Impact of stool transplantation and metformin on polyp reduction and inflammation in an APC Min mouse model

Yeon Ji Kim; Jiwon Lee; Eunmi Lee; Seun Ja Park; Jae Hyun Kim

doi:10.5217/ir.2025.00011

Articles

Page Path: HOME > Intest Res > Ahead-of print articles > Article

Original Article Impact of stool transplantation and metformin on polyp reduction and inflammation in an APC Min mouse model: Yeon Ji Kim¹, Jiwon Lee², Eunmi Lee³, Seun Ja Park³, Jae Hyun Kim^3,; DOI: https://doi.org/10.5217/ir.2025.00011
Published online: May 19, 2025

¹Institute of Gastroenterology, Kosin University College of Medicine, Busan, Korea

²Kosin University College of Medicine, Busan, Korea

³Department of Internal Medicine, Kosin University College of Medicine, Busan, Korea

Correspondence to Jae Hyun Kim, Department of Internal Medicine, Kosin University College of Medicine, Gamcheon-ro, Seo-gu, Busan 49267, Korea. E-mail: kjh8517@daum.net

• Received: January 30, 2025 • Revised: February 27, 2025 • Accepted: March 18, 2025

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

443 Views
45 Download

Full Article

Download PDF

Abstract
INTRODUCTION
METHODS
RESULTS
DISCUSSION
NOTES
REFERENCES

Abstract

Background/Aims
Familial adenomatous polyposis is a hereditary condition characterized by numerous adenomatous polyps in the colon and rectum, significantly increasing colorectal cancer risk. Current management strategies, such as prophylactic colectomy, are invasive and have long-term consequences, highlighting the need for alternative therapies. This study aimed to evaluate whether stool transplantation and metformin therapy synergistically reduce polyp formation and inflammation.
Methods
APC Min mice were divided into 4 groups: control, anti-control (antibiotic pretreatment), stool (stool transplantation), and stool+metformin. Polyp burden, bacterial abundance, inflammatory cytokines (interleukin [IL]-6, tumor necrosis factor [TNF]-α, IL-10), and tumorigenic markers (NF-κB, Cox2, c-myc, β-catenin) were assessed using messenger RNA (mRNA) and protein analyses of intestinal tissues, along with serum and fecal microbiota evaluations.
Results
Stool transplantation combined with metformin significantly reduced bacterial abundance and polyp burden. The anti-control group showed similar reductions, suggesting suppression of gut microbiota re-establishment. TNF-α and IL-10 levels remained unchanged, but a significant increase in IL-6 was observed in the stool+metformin group’s intestinal tissues, indicating localized immune activation. Intestinal Cox2 mRNA expression was reduced in the combination group, correlating with polyp suppression. Protein levels of NF-κB, Cox2, and β-catenin showed no significant changes in vivo, while in vitro experiments revealed a decrease in NF-κB and an increase in Cox2, suggesting complex regulation of inflammation-related pathways.
Conclusions
Stool transplantation combined with metformin reduces polyp burden in APC Min mice through gut microbiota modulation and localized immune activation. These findings support the therapeutic potential of this combination treatment for familial adenomatous polyposis.
Keywords: Familial adenomatous polyposis; Gastrointestinal microbiome; Fecal microbiota transplantation; Metformin

INTRODUCTION

Familial adenomatous polyposis (FAP) is a hereditary condition characterized by hundreds to thousands of adenomatous polyps in the colon and rectum [1]. If left untreated, FAP almost invariably progresses to colorectal cancer (CRC). Current management strategies for FAP, such as prophylactic colectomy, are invasive and carry significant long-term consequences [2-4]. Therefore, there is a critical need for alternative or adjunct therapies that can reduce polyp burden and delay disease progression.

The gut microbiota plays a pivotal role in intestinal homeostasis and disease, with dysbiosis linked to the development and progression of polyps in FAP and CRC [5]. Restoration of gut microbial balance through stool transplantation has shown promise in reducing inflammation and modulating immune responses, suggesting its potential to reduce polyp burden in FAP patients [6,7].

