL-17-driven signaling contributed to its anti-CRC effects.

To identify the bioactive constituents underlying these effects, UHPLC-MS/MS analysis was performed on the standardized concentrated HD extract used in this study. Thirty phytochemicals were detected, with quercetin, sinapinic acid, kaempferol, and FA representing the most abundant components. Notably, this phytochemical profile differed from that reported for the dried whole herb documented in the Chinese Pharmacopoeia, highlighting the importance of characterizing clinically relevant formulations. Quercetin and kaempferol are well-characterized flavonoids with extensively documented anti-cancer activities in CRC models [27,28], limiting their novelty for further mechanistic exploration in this context. Sinapinic acid, although relatively abundant, remains poorly studied in CRC and lacks sufficient preclinical evidence. In contrast, FA has been reported to exert both anti-inflammatory and anti-cancer effects and is mechanistically distinct from flavonoids [29,30,31,32]. Previous studies have shown that FA effectively reduces inflammation and mucosal damage in rats with ulcerative colitis by activating the nuclear factor erythroid 2-related factor 2/heme oxygenase 1 pathway or by inhibiting the thioredoxin-interacting protein/NOD-like receptor pyrin domain-containing 3 and toll-like receptor4/NF-κB pathways [33,34,35]. FA suppresses CRC cell proliferation through regulation of autophagy via the miRNA-221/p53-inducible protein 1 axis and inhibition of glycolytic metabolism through modulation of the lncRNA 495810/PKM2 pathway and epidermal growth factor receptor signaling [36,37]. FA has also demonstrated chemopreventive activity in chemically induced CRC models in rats by modulating detoxification enzyme systems [38,39]. Here, we extend these findings by demonstrating, for the first time, that FA significantly mitigated inflammation-driven CRC in vivo. Importantly, our molecular docking analysis revealed that among the major HD constituents, FA exhibited both favorable binding affinity and the highest binding-site consistency at the IL-17A/IL-17RA interface, supporting its potential to interfere with IL-17-mediated inflammatory signaling. In comparison, quercetin, despite a lower minimum ΔG, bound less consistently to the interface, while kaempferol did not bind at the critical site. These findings provided a mechanistic rationale for prioritizing FA as the key bioactive constituent of HD contributing to its anti-CRC and immunomodulatory effects.

IL-17A is a pleiotropic cytokine critically involved in inflammatory, autoimmune, and malignant diseases, including CRC [7,40]. IL-17A expression increases progressively during the adenoma-carcinoma sequence and correlates with dysplasia severity and poor clinical prognosis [41,42]. Experimental studies have further shown that while depletion of Th17 cells delays tumor development, complete neutralization of IL-17A more effectively suppresses colitis-associated tumorigenesis, underscoring the contribution of IL-17A derived from multiple immune cell sources [43]. Consistently, our transcriptomic data revealed a 14-fold induction of IL-17A following AOM/DSS treatment.

Mechanistically, engagement of IL-17A with the IL-17RA/RC receptor complex initiates recruitment of the adaptor protein Act1, which subsequently recruits and ubiquitinates tumor necrosis factor (TNF) receptor-associated factor 6, leading to activation of the IκB kinase (IKK) complex. This activation of IKK promotes phosphorylation and degradation of IκB, thereby allowing NF-κB to translocate into the nucleus. This signaling cascade drives transcription of pro-survival and pro-inflammatory genes, including IL-6, TNF, and chemokine (C-X-C motif) ligand 1, which contribute to immune cell recruitment, epithelial hyperproliferation, and inflammation-associated tumor progression [41,43,44]. In the present study, molecular docking analysis predicted that FA could occupy the IL-17A-IL-17RA interface and form hydrogen bond interactions with Pro59 and Arg101, residues previously implicated in IL-17A receptor engagement [17]. Importantly, these in silico findings were not intended to imply direct destabilization or degradation of the IL-17A protein. Rather, they suggested a potential for FA to interfere with IL-17A-IL-17RA signaling at the receptor interaction level. Consistent with attenuation of IL-17A signaling rather than altered protein stability, transcriptomic profiling revealed marked downregulation of Il17a mRNA (-16-fold change vs. CRC) and multiple NF-κB-associated inflammatory genes in FA-treated mice (Table S1), indicating suppression at the transcriptional level. The observed reduction in IL-17A protein expression and NF-κB activation in colonic tissues, therefore, likely reflects downstream consequences of dampened inflammatory signaling and disrupted positive feedback loops that sustain IL-17A production in chronic colitis. Together, these findings supported a model in which FA modulated the IL-17A-IL-17RA-NF-κB axis primarily through regulation of inflammatory signaling networks, with molecular docking providing hypothesis-generating evidence for a potential site of interaction and pathway interference rather than definitive evidence of direct protein inhibition. Nevertheless, further biochemical and biophysical studies will be required to determine whether FA directly disrupts IL-17A–IL-17RA binding in vivo or acts predominantly through modulation of upstream immune cell responses.

