Multi-Targeted Anti-Cancer Mechanisms of Li-Ginseng Powder in Colitis-Associated Colorectal Cancer: Integrating Inflammation, Apoptosis and Immunity

Multi-Targeted Anti-Cancer Mechanisms of Li-Ginseng Powder in Colitis-Associated Colorectal Cancer: Integrating Inflammation, Apoptosis and Immunity | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Multi-Targeted Anti-Cancer Mechanisms of Li-Ginseng Powder in Colitis-Associated Colorectal Cancer: Integrating Inflammation, Apoptosis and Immunity Yinghua Jin, Kwang-Il To, Hai-Lun Ye, Gang-Ao Li, Zhen-Xing Zhu, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6343012/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The incidence of colitis-associated colorectal cancer (CAC) is increasing, while conventional single-target therapies often demonstrate limited efficacy and long-term adverse effects. As a result, multi-target natural compounds have emerged as promising alternatives. This study investigates the anti-CAC potential of Li-Ginseng Powder (LGP), a specially processed functional food derived from Panax ginseng and enriched with rare ginsenosides (Rk1, Rk3, Rh4, Rg3, and Rg5), demonstrates strong preventive potential and characterized by minimal toxicity. Multi-omics analyses revealed that CAC model mice exhibited key tumor-promoting features, including heightened inflammation, impaired apoptosis, and immune suppression. Notably, LGP displayed significant anti-CAC activity and reversed 98.14% of dysregulated protein expression (fold-change > 1.5, p < 0.05). It effectively mitigated inflammation by inhibiting STAT3/NF-κB signaling and modulating inflammatory gene expression. LGP induced apoptosis by downregulating anti-apoptotic proteins (Bcl-2, Bcl-XL, and x-IAP), upregulating pro-apoptotic Bax and Granzyme B, and promoting PARP and Caspase-9 cleavage to facilitate the elimination of damaged cells. Moreover, it enhanced immune responses by increasing NK cell and CD3 + T cell infiltration while activating CD4 + and CD8 + T cells. Additionally, LGP modulated serum metabolites and gut microbiota composition, fostering a favorable disease trajectory. These findings elucidate LGP’s comprehensive anti-CAC mechanism, integrating inflammation suppression, apoptosis induction, immune modulation, and microbiota regulation. This study addresses a critical gap in ginseng-derived CAC therapies, offering a promising multi-targeted and low-risk therapeutic strategy for clinical application. Biological sciences/Cancer/Gastrointestinal cancer/Colorectal cancer Biological sciences/Cell biology Colitis-associated colorectal cancers Rare ginsenosides inflammation apoptosis immune response proteomics network pharmacology gut microbiota Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Colorectal cancer (CRC) is among the most prevalent malignancies worldwide and the third leading cause of cancer-related mortality [ 1 ] . Unlike sporadic CRC, colitis-associated colorectal cancer (CAC) follows a distinct inflammation-dysplasia-carcinoma sequence and is a major complication of inflammatory bowel disease (IBD) [ 2 ] . With the rising global incidence of IBD [ 3 ] , CAC has become an increasingly urgent public health concern [ 4 ] . Compared to sporadic CRC, CAC typically manifests at a younger age, involves more extensive colonic damage, and is associated with a poorer prognosis [ 5 ] . While conventional treatments initially show efficacy [ 6 ] , major challenges, including high chemoresistance [ 7 ] , treatment-associated toxicity, and the immunosuppressive effects of anti-inflammatory agents on tumor immunity [ 8 ] continue to hinder CAC management. Consequently, there is growing interest in identifying safe and effective therapeutic alternatives, particularly those derived from natural compounds [ 9 , 10 ] . Natural bioactive compounds, such as alkaloids and terpenoids, have demonstrated anti-inflammatory and pro-apoptotic effects against CAC [ 11 , 12 ] . However, their clinical translation remains limited due to poor bioavailability, insufficient therapeutic potency, unclear molecular targets, and a lack of robust clinical validation. In contrast, ginseng extracts, particularly ginsenosides exhibit superior bioavailability, potent bioactive metabolites, and dual anti-inflammatory and immunomodulatory properties [ 13 , 14 ] , making them promising candidates for CAC therapy. Ginsenosides, the primary bioactive constituents of ginseng, include rare ginsenosides that possess enhanced bioavailability and potent biological activity. Notably, rare ginsenosides such as Rh4 [ 15 ] and Rg3 [ 16 ] have been shown to inhibit inflammatory cascades via suppression of the NF-κB signaling pathway and NLRP3 inflammasome activation. Additionally, Rg5 has been reported to induce apoptosis and inhibit the proliferation of cancer cells [ 17 ] . Li-Ginseng Powder (LGP), a specially processed functional food, is enriched with rare ginsenosides, including Rg3, Rg5, Rk1, Rk3, and Rh4. This study explores the therapeutic potential of LGP in CAC, revealing through multi-omics analysis and conventional biological study that LGP exerts its effects by inducing apoptosis, suppressing inflammation, and modulating immune responses. Additionally, its impact on gut microbiota composition and metabolite regulation was investigated. These findings offer new insights into CAC pathophysiology and position LGP as a promising multi-target, low-toxicity therapeutic strategy. Results 1. LGP ameliorates pathological damage and tumorigenesis in AOM/DSS-induced CAC mice To evaluate the therapeutic potential of LGP against CAC, we employed an azoxymethane/dextran sulfate sodium (AOM/DSS)-induced murine model. Severe body weight loss was observed in both the AOM/DSS model and treatment groups following each DSS cycle. Notably, LGP administration, particularly at high doses, significantly attenuated DSS-induced weight loss ( Figure 1 B). Disease activity index (DAI) scores exhibited a trend consistent with body weight changes (Figure. S3). Importantly, the AOM/DSS model group exhibited pronounced tumorigenesis and colon shortening ( Figure 1 C). LGP treatment led to a significant restoration of colon length and inhibition of tumor formation ( Figure 1 D, E). Histopathological examination via hematoxylin and eosin (H&E) staining revealed severe structural disruption and mucosal damage in the AOM/DSS group, characterized by submucosal connective tissue damage and crypt deformation ( Figure 1 F). In contrast, LGP-treated mice exhibited reduced mucosal lesions, improved crypt integrity, and significantly fewer dysplastic and adenomatous formations, particularly in the high-dose group, highlighting its protective role against CAC-induced histopathological alterations. 2. Identification of Core Targets and Pathway via Network Pharmacology To explore LGP’s molecular targets, network pharmacology analysis was conducted. A total of 348 putative LGP targets intersected with 709 known CAC-related genes, yielding 49 shared targets ( Figure 2 A). Protein-protein interaction (PPI) network analysis using Cytoscape identified key regulatory nodes, including TNF-α, CASP3, IL-6, and STAT3 ( Figure 2 B, C). To further investigate the signaling pathways and biological processes associated with the selected key genes, we performed Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) pathway analyses. KEGG analysis revealed significant enrichment of key targets in the NF-κB signaling pathway, apoptosis pathway，TNF signaling pathway, inflammatory bowel disease (IBD), JAK-STAT signaling pathway ( Figure 2 D). Similarly, GO analysis indicated that the anti-CAC targets of LGP were primarily involved in apoptosis regulation, nitric oxide biosynthesis, inflammatory response, IL-6 expression regulation, NF-κB binding ( Figure 2 E). 3. Proteomic Analysis to Elucidate Targets and Key Enrichment Pathways in LGP-Mediated Inhibition of CAC Proteomic profiling of colon tissues from LGP-treated mice was performed to further elucidate its molecular effects. Principal component analysis (PCA) demonstrated that the global protein expression patterns in LGP-treated groups closely resembled those of healthy controls ( Figure 3 A). A heatmap of differentially expressed proteins further substantiated this observation, showing a trend toward normalization ( Figure 3 B). Specifically, LGP treatment restored 98.14% of the abnormally expressed proteins in the AOM/DSS model (Figure S4). Gene Set Enrichment Analysis (GSEA) based on Gene Ontology (GO) annotations revealed that inflammatory bowel disease (IBD)-related pathways were significantly upregulated in the AOM/DSS model group but markedly downregulated upon LGP treatment ( Figure 3 C). LGP administration also inhibited NF-κB signaling and suppressed TNF family cytokine production ( Figure 3 D). Apoptosis-related protein expression, which was significantly downregulated in the AOM/DSS group, was restored following LGP treatment, aligning with the network pharmacology predictions ( Figure 3 E). Furthermore, GSEA indicated suppression of both innate and adaptive immune pathways in the AOM/DSS group, particularly those governing lymphocyte and NK cell activity. LGP treatment reinstated immune regulatory functions, reversing the suppression of innate, adaptive, and humoral immune responses ( Figure 3 F, G). KEGG pathway analysis revealed enrichment in complement cascade pathways post-LGP treatment, underscoring its role in immune modulation ( Figure 3 H). 