Metformin, a widely used antidiabetic drug, has gained attention for its pleiotropic effects, including anti-inflammatory and antitumor properties. Mechanistically, metformin alters microbial composition, promotes the production of short-chain fatty acids, and reduces systemic inflammation by modulating pathways such as AMPK (AMP-activated protein kinase), mTOR (mammalian target of rapamycin) [8-10]. Additionally, metformin has been shown to suppress tumorigenic pathways, including NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) and Cox2 (cyclooxygenase-2) signaling, which are implicated in polyp formation and progression [11].

This study investigates the combined effects of stool transplantation and metformin on polyp burden and inflammation in APC Min mice, a well-established model for FAP. We aim to identify whether this combination therapy can synergistically reduce polyp formation and suppress inflammation-related pathways, providing a potential therapeutic strategy for managing FAP.

METHODS

1. Animals

APC Min male mice were purchased from the Jackson Laboratory and bred in-house under specific-pathogen-free conditions. Male APC Min mice were mated with wild-type female mice to produce offspring. The resulting APC Min female mice were used for experiments, while APC Min male mice were retained exclusively for breeding. Experimental APC Min mice used in this study carried a Mom2 wild-type genotype and a Mom1 heterozygous genotype. Due to survival issues with Mom2 mutation carriers, only Mom2 wild-type mice were included. The mice selected for this study were 11-week-old females at the start of the experiment. All procedures were conducted with the approval of the Animal Experimentation Ethics Committee of Kosin University College of Medicine (IACUC protocol number: KMAP-20-07).

2. Experimental Groups

Mice were randomly assigned into 4 groups (n=5 per group): (1) control, (2) anti-control, (3) stool, and (4) stool+metformin. The control group received no treatment. The anti-control group, stool group, and stool+metformin group were treated with a cocktail of broad-spectrum antibiotics (0.2 g/L ampicillin, 0.1 g/L vancomycin, 0.2 g/L neomycin, and 0.2 g/L metronidazole) in their drinking water for 2 weeks to establish a flora-deficient status. Following the 2-week antibiotic pretreatment, the stool group received stool transplantation, and the stool+metformin group received both stool transplantation and metformin injections.

3. Stool Transplantation and Metformin Administration

Fresh stool samples were collected from 10 healthy volunteers (4 men and 6 women, aged 40.9 ±9.8 years) and stored in a deep freezer.12 For transplantation, the stools were homogenized in phosphate-buffered saline (PBS) at a concentration of 1 g/5 mL, filtered to remove particulates, and administered to each mouse as 200 μL of oral gavage 3 times weekly for 8 weeks. Metformin (250 mg/kg) was dissolved in sterile PBS and administered intraperitoneally immediately after each stool gavage, ensuring synchronized treatment throughout the 8-week experimental period.

4. Sample Collection and Processing

After 8 weeks of treatment, mice were sacrificed via carbon dioxide inhalation. Blood was collected via cardiac puncture and centrifuged to isolate serum. Intestinal tissues (small intestine and colon) were excised, and sections were preserved in 10% formalin for histological analysis or snap-frozen in liquid nitrogen for molecular analyses. Fecal samples were collected weekly throughout the experimental period for microbiota analysis.

5. Analysis and Evaluation

The total number of polyps was visually counted under a stereomicroscope by 2 independent observers blinded to the group assignments, and their distribution along the intestinal tract was documented.

Cytokine levels of interleukin (IL)-6, tumor necrosis factor (TNF)-α, IL-10 in serum and intestinal tissue lysates were quantified using commercially available enzyme-linked immunosorbent assay (ELISA) kits, following the manufacturer’s protocols. Absorbance was measured at the specified wavelength using a microplate reader.

Total RNA was extracted from intestinal tissues using TRIzol reagent and reverse-transcribed into complementary DNA. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed to measure the expression levels of NF-κB, Cox2, c-myc, and β-catenin using specific primers, with relative expression levels normalized to GAPDH as a housekeeping gene.

Protein levels of NF-κB, Cox2, c-myc, and β-catenin in intestinal tissues were measured using commercially available ELISA kits. Tissue samples were homogenized in lysis buffer, and supernatants were collected after centrifugation. Protein concentrations were determined using the Bradford assay to ensure consistent sample loading. ELISA was performed according to the manufacturer’s instructions, and absorbance was measured using a microplate reader. Protein levels were expressed as concentrations normalized to tissue weight.

