Natural triterpenoid Ardisiacrispin B attenuates colitis-associated cancer via JAK2/STAT3 pathway and gut microbiota modulation

1 Introduction

Inflammatory bowel disease (IBD) is a chronic and refractory gastrointestinal disease comprising Crohn's disease (CD) and ulcerative colitis (UC) [1]. The global burden of inflammatory bowel disease (IBD) is considerable, affecting over 6.8 million individuals worldwide, with incidence and prevalence continuing to rise across both developed and developing regions [2]. The prevalence of IBD is rising annually across many developing regions, with emerging epidemiological patterns suggesting that this increase parallels the rapid socioeconomic and technological transition in parts of Africa and Asia [3]. The role of chronic inflammation in the colon as a central contributor to colorectal tumorigenesis, especially in chronic colitis cohorts that demonstrate a dramatically elevated risk of developing Colorectal Cancer (CRC) [4, 5]. The exact molecular mechanisms of inflammation-associated colorectal carcinogenesis are not fully elucidated thus far; however, gut microbiota, aberrant immune response, and chronic persistent inflammation have been implicated as key factors in the development of colorectal cancer [6]. Chronic gut inflammation creates a tumor-permissive environment that contributes to multistage cellular transformation through increased cytokines and chemokines, leading to the progression of chronic inflammation into Colorectal Cancer [7]. Meanwhile, colonic endothelial cells under an inflammatory tumor microenvironment contribute to the proliferation, migration, and metastasis of tumor cells.

The IL-6/JAK2/STAT3 and associated signaling pathways are the most crucial and multiple signals to the tumorigenesis of chronic inflammation. This cascade of signaling networks, influenced in part by the acidic tumor microenvironment as the most crucial feature, composes many physiological and pathological processes such as angiogenesis, immune modulation, cellular proliferation, and differentiation. It plays a critical role throughout the tumorigenic continuum, encompassing tumor initiation, progression, and metastasis. As such, sustained or aberrant activation of the IL-6/JAK2/STAT3 pathway is thought to be one of the key events during the inflammation–to–cancer transition [8]. In colonic inflammation, the epithelial and immune cells release elevated levels of interleukin-6 (IL-6), which binds to its membrane receptor complex, leading to the activation of non-receptor tyrosine kinases such as JAK2 and a subsequent series of phosphorylation reactions downstream target genes [9]. After tyrosine phosphorylation by JAK2, STAT3 monomers dimerize and translocate to the nucleus, where they bind to DNA response elements specific for STAT3 to induce transcription of target genes. Concurrently, Toll-like receptor 4 (TLR4), which is involved in the activation of innate immunity, can recognize lipopolysaccharide (LPS) from Gram-negative bacteria and drive downstream inflammatory responses [9]. TLR4, a key component of the innate immune system, recognizes LPS derived from Gram-negative bacteria and subsequently initiates and triggers proinflammatory signaling cascades [10]. Loss of intestinal integrity of the intestinal mucosal barrier leads to bacterial translocation. Lipopolysaccharide (LPS) binds to TLR4 with myeloid differentiation factor 88 (MyD88), and IL-1 receptor-associated kinases (IRAKs) are induced to activate signaling pathways involving inflammation at downstream processes [11]. Simultaneously, downstream of IRAK, the adaptor protein TRAF6 induces NF-κB and MAP kinase family protein activation that enhances inflammatory signaling by promoting the transcriptional activity of NF-κB target genes [12]. Thus, continued upregulation of TLR4 in the context of a chronic inflammatory microenvironment promotes malignant cell proliferation, resistance to apoptosis, invasion, and metastasis or generation of a pro-tumorigenic niche, collectively leading to inflammation-associated carcinogenesis [13].

The gut harbors trillions of microorganisms, which include bacteria, fungi, and other complex communities in a symbiotic and mutually beneficial relationship with the host, while controlling important physiological, immunological, and metabolic activities [14]. Among these microbial communities, Firmicutes and Bacteroidetes are especially important, as they have the potential to modulate inflammatory responses involved in CRC development [15]. A recognized gut-protective bacterium, Barnesiella, has been reported to exhibit a negative association with LPS-induced interferon-γ production and may therefore play a role in the regulation of the TLR4 signaling pathway, thus keeping up an intestinal homeostasis [16].

Given that chronic inflammation, dysregulated immune signaling, and gut microbiota imbalance collectively drive the initiation and progression of colitis-associated colorectal cancer (CAC), therapeutic strategies capable of concurrently modulating these interconnected pathways are of considerable translational interest. Natural products possessing anti-inflammatory as well as anti-tumor activities are being considered as potential candidates for intervention in inflammation-induced tumorigenesis. Triterpenoid saponins (TS) are glycosylated large molecules, reported to exhibit diverse bioactivities including immune modulation, inhibition of metastasis, and suppression of intestinal tract proliferative cells. Ardisiacrispin B (AB), a triterpenoid saponin extracted from ancient traditional Chinese medicinal herbs, presents anti-inflammation and antitumor activities, and has been applied to typical classical Chinese medicinal formulas for centuries.