4. Materials and Methods

4.1. Herb, Chemicals, and Reagents

Standardized concentrated Hedyotidis Diffusae Herba (Bai Hua She She Cao Herbal Extract Granules), extracted from the dried whole part of Hedyotis diffusa Willd, was obtained from a GMP-certified Chinese medicine manufacturer (Sun Ten Pharmaceutical Co., Taipei, Taiwan). All other chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA), unless otherwise specified. DSS (molecular weight 36,000–50,000 Da) was obtained from MP Biomedicals (Irvine, CA, USA). FA (purity ≥ 99%) was purchased from Alfa Aesar (Ward Hill, MA, USA). Mouse monoclonal antibody against p65 (MAB3026), rabbit polyclonal antibody against IL-17A (bs-1183R), and rabbit monoclonal antibody against CD11b (ab133357) were obtained from Millipore (Temecula, CA, USA), Bioss Antibodies (Woburn, MA, USA), and abcam (Cambridge, UK), respectively.

4.2. UHPLC-MS/MS Analysis of HD Extract

The aqueous extract of HD was prepared by soaking standardized concentrated Hedyotidis Diffusae Herba powder in double-distilled water at a ratio of 1:5 (w/v) for 24 h at 4 °C, followed by centrifugation at 3000× g for 20 min. The supernatant was collected for further analysis. UHPLC analysis was performed using an UltiMate 3000 UHPLC system equipped with a Variable Wavelength Detector (ThermoFisher Scientific, Waltham, MA, USA). Chromatographic separation was achieved using a Luna Omega Polar C18 column (2.1 mm × 100 mm, 1.6 μm particle size; Phenomenex, Torrance, CA, USA). The mobile phase consisted of solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile), with the following gradient program: 0–5 min, 0–2% B; 5–59 min, 2–35% B; 59–70 min, 2% B. The flow rate was maintained at 0.2 mL/min, the injection volume was 30 μL, and the column temperature was set at 30 °C. Mass spectrometric detection was conducted on an Orbitrap Fusion™ Lumos™ Tribrid™ Mass Spectrometer (ThermoFisher Scientific, Waltham, MA, USA) equipped with a heated electrospray ionization source operating in positive ion mode. The instrument parameters were as follows: spray voltage, 3500 V; sheath gas, 25 psi; auxiliary gas, 15 psi; sweep gas, 1 psi; ion transfer tube temperature, 285 °C; vaporizer temperature, 350 °C.

4.3. Animals

Male transgenic NF-κB-luciferase reporter mice (FVB background), aged 6–8 weeks and weighing 20–25 g, were used in this study. These mice carry five tandem copies of the NF-κB responsive element (5′-GGGACTTTCC-3′) upstream of a luciferase gene, as previously described [16]. Female BALB/cByJ mice, aged 5–6 weeks and weighing 20 ± 1 g, were purchased from the National Center for Biomodels (Taipei, Taiwan). All animals were housed at the China Medical University Animal Center under standard conditions (12 h light/dark cycle, controlled temperature and humidity) with free access to food and water. All experimental procedures were approved by the Institutional Animal Care and Use Committee of China Medical University (Permit No. CMUIACUC-2021-294) and conducted in accordance with the U.S. National Institutes of Health Guidelines for the Care and Use of Laboratory Animals (NIH Publication No. 85–23, revised 1996).