4. LGP Suppresses Inflammation in Colon Tissue via Inhibition of the STAT3/NF-κB Pathway To determine whether the anti-inflammatory effects of LGP in AOM/DSS-induced CAC are associated with the regulation of inflammatory cytokines, we quantified the levels of IL-6, TNF-α, and IL-1β in colon tissues using ELISA. LGP treatment significantly reduced the expression of these pro-inflammatory cytokines compared to the AOM/DSS model group ( Figure 4 A). Consistently, RT-PCR analysis revealed a marked downregulation of IL-6, TNF-α, and IL-1β mRNA levels following LGP treatment, with the most pronounced effects observed in the high-dose group ( Figure 4 B). Inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) are key enzymes involved in the synthesis of nitric oxide (NO) and prostaglandin E2 (PGE2), respectively, both of which contribute to inflammation and tumorigenesis. Western blot analysis demonstrated a significant upregulation of iNOS and COX-2 in the AOM/DSS model group, whereas their expression was markedly suppressed in both low- and high-dose LGP treatment groups ( Figure 4 C). Furthermore, LGP treatment effectively reduced the elevated levels of NO and PGE2 observed in the AOM/DSS model group ( Figure 4 D), consistent with predictions from network pharmacology. To elucidate the molecular mechanism underlying LGP’s anti-inflammatory effects, we examined key components of the NF-κB and STAT3 signaling pathways in colon tissue. Western blot analysis revealed that phosphorylation levels of NF-κB p50 (Ser337) were significantly elevated in the AOM/DSS model group but were strongly suppressed by LGP treatment in a dose-dependent manner. Additionally, we assessed phosphorylation levels of IκBα, an upstream regulator of NF-κB ( Figure 4 E), as well as STAT3 (Tyr705). LGP treatment significantly downregulated the phosphorylation of both proteins compared to the AOM/DSS model group ( Figure 4 F). These findings confirm that LGP inhibits inflammation in CAC mice by suppressing the STAT3/NF-κB signaling axis. 5. LGP Potently Induces Apoptosis in Colon Tissue of CAC Mice To assess LGP’s pro-apoptotic effects, TUNEL staining revealed a significant increase in apoptotic cells in LGP-treated colon tissues, with the high-dose group exhibiting the most pronounced effect ( Figure 5 A). Western blot and immunohistochemistry (IHC) analyses demonstrated increased PARP cleavage and caspase-3 activation following LGP treatment ( Figure 5 B, C). Additionally, anti-apoptotic proteins Bcl-2, Bcl-xL, cIAP-2, and x-IAP were upregulated in AOM/DSS model mice, indicating impaired apoptosis. LGP treatment significantly downregulated these proteins while upregulating the pro-apoptotic protein Bax and cleaved caspase-9, indicating caspase-9-mediated apoptosis activation ( Figure 5 D-F). LGP treatment also markedly reduced Ki-67 expression, a proliferation marker highly upregulated in the AOM/DSS group, suggesting that LGP inhibits excessive proliferation while promoting apoptosis ( Figure 5 G). 6. LGP Potently Enhances the Immune Response in CAC Mice Gene Set Enrichment Analysis (GSEA) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that LGP treatment restored both innate and adaptive immune functions in CAC mice while suppressing negative immune regulation. Western blot (WB) and immunohistochemistry (IHC) analyses demonstrated a significant upregulation of granzyme B (GZMB), a pro-apoptotic protein derived from cytotoxic immune cells, in LGP-treated mice, whereas its expression was markedly reduced in the AOM/DSS model group ( Figure 6 A). These findings indicate severe immune impairment in the model group, which was effectively reversed by LGP treatment, leading to immune system reactivation and therapeutic involvement in CAC progression. To further investigate these immunomodulatory effects, we performed flow cytometric analysis of immune cell populations in colon tissues. LGP treatment significantly increased the proportion of NK cells and activated CD69+ NK cells ( Figure 6 B). Additionally, the percentage of CD3+ T cells in the colon was markedly elevated, with a dose-dependent enhancement observed in the high-dose LGP group. Notably, high-dose LGP administration resulted in a significant increase in both the proportion and absolute number of CD4+ and CD8+ T cells compared to the AOM/DSS model group, with CD8+ T cells and their activated subsets increasing by more than 200% ( Figure 6 C). To assess the impact of LGP on humoral immunity, we conducted H&E staining of spleens from CAC mice across different groups. In the AOM/DSS model group, the white pulp appeared atrophic with disrupted architecture, poorly defined margins, and the absence of germinal centers. In contrast, LGP-treated mice exhibited a notable restoration of spleen architecture, characterized by an expanded white pulp and a well-defined marginal zone, with the most pronounced recovery observed in the high-dose group. Furthermore, fluorescence labeling revealed a significant reduction in B cell populations within the spleens of AOM/DSS model mice, which was effectively reversed following LGP treatment ( Figure 6 D). These findings suggest that LGP plays a pivotal role in restoring immune homeostasis and enhancing B cell-mediated immune responses in colitis-associated colorectal cancer. 7. LGP Modulates Intestinal Microbiota Composition and Metabolite Profiles in CAC Mice Colitis-associated colorectal cancer (CAC) is closely linked to alterations in gut microbiota composition. To investigate these changes, stool samples were collected at the end of the experiment, and comprehensive analyses of gut microbiota and metabolite profiles were performed. As illustrated in Figure 7 A, significant differences were observed between the AOM/DSS group and the LGP-treated groups across multiple parameters. To further characterize these changes, community composition analysis was conducted (Figure. S5). Gate-level taxonomic profiling revealed an increased abundance of Bacteroides in the LGP-treated groups compared to the AOM/DSS model group, whereas the relative abundance of sessile bacteria was reduced ( Figure 7 B). Specifically, the genera Turicibacter , Alistipes , and Helicobacter were significantly elevated in the AOM/DSS group compared to both LGP-treated groups. In contrast, beneficial taxa such as Bifidobacterium , Akkermansia muciniphila , Roseburia intestinalis , and Parabacteroides distasonis exhibited increased abundance following LGP administration. Notably, Butyricimonas levels were elevated in the low-dose group, while Butyricicoccus pullicaecorum and Lactobacillus were significantly enriched in the high-dose group ( Figure 7 C). Metabolomic profiling revealed substantial metabolic perturbations in the AOM/DSS model group, as indicated by the pronounced separation from the control group in principal component analysis. In contrast, LGP treatment mitigated these disturbances, with the metabolomic profiles of LGP-treated mice exhibiting greater similarity to those of the control group (Figure. S6). Specifically, 15 differential metabolites, including creatine and L-aspartic acid, were identified when comparing the AOM/DSS and low-dose LGP groups ( Figure 7 D), while 14 metabolites, including creatine and azelaic acid, were differentially expressed between the AOM/DSS and high-dose LGP groups ( Figure 7 E). Further correlation analysis between differential microbiota and metabolite expression indicated strong associations between specific microbial taxa and key metabolites. Notably, creatine exhibited significant negative and positive correlations with specific bacterial genera, while L-aspartic acid and azelaic acid displayed similarly distinct association patterns (Figure. S6). Moreover, these differentially abundant microbial taxa and metabolites demonstrated strong correlations with apoptosis, inflammatory responses, and immune modulation pathways ( Figure 7 F). These findings suggest that LGP treatment beneficially alters gut microbiota composition and metabolic profiles, thereby exerting a protective effect in AOM/DSS-induced CAC mice. Materials and methods 1 . Mice Male C57BL/6 mice (7-8 weeks old, SPF grade) were purchased from Changsheng Biotechnology (Liaoning, China). The animals were housed in the Animal Experiment Center of the College of Life Sciences, Jilin University, China. The conditions are SPF level, free access to food and water, the temperature is 22±2°C and the humidity is 50±5%. Mice were randomized and then experimented as planned in Figure.1A. Intragastric administration is administered daily at a low dose of 167 mg/kg and a high dose of 334 mg/kg 2. Li-ginseng Powder （ LGP ） Li-ginseng Powder is a specially processed ginseng provided by Yanbian Anti Kanghua Biotechnology Development Company. which is rich in rare ginsenosides Rh4, Rg3, Rg5, Rk1 and Rk3, accounting for 60% of the total saponins (Figure. S1, S2). 3. Histopathological analysis Fresh colon tissue was fixed in 4% paraformaldehyde for 24 h. After fixation, dehydrate colonic tissue with gradient sucrose solution (15%, 20%, 25%) and incubate at 4°C for 24 h. Immobilized colonic segments were embedded in paraffin using standard procedures and 5 μm sections were stained with H&E. Histopathological changes in colon tissue were observed using an Olympus microscope (Tokyo, Japan). 4. Western blot Assay Intestinal tissue (25mg) was added to 800 ml RIPA lysate, and homogenized by tissue homogenizer for 55min. After centrifugation and boiling, the samples were added to the SDS-PAGE gel for electrophoresis. After transferring the protein to the PVDF membrane, blocking for 1 hour, the protein bands were detected by ECL after the primary and secondary antibody incubation was completed, and the protein levels were quantified using ImageJ. All experiments were performed at least 3 times, and the mean values were compared. 5. ELISA assays levels of IL-6, TNF-α, IL-1β, and PGE2. Place the colon tissue sample (30 mg) in a pre-chilled 5 mL EP tube and add 900 μl of precast RIPA lysate containing protease inhibitors to the EP tube. Homogenize the sample and place it on ice for 20 min one last time. Transfer the homogenate to a 1.5 mL EP tube, centrifuge at 12,000 rpm, 4°C for 20 minutes, and collect the supernatant. The levels of IL-6, IL-1β, TNF-α, and PGE2 in the supernatant were measured according to the manufacturer's instructions of the ELISA kit (CLOUD-CLONE CORP., Wuhan, China). 6. TUNEL Assay Sections were mounted on poly-L-lysine-coated glass slides. Sections were incubated in 20 μg/ml proteinase K in PBS for 15 min at room temperature, washed with double distilled water, and treated with 3% H 2 O 2 in PBS for 5 min at room temperature to block endogenous peroxidase. Then 50 μL labeling reaction mixture consisting of 5 μL TdT enzyme (Takara Bio Inc., Shiga, Japan) + 45 μL Labeling Safe buffer (Takara Bio Inc., Shiga, Japan) prepared and cooled on ice before use was applied to the sections and incubated for 90 min at 37 °C. The reaction was terminated by washing the slides three times in PBS for 5 min each. Staining was visualized using DAB as chromogen and sections were counterstained with hematoxylin. 7. Proteomic analysis 20mg of colon tissue from 3 mice in AOM/DSS group, CTRL group and LGP high-dose group were randomly selected, stored in liquid nitrogen, transported on dry ice, and detected and analyzed by Jingjie Biotechnology. 8. Flow cytometry analysis The intestinal tissue from which the mucus has been removed is minced in a mixture of 1 mg/ml of collagenase I and collagenase IV. The intestinal tissue was placed at 37°C, 5% CO2 environment and cultured for 1h. After centrifugation, the pellet is resuspended with 1640 medium containing 2.5% fetal bovine. The resuspended cells were stained with the desired dye and then entered into the flow cytometer for detection. The test results were analyzed using CytExpert software. 