Recent studies demonstrate that Ardisiacrispin B inhibits tumor cell proliferation and possesses antibacterial and anti-inflammatory activities [17, 18]. In addition, Ardisiacrispin B has been shown to exert anti-inflammatory effects by inhibiting pro-inflammatory mediator release in LPS-stimulated macrophages and influencing signaling pathways such as PI3K-AKT [19]. Previous studies have shown that ardisiacrispin (A + B), a mixture of ardisiacrispins A and B from Ardisia crenata, exerts direct anticancer activity in vitro by inhibiting proliferation and inducing apoptosis in human cancer cells, including Bel-7402 cells, partly through mitochondrial dysfunction and microtubule disassembly [20]. Moreover, ardisiacrispin B and other steroidal or triterpenoid saponins have been reported to trigger iron-dependent programmed cell death in cancer cells [21]. However, these studies were largely conducted in isolated tumor models or non-chronic inflammatory conditions and did not address the pathological context of colitis-associated colorectal cancer (CAC). CAC is mechanistically distinct from sporadic colorectal cancer or experimental colitis alone, as it arises from sustained intestinal inflammation and involves persistent activation of pro-inflammatory and pro-tumorigenic signaling pathways such as IL-6/STAT3 and NF-κB, together with gut microbiota dysbiosis [22]. To date, whether Ardisiacrispin B can prevent or attenuate inflammation-driven colorectal tumorigenesis and modulate the inflammatory microbial axis in CAC remains unknown. Therefore, the present study is the first to systematically investigate the protective effects of Ardisiacrispin B in an experimental model of colitis-associated cancer, with particular emphasis on its ability to suppress chronic intestinal inflammation, inhibit key pro-tumorigenic signaling pathways, and restore gut microbiota homeostasis.

2 Materials and methods

2.1 Materials and reagents

The Ardisiacrispin B (AB, 200, 116) was purchased from Chengdu Biopurify Phytochemicals Ltd. (Chengdu, China), and the positive control drug Sulfasalazine (SASP, 33, 816) was bought from MedChemExpress (New Jersey, USA). Dextran sulfate sodium (DSS, MW: 36⁓50 kDa, S5036) was acquired from MP Biomedicals (California, USA), and Azoxymethane (AOM, A5486) was purchased from Sigma-Aldrich (Saint Louis, USA). The antibodies used for western blot were from Affinity Biosciences: JAK2 (AF6022), STAT3 (AF6294), MyD88 (AF5195), β-Actin (T0022), JNK1/2/3 (AF6318), p-JNK1/2/3 (AF3318), ERK1/2 (BF8004), p-ERK1/2 (AF1015), P38 (BF8015), p-P38 (AF4001), P53 (BF8013), cleaved caspase 3 (AF7022), cleaved PARP (AF7023), BCL2 (AF6139), BAX (AF0120) and ki67 (AF0198). The TLR4 (293, 072) was purchased from ABclonal, and NF-κB (8242 s), phospho-NF-κB (p-NF-κB, 3033t), phospho-JAK2 (p-JAK2, 3771 s), and phospho-STAT3 (p-STAT3, 9145t) were bought from Cell Signal Technology.

2.2 Animal experiment design and treatment protocol

Male C57BL/6 J mice (6–8 weeks old) were obtained from Guangdong Experimental Animal Center (Foshan, China) and acclimated for one week under specific pathogen-free conditions (temperature: 22 ± 2 ℃; humidity: 70 ± 5%). All procedures were approved by the Ethics and Welfare Committee of Zhongshan Hospital of Traditional Chinese Medicine and conducted in accordance with institutional guidelines. Mice were randomly assigned to five groups (n = 8 per group) based on body weight: Control (distilled water + PBS), Model (AOM + 1.5% DSS), SASP (AOM + 1.5% DSS + sulfasalazine, 200 mg/kg, p.o.), AB low-dose (AOM + 1.5% DSS + Ardisiacrispin B 0.2 mg/kg, i.p.), and AB high-dose (AOM + 1.5% DSS + Ardisiacrispin B 0.6 mg/kg, i.p.). The selected AB doses were determined from a preliminary dose-escalation study designed to assess safety, tolerability, and potential therapeutic effects, ensuring that the doses used in this study were both effective and non-toxic. Except for the Control group, all mice received a single intraperitoneal injection of AOM (10 mg/kg) dissolved in PBS, while Control mice were injected with an equivalent volume of PBS. Seven days after azoxymethane (AOM) administration, mice received 1.5% dextran sodium sulfate (DSS) in drinking water for 7 days, followed by 14 days of distilled water; this cycle was repeated twice to induce colitis-associated cancer. AB or SASP treatments were administered according to group allocation throughout the experimental period. Body weight was recorded weekly, and disease activity index (DAI) scores were assessed throughout the experiment. On day 70, fecal samples were collected for gut microbiota analysis. Following fecal collection, mice were fasted for 8 h before sacrifice, after which tissues were systematically harvested for downstream histological, molecular, and biochemical analyses.

2.3 Disease activity index score (DAI)

Mice were monitored daily for general health and clinical signs of colitis as well as colitis-associated tumor progression. Body weight, stool consistency, and feces with blood were collected and scored based on a previous study (Table 1). These combined parameters allowed the determination of the disease activity index (DAI), which quantitatively estimated the Disease severity at all experimental time points [23].

2.4 Western blot assay

Colonic tissues were homogenized, and the total protein (40 µg) was run on 8–12% gels and transferred onto polyvinylidene fluoride (PVDF) membranes. Membranes were blocked and incubated overnight at 4 ℃ with primary antibodies against JAK2, p-JAK2, STAT3, p-STAT3, MyD88, TLR4, NF-κB, p-NF-κB, JNK1/2/3, p-JNK1/2/3, ERK1/2, p-ERK1/2, P38, p-P38, P53, cleaved PARP, BAX, BCL2, and β-Actin. Membranes were then incubated with species-specific horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature, and immunoreactive bands were detected using the ECL detection kit. Relative protein expression was calculated from densitometric analysis of protein bands using ImageJ.