Fecal DNA was extracted using a commercially available kit, and total bacterial abundance was determined using quantitative PCR (qPCR) of the 16S rRNA gene. Relative changes in microbial composition were analyzed to evaluate the effects of stool transplantation and metformin. All experiments were conducted in biological triplicates to ensure the reproducibility of the findings.

6. Cell Culture

For in vitro studies, CRC cell lines HCT 116 and HT29 were cultured in minimum essential medium (Sigma St. Louis, MO, USA) at 37℃ in 5% CO₂. Cells were treated with stool (1:100 or 1:500 dilution) and metformin (40 mM) for 6 hours, followed by Western blot analysis. Equivalent amounts of total protein (15 μg) were loaded onto a 12% SDS/PAGE gel, separated, and transferred to a nitrocellulose membrane using an electroblotting apparatus (Bio-Rad, Richmond, CA, USA). The proteins then were incubated with antibodies targeting β-catenin, c-myc, NF-κB, and Cox-2 (diluted 1:100, Santa Cruz Biotechnology, Dallas, CA, USA) according to standard protocols. β-Actin was used as an internal control to ensure equal protein loading. All experiments were performed in biological triplicates to ensure the reproducibility of the findings.

7. Statistical Analysis and Ethics Approval

Data were analyzed using one-way analysis of variance followed by Tukey’s post hoc test for multiple comparisons. Results were expressed as mean± standard deviation, and P-values <0.05 were considered statistically significant. This study was approved by the Institutional Animal Care and Use Committee of Kosin University College of Medicine. For the collection of human stool samples, the study protocol was reviewed and approved by the Institutional Review Board of Kosin University Gospel Hospital (approval no. KUGH 2020-03-023). All experiments were conducted in biological triplicates to ensure reproducibility of the findings.

RESULTS

1. Changes in Gut Microbial Abundance

The relative levels of total bacteria were assessed throughout the experimental period (Fig. 1). The anti-control group exhibited a significant reduction in bacterial abundance, as expected due to the depletion of gut microbiota by antibiotics. Interestingly, the stool+metformin group demonstrated a similar reduction in bacterial abundance, suggesting that metformin in combination with stool transplantation may suppress the re-establishment of gut microbiota after antibiotic treatment.

2. Reduction in Polyp Burden

Polyp counts were significantly reduced in both the anti-control and stool+metformin groups compared to the control group (Fig. 2A). The reduction in polyp burden observed in the anti-control group aligns with the decrease in bacterial abundance induced by antibiotics. Similarly, the stool+metformin group demonstrated a significant reduction in both bacterial abundance and polyp burden. Consistent with the pathology of APC Min mice, polyps were primarily confined to the small intestine (Fig. 2B). These findings suggest a potential link between gut microbiota suppression and reduced polyp formation. Although the exact mechanism underlying the decrease in bacterial abundance in the stool+metformin group remains unclear, it is possible that stool transplantation combined with metformin treatment inhibits bacterial re-establishment following antibiotic treatment.

3. Inflammatory Cytokine Profiles in Serum and Intestinal Tissue

Cytokine levels of IL-6, TNF-α, and IL-10 were measured in serum and intestinal tissue lysates to assess systemic and local inflammation (Fig. 3). In the anti-control group, cytokine levels in both serum and intestinal tissues were comparable to those in the control group, suggesting that antibiotic-induced suppression of gut microbiota does not significantly impact systemic or local inflammation.

In the stool+metformin group, a modest but significant increase in IL-6 level was observed in intestinal tissues, indicating localized immune activation potentially linked to the combined treatment. TNF-α and IL-10 levels remained unchanged in both serum and intestinal tissues across all groups. These findings suggest that the observed reduction in polyp burden in the anti-control and stool+metformin groups may not be primarily mediated by systemic inflammatory cytokines, although localized immune modulation could contribute to the effects of the combined treatment.