9. Correlation analysis of gut microbiome-serum metabolome Pearson correlation or Spearman rank correlation is used to calculate the correlation and p-value between two histological data. The correlation coefficient and p-value for each species-metabolite pair were calculated and considered to be significantly correlated with the cut-off value of p≤1E-3. Heat maps based on these data show significant positive or significant negative correlations between metabolites and the microbiome. 10. Statistical Analysis Experimental data were obtained from independent triple-replicate experiments and were expressed as mean ± standard deviation (mean ± SD). GraphPad Prism 8 software was used for statistical analysis, and Student t-test statistical analysis was used for comparison between groups, and P<0.5 indicated that the difference between groups was statistically significant. Discussion In this study, we observed that LGP treatment significantly increased colonic length and markedly reduced tumor burden in AOM/DSS-induced CAC mice, indicating its potential to suppress the progression of inflammatory colorectal cancer. Integrative multi-omics analyses combining network pharmacology and proteomics, validated through subsequent experiments, revealed that LGP exerts its anti-CAC effects primarily by modulating inflammatory responses, promoting apoptosis, activating immune responses, and regulating gut microbiota. The application of multi-omics approaches has become a standard strategy for elucidating the mechanisms of anti-cancer drug [ 18 , 19 ] . Network pharmacology effectively identifies potential targets and mechanisms of multi-component drugs [ 20 ] , while proteomics provides insight into protein composition and dynamic alterations within biological systems [ 21 ] , enhancing our understanding of disease mechanisms and therapeutic interventions. In this study, we employed a multi-omics strategy to delineate the therapeutic targets and molecular mechanisms underlying LGP's anti-CAC effects. Network pharmacology analysis identified TNF-α, IL-6, CASP3, and STAT3 as key targets mediating LGP's effects in CAC, implicating pathways involved in apoptosis regulation, inflammatory response, cell proliferation, and NF-κB signaling. Proteomics analysis revealed that LGP-treated CAC mice exhibited protein expression profiles closely resembling those of healthy controls, suggesting a restoration of intestinal homeostasis. Gene Set Enrichment Analysis (GSEA) based on the Gene Ontology (GO) database demonstrated that LGP significantly downregulated inflammatory bowel disease (IBD)-associated proteins, which were markedly upregulated in the model group. LGP effectively suppressed inflammatory responses while restoring apoptotic processes, as evidenced by the activation of apoptosis. Furthermore, the immunosuppressive effects observed in the model group, characterized by inhibited innate and adaptive immune responses, were reversed by LGP treatment. Specifically illustrated in Figure. S7. proteins involved in the suppression of adaptive, humoral, and innate immunity were significantly downregulated following LGP administration, highlighting its immunomodulatory potential. The interplay between inflammation and cancer has garnered significant attention, particularly in the context of colitis-associated colorectal cancer (CAC) [ 22 – 25 ] . Chronic inflammation is a critical driver of CAC pathogenesis [ 2 , 26 ] , and mitigating inflammatory responses is a pivotal factor influencing therapeutic outcomes. This study demonstrated that LGP significantly inhibits inflammatory responses in CAC mice by suppressing the STAT3/NF-κB signaling pathway. Given the established role of NF-κB in colorectal carcinogenesis, the observed suppression of NF-κB and STAT3 activation by LGP supports its anti-inflammatory and anti-tumorigenic properties. Key bioactive components of LGP, such as ginsenoside Rh4, (20S) G-Rh2, Rg5, and Rk1 have been previously reported to exert anti-inflammatory effects by targeting NF-κB and STAT3 signaling pathways [ 13 , 17 , 27 ] . The pathogenesis of CAC is driven by multiple interconnected pathways, among which apoptosis is a fundamental mechanism of tumor suppression [ 28 , 29 ] . LGP effectively induced endogenous apoptosis in CAC mice, as evidenced by TUNEL assay results showing a significant increase in apoptotic cell populations following LGP treatment. Further analysis revealed that LGP promoted apoptosis by enhancing caspase-3 activation and inducing PARP cleavage. Additionally, LGP downregulated anti-apoptotic proteins (Bcl-2, Bcl-xL, x-IAP, and cIAP-2) while upregulating the pro-apoptotic protein Bax and cleaved caspase-9, confirming the activation of the intrinsic apoptotic pathway. These findings highlight LGP’s role in suppressing tumorigenesis by promoting apoptosis to eliminate AOM/DSS-induced damaged cells. Immune system regulation plays a crucial role in cancer therapy, as it facilitates tumor cell recognition and elimination [ 30 , 31 ] . Accumulating evidence supports the significance of immunomodulation in colorectal cancer (CRC) treatment [ 24 , 32 ] . Our findings demonstrated that LGP significantly enhanced immune activation in the AOM/DSS-induced CAC model. Western blot analysis revealed that LGP treatment restored Granzyme B (GZMB) levels, a key marker of cytotoxic lymphocyte activation [ 33 ] , indicating enhanced anti-tumor immune responses. Flow cytometry analysis further confirmed that LGP promoted immune cell infiltration in colonic tissues, particularly NK cells, CD3 + T cells, and tumor-associated macrophages (TAMs). Notably, LGP exhibited dose-dependent effects on immune activation. Low-dose LGP primarily enhanced NK cell and TAM activity, whereas high-dose LGP more effectively activated CD4 + and CD8 + T cells. B cell activation also followed a similar pattern, with high-dose LGP promoting B cell enrichment and differentiation, as evidenced by increased CD19 + cell populations and improved germinal center structure in the spleen. Notably, we observed that LGP induces distinct macrophage polarization patterns depending on its administered concentration. This dose-dependent effect warrants further in-depth investigation (Figure.S8).These findings highlight LGP’s dual role in modulating both innate and adaptive immunity, reinforcing its potential as an immunotherapeutic agent against CAC. Gut microbiota dysbiosis is increasingly recognized as a contributing factor to CAC pathogenesis [ 34 , 35 ] . Our study revealed significant alterations in microbial composition following LGP treatment, with reductions in pathogenic bacteria such as Helicobacter pylori, which is known to induce chronic inflammation and impair immune surveillance [ 36 , 37 ] . Additionally, LGP treatment increased azelaic acid levels, a metabolite associated with anti-inflammatory and pro-apoptotic effects. These findings suggest that LGP exerts its anti-CAC effects, in part, by modulating the gut microbiota and its associated metabolic pathways. Given the complexity of tumorigenesis, effective cancer treatment necessitates a multifaceted approach [ 38 , 39 ] . Chronic inflammation promotes genetic mutations, immune evasion, and tumor microenvironment alterations, increasing the risk of malignancy [ 23 , 38 , 40 ] . Inducing apoptosis provides a critical defense against potentially malignant cells [ 41 ] . The timely removal of cells with genetic damage or mutations is crucial for preventing cancer by eliminating precancerous lesions. Similarly, immune activation plays a vital role in cancer suppression by enhancing the immune system’s ability to recognize and eliminate tumor cells, thus providing a robust tumor surveillance mechanism [ 42 – 44 ] . Our findings demonstrate that LGP suppresses CAC through a multi-pronged mechanism: reducing inflammation, inducing apoptosis, enhancing immune responses, and modulating gut microbiota composition. This integrative strategy not only enhances therapeutic efficacy but also mitigates cancer recurrence risks. In addition, according to the commissioned acute toxicity test report, the LD 50 of LGP was not detected, and administering approximately 134 times the standard LGP (H) treatment dose (22.9 g/kg body weight) had no adverse effects on mice. Therefore, LGP, enriched with rare ginsenosides and exhibiting minimal side effects, may serve as an effective medicinal natural product for CAC therapy. Future studies are warranted to further elucidate the precise molecular mechanisms underlying LGP’s effects on gut microbiota regulation and epithelial-mesenchymal transition (EMT) (Figure.S9). Additionally, the observed dose-dependent immunomodulatory effects necessitate further exploration to optimize therapeutic regimens. Conclusion This study offers novel insights into the therapeutic potential of ginseng-derived products for CAC treatment by elucidating the molecular mechanisms through which LGP regulates apoptosis, inflammation, immunity, and gut microbiota. Our findings support the development of natural, low-toxicity therapies for inflammatory colorectal cancer. Moreover, this research contributes to the modernization of traditional Chinese medicine and provides promising strategies for preventing and treating inflammation-associated cancers. Declarations Funding : This work was supported by Specific Funding of Development and Reform Commission of Jilin Province [grant numbers 2021FGWCXNLJSSZ01] and The Leading Team of the Changbai Mountain Talent Engineering Project (grant numbers 000009). Conflict of interest: The authors declare no competing interests. Ethics approval and consent to participate: This study was approved by the Institutional Animal Care and Use Committee (IACUC) of Jilin University (Approval Number: S2021006). Consent for publication: All authors have seen and approved the final version of the manuscript being submitted. All authors warrant that the article is the authors' original work, hasn't received prior publication and isn't under consideration for publication elsewhere. Data availability: The authors confirm that the data supporting the findings of this study are available within the article or its supplementary materials. For more specific data requirements, please contact Ying-Hua Jin. Materials availability: Correspondence and requests for materials should be addressed to Ying-Hua Jin. Code availability : Not applicable. Author contribution: Ying-Hua Jin conceived the concept, designed the experiments, revised the manuscript，and provided fund support. Kwang-Il To and Hai-Lun Ye were responsible for designing and conducting the experiments. Gang-Ao Li, Zhen-Xing Zhu and Ya-Ni Wang provided Network pharmacology analysis. Xing-Hui Jin, Xin-Hao Cai and Shi-Yin Zhang contributed to the analysis of omics data. Yao-Yang Ma, Xing-Chen Zhu, Xiao-Shi Zheng offered experimental support for molecular and animal studies. Yang Li provided essential resources and experimental guidance for the study. Kwang-Il To and Hai-Lun Ye drafted and revised the manuscript. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6343012","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":461461819,"identity":"f7c578d3-7dbf-45e1-adc8-6a7f70347632","order_by":0,"name":"Yinghua Jin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuElEQVRIiWNgGAWjYDACCYaEAwwVbDIQNvFazrDxkKSFgYGxjYEELQa3Gx4eLpzHx2NwgPngbR4GuzzCWu4cSDg8cxsbUAtbsjUPQ3IxYS03EhIO84K18JhJ8zAcSGwgTssckBb+b6RoaQDbwkacFkmQFp5jbDySh9mMLecYJBPWwncjJ/kzT80xOb7jzQ9vvKmwI6xF4QBPApA6xsDADHYnIfVAIN/AfgBI1RChdBSMglEwCkYsAAAOqTsX0qnoWQAAAABJRU5ErkJggg==","orcid":"","institution":"jilin university","correspondingAuthor":true,"prefix":"","firstName":"Yinghua","middleName":"","lastName":"Jin","suffix":""},{"id":461461820,"identity":"96db3379-ad15-4792-a82c-719ecde3fbc2","order_by":1,"name":"Kwang-Il To","email":"","orcid":"","institution":"jilin university","correspondingAuthor":false,"prefix":"","firstName":"Kwang-Il","middleName":"","lastName":"To","suffix":""},{"id":461461821,"identity":"1fb3c46c-6204-487d-abb7-23a5ed2f8b23","order_by":2,"name":"Hai-Lun Ye","email":"","orcid":"","institution":"jilin university","correspondingAuthor":false,"prefix":"","firstName":"Hai-Lun","middleName":"","lastName":"Ye","suffix":""},{"id":461461822,"identity":"af8a3a00-ba5e-4290-ad8e-b555515e8004","order_by":3,"name":"Gang-Ao Li","email":"","orcid":"","institution":"jilin university","correspondingAuthor":false,"prefix":"","firstName":"Gang-Ao","middleName":"","lastName":"Li","suffix":""},{"id":461461823,"identity":"c46e58a9-05af-40a6-ac0a-6cdaea98f035","order_by":4,"name":"Zhen-Xing Zhu","email":"","orcid":"","institution":"jilin university","correspondingAuthor":false,"prefix":"","firstName":"Zhen-Xing","middleName":"","lastName":"Zhu","suffix":""},{"id":461461824,"identity":"2774d0db-6d9d-4e70-93eb-18ca6d39d9ad","order_by":5,"name":"Ya-Ni Wang","email":"","orcid":"","institution":"jilin university","correspondingAuthor":false,"prefix":"","firstName":"Ya-Ni","middleName":"","lastName":"Wang","suffix":""},{"id":461461825,"identity":"ef5ff961-750f-4404-a7f4-8f3ec28085c1","order_by":6,"name":"Xing-Hui Jin","email":"","orcid":"","institution":"jilin university","correspondingAuthor":false,"prefix":"","firstName":"Xing-Hui","middleName":"","lastName":"Jin","suffix":""},{"id":461461826,"identity":"ac71a7b7-3303-481c-841b-8683aec45d97","order_by":7,"name":"Xin-Hao Cai","email":"","orcid":"","institution":"jilin university","correspondingAuthor":false,"prefix":"","firstName":"Xin-Hao","middleName":"","lastName":"Cai","suffix":""},{"id":461461827,"identity":"855d5597-d16c-4961-acf2-34b86213e6ce","order_by":8,"name":"Shi-Yin Zhang","email":"","orcid":"","institution":"jilin university","correspondingAuthor":false,"prefix":"","firstName":"Shi-Yin","middleName":"","lastName":"Zhang","suffix":""},{"id":461461828,"identity":"3fb1f824-cf7f-4e31-9c39-2cb405f43851","order_by":9,"name":"Yao-Yang Ma","email":"","orcid":"","institution":"jilin university","correspondingAuthor":false,"prefix":"","firstName":"Yao-Yang","middleName":"","lastName":"Ma","suffix":""},{"id":461461829,"identity":"82aafba9-8e63-4110-9117-b65ffe35a605","order_by":10,"name":"Xing-Chen Zhu","email":"","orcid":"","institution":"jilin university","correspondingAuthor":false,"prefix":"","firstName":"Xing-Chen","middleName":"","lastName":"Zhu","suffix":""},{"id":461461830,"identity":"31375032-69cb-41fc-8b1c-a1f717a5f570","order_by":11,"name":"Xiao-Shi Zheng","email":"","orcid":"","institution":"jilin university","correspondingAuthor":false,"prefix":"","firstName":"Xiao-Shi","middleName":"","lastName":"Zheng","suffix":""},{"id":461461831,"identity":"0a810466-89dd-48ec-acd6-192a4f02d05c","order_by":12,"name":"Yang Li","email":"","orcid":"","institution":"jilin university","correspondingAuthor":false,"prefix":"","firstName":"Yang","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2025-03-31 08:35:40","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6343012/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6343012/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":83532874,"identity":"6942af0d-a630-4940-8782-01694fbcc860","added_by":"auto","created_at":"2025-05-28 05:34:00","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1342569,"visible":true,"origin":"","legend":"\u003cp\u003eLGP significantly ameliorates pathological damage and tumorigenesis in AOM/DSS-induced CAC mice.\u003c/p\u003e\n\u003cp\u003e(A) Experimental design for AOM/DSS-induced CAC model development. DSS, Dextran sodium sulfate (2.5%); AOM, Azomethane (10 mg/kg), (B) AOM/DSS-induced weight changes in CAC mice (n=8), (C) Pathology of the colon, (D) Change in tumor incidence(n=8), (E) Change in colon length(n=8), (F) H\u0026amp;E staining of colon tissue. Data are presented as the mean of SD values performed in triplicate± (##p \u0026lt; 0.01 vs. control group, *p \u0026lt; 0.05, **p \u0026lt; 0.01 vs. AOM/DSS group).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6343012/v1/e5ab7bfec51a69b26c1d9629.png"},{"id":83532873,"identity":"b852d14a-64e6-493e-89fa-177273985077","added_by":"auto","created_at":"2025-05-28 05:34:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1026840,"visible":true,"origin":"","legend":"\u003cp\u003eNetwork pharmacology predicts potential targets and key pathways involved in the inhibition of CAC by LGP.\u003c/p\u003e\n\u003cp\u003e(A) Forty-nine overlapping target proteins between CAC-related proteins and targets of LGP. (B) Protein-protein networks of overlapping 49 target proteins. Edges: Interactions between protein(s) and protein(s). (C) Network topology analysis of LGP to treat Colitis-associated colorectal cancer. (D) KEGG pathway analysis of the potential target genes of LGP against Colitis-associated colorectal cancer. (E) Gene Ontology analysis of the target genes of LGP against Colitis-associated colorectal cancer.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6343012/v1/422fdcbda289afa0b376f115.png"},{"id":83533444,"identity":"8fd5e7e0-a3c8-49d7-a24d-ba8b86f53b3a","added_by":"auto","created_at":"2025-05-28 05:42:00","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":445341,"visible":true,"origin":"","legend":"\u003cp\u003eProteomic analysis revealed changes in protein expression of key pathways involved in the inhibition of CAC process by LGP.\u003c/p\u003e\n\u003cp\u003e(A) 2D- PCA distribution of colon tissue protein in healthy control group, AOM/DSS group and LGP treatment group. (B) Differential protein heat map of colon tissue in healthy control group, AOM/DSS group, and LGP-treated group. (C) The GSEA analysis of inflammatory bowel disease pathways based on the GO database. (D) The GSEA analysis of inflammation regulation-related pathways based on the GO database. (E) The GSEA analysis of Apoptosis pathways based on the GO database. (F) The GSEA analysis of immunity regulation-related pathways based on the GO database. (G) Heat map of differential proteins enriched in the immunity negative regulation. (H) Bubble diagram of differential proteins in the LGP-treated group relative to the AOM/DSS group. (Red is up, blue is down.)\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6343012/v1/d9fda7b7e062871243b20314.png"},{"id":83532877,"identity":"24b06f51-696e-440a-ae2d-3e704a08684f","added_by":"auto","created_at":"2025-05-28 05:34:00","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":373262,"visible":true,"origin":"","legend":"\u003cp\u003eLGP directly inhibits the NF-kB and STAT3 pathways to attenuate inflammation.\u003c/p\u003e\n\u003cp\u003e(A) LGP treatment inhibits the expression of pro-inflammatory cytokines, (B) LGP treatment inhibits mRNA expression of pro-inflammatory factors, (C) LGP effects on iNOS and COX-2 expression, (D) LGP effects on NO and PGE2 release, (E) determination of p-IκBα (Ser32) and p-p50 using Western blot analysis (Ser337) protein, (F) Effect of LGP on AOM/DSS-induced STAT-3 activation in mice. Data are presented as average SD values performed in triplicate± (##p \u0026lt; 0.01 and ###p \u0026lt; 0.001 vs. control, *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001 vs. AOM/DSS group).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6343012/v1/cdfa6bab3d087a81cef22149.png"},{"id":83533446,"identity":"fd76eab2-71d3-4346-8d9e-6af848051072","added_by":"auto","created_at":"2025-05-28 05:42:01","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1546945,"visible":true,"origin":"","legend":"\u003cp\u003eLGP promotes apoptosis and inhibits malignant proliferation\u003c/p\u003e\n\u003cp\u003e(A) Effect of LGP on apoptosis in colon tissues of CAC mice measured by TUNEL, (B) Effect of LGP on PARP cleavage, (C) Up-regulation of caspase-3 expression in lysed caspase-3 after LGP treatment, and (D) changes in expression of Bcl-2 family proteins after LGP treatment. (E) Changes in the expression of x-IAP family proteins after LGP treatment(F) WB assay of cleaved Caspase-9 and Caspase-9, (G) WB analysis and IHC staining of Ki-67 in colorectal tissue. Data were presented as the average of SD values performed in triplicate± (ns p\u0026gt;0.05 no significance, **p \u0026lt; 0.01 vs AOM/DSS group).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6343012/v1/7bdf314b591b4a860bf855af.png"},{"id":83532885,"identity":"22c82e6b-b841-4163-b098-118d5d07bc13","added_by":"auto","created_at":"2025-05-28 05:34:01","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1562361,"visible":true,"origin":"","legend":"\u003cp\u003eImmune response activated by LGP treatment in AOM/DSS mice\u003c/p\u003e\n\u003cp\u003e(A) WB and IHC staining results of granzyme B changes after LGP treatment, and (B) Enrichment and activation of NK cells in colon tissue of CAC mice after LGP treatment, (C) Enrichment and activation of CD4\u003csup\u003e+ \u003c/sup\u003eT cells and CD8\u003csup\u003e+\u003c/sup\u003e T cells in colon tissues of CAC mice after LGP treatment, (D) HE staining of B cell-producing regions in the spleen of CAC mice after LGP treatment and IHC results.\u0026nbsp; Data is presented as the average ± SD value performed in triplicate (*p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001 vs. AOM/DSS group).