2.5 Enzyme-linked immunosorbent assay (ELISA)

Colon tissues were homogenized for the assessment of cytokine levels, including IL-6, LPS, G-CSF, CXCR4, and MCP-1/CCL2. Quantification was performed using commercially available ELISA kits (Tianjin Anoric Biotechnology Co., Ltd., Tianjin, China) with the following catalog numbers: IL-6 (Cat. No.385210914), LPS (Cat. No.261210607), G-CSF (Cat. No.317220321), CXCR4 (Cat. No.894220321), and MCP-1/CCL2 (Cat. No.433220322), according to the manufacturer's instructions. Cytokine concentrations were quantified to evaluate inflammatory status and treatment effects in colitis-associated cancer mice.

2.6 Histopathology

Tumor tissues and adjacent colonic tissues were fixed in 4% paraformaldehyde for 24 h, subsequently embedded in paraffin, and dehydrated through a graded ethanol series (70–95%). Paraffin-embedded tissue Sects. (4 µm) were stained with hematoxylin and eosin (H&E) to assess histopathological alterations, including inflammatory infiltration, epithelial injury, and tumor morphology, and were imaged using an inverted microscope (Nikon Corporation, Tokyo, Japan).

2.7 Immunohistochemistry (IHC)

Immunohistochemistry was performed on 4 µm sections of paraffin-embedded colon tissues to assess the expression of NF-κB, STAT3, P53, cleaved PARP, cleaved caspase-3, and Ki67. Sections were deparaffinized, and endogenous peroxidase activity was blocked with 3% H₂O₂. Antigen retrieval was performed by heating tissue sections in 10 mM sodium citrate buffer (pH 6.0) for 10 min, followed by gradual cooling to room temperature. The slides were blocked and incubated overnight at 4 ℃ with primary antibodies (1:200, Proteintech) targeting the target proteins. Counterstaining of nuclei was performed with DAPI after incubation with secondary antibodies specific for each species. The protein expression and localization were examined using light microscopy at different magnifications, and representative images were captured.

2.8 Gut microbiota profiling

Fresh fecal specimens were collected from each experimental group and promptly preserved at− 80 ℃, followed by the extraction of genomic DNA in colon contents using the TIANamp Fecal DNA kit following the manufacturer's instructions. The integrity of extracted DNA was evaluated using 0.8% agarose gel electrophoresis, with concentration determined through a Nanodrop spectrophotometer (Thermo Scientific, New York, USA). The V3–V4 hypervariable region of the bacterial 16S rRNA gene was amplified by PCR using specific primers (5′-ACTCCTACGGGAGGCAGCA-3′ and 5′-GGACTACHVGGGTWTCTAAT-3′) with Phusion^® High-Fidelity PCR Master Mix. The amplicons were confirmed by 2% agarose gel electrophoresis, and the target fragments were extracted with a DNA gel extraction kit (AXYGEN, USA). Libraries were prepared using the TruSeq^® DNA PCR-Free Sample Preparation Kit (SanDiego, USA) according to the manufacturer's instructions with a unique 6-bp index. Sequencing of libraries was performed using an Illumina HiSeq 2500 (pair-end reads of 250 bp), and later analyzed for microbial composition and diversity.

2.9 Statistical analysis

All quantitative data are presented as mean ± standard error (SEM). Group comparisons were performed using one-way analysis of variance (ANOVA) followed by Dunnett's post hoc test to assess statistical significance. A p-value < 0.05 was considered statistically significant. Statistical analyses were conducted using SPSS version 23.

3 Result

3.1 AB reduces tumor growth in AOM/DSS-Induced CAC Mice

The antitumor and chemopreventive effects of Ardisiaspin B (AB) on colitis-associated colorectal cancer (CAC) were investigated in a murine model induced by azoxymethane (AOM)/dextran sulfate sodium (DSS). Mice in the AOM/DSS group showed obvious clinical signs of colitis, including diarrhea, rectal bleeding, and perianal swelling, all of which were significantly attenuated after treatment with AB (Fig. 1A). AOM/DSS-treated mice exhibited steady weight loss and a dramatic rise in the disease activity index (DAI) as a sign of severe inflammation in the gut during the development of disease (Fig. 1B, C). In contrast, treatments with low and high-dose AB improved body weight gain and decreased its effects comparable to those observed with Sulfasalazine (SASP). Macroscopic examination of colons from AOM/DSS-treated mice revealed shortening and multiple tumors, whereas AB treatment reduced both tumor number and size and largely preserved normal tissue morphology (Fig. 1D). Quantitative analysis indicated that AB significantly decreased the colon weight p < 0.05 (Fig. 1E), tumor number p < 0.01 (Fig. 1F), and tumor load p < 0.05 (Fig. 1G), with a smaller percentage of large tumors (> 4 mm) as compared to untreated CAC mice (Fig. 1H). These results indicate that AB effectively inhibits AOM/DSS-induced tumorigenesis and alleviates colitis symptoms, demonstrating a potent chemopreventive effect during the inflammation-to-cancer transition.