4. Expression of Tumorigenic Markers in Intestinal Tissue

The messenger RNA (mRNA) expression levels of NF-κB, Cox2, c-myc, and β-catenin in intestinal tissues were evaluated to assess the impact of the interventions on tumorigenic pathways (Fig. 4A). Cox2 expression was significantly reduced in both the stool and stool+metformin groups compared to the control group (P<0.05), indicating a potential anti-inflammatory effect of stool transplantation and the combination treatment. While the stool+metformin group also showed a modest reduction in NF-κB expression, this change was not significant. In contrast, c-myc and β-catenin mRNA expression levels remained unchanged across all groups, suggesting that these pathways are not directly affected by the interventions at the transcriptional level.

5. Protein Expression of Tumorigenic Pathways

Protein levels of NF-κB, Cox2, c-myc, and β-catenin were assessed using ELISA to evaluate the effects of the interventions on tumorigenic pathways (Fig. 4B). The stool group displayed a significant increase in NF-κB protein level compared to the control group (P<0.05), suggesting a potential inflammatory response induced by stool transplantation. In contrast, the stool+metformin group showed slight but non-significant reductions in Cox2, c-myc, and β-catenin protein levels. These findings suggest that, while stool transplantation alone may activate certain inflammatory pathways, the combination of stool transplantation and metformin appears to mitigate these effects, potentially modulating tumorigenic signaling.

6. Microbiota Composition and Alterations

As described in the previous section, “Changes in Gut Microbial Abundance,” antibiotic treatment caused a significant reduction in bacterial abundance. Interestingly, the combination of stool transplantation and metformin also resulted in a marked reduction in bacterial abundance, despite the introduction of stool. This reduction may be attributed to metformin’s effects in inhibiting microbial metabolism and disrupting re-establishment dynamics following antibiotic pretreatment. These microbial alterations likely play a critical role in reducing inflammation and tumorigenesis, aligning with the observed reductions in polyp burden.

7. In vitro Effects of Stool and Metformin on Tumorigenic Pathways

To further investigate the observed in vivo effects, in vitro experiments were conducted using CRC cell lines (HCT116 and HT29). Cells were treated with stool (1:100 or 1:500 dilution) and metformin (40 mM) for 6 hours, followed by Western blot analysis of key tumorigenic markers (Fig. 5). Unlike the in vivo experiments, an anti-control group was not included in the in vitro setup, as microbiota removal was unnecessary in this controlled cellular environment.

The combination treatment resulted in significant reduction in NF-κB expression compared to the control, supporting its potential anti-inflammatory role as suggested by the in vivo findings. However, β-catenin and c-myc protein levels remained unchanged across all treatment groups, indicating that these pathways may not be directly influenced by the treatments. Notably, Cox-2 expression was increased in the combination treatment group, suggesting a complex regulatory response involving both pro- and anti-inflammatory signaling pathways. These findings highlight the intricate interactions between stool transplantation and metformin in modulating tumorigenic pathways. Further studies are warranted to elucidate their mechanistic roles and to assess how these effects could inform therapeutic strategies for CRC.

DISCUSSION

This study demonstrates that the combination of stool transplantation and metformin effectively reduces polyp burden and modulates inflammation-related pathways in APC Min mice, providing insights into potential therapeutic strategies for managing FAP.

The significant reduction in polyp burden observed in both the anti-control and stool+metformin groups underscores the critical role of gut microbiota in tumorigenesis. Antibiotic-induced bacterial depletion in the anti-control group led to reduced polyp formation, consistent with prior studies demonstrating the involvement of microbiota in promoting inflammation and tumorigenesis in FAP models [13,14]. Interestingly, the stool+metformin group exhibited similar bacterial depletion despite stool transplantation, likely attributable to metformin’s inhibitory effects on microbial re-establishment. These findings suggest that metformin enhances the efficacy of microbiota- modulating interventions by suppressing gut microbial recolonization [8,15].

The significant reduction in Cox2 expression at the intestinal mRNA level in both the stool and stool+metformin groups highlights a key mechanism of inflammation attenuation, contributing to polyp suppression. As Cox2 is a well-documented mediator of inflammation and tumorigenesis in intestinal tissues, its suppression underscores the anti-inflammatory potential of these interventions [16]. Furthermore, while NF-κB protein level was elevated in the Stool group, indicative of a pro-inflammatory response, the combination treatment mitigated this effect, reinforcing the anti-inflammatory synergy between stool transplantation and metformin [17].