4.4. DSS-Induced Colitis Model and Treatment Protocol

The DSS-induced mouse colitis model was established as previously described [45,46]. Briefly, colitis was induced by administering 3% DSS in drinking water ad libitum for 7 consecutive days. A total of 25 male transgenic NF-κB-luciferase reporter mice were randomly assigned to five groups (n = 5 per group): (1) mock (no DSS, no treatment), (2) DSS, (3) 50 mg/kg HD (DSS + 50 mg/kg HD), (4) 100 mg/kg HD (DSS + 100 mg/kg HD), and (5) 200 mg/kg HD (DSS + 200 mg/kg HD). HD granules were freshly suspended in distilled water each day and administered by oral gavage in a volume of 100 μL once daily for 7 days, starting concurrently with DSS exposure. The mock group received normal drinking water and a vehicle only. All treatments were given at the same time each day to minimize diurnal variability.

At the end of the 7-day treatment period, mice were intraperitoneally injected with D-luciferin (150 mg/kg body weight). After 10 min, mice were euthanized, and the entire colon was excised from the rectum to the anal verge. Colon tissues were imaged for bioluminescent signals using the IVIS Imaging System 100 Series (Xenogen, Alameda, CA, USA) at the highest sensitivity setting for 1 min. Photon emission from tissues was quantified using Living Image software version 4.8.4 (Xenogen, Alameda, CA, USA). Signal intensity was calculated as the total photon flux per second within a defined region of interest, with background luminescence substrated, and expressed as photons per second per square centimeter per steradian (p/s/cm^2^/sr).

4.5. AOM/DSS-Induced CRC Model and Treatment Protocol

The AOM/DSS-induced CRC model was established as previously described [15,47]. Briefly, on Day 1, female BALB/c mice were administered an intraperitoneal injection of either phosphate-buffered saline (PBS; 137 mM NaCl, 1.4 mM KH_2_PO_4_, 4.3 mM Na_2_HPO_4_, 2.7 mM KCl, pH 7.2) (mock group) or 10 mg/kg AOM dissolved in PBS (CRC and treatment groups). On Day 8, AOM-injected mice received 2.5% DSS in drinking water for 7 consecutive days, followed by regular drinking water for 14 days. This cycle was repeated for three additional rounds.

Mice were randomly divided into four groups (n = 10 per group): (1) mock (no AOM/DSS, no treatment), (2) CRC (AOM/DSS without treatment), (3) HD (AOM/DSS + 200 mg/kg HD), and (4) FA (AOM/DSS + 100 mg/kg FA). HD granules and FA were freshly suspended in distilled water each day and administered by oral gavage (100 μL/mouse/day), beginning on Day 1 and continuing daily for 91 days. Body weight and food intake were measured weekly. On Day 91, all mice were euthanized. Colons were excised, opened longitudinally, and inspected for observation. Tumor number and size were measured using a digital caliper. Colon tissues were collected for histopathological examination, IHC, and RNA-Seq analysis.

4.6. RNA-Seq Analysis

Total RNA was extracted from 30 mg of colon tissue using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA). RNA concentration and integrity were assessed using a spectrophotometer and the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). cDNA libraries were constructed by enriching mRNA with oligo(dT) beads, followed by reverse transcription, adaptor ligation, and polymerase chain reaction amplification. Library quality was validated using the Agilent 2100 Bioanalyzer, and 150 bp paired-end sequencing was performed on the NovaSeq 6000 System (Illumina, San Diego, CA, USA). Raw reads were filtered and aligned to the reference genome using Bowtie 2. Gene expression levels were quantified with RSEM (http://deweylab.github.io/RSEM/) (accessed on 22 May 2021). DEGs were identified using the EBSeq package (https://www.biostat.wisc.edu/~kendzior/EBSEQ/) (accessed on 22 May 2021), and statistical significance was assessed using the posterior probability of differential expression (PPDE). Genes with fold change ≥ 2 and PPDE ≥ 0.95 (equivalent to adjusted p ≤ 0.05) were considered significantly differentially expressed. GO annotation and KEGG pathway analyses were performed using NetworkAnalyst (https://www.networkanalyst.ca/) (accessed on 22 May 2021) [48].