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3.2 AB dampens colonic inflammation in colitis-associated cancer

To expand our examination to tissue-level alterations associated with colitis and tumorigenesis, colon sections were subjected to microscopic analyses. Histopathological examination of the colon tissue revealed marked destruction of epithelium, crypt distortion and massive infiltration by inflammatory cells in the mucosal and submucosal layers (Fig. 2A). Interestingly, the architecture of mucosae in AB-treated mice was largely restored, with improved epithelial integrity and significantly reduced inflammatory cell infiltration in a dose-dependent manner, notably tissue morphology in the high dose treatment group closely resembled that observed in normal control morphology as depicted in Fig. 2A. Given the vital roles of iNOS and COX-2 in inflammation driven tumorigenesis, their levels in colonic tissue were assessed using western blot analysis. Findings showed a significant upregulation of iNOS and COX-2 in AOM/DSS-treated mice (p < 0.05), which were dramatically reduced after treatment with AB (p < 0.01), as shown in Fig. 2B, C. These results reveal that AB displays strong anti-inflammatory effects in CAC by protecting intestinal tissue integrity and decreasing major proinflammatory markers associated with tumorigenic inflammation.

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3.3 AB Targets JAK2/STAT3 and TLR4/NF-κB to Block CAC Progression

Ardisiacrispin B ameliorates colitis-associated inflammation by regulating the JAK2/STAT3 and TLR4/MyD88/NF-κB pathways. To further clarify the molecular mechanisms responsible for the anti-inflammatory function of Ardisiacrispin B (AB) on AOM/DSS-induced colitis, the levels of major proinflammatory mediators were detected. Colonic levels of IL-6 and LPS were evidently elevated in the AOM/DSS group than those in control mice (p < 0.01), as demonstrated by Fig. 3A, B. Following AB intervention, both low- and high-dose AB groups exhibited a significant reduction in the expression of these parameters, with IL-6 (p < 0.05) and LPS (p < 0.01) in a dose-dependent manner as compared to the positive control drug (SASP), thereby reflecting effective inhibition of both systemic and mucosal inflammation. Moreover, western blotting results showed that the JAK2/STAT3 signaling pathway was markedly activated by AOM/DSS treatment, as noted by increased phosphorylation of JAK2 and STAT3 (p < 0.05), as indicated by Fig. 3C, D. Treatment with AB effectively reversed these alterations, restoring phosphorylation levels nearly to those observed in the control group (p < 0.05). Concurrently, AB attenuated the AOM/DSS-induced upregulation of TLR4 and MyD88 expression, as well as NF-κB (p65) phosphorylation, indicating suppression of the classical TLR4/MyD88/NF-κB signaling pathway (p < 0.05) as depicted by Fig. 3E, F. Consistent with Western blot results, immunohistochemical analysis further revealed enhanced nuclear translocation of NF-κB within the colonic epithelium from AOM/DSS-treated mice, which was significantly reduced in both AB-treated groups (Fig. 3G). In parallel with tumor suppression, AB treatment significantly reduced endotoxin-associated inflammation and the expression of pro-inflammatory mediators implicated in tumor-promoting chronic inflammation. Ardisiacrispin B ameliorates colitis-associated inflammation and correlates with alterations in JAK2/STAT3 and TLR4/MyD88/NF-κB signaling. However, direct mechanistic involvement of these pathways remains to be established.

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3.4 AB inhibits CAC tumor progression via chemokines and MAPK signaling

To better elucidate the molecular mechanism underlying AB-mediated protection. Subsequently, its impact on chemokine production and MAPK signaling pathway activation in AOM/DSS-treated mice was investigated. Colonic expression of CXCR4 (p < 0.01), MCP-1/CCL2 (p < 0.05), and G-CSF (p < 0.01) was dramatically elevated in AOM/DSS-administered mice compared with the control group, as demonstrated in (Fig. 4A–C). Nevertheless, AB treatment significantly suppressed the elevated levels of these mediators, achieving effects comparable to those of SASP, thereby showing potent inhibition of chemokine-driven immune cell recruitment. Considering the important role of MAPK signaling in inflammation, the study further examined the phosphorylation levels of c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated kinases ERK1/2, and p38-MAPK. As shown in Fig. 4D, E, western blot analysis revealed marked activation of the three MAPK members in AOM/DSS-treated mice, with higher expression of p-JNK1/2/3, p-ERK1/2, and p-p38 (p < 0.05). On the other hand, the phosphorylation level of such kinases was noticeably decreased (p < 0.05) in response to AB treatment without affecting the total protein expression, suggesting that AB selectively inhibits their MAPK signal pathway. These findings indicate that Ardisiacrispin B exerts its anti-inflammatory activity, at least in part, by suppressing chemokine production and subsequent MAPK pathway activation, thereby inhibiting the downstream inflammation associated with colitis-associated carcinoma. Activated MAPK can transmit extracellular stimuli, regulate cell growth, development, and migration, and is associated with apoptosis [24].

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3.5 AB promotes apoptosis to inhibit tumor growth in CAC Mice

To investigate whether Ardisiacrispin B (AB) suppresses colitis-associated tumorigenesis through modulation of cell survival and apoptosis, the expression of apoptosis-and proliferation-related proteins in colon tissues was analyzed. Analysis of protein expression revealed that tumor suppressor P53 and pro-apoptotic proteins, cleaved PARP, and BAX were increased in AOM/DSS-treated mice compared with controls (p < 0.05), whereas anti-apoptotic protein BCL2 was decreased (Fig. 5A, B). Interestingly, AB significantly increased cleaved PARP, p53, and BAX expression (p < 0.05) while reducing BCL2 expression, reflecting a pro-apoptotic tendency at the molecular level. These findings were further evaluated and supported by immunohistochemical staining, as shown in Fig. 5C. The staining results exhibited weak cleaved PARP and P53 signals but strong nuclear Ki67 expression, indicating enhanced cellular proliferation activity. In contrast, AB-treated mice exhibited markedly increased expression of cleaved PARP, P53, and cleaved caspase-3, accompanied by a significant reduction in Ki67-positive cells, indicating enhanced apoptotic activity and suppressed cellular proliferation (Fig. 5C).