The in vitro findings provided additional mechanistic insights, revealing a reduction in NF-κB expression under combination treatment, supporting its anti-inflammatory role. However, the observed increase in Cox2 expression in vitro highlights the complexity of inflammatory pathways and suggests potential differences between in vivo and in vitro environments. These discrepancies may arise from differences in the cellular microenvironment, systemic immune interactions, or microbiota-related effects absent in vitro [18].

FAP patients harbor colonic biofilms enriched with tumorigenic bacteria and mucosal immune dysfunction, both contributing to microbial dysbiosis, impaired tumor surveillance, and accelerated tumorigenesis [19]. In this context, metformin’s anti-tumorigenic effects, mediated through AMPK activation and inhibition of NF-κB signaling, have been demonstrated in various cancer models [11,20]. This study builds on these findings by showing that combining stool transplantation and metformin produces complementary effects, reducing bacterial abundance and suppressing inflammation to attenuate polyp formation.

Despite these promising findings, several limitations warrant consideration. First, while reductions in bacterial abundance were associated with polyp suppression, the precise mechanisms linking microbiota modulation to tumorigenesis remain unclear. Future studies employing advanced microbiome analyses, such as 16S rRNA sequencing or metagenomics, are needed to identify specific microbial taxa or functional pathways involved in these effects. Second, the increase in Cox2 expression in vitro highlights the complexity of inflammatory signaling and suggests potential context-specific differences between in vivo and in vitro environments. Further studies are needed to reconcile these differences and clarify the interplay between microbiota and inflammatory pathways. Third, this study did not assess the long-term effects of these interventions on polyp progression to malignancy. Extended follow-up studies are necessary to evaluate whether stool transplantation and metformin can prevent CRC development in FAP models.

In conclusion, stool transplantation combined with metformin effectively reduces polyp burden in APC Min mice, likely through modulation of gut microbiota and inflammatory pathways. These findings provide a strong foundation for further exploration of combination therapies targeting the gut microbiota to manage FAP and potentially prevent CRC.

NOTES

Funding Source

This study was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (#2020R1C1C1012694).

Conflict of Interest

No potential conflict of interest relevant to this article was reported.

Data Availability Statement

The technical appendix and dataset are available from the corresponding author.

Author Contributions

Conceptualization: Kim YJ, Lee J, Kim JH. Data curation/Formal analysis/Investigation: KIM YJ, Kim JH. Methodology/Visualization: Kim YJ. Supervision: Lee E, Park SJ. Writing - original draft: Kim JH, Kim YJ. Writing - review & editing: Lee J, Lee E, Park SJ. Approval of final manuscript: all authors.

Fig. 1.

Changes in gut microbial abundance. Relative levels of total bacteria were assessed across experimental groups using 16S rRNA gene quantification by qPCR. Fecal samples were collected weekly throughout the experimental period, and DNA was extracted using a commercially available kit. qPCR, quantitative polymerase chain reaction.

Fig. 2.

Reduction in polyp burden. (A) Total polyp counts in the small intestine and colon were determined under a stereomicroscope by 2 independent observers blinded to group assignments. (B) Representative images of the small intestine and colon were captured to visualize polyp distribution. ^aP<0.01. NS, not significant.

Fig. 3.

Inflammatory cytokine profiles in serum and intestinal tissue. (A) Cytokine levels of IL-6, TNF-α, and IL-10 in serum were measured using ELISA, with concentrations expressed as pg/mL on the y-axis. (B) Cytokine levels of IL-6, TNF-α, and IL-10 in intestinal tissue lysates were assessed using ELISA, with concentrations expressed as pg/mg of tissue on the y-axis. ^aP<0.01. NS, not significant.

Fig. 4.

Evaluation of tumorigenic markers in intestinal tissues. (A) The expression levels of NF-κB, Cox2, c-myc, and β-catenin mRNA in intestinal tissues were evaluated using real-time PCR. Total RNA was extracted with TRIzol, reverse-transcribed into cDNA, and amplified using gene-specific primers. (B) Protein levels of NF-κB, Cox2, c-myc, and β-catenin in intestinal tissues were assessed using ELISA to evaluate the effects of the interventions on tumorigenic pathways. ^aP<0.05. NS, not significant; mRNA, messenger RNA; cDNA, complementary DNA; PCR, polymerase chain reaction; ELISA, enzyme-linked immunosorbent assay.