\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6343012/v1/5e13bfea64d19c5057e61083.png"},{"id":83534378,"identity":"75af767d-7647-45e5-9751-29f4668488d5","added_by":"auto","created_at":"2025-05-28 06:06:35","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":630635,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in microbial communities and metabolites in CAC mice after LGP administration\u003c/p\u003e\n\u003cp\u003e(A) Comparison of α diversity indices between the four groups of specimens, (B) Community bar graph at gate level, and (C) Microbial community heat map analysis at the species level(D), Heat map of the relative abundance of differential metabolites after LGP treatment (AOM/DSS-Low) (E) Heat map of the relative abundance of differential metabolites after LGP treatment (AOM/DSS-High). Red is up, blue is down. (F) Differential microbiota and potential associations of differential metabolites with apoptosis, inflammatory responses, and immune responses. Data are presented as the average of the SD values performed in triplicate± (*p \u0026lt; 0.05 vs. AOM/DSS group).\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-6343012/v1/5ced07f2101d46ac782bf901.png"},{"id":83534938,"identity":"de1a5ae3-1a12-45c4-af4e-a25b41fc0e34","added_by":"auto","created_at":"2025-05-28 06:14:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7314797,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6343012/v1/e1b6568a-4830-4af5-b3f2-2b7358a0f59c.pdf"},{"id":83533445,"identity":"d00868d6-7c03-4ca4-aef5-b9b383911640","added_by":"auto","created_at":"2025-05-28 05:42:00","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":5536652,"visible":true,"origin":"","legend":"supplement material","description":"","filename":"supplementmaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-6343012/v1/5fb3c3f6589a2b3ae3c0b674.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Multi-Targeted Anti-Cancer Mechanisms of Li-Ginseng Powder in Colitis-Associated Colorectal Cancer: Integrating Inflammation, Apoptosis and Immunity","fulltext":[{"header":"Introduction","content":"\u003cp\u003eColorectal cancer (CRC) is among the most prevalent malignancies worldwide and the third leading cause of cancer-related mortality\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Unlike sporadic CRC, colitis-associated colorectal cancer (CAC) follows a distinct inflammation-dysplasia-carcinoma sequence and is a major complication of inflammatory bowel disease (IBD) \u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. With the rising global incidence of IBD\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e, CAC has become an increasingly urgent public health concern\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Compared to sporadic CRC, CAC typically manifests at a younger age, involves more extensive colonic damage, and is associated with a poorer prognosis\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. While conventional treatments initially show efficacy\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e, major challenges, including high chemoresistance\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e, treatment-associated toxicity, and the immunosuppressive effects of anti-inflammatory agents on tumor immunity\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e continue to hinder CAC management. Consequently, there is growing interest in identifying safe and effective therapeutic alternatives, particularly those derived from natural compounds\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eNatural bioactive compounds, such as alkaloids and terpenoids, have demonstrated anti-inflammatory and pro-apoptotic effects against CAC\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. However, their clinical translation remains limited due to poor bioavailability, insufficient therapeutic potency, unclear molecular targets, and a lack of robust clinical validation. In contrast, ginseng extracts, particularly ginsenosides exhibit superior bioavailability, potent bioactive metabolites, and dual anti-inflammatory and immunomodulatory properties\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e, making them promising candidates for CAC therapy.\u003c/p\u003e \u003cp\u003eGinsenosides, the primary bioactive constituents of ginseng, include rare ginsenosides that possess enhanced bioavailability and potent biological activity. Notably, rare ginsenosides such as Rh4\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e and Rg3\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e have been shown to inhibit inflammatory cascades via suppression of the NF-κB signaling pathway and NLRP3 inflammasome activation. Additionally, Rg5 has been reported to induce apoptosis and inhibit the proliferation of cancer cells\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eLi-Ginseng Powder (LGP), a specially processed functional food, is enriched with rare ginsenosides, including Rg3, Rg5, Rk1, Rk3, and Rh4. This study explores the therapeutic potential of LGP in CAC, revealing through multi-omics analysis and conventional biological study that LGP exerts its effects by inducing apoptosis, suppressing inflammation, and modulating immune responses. Additionally, its impact on gut microbiota composition and metabolite regulation was investigated. These findings offer new insights into CAC pathophysiology and position LGP as a promising multi-target, low-toxicity therapeutic strategy.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e1. LGP ameliorates pathological damage and tumorigenesis in AOM/DSS-induced CAC mice\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the therapeutic potential of LGP against CAC, we employed an azoxymethane/dextran sulfate sodium (AOM/DSS)-induced murine model. Severe body weight loss was observed in both the AOM/DSS model and treatment groups following each DSS cycle. Notably, LGP administration, particularly at high doses, significantly attenuated DSS-induced weight loss (\u003cstrong\u003eFigure 1\u003c/strong\u003eB). Disease activity index (DAI) scores exhibited a trend consistent with body weight changes (Figure. S3).\u003c/p\u003e\n\u003cp\u003eImportantly, the AOM/DSS model group exhibited pronounced tumorigenesis and colon shortening (\u003cstrong\u003eFigure 1\u003c/strong\u003eC). LGP treatment led to a significant restoration of colon length and inhibition of tumor formation (\u003cstrong\u003eFigure 1\u003c/strong\u003eD, E). Histopathological examination via hematoxylin and eosin (H\u0026amp;E) staining revealed severe structural disruption and mucosal damage in the AOM/DSS group, characterized by submucosal connective tissue damage and crypt deformation (\u003cstrong\u003eFigure 1\u003c/strong\u003eF). In contrast, LGP-treated mice exhibited reduced mucosal lesions, improved crypt integrity, and significantly fewer dysplastic and adenomatous formations, particularly in the high-dose group, highlighting its protective role against CAC-induced histopathological alterations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2. Identification of Core Targets and Pathway via Network Pharmacology\u003c/strong\u003e\u003cbr\u003eTo explore LGP\u0026rsquo;s molecular targets, network pharmacology analysis was conducted. A total of 348 putative LGP targets intersected with 709 known CAC-related genes, yielding 49 shared targets (\u003cstrong\u003eFigure 2\u003c/strong\u003eA). Protein-protein interaction (PPI) network analysis using Cytoscape identified key regulatory nodes, including TNF-\u0026alpha;, CASP3, IL-6, and STAT3 (\u003cstrong\u003eFigure 2\u003c/strong\u003eB, C). To further investigate the signaling pathways and biological processes associated with the selected key genes, we performed Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) pathway analyses. KEGG analysis revealed significant enrichment of key targets in the NF-\u0026kappa;B signaling pathway, apoptosis pathway，TNF signaling pathway, inflammatory bowel disease (IBD), JAK-STAT signaling pathway (\u003cstrong\u003eFigure 2\u003c/strong\u003eD). Similarly, GO analysis indicated that the anti-CAC targets of LGP were primarily involved in apoptosis regulation, nitric oxide biosynthesis, inflammatory response, IL-6 expression regulation, NF-\u0026kappa;B binding (\u003cstrong\u003eFigure 2\u003c/strong\u003eE).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. Proteomic Analysis to Elucidate Targets and Key Enrichment Pathways in LGP-Mediated Inhibition of CAC\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProteomic profiling of colon tissues from LGP-treated mice was performed to further elucidate its molecular effects. Principal component analysis (PCA) demonstrated that the global protein expression patterns in LGP-treated groups closely resembled those of healthy controls (\u003cstrong\u003eFigure 3\u003c/strong\u003eA). A heatmap of differentially expressed proteins further substantiated this observation, showing a trend toward normalization (\u003cstrong\u003eFigure 3\u003c/strong\u003eB). Specifically, LGP treatment restored 98.14% of the abnormally expressed proteins in the AOM/DSS model (Figure S4).\u003c/p\u003e\n\u003cp\u003eGene Set Enrichment Analysis (GSEA) based on Gene Ontology (GO) annotations revealed that inflammatory bowel disease (IBD)-related pathways were significantly upregulated in the AOM/DSS model group but markedly downregulated upon LGP treatment (\u003cstrong\u003eFigure 3\u003c/strong\u003eC). LGP administration also inhibited NF-\u0026kappa;B signaling and suppressed TNF family cytokine production (\u003cstrong\u003eFigure 3\u003c/strong\u003eD). Apoptosis-related protein expression, which was significantly downregulated in the AOM/DSS group, was restored following LGP treatment, aligning with the network pharmacology predictions (\u003cstrong\u003eFigure 3\u003c/strong\u003eE).\u003c/p\u003e\n\u003cp\u003eFurthermore, GSEA indicated suppression of both innate and adaptive immune pathways in the AOM/DSS group, particularly those governing lymphocyte and NK cell activity. LGP treatment reinstated immune regulatory functions, reversing the suppression of innate, adaptive, and humoral immune responses (\u003cstrong\u003eFigure 3\u003c/strong\u003eF, G). KEGG pathway analysis revealed enrichment in complement cascade pathways post-LGP treatment, underscoring its role in immune modulation (\u003cstrong\u003eFigure 3\u003c/strong\u003eH).