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3.6 AB modulates gut microbiota to suppress tumor growth in CAC Mice

To extend our investigation of the modulatory effects of Ardisiacrispin B (AB) on gut microbial diversity in colitis-associated cancer (CAC), 16S rRNA sequencing analysis was carried out. The Venn diagram showed that 453 Operational Taxonomic Units (OTUs) were shared between the groups, and every group was characterized by its unique microbial communities (Fig. 6A). The model group exhibited a substantial decrease in unique OTUs compared with the control, and the AB-treated groups had more similarities to the control microbiota, particularly at high doses, suggesting, that the richness of the Microbiota was partially increased. Sob index was used for α-diversity analysis. Results showed that microbial richness in the CAC group was significantly lower than that of the control (Fig. 6B). Both low and high doses of AB caused a significant increase in the Sob index, demonstrating that AB treatment could effectively restore CAC microbial loss and rise in diversity of gut microbiota.

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In addition, β-Diversity parameters were analyzed, and the results confirmed that AB treatment affected the microbial structure. Principal Coordinate Analysis (PCoA) and Partial Least Squares Discriminant Analysis (PLS-DA) plots demonstrated clear segregation between the four groups (Fig. 6C, D). The microecology of the CAC group was markedly different from that of the control group. In contrast, following AB treatment, particularly those in the high dose group, exhibited a microbial profile that clustered closer to the healthy control group, suggesting a restorative effect of AB on the gut microbial community structure. At the phylum level, Firmicutes and Bacteroidetes remained dominant taxa across all groups; however, Proteobacteria were relatively more enriched in the CAC model group, and accompanied by a notable decrease in the proportion of Bacteroidetes (Fig. 6E). These shifts were partially restored with AB treatment, restoring a more favorable microbiome profile. At the genus level, the CAC group showed strongly increased relative abundance of Firmicutes and decreased Bacteroidetes (Fig. 6F, G) as quantified. A notable decrease in Firmicutes and an increase in Bacteroidetes were noted in both AB AB-treated groups as compared to the model group, indicating that Ardisiacrispin B reverses colitis-associated dysbiosis by restoring gut microbial diversity and rebalancing microbial community composition.

3.7 Taxonomic shifts in the gut microbiota induced by Ardisiacrispin B treatment

Furthermore, the gut microbiota composition was analyzed at the genus and species level to further identify specific microbial taxa responsive to Ardisiacrispin B treatment. The potential abundance at the genus-level was significantly different between groups (Fig. 7A). DSS exposure contributed to the significant enrichment of Clostridium XIVa and Olsenella, with a simultaneous depletion of prebiotically beneficial genera, such as Lactobacillus and Akkermansia. AB administration, particularly at the high dose, partially restored the abundance of beneficial probiotic genera while reducing the prevalence of pathogenic taxa, indicating a corrective effect on dysbiosis. At the species-level, A higher abundance of Clostridium scindens and Desulfovibrio simplex was observed in CAC group mice with a lower ratio of Akkermansia muciniphila and Alcaligenes faecalis (Fig. 7B). These alterations were reversed by both low and high doses of AB, with notable restoration of Akkermansia muciniphila and Bacteroides uniformis following AB treatment. These results were further confirmed by quantitative comparisons, which revealed that the relative abundance of Akkermansia was markedly reduced in the model group and partially restored following AB treatment, indicating an improvement toward, resulting in a partial reconstitution of the gut microbial community (Fig. 7C). The levels of Bacteroides and Butyricicoccus in AB-treated groups were similarly dramatically higher than those in the model group (Fig. 7E, F), indicating that SCFA-producing bacteria had also been recovered. Additionally, LEfSe analysis identified discriminative taxa contributing to group differences (Fig. 7G). The DSS group was characterized by enrichment of Olsenella and Clostridium III, while the AB-H group was enriched in Lactobacillus, Lactobacillaceae, and Butyricicoccus. These alterations highlight that Ardisiacrispin B modulates key microbial taxa associated with mucosal health and anti-inflammatory activity, thereby contributing to its protective effects against colitis-associated dysbiosis.

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Moreover, Genus-level heatmap and hierarchical clustering analyses demonstrated a clear separation between the Control and Model groups, with the Model group exhibiting reduced abundances of beneficial bacteria and enrichment of inflammation-associated taxa. AB administration markedly reshaped the gut microbial community structure, with the AB-treated groups, particularly the AB-H group, showing a microbial profile more closely resembling that of the Control group. Specifically, AB treatment significantly increased the relative abundances of short-chain fatty acid-producing and gut barrier-supporting genera, including Ruminococcus, Lachnospiraceae_incertae_sedis, Butyricicoccus, Lactobacillus, Bifidobacterium, Alloprevotella, and Barnesiella, while reducing the abundance of potentially pathogenic and pro-inflammatory genera such as Desulfovibrio and Helicobacter as depicted in Fig. 8A. Consistent with these compositional changes, functional prediction analysis further revealed that microbial metabolic pathways were markedly altered following AB intervention. Compared with the Model group, AB-treated groups showed a significant suppression of pathways associated with lipopolysaccharide biosynthesis, bacterial invasion of epithelial cells, bacterial motility proteins, two-component systems, and infectious disease-related functions, indicating an attenuation of pro-inflammatory and pathogenic microbial activity. In parallel, pathways related to carbohydrate metabolism, energy metabolism, amino acid metabolism, and glycan biosynthesis and metabolism were significantly enriched in the AB-treated groups, with the most pronounced effects observed in the AB-H group as shown in Fig. 8B. Together, these results indicate that AB not only restores gut microbial composition but also reprograms microbial functional potential toward a metabolically beneficial and less inflammatory profile, which may contribute to its protective effects against intestinal inflammation and tumor progression.