4.7. Molecular Docking Analysis

The crystal structure of the human IL-17A/IL-17RA complex (PDB ID: 4HSA) was retrieved from the Protein Data Bank (https://www.rcsb.org/) (accessed on 2 October 2022). The three-dimensional structures of the major compounds identified in HD were obtained from the ZINC database (https://zinc.docking.org/) (accessed on 2 October 2022) in ready-to-dock format, including FA (ZINC ID: 58258), quercetin (ZINC ID: 3869685), sinapinic acid (ZINC ID: 153654), and 4-coumaric acid (ZINC ID: 39811). Molecular docking was performed using AutoDock Vina version 1.1.2 [49], with the IL-17A/IL-17RA complex serving as the docking target. For each compound, nine independent docking runs were conducted to evaluate both predicted binding free energy (ΔG) and binding-site preference. Docking poses were analyzed to determine binding frequency at the IL-17A/IL-17RA interface (region i). Representative docking conformations were visualized using PyMOL version 3.1 for figure generation.

4.8. Histopathological and IHC Staining Analysis

Colon tissues were fixed, paraffin-embedded, and sectioned at 5 μm thickness. H&E staining was performed, and images were captured at 20× magnification using the Snapshot tool in Aperio ImageScope (v12.4.6).

For IHC staining, deparaffinized sections were subjected to antigen retrieval in citrate buffer, treated with 3% H_2_O_2_ in PBS to block endogenous peroxidase, and incubated with blocking solution. Slides were then incubated overnight at 4 °C with primary antibodies: anti-p65 (1:100), anti-IL-17A (1:200), or anti-CD11b (1:400) in a humidity chamber. Detections were performed using Post Primary reagent (rabbit anti-mouse IgG), Novolink^TM^ Polymer (anti-rabbit poly-horseradish peroxidase IgG), and 3,3’-diaminobenzidine chromogen (Leica Biosystems, Wetzlar, Germany) according to the manufacturer’s protocol. IHC images were analyzed using ImageJ version 1.54p (Media Cybernetics, Bethesda, MD, USA). The percentage of stained area was calculated as (brown-stained area/total tissue area) × 100, and the stained cell percentage as (number of brown-stained cells/total number of cells) × 100. A total of 100 cells were counted per field.

4.9. Statistics Analysis

Data are presented as mean ± standard error. One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was performed using GraphPad Prism version 10.2.0 (GraphPad Software, San Diego, CA, USA). A p-value < 0.05 was considered statistically significant.

5. Conclusions

In conclusion, this study demonstrated that HD suppressed colitis-associated colorectal carcinogenesis by targeting the IL-17A-driven inflammatory signaling cascade. Integrative transcriptomic, molecular docking, and in vivo analyses revealed that HD attenuated IL-17A expression and downstream NF-κB-associated inflammatory networks, resulting in reduced immune cell infiltration, epithelial hyperproliferation, and tumor burden in AOM/DSS-treated mice. Among the identified phytochemicals, FA emerged as a potent active component with docking simulations indicating binding to conserved IL-17A residues (Pro59 and Arg101), potentially disrupting IL-17A-IL-17RA engagement and subsequent inflammatory signal transduction. Consistently, FA treatment recapitulated the anti-tumor and anti-inflammatory effects of HD in vivo. Collectively, these findings provided the first mechanistic evidence linking HD to modulation of the IL-17A-NF-κB axis in inflammation-driven CRC and identified FA as a molecular mediator underlying its therapeutic efficacy.