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eLGP Suppresses Inflammation in Colon Tissue via Inhibition of the STAT3/NF-\u0026kappa;B Pathway\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine whether the anti-inflammatory effects of LGP in AOM/DSS-induced CAC are associated with the regulation of inflammatory cytokines, we quantified the levels of IL-6, TNF-\u0026alpha;, and IL-1\u0026beta; in colon tissues using ELISA. LGP treatment significantly reduced the expression of these pro-inflammatory cytokines compared to the AOM/DSS model group (\u003cstrong\u003eFigure 4\u003c/strong\u003eA). Consistently, RT-PCR analysis revealed a marked downregulation of IL-6, TNF-\u0026alpha;, and IL-1\u0026beta; mRNA levels following LGP treatment, with the most pronounced effects observed in the high-dose group (\u003cstrong\u003eFigure 4\u003c/strong\u003eB).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eInducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) are key enzymes involved in the synthesis of nitric oxide (NO) and prostaglandin E2 (PGE2), respectively, both of which contribute to inflammation and tumorigenesis. Western blot analysis demonstrated a significant upregulation of iNOS and COX-2 in the AOM/DSS model group, whereas their expression was markedly suppressed in both low- and high-dose LGP treatment groups (\u003cstrong\u003eFigure 4\u003c/strong\u003eC). Furthermore, LGP treatment effectively reduced the elevated levels of NO and PGE2 observed in the AOM/DSS model group (\u003cstrong\u003eFigure 4\u003c/strong\u003eD), consistent with predictions from network pharmacology.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo elucidate the molecular mechanism underlying LGP\u0026rsquo;s anti-inflammatory effects, we examined key components of the NF-\u0026kappa;B and STAT3 signaling pathways in colon tissue. Western blot analysis revealed that phosphorylation levels of NF-\u0026kappa;B p50 (Ser337) were significantly elevated in the AOM/DSS model group but were strongly suppressed by LGP treatment in a dose-dependent manner. Additionally, we assessed phosphorylation levels of I\u0026kappa;B\u0026alpha;, an upstream regulator of NF-\u0026kappa;B (\u003cstrong\u003eFigure 4\u003c/strong\u003eE), as well as STAT3 (Tyr705). LGP treatment significantly downregulated the phosphorylation of both proteins compared to the AOM/DSS model group (\u003cstrong\u003eFigure 4\u003c/strong\u003eF). These findings confirm that LGP inhibits inflammation in CAC mice by suppressing the STAT3/NF-\u0026kappa;B signaling axis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5. LGP Potently Induces Apoptosis in Colon Tissue of CAC Mice\u003c/strong\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess LGP\u0026rsquo;s pro-apoptotic effects, TUNEL staining revealed a significant increase in apoptotic cells in LGP-treated colon tissues, with the high-dose group exhibiting the most pronounced effect (\u003cstrong\u003eFigure 5\u003c/strong\u003eA). Western blot and immunohistochemistry (IHC) analyses demonstrated increased PARP cleavage and caspase-3 activation following LGP treatment (\u003cstrong\u003eFigure 5\u003c/strong\u003eB, C). Additionally, anti-apoptotic proteins Bcl-2, Bcl-xL, cIAP-2, and x-IAP were upregulated in AOM/DSS model mice, indicating impaired apoptosis. LGP treatment significantly downregulated these proteins while upregulating the pro-apoptotic protein Bax and cleaved caspase-9, indicating caspase-9-mediated apoptosis activation (\u003cstrong\u003eFigure 5\u003c/strong\u003eD-F).\u003c/p\u003e\n\u003cp\u003eLGP treatment also markedly reduced Ki-67 expression, a proliferation marker highly upregulated in the AOM/DSS group, suggesting that LGP inhibits excessive proliferation while promoting apoptosis (\u003cstrong\u003eFigure 5\u003c/strong\u003eG).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6. LGP Potently Enhances the Immune Response in CAC Mice\u003c/strong\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGene Set Enrichment Analysis (GSEA) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that LGP treatment restored both innate and adaptive immune functions in CAC mice while suppressing negative immune regulation. Western blot (WB) and immunohistochemistry (IHC) analyses demonstrated a significant upregulation of granzyme B (GZMB), a pro-apoptotic protein derived from cytotoxic immune cells, in LGP-treated mice, whereas its expression was markedly reduced in the AOM/DSS model group (\u003cstrong\u003eFigure 6\u003c/strong\u003eA). These findings indicate severe immune impairment in the model group, which was effectively reversed by LGP treatment, leading to immune system reactivation and therapeutic involvement in CAC progression.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo further investigate these immunomodulatory effects, we performed flow cytometric analysis of immune cell populations in colon tissues. LGP treatment significantly increased the proportion of NK cells and activated CD69+ NK cells (\u003cstrong\u003eFigure 6\u003c/strong\u003eB). Additionally, the percentage of CD3+ T cells in the colon was markedly elevated, with a dose-dependent enhancement observed in the high-dose LGP group. Notably, high-dose LGP administration resulted in a significant increase in both the proportion and absolute number of CD4+ and CD8+ T cells compared to the AOM/DSS model group, with CD8+ T cells and their activated subsets increasing by more than 200% (\u003cstrong\u003eFigure 6\u003c/strong\u003eC).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo assess the impact of LGP on humoral immunity, we conducted H\u0026amp;E staining of spleens from CAC mice across different groups. In the AOM/DSS model group, the white pulp appeared atrophic with disrupted architecture, poorly defined margins, and the absence of germinal centers. In contrast, LGP-treated mice exhibited a notable restoration of spleen architecture, characterized by an expanded white pulp and a well-defined marginal zone, with the most pronounced recovery observed in the high-dose group. Furthermore, fluorescence labeling revealed a significant reduction in B cell populations within the spleens of AOM/DSS model mice, which was effectively reversed following LGP treatment (\u003cstrong\u003eFigure 6\u003c/strong\u003eD). These findings suggest that LGP plays a pivotal role in restoring immune homeostasis and enhancing B cell-mediated immune responses in colitis-associated colorectal cancer.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e7. LGP Modulates Intestinal Microbiota Composition and Metabolite Profiles in CAC Mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eColitis-associated colorectal cancer (CAC) is closely linked to alterations in gut microbiota composition. To investigate these changes, stool samples were collected at the end of the experiment, and comprehensive analyses of gut microbiota and metabolite profiles were performed. As illustrated in\u0026nbsp;\u003cstrong\u003eFigure 7\u003c/strong\u003eA, significant differences were observed between the AOM/DSS group and the LGP-treated groups across multiple parameters.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo further characterize these changes, community composition analysis was conducted (Figure. S5). Gate-level taxonomic profiling revealed an increased abundance of Bacteroides in the LGP-treated groups compared to the AOM/DSS model group, whereas the relative abundance of sessile bacteria was reduced (\u003cstrong\u003eFigure 7\u003c/strong\u003eB). Specifically, the genera \u003cem\u003eTuricibacter\u003c/em\u003e, \u003cem\u003eAlistipes\u003c/em\u003e, and \u003cem\u003eHelicobacter\u003c/em\u003e were significantly elevated in the AOM/DSS group compared to both LGP-treated groups. In contrast, beneficial taxa such as \u003cem\u003eBifidobacterium\u003c/em\u003e, \u003cem\u003eAkkermansia muciniphila\u003c/em\u003e, \u003cem\u003eRoseburia intestinalis\u003c/em\u003e, and \u003cem\u003eParabacteroides distasonis\u003c/em\u003e exhibited increased abundance following LGP administration. Notably, \u003cem\u003eButyricimonas\u003c/em\u003e levels were elevated in the low-dose group, while \u003cem\u003eButyricicoccus pullicaecorum\u003c/em\u003e and \u003cem\u003eLactobacillus\u003c/em\u003e were significantly enriched in the high-dose group (\u003cstrong\u003eFigure 7\u003c/strong\u003eC). Metabolomic profiling revealed substantial metabolic perturbations in the AOM/DSS model group, as indicated by the pronounced separation from the control group in principal component analysis. In contrast, LGP treatment mitigated these disturbances, with the metabolomic profiles of LGP-treated mice exhibiting greater similarity to those of the control group (Figure. S6). Specifically, 15 differential metabolites, including creatine and L-aspartic acid, were identified when comparing the AOM/DSS and low-dose LGP groups (\u003cstrong\u003eFigure 7\u003c/strong\u003eD), while 14 metabolites, including creatine and azelaic acid, were differentially expressed between the AOM/DSS and high-dose LGP groups (\u003cstrong\u003eFigure 7\u003c/strong\u003eE).\u003c/p\u003e\n\u003cp\u003eFurther correlation analysis between differential microbiota and metabolite expression indicated strong associations between specific microbial taxa and key metabolites. Notably, creatine exhibited significant negative and positive correlations with specific bacterial genera, while L-aspartic acid and azelaic acid displayed similarly distinct association patterns (Figure. S6). Moreover, these differentially abundant microbial taxa and metabolites demonstrated strong correlations with apoptosis, inflammatory responses, and immune modulation pathways (\u003cstrong\u003eFigure 7\u003c/strong\u003eF).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThese findings suggest that LGP treatment beneficially alters gut microbiota composition and metabolic profiles, thereby exerting a protective effect in AOM/DSS-induced CAC mice.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003cstrong\u003e. Mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMale\u0026nbsp;C57BL/6 mice (7-8 weeks old, SPF grade) were purchased from Changsheng Biotechnology (Liaoning, China). The animals were housed in the Animal Experiment Center of the College of Life Sciences, Jilin University, China. The conditions are SPF level, free access to food and water, the temperature is 22\u0026plusmn;2\u0026deg;C and the humidity is 50\u0026plusmn;5%.