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3.8 Proposed mechanism of Ardisiacrispin B in CAC attenuation

Based on the integrated microbiome and molecular analyses, a novel mechanistic model was suggested, which demonstrated for the first time how AB exhibited its protective effect against CAC (Fig. 8C). AB treatment restructured the gut microbiota composition, leading to the enrichment of beneficial bacteria, including Barnesiella intestinihominis and Enterorhabdus caecimuris, that contribute to mucosal barrier repair and anti-inflammatory effects. The increased/decreased gut microbial balance was accompanied by the inhibition of inflammatory signaling in intestinal epithelial cells. At the molecular level, AB suppressed the expression of TLR4/MyD88/NF-κB and JAK2/STAT3 pathways, as well as pro-inflammatory cytokines (IL-6, LPS, CXCR4 serine hydrolase), such as CCL2. Meanwhile, AB suppressed MAPK-driven oncogenic signaling and promoted apoptosis in colonic epithelial cells, accompanied by enhanced cleaved PARP/cleaved caspase-3/Bax expression and the repression of Bcl-2. By these synergically interactive mechanisms, including microbial modulation, inflammation suppression, and apoptosis induction, AB efficiently intercepts the inflammatory–dysbiosis–tumor progression axis in colitis-associated tumorigenesis.

4 Discussion

Chronic colonic inflammation of the colon leads to a complex interplay between immune dysregulation, epithelial injury, and microbial perturbation, all of which collectively contribute to tumor formation. In patients with IBD, the increased lifetime risk of CAC emphasizes the importance of persistent mucosal inflammation in shaping a microenvironment that promotes tumorigenesis [24]. A meta-analysis study of patients with UC in Asia showed a rapid rise in the incidence of colorectal cancer (CRC) around 30 years after disease diagnosis. Chronic enteritis and prolonged disease duration, extensive inflammation, disease-related complications, and dietary factors are considered the major contributors to the increased incidence of colorectal cancer in these populations [25, 26]. Nevertheless, Conventional therapies, including surgery and radiotherapy, are cytotoxic in nature and not only cause damage to the malignant cells but also affect normal proliferating tissue, a limitation that can lead to tumor regrowth. In contrast, some natural Chinese herbs exhibit favorable safety profiles and distinct pharmacological activities, with several having been investigated for their anti-tumor and anti-inflammatory properties [27, 28]. There is growing interest in developing interventions that can suppress inflammation while maintaining mucosal homeostasis and balanced gut microbiota, thereby preventing inflammatory bowel disease and colitis-associated disorders. The linkage between chronic inflammation and epithelial hyperplasia or neoplastic transformation in CAC is well recognized, as persistent intestinal inflammation has been identified as a central factor for the initiation of colorectal cancer development [29]. At present, the standard treatment for colorectal cancer is surgery, radiotherapy, chemotherapy, and immunotherapy [30, 31]. In this context, the safety of Ardisiacrispin B was considered based on preliminary dose-finding and tolerability observations conducted before the present experiments. Observations from these studies indicated that the selected doses were suitable for in vivo use, providing confidence that the therapeutic effects of Ardisiacrispin B are attributable to its pharmacological activity rather than nonspecific toxicity. Comprehensive toxicological evaluations, including long-term studies, will be valuable in future investigations to further establish its safety profile. In this study, Ardisiacrispin B (AB) significantly reduced tumor burden, preserved colonic architecture, and suppressed inflammatory enzyme activities in a murine model of AOM/DSS-induced CAC. Histopathological analysis revealed reduced inflammatory cell infiltration and restoration of crypt architecture. These results demonstrate that AB effectively interferes with inflammation-driven tumorigenesis and gut dysbiosis.

Herein, we showed that Ardisiacrispin B (AB) significantly reduced tumor load, maintained colonic architecture, and inhibited inflammatory enzyme activities in a murine model of AOM/DSS-induced CAC. Histopathological analysis showed a significant reduction in inflammatory cell infiltration accompanied by restoration of crypt architecture. These observations are in line with previous evidence indicating that repeated crypt injury and epithelial regeneration contribute to CAC development [32]. Most importantly, AB treatment significantly reduced tumor numbers and size, showing the potential for this agent to interfere with inflammation-associated carcinogenesis at various mechanistic levels.

Key signaling networks, including NF-κB and STAT3, along with pro-inflammatory mediators such as iNOS and COX-2, represent a vital mechanism through which chronic inflammation promotes neoplastic progression [33]. At a molecular level, AB significantly suppressed the expression of iNOS and COX-2, enzymes known to facilitate nitrosative stress and prostaglandin synthesis, leading to DNA damage and abnormal epithelial proliferation. Overexpression of iNOS and COX-2 is known to drive inflammation-induced tumorigenesis by promoting oxidative DNA damage, enhancing pro-survival prostaglandin signaling [33]. Similar anti-inflammatory and chemopreventive effects have been observed with other plant-derived bioactive compounds, such as polysaccharides and flavonoids. Importantly, AB exhibits a broader inhibitory effect across multiple inflammatory pathways, indicating a coordinated mechanism that can restore epithelial homeostasis and suppress localized, cancer-promoting inflammation.