Fig. 5.

Analysis of tumorigenic markers in colorectal cancer cell lines. Colorectal cancer cell lines (HCT116 and HT29) were treated with stool (1:100 or 1:500 dilution) and metformin (40 mM) for 6 hours. Western blot analysis was performed to evaluate the expression of β-catenin, c-myc, NF-κB, and Cox-2. β-Actin was used as a loading control.

REFERENCES

1. Galiatsatos P, Foulkes WD. Familial adenomatous polyposis. Am J Gastroenterol 2006;101:385–398.Article PubMed
2. Zaffaroni G, Mannucci A, Koskenvuo L, et al. Updated European guidelines for clinical management of familial adenomatous polyposis (FAP), MUTYH-associated polyposis (MAP), gastric adenocarcinoma, proximal polyposis of the stomach (GAPPS) and other rare adenomatous polyposis syndromes: a joint EHTG-ESCP revision. Br J Surg 2024;111–znae070.Article PDF
3. Monahan KJ, Bradshaw N, Dolwani S, et al. Guidelines for the management of hereditary colorectal cancer from the British Society of Gastroenterology (BSG)/Association of Coloproctology of Great Britain and Ireland (ACPGBI)/United Kingdom Cancer Genetics Group (UKCGG). Gut 2020;69:411–444.Article PubMed PMC
4. Church J, Simmang C, Standards Task Force; American Society of Colon and Rectal Surgeons; Collaborative Group of the Americas on Inherited Colorectal Cancer and the Standards Committee of The American Society of Colon and Rectal Surgeons. Practice parameters for the treatment of patients with dominantly inherited colorectal cancer (familial adenomatous polyposis and hereditary nonpolyposis colorectal cancer). Dis Colon Rectum 2003;46:1001–1012.Article PubMed
5. Garrett WS. Cancer and the microbiota. Science 2015;348:80–86.Article PubMed PMC
6. Yadegar A, Bar-Yoseph H, Monaghan TM, et al. Fecal microbiota transplantation: current challenges and future landscapes. Clin Microbiol Rev 2024;37:e0006022.Article PubMed PMC PDF
7. Paramsothy S, Kamm MA, Kaakoush NO, et al. Multidonor intensive faecal microbiota transplantation for active ulcerative colitis: a randomised placebo-controlled trial. Lancet 2017;389:1218–1228.Article PubMed
8. Wu H, Esteve E, Tremaroli V, et al. Metformin alters the gut microbiome of individuals with treatment-naive type 2 diabetes, contributing to the therapeutic effects of the drug. Nat Med 2017;23:850–858.Article PubMed PDF
9. Seo Y, Kim J, Park SJ, et al. Metformin suppresses cancer stem cells through AMPK activation and inhibition of protein prenylation of the mevalonate pathway in colorectal cancer. Cancers (Basel) 2020;12:2554.Article PubMed PMC
10. Kim JH, Lee KJ, Seo Y, et al. Effects of metformin on colorectal cancer stem cells depend on alterations in glutamine metabolism. Sci Rep 2018;8:409.Article PubMed PMC PDF
11. Pollak M. Metformin and other biguanides in oncology: advancing the research agenda. Cancer Prev Res (Phila) 2010;3:1060–1065.Article PubMed PMC PDF
12. Kim JH, Kim YJ, Oh GM, Jung W, Park SJ. How is gut microbiome of patients with familial adenomatous polyposis different from healthy people? Medicine (Baltimore) 2022;101:e32194.Article PubMed PMC
13. Sears CL, Garrett WS. Microbes, microbiota, and colon cancer. Cell Host Microbe 2014;15:317–328.Article PubMed PMC
14. O’Keefe SJ. Diet, microorganisms and their metabolites, and colon cancer. Nat Rev Gastroenterol Hepatol 2016;13:691–706.Article PubMed PMC PDF
15. Forslund K, Hildebrand F, Nielsen T, et al. Disentangling type 2 diabetes and metformin treatment signatures in