\u003c/p\u003e\n\u003cp\u003eMice were randomized and then experimented as planned in Figure.1A. Intragastric administration is administered daily at a low dose of 167 mg/kg and a high dose of 334 mg/kg\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2. Li-ginseng Powder\u003c/strong\u003e\u003cstrong\u003e（\u003c/strong\u003e\u003cstrong\u003eLGP\u003c/strong\u003e\u003cstrong\u003e）\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLi-ginseng Powder is a specially processed ginseng provided by Yanbian Anti Kanghua Biotechnology Development Company. which is rich in rare ginsenosides Rh4, Rg3, Rg5, Rk1 and Rk3, accounting for 60% of the total saponins (Figure. S1, S2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. Histopathological analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFresh colon tissue\u0026nbsp;was fixed in 4% paraformaldehyde for 24 h. After fixation, dehydrate colonic tissue with gradient sucrose solution (15%, 20%, 25%) and incubate at 4\u0026deg;C for 24 h. Immobilized colonic segments were embedded in paraffin using standard procedures and 5 \u0026mu;m sections were stained with H\u0026amp;E. Histopathological changes in colon tissue were observed using an Olympus microscope (Tokyo, Japan).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eWestern blot Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIntestinal tissue (25mg) was added to\u0026nbsp;800\u0026nbsp;ml RIPA lysate, and homogenized by tissue homogenizer for 55min. After centrifugation and boiling, the samples were added to the SDS-PAGE gel for electrophoresis. After transferring the protein to the PVDF membrane, blocking for 1 hour, the protein bands were detected by ECL after the primary and secondary antibody incubation was completed, and the protein levels were quantified using ImageJ. All experiments were performed at least 3 times, and the mean values were compared.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eELISA assays levels of IL-6, TNF-\u0026alpha;, IL-1\u0026beta;, and PGE2.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePlace the colon tissue sample (30 mg) in a pre-chilled 5 mL EP tube and add 900 \u0026mu;l of precast RIPA lysate containing protease inhibitors to the EP tube. Homogenize the sample and place it on ice for 20 min one last time. Transfer the homogenate to a 1.5 mL EP tube, centrifuge at 12,000 rpm, 4\u0026deg;C for 20 minutes, and collect the supernatant. The levels of IL-6, IL-1\u0026beta;, TNF-\u0026alpha;, and PGE2 in the supernatant were measured according to the manufacturer\u0026apos;s instructions of the ELISA kit (CLOUD-CLONE CORP., Wuhan, China).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6. TUNEL Assay\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSections were mounted on poly-L-lysine-coated glass slides. Sections were incubated in 20 \u0026mu;g/ml proteinase K in PBS for 15 min at room temperature, washed with double distilled water, and treated with 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in PBS for 5 min at room temperature to block endogenous peroxidase. Then 50 \u0026mu;L labeling reaction mixture consisting of 5 \u0026mu;L TdT enzyme (Takara Bio Inc., Shiga, Japan) + 45 \u0026mu;L Labeling Safe buffer (Takara Bio Inc., Shiga, Japan) prepared and cooled on ice before use was applied to the sections and incubated for 90 min at 37 \u0026deg;C. The reaction was terminated by washing the slides three times in PBS for 5 min each. Staining was visualized using DAB as chromogen and sections were counterstained with hematoxylin.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e7. Proteomic analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e20mg of colon tissue from 3 mice in AOM/DSS group, CTRL group and LGP high-dose group were randomly selected, stored in liquid nitrogen, transported on dry ice, and detected and analyzed by Jingjie Biotechnology.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e8. Flow cytometry analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe intestinal tissue from which the mucus has been removed is minced in a mixture of 1 mg/ml of collagenase I and collagenase IV. The intestinal tissue was placed at 37\u0026deg;C, 5% CO2 environment and cultured for 1h. After centrifugation, the pellet is resuspended with 1640 medium containing 2.5% fetal bovine. The resuspended cells were stained with the desired dye and then entered into the flow cytometer for detection. The test results were analyzed using CytExpert software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e9. Correlation analysis of gut microbiome-serum metabolome\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePearson correlation or Spearman rank correlation is used to calculate the correlation and p-value between two histological data. The correlation coefficient and p-value for each species-metabolite pair were calculated and considered to be significantly correlated with the cut-off value of p\u0026le;1E-3. Heat maps based on these data show significant positive or significant negative correlations between metabolites and the microbiome.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e10. Statistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExperimental data were obtained from independent triple-replicate experiments and were expressed as mean \u0026plusmn; standard deviation (mean \u0026plusmn; SD). GraphPad Prism 8 software was used for statistical analysis, and Student t-test statistical analysis was used for comparison between groups, and P\u0026lt;0.5 indicated that the difference between groups was statistically significant.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we observed that LGP treatment significantly increased colonic length and markedly reduced tumor burden in AOM/DSS-induced CAC mice, indicating its potential to suppress the progression of inflammatory colorectal cancer. Integrative multi-omics analyses combining network pharmacology and proteomics, validated through subsequent experiments, revealed that LGP exerts its anti-CAC effects primarily by modulating inflammatory responses, promoting apoptosis, activating immune responses, and regulating gut microbiota.\u003c/p\u003e \u003cp\u003eThe application of multi-omics approaches has become a standard strategy for elucidating the mechanisms of anti-cancer drug\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Network pharmacology effectively identifies potential targets and mechanisms of multi-component drugs\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e, while proteomics provides insight into protein composition and dynamic alterations within biological systems\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e, enhancing our understanding of disease mechanisms and therapeutic interventions. In this study, we employed a multi-omics strategy to delineate the therapeutic targets and molecular mechanisms underlying LGP's anti-CAC effects. Network pharmacology analysis identified TNF-α, IL-6, CASP3, and STAT3 as key targets mediating LGP's effects in CAC, implicating pathways involved in apoptosis regulation, inflammatory response, cell proliferation, and NF-κB signaling. Proteomics analysis revealed that LGP-treated CAC mice exhibited protein expression profiles closely resembling those of healthy controls, suggesting a restoration of intestinal homeostasis.\u003c/p\u003e \u003cp\u003eGene Set Enrichment Analysis (GSEA) based on the Gene Ontology (GO) database demonstrated that LGP significantly downregulated inflammatory bowel disease (IBD)-associated proteins, which were markedly upregulated in the model group. LGP effectively suppressed inflammatory responses while restoring apoptotic processes, as evidenced by the activation of apoptosis. Furthermore, the immunosuppressive effects observed in the model group, characterized by inhibited innate and adaptive immune responses, were reversed by LGP treatment. Specifically illustrated in Figure. S7. proteins involved in the suppression of adaptive, humoral, and innate immunity were significantly downregulated following LGP administration, highlighting its immunomodulatory potential.\u003c/p\u003e \u003cp\u003eThe interplay between inflammation and cancer has garnered significant attention, particularly in the context of colitis-associated colorectal cancer (CAC) \u003csup\u003e[\u003cspan additionalcitationids=\"CR23 CR24\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. Chronic inflammation is a critical driver of CAC pathogenesis\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e, and mitigating inflammatory responses is a pivotal factor influencing therapeutic outcomes. This study demonstrated that LGP significantly inhibits inflammatory responses in CAC mice by suppressing the STAT3/NF-κB signaling pathway. Given the established role of NF-κB in colorectal carcinogenesis, the observed suppression of NF-κB and STAT3 activation by LGP supports its anti-inflammatory and anti-tumorigenic properties. Key bioactive components of LGP, such as ginsenoside Rh4, (20S) G-Rh2, Rg5, and Rk1 have been previously reported to exert anti-inflammatory effects by targeting NF-κB and STAT3 signaling pathways\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe pathogenesis of CAC is driven by multiple interconnected pathways, among which apoptosis is a fundamental mechanism of tumor suppression\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. LGP effectively induced endogenous apoptosis in CAC mice, as evidenced by TUNEL assay results showing a significant increase in apoptotic cell populations following LGP treatment. Further analysis revealed that LGP promoted apoptosis by enhancing caspase-3 activation and inducing PARP cleavage. Additionally, LGP downregulated anti-apoptotic proteins (Bcl-2, Bcl-xL, x-IAP, and cIAP-2) while upregulating the pro-apoptotic protein Bax and cleaved caspase-9, confirming the activation of the intrinsic apoptotic pathway. These findings highlight LGP\u0026rsquo;s role in suppressing tumorigenesis by promoting apoptosis to eliminate AOM/DSS-induced damaged cells.