High expression of IL-6 has been associated with an increased risk of CAC and activation/phosphorylation of the JAK2/STAT3 signaling pathway [34]. Upon activation, STAT3 regulates tumor cell survival and proliferation by modulating key apoptotic regulators, including BAX and BCL-2, thereby inhibiting cell death and promoting cell survival [35]. Persistent colonic inflammation culminates in tissue damage with loss of columnar and goblet cells, impaired intestinal barrier function, and gut dysbiosis. These alterations enhance the translocation of LPS across the damaged barrier, leading to enhanced local and systemic inflammatory reactions [36]. Toll-like receptor 4 (TLR4), a member of the TLR family, plays a central role in initiating pro-inflammatory responses in response to microbial stimuli. Lipopolysaccharide (LPS) activates TLR4, which recruits the adaptor protein MyD88 and subsequently triggers NF-κB signaling through enhanced phosphorylation, ultimately linking chronic inflammation with tumorigenesis [36]. Persistent activation of the IL-6/JAK2/STAT3 and TLR4/MyD88/NF-κB signaling pathways, which links chronic inflammation with tumor development key event in the pathogenesis of colitis-associated colorectal cancer (CAC). A central feature of colitis-associated colorectal cancer (CAC) IL-6/JAK2/STAT3 signaling promotes epithelial growth and suppresses anti-tumor immunity; TLR4 surveillance of microbial LPS sustains current immune activation. Our data indicate that AB is associated with downregulation of JAK2/STAT3/NF-κB phosphorylation, consistent with modulation of these pathways, which may contribute to decreased inflammatory and proliferative signals, although direct pathway-specific validation was not conducted. Inhibiting these nodes, AB may partially interfere with the feedback loops elicited by inflammation-driven carcinogenesis, although the precise pathway dependency remains to be confirmed. It should be noted that the antitumor effects observed following Ardisiacrispin B treatment are predominantly supported by indirect mechanisms, particularly through suppression of chronic, tumor-promoting intestinal inflammation. The signaling pathways inhibited in this study, including IL-6/JAK2/STAT3 and TLR4/MyD88/NF-κB, are well established as central drivers of inflammation-associated colorectal tumorigenesis and the maintenance of a tumor-permissive microenvironment, rather than as mediators of effective antitumor immune surveillance. Accordingly, the anti-inflammatory activity of Ardisiacrispin B appears to preferentially target pathological inflammatory signaling that supports tumor initiation and progression. While modulation of apoptosis-related markers was observed in vivo, these findings are indicative of pro-apoptotic tendencies rather than direct evidence of apoptosis. These findings do not provide direct evidence of tumor cell-intrinsic effects, and further mechanistic studies will be required to clarify the potential contribution of direct tumor cell regulation to the overall antitumor efficacy of Ardisiacrispin B.

The CXC chemokine receptor CXCR4, which binds stromal cell-derived factor-1 (CXCL12), is frequently expressed in several types of tumors, including breast cancer, melanoma, and colorectal carcinoma, where its abnormal expression is associated with enhanced tumor cell survival and migratory capacity [37]. In the present study, we observed that AB significantly suppressed CXCR4-related chemokines, which promoted the expression of pro-apoptotic proteins and inhibited tumor cell growth. Among the key signaling pathways involved, the mitogen-activated protein kinase (MAPK) family includes four major cascades: extracellular signal-regulated kinases 1/2 (ERK1/2), c-Jun N-terminal kinases 1/2 (JNK1/2), p38-MAPK, and ERK5 [37]. Notably, the MAPK/ERK pathway broadly participates in the regulation of cell proliferation, differentiation, migration, senescence, and apoptosis [38]. These data indicate that AB could inhibit colonic tumorigenesis by inhibiting the MAPK signaling pathway and interfering with the important oncogenic actions. Moreover, an increasing body of evidence underpins the crucial contribution of gut microbiota in shaping colonic inflammation and tumorigenesis.

Accumulating evidence indicates that gut microbiota dysbiosis can actively regulate intestinal inflammatory signaling through microbial-derived products, particularly lipopolysaccharide (LPS). In colitis and colitis-associated colorectal cancer, an increased abundance of Gram-negative bacteria elevates luminal and systemic LPS levels, which in turn activate Toll-like receptor 4 (TLR4) on intestinal epithelial and immune cells [38]. Persistent LPS–TLR4 engagement triggers MyD88-dependent NF-κB signaling, sustaining chronic inflammation, epithelial injury, and the formation of a tumor-promoting microenvironment [39]. Consistent with this concept, AB treatment reshaped the gut microbiota composition, partially restoring microbial diversity and enriching multiple genera previously linked to lower endotoxin burden and suppression of LPS-mediated inflammatory signaling, which may contribute to the anti-inflammatory and antitumor effects of AB [40]. These microbiota changes may help reduce microbial-derived LPS translocation across the impaired intestinal barrier, potentially contributing to the anti-inflammatory effects of AB, and are associated with modulation of TLR4/MyD88/NF-κB signaling and downstream pro-inflammatory pathways, although direct causality remains to be established [41]. Collectively, these findings support an integrated host–microbe mechanism in which AB-mediated modulation of gut microbiota is not merely a secondary consequence of reduced inflammation, but may actively participate in dampening LPS/TLR4-driven inflammatory cascades that contribute to colitis-associated colorectal cancer progression. AB directly suppressed pro-inflammatory signaling and subsequently mediated the gut microbiome, which further increased systemic anti-inflammatory effects. The Firmicutes to Bacteroidetes ratio plays a crucial role in the homeostasis of gut immunity; Firmicutes dominance can be associated with elevated endotoxin release and increased inflammatory cytokines. AB intervention appeared to restore this imbalance by significantly increasing Barnesiella, known as a flagellated genus that inhibits LPS-induced IFN-γ production, which inversely associates with LPS levels and is involved in the maintenance of intestinal homeostasis through the modulation of TLR4 signaling [42]. This reconfiguration of the gut microbiota is probably a result of lower endotoxin load, decreased levels of TLR4 stimulation, and associated down-regulation in inflammatory mediators, therefore establishing mechanisms for the anti-inflammatory effects of AB. These findings suggest that AB-mediated modulation of the gut microbiota may actively influence inflammatory signaling. By increasing beneficial genera such as Barnesiella and restoring the Firmicutes/Bacteroidetes balance, AB likely reduces translocation of microbial-derived LPS across the damaged intestinal barrier, thereby attenuating TLR4/MyD88/NF-κB activation. This mechanistic link indicates that changes in microbial composition may not only reflect a secondary consequence of reduced inflammation but also contribute directly to the suppression of chronic, tumor-promoting inflammatory pathways, providing an integrated host-microbe mechanism for AB's protective effects in colitis-associated colorectal cancer. This pleiotropic action indicates that AB acts not only directly as an anti-inflammatory molecule, but also as a regulator of host-microbiota interaction and epithelial signal. Natural products that exerted multi-organ regulatory effects are viewed as candidates for chemoprevention of cancers associated with inflammation [43].

Collectively, our study demonstrates that Ardisiacrispin B is an unexplored triterpenoid with dual modulation of host and microbial signaling to target the central inflammatory and microbial circuits, reprogramming the colitis-to-carcinoma progression. By parallel repair of epithelial integrity, alteration of gut microbiota profile, as well as suppression of IL-6/JAK2/STAT3 and TLR4/NF-κB signaling, AB may be a potential multitarget natural agent for the prevention and treatment of colitis-associated colorectal cancer. Although KEGG-based functional prediction indicates that Ardisiacrispin B modulates microbial metabolic pathways, a direct causal link between these microbiota changes and antitumor effects was not established. Functional validation experiments, such as fecal microbiota transplantation or metabolite profiling, will be required to determine whether these microbial shifts directly contribute to AB's therapeutic effects. Similarly, while AB significantly suppressed IL-6/JAK2/STAT3 and TLR4/MyD88/NF-κB signaling in colitis-associated colorectal cancer, the mechanistic involvement of these pathways was inferred primarily from phosphorylation and expression analyses. Direct causal validation using pathway-specific inhibitors, genetic manipulation, or rescue experiments was not performed, and future studies will be necessary to confirm pathway dependency and clarify the precise molecular targets of Ardisiacrispin B. In addition, the present study did not directly assess the effects of Ardisiacrispin B on tumor cells independent of the inflammatory microenvironment; therefore, a direct tumor cell-intrinsic effect cannot be excluded and warrants further investigation.

5 Conclusion

Ardisiacrispin B (AB), an active triterpenoid saponin isolated from medicinal plants, exhibits anti-inflammatory and antitumor properties. AB treatment is associated with improvement in disease activity and colon histology, including reduced inflammation, decreased tumor number and size, and partial restoration of epithelial architecture. Mechanistically, AB is associated with modulation of the gut microbiota, characterized by enrichment of beneficial microbes and reduction of proinflammatory bacteria; it is also linked to increased expression of pro-apoptotic markers, including cleaved PARP, caspase-3, P53, and BAX, along with reduced proliferative activity as indicated by Ki67 downregulation. In addition, AB treatment is associated with attenuation of key inflammatory and proliferative signaling pathways, such as IL-6/JAK2/STAT3, LPS/TLR4/MyD88/NF-κB, and MAPK, which may contribute to disruption of the colitis–carcinoma sequence. Taken together, these findings suggest that AB has potential as a natural multitarget agent for CAC chemoprevention, while further studies are required to establish direct causal mechanisms.

Notes

Acknowledgements

Not applicable

Author contributions

Hidayat Ullah contributed to the methodology and wrote the original draft. Huanli Cui contributed to the method. Yu Li contributed to data interpretation and manuscript revision. Weijie Peng and Binghuang Ye performed the experimental work. Weijie Peng analyzed the data and revised the manuscript. Xianjing Hu and Weibo Dai conceived and designed the study and directed manuscript development. Xianjing Hu supervised the study. Yongdui Ruan and Weibo Dai provided resources. Chunling Ma and Weijie Peng participated in manuscript revision.

Funding

State Key Laboratory of Pathogenesis, Prevention, Treatment of Central Asian High Incidence Diseases Fund, SKL-HIDCA-2025-GD1, SKL-HIDCA-2025-GD2, Natural Science Foundation of Guangdong Province, 2023A1515011116, Dongguan Science and Technology of Social Development Program, 20, 231, 800, 939, 832, Student Innovation Research and Entrepreneurship Training of Guangdong Medical University, GDMUCX2024214, GDMUCX2024168, Guangdong Medical University Undergraduate Innovation and Entrepreneurship Education Base Project, JDXM2024187, JDXM2024067F, Key Laboratory Construction Project of Dongguan Science and Technology Plan, 20, 231, 600, 401, 471, 20, 241, 600, 403, 291, Special Project for Clinical and Basic Sci &Tech Innovation of Guangdong Medical University, 4SG25302G、GDMULCJC2025132.

Data availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

All procedures were approved by the Ethics and Welfare Committee of Zhongshan Hospital of Traditional Chinese Medicine under approval number (AEWC-2021014) and conducted in accordance with institutional guidelines.

Consent for publication

All authors have read this manuscript and would like it to be considered exclusively for publication.

Competing interests

All authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest or non-financial interest in the subject matter or materials discussed in this manuscript.