\u003c/p\u003e \u003cp\u003eImmune system regulation plays a crucial role in cancer therapy, as it facilitates tumor cell recognition and elimination\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. Accumulating evidence supports the significance of immunomodulation in colorectal cancer (CRC) treatment\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. Our findings demonstrated that LGP significantly enhanced immune activation in the AOM/DSS-induced CAC model. Western blot analysis revealed that LGP treatment restored Granzyme B (GZMB) levels, a key marker of cytotoxic lymphocyte activation\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e, indicating enhanced anti-tumor immune responses. Flow cytometry analysis further confirmed that LGP promoted immune cell infiltration in colonic tissues, particularly NK cells, CD3\u0026thinsp;+\u0026thinsp;T cells, and tumor-associated macrophages (TAMs). Notably, LGP exhibited dose-dependent effects on immune activation. Low-dose LGP primarily enhanced NK cell and TAM activity, whereas high-dose LGP more effectively activated CD4\u0026thinsp;+\u0026thinsp;and CD8\u0026thinsp;+\u0026thinsp;T cells. B cell activation also followed a similar pattern, with high-dose LGP promoting B cell enrichment and differentiation, as evidenced by increased CD19\u0026thinsp;+\u0026thinsp;cell populations and improved germinal center structure in the spleen. Notably, we observed that LGP induces distinct macrophage polarization patterns depending on its administered concentration. This dose-dependent effect warrants further in-depth investigation (Figure.S8).These findings highlight LGP\u0026rsquo;s dual role in modulating both innate and adaptive immunity, reinforcing its potential as an immunotherapeutic agent against CAC.\u003c/p\u003e \u003cp\u003eGut microbiota dysbiosis is increasingly recognized as a contributing factor to CAC pathogenesis\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. Our study revealed significant alterations in microbial composition following LGP treatment, with reductions in pathogenic bacteria such as Helicobacter pylori, which is known to induce chronic inflammation and impair immune surveillance\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e. Additionally, LGP treatment increased azelaic acid levels, a metabolite associated with anti-inflammatory and pro-apoptotic effects. These findings suggest that LGP exerts its anti-CAC effects, in part, by modulating the gut microbiota and its associated metabolic pathways.\u003c/p\u003e \u003cp\u003eGiven the complexity of tumorigenesis, effective cancer treatment necessitates a multifaceted approach\u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e. Chronic inflammation promotes genetic mutations, immune evasion, and tumor microenvironment alterations, increasing the risk of malignancy\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e. Inducing apoptosis provides a critical defense against potentially malignant cells\u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e. The timely removal of cells with genetic damage or mutations is crucial for preventing cancer by eliminating precancerous lesions. Similarly, immune activation plays a vital role in cancer suppression by enhancing the immune system\u0026rsquo;s ability to recognize and eliminate tumor cells, thus providing a robust tumor surveillance mechanism\u003csup\u003e[\u003cspan additionalcitationids=\"CR43\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e. Our findings demonstrate that LGP suppresses CAC through a multi-pronged mechanism: reducing inflammation, inducing apoptosis, enhancing immune responses, and modulating gut microbiota composition. This integrative strategy not only enhances therapeutic efficacy but also mitigates cancer recurrence risks. In addition, according to the commissioned acute toxicity test report, the LD\u003csub\u003e50\u003c/sub\u003e of LGP was not detected, and administering approximately 134 times the standard LGP (H) treatment dose (22.9 g/kg body weight) had no adverse effects on mice. Therefore, LGP, enriched with rare ginsenosides and exhibiting minimal side effects, may serve as an effective medicinal natural product for CAC therapy.\u003c/p\u003e \u003cp\u003eFuture studies are warranted to further elucidate the precise molecular mechanisms underlying LGP\u0026rsquo;s effects on gut microbiota regulation and epithelial-mesenchymal transition (EMT) (Figure.S9). Additionally, the observed dose-dependent immunomodulatory effects necessitate further exploration to optimize therapeutic regimens.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study offers novel insights into the therapeutic potential of ginseng-derived products for CAC treatment by elucidating the molecular mechanisms through which LGP regulates apoptosis, inflammation, immunity, and gut microbiota. Our findings support the development of natural, low-toxicity therapies for inflammatory colorectal cancer. Moreover, this research contributes to the modernization of traditional Chinese medicine and provides promising strategies for preventing and treating inflammation-associated cancers.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e: This work was supported by Specific Funding of Development and Reform Commission of Jilin Province [grant numbers 2021FGWCXNLJSSZ01] and The Leading Team of the Changbai Mountain Talent Engineering Project (grant numbers 000009).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest: \u003c/strong\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate:\u003c/strong\u003e This study was approved by the Institutional Animal Care and Use Committee (IACUC) of Jilin University (Approval Number: S2021006).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u003c/strong\u003e All authors have seen and approved the final version of the manuscript being submitted. All authors warrant that the article is the authors\u0026apos; original work, hasn\u0026apos;t received prior publication and isn\u0026apos;t under consideration for publication elsewhere.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability:\u003c/strong\u003e The authors confirm that the data supporting the findings of this study are available within the article or its supplementary materials. For more specific data requirements, please contact Ying-Hua Jin.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterials availability:\u003c/strong\u003e Correspondence and requests for materials should be addressed to Ying-Hua Jin.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode availability\u003c/strong\u003e: Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution: \u003c/strong\u003eYing-Hua Jin conceived the concept, designed the experiments, revised the manuscript，and provided fund support. Kwang-Il To and Hai-Lun Ye were responsible for designing and conducting the experiments. Gang-Ao Li, Zhen-Xing Zhu and Ya-Ni Wang provided Network pharmacology analysis. Xing-Hui Jin, Xin-Hao Cai and Shi-Yin Zhang contributed to the analysis of omics data. Yao-Yang Ma, Xing-Chen Zhu, Xiao-Shi Zheng offered experimental support for molecular and animal studies. Yang Li provided essential resources and experimental guidance for the study. Kwang-Il To and Hai-Lun Ye drafted and revised the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eDeDecker, L., Coppedge, B., Avelar-Barragan, J.\u003cem\u003e, et al.\u003c/em\u003e (2021) Microbiome distinctions between the CRC carcinogenic pathways. \u003cem\u003eGut Microbes\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, e1854641.\u003c/li\u003e\n\u003cli\u003eRogler, G. 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(2011) Cancer Immunoediting: Integrating Immunity\u0026apos;s Roles in Cancer Suppression and Promotion. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e331\u003c/strong\u003e, 1565-1570.\u003c/li\u003e\n\u003cli\u003eBinnewies, M., Roberts, E.W., Kersten, K.\u003cem\u003e, et al.\u003c/em\u003e (2018) Understanding the tumor immune microenvironment (TIME) for effective therapy. \u003cem\u003eNat Med\u003c/em\u003e \u003cstrong\u003e24\u003c/strong\u003e, 541-550.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Colitis-associated colorectal cancers, Rare ginsenosides, inflammation, apoptosis, immune response, proteomics, network pharmacology, gut microbiota","lastPublishedDoi":"10.21203/rs.3.rs-6343012/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6343012/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe incidence of colitis-associated colorectal cancer (CAC) is increasing, while conventional single-target therapies often demonstrate limited efficacy and long-term adverse effects. As a result, multi-target natural compounds have emerged as promising alternatives. This study investigates the anti-CAC potential of Li-Ginseng Powder (LGP), a specially processed functional food derived from Panax ginseng and enriched with rare ginsenosides (Rk1, Rk3, Rh4, Rg3, and Rg5), demonstrates strong preventive potential and characterized by minimal toxicity. Multi-omics analyses revealed that CAC model mice exhibited key tumor-promoting features, including heightened inflammation, impaired apoptosis, and immune suppression. Notably, LGP displayed significant anti-CAC activity and reversed 98.14% of dysregulated protein expression (fold-change\u0026thinsp;\u0026gt;\u0026thinsp;1.5, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). It effectively mitigated inflammation by inhibiting STAT3/NF-κB signaling and modulating inflammatory gene expression. LGP induced apoptosis by downregulating anti-apoptotic proteins (Bcl-2, Bcl-XL, and x-IAP), upregulating pro-apoptotic Bax and Granzyme B, and promoting PARP and Caspase-9 cleavage to facilitate the elimination of damaged cells. Moreover, it enhanced immune responses by increasing NK cell and CD3\u0026thinsp;+\u0026thinsp;T cell infiltration while activating CD4\u0026thinsp;+\u0026thinsp;and CD8\u0026thinsp;+\u0026thinsp;T cells. Additionally, LGP modulated serum metabolites and gut microbiota composition, fostering a favorable disease trajectory. These findings elucidate LGP\u0026rsquo;s comprehensive anti-CAC mechanism, integrating inflammation suppression, apoptosis induction, immune modulation, and microbiota regulation. This study addresses a critical gap in ginseng-derived CAC therapies, offering a promising multi-targeted and low-risk therapeutic strategy for clinical application.\u003c/p\u003e","manuscriptTitle":"Multi-Targeted Anti-Cancer Mechanisms of Li-Ginseng Powder in Colitis-Associated Colorectal Cancer: Integrating Inflammation, Apoptosis and Immunity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-28 05:33:55","doi":"10.21203/rs.3.rs-6343012/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d5552e28-0c9e-4b78-bd02-5e4bc598f89b","owner":[],"postedDate":"May 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":49009964,"name":"Biological sciences/Cancer/Gastrointestinal cancer/Colorectal cancer"},{"id":49009965,"name":"Biological sciences/Cell biology"}],"tags":[],"updatedAt":"2025-05-28T05:33:55+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-28 05:33:55","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6343012","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6343012","identity":"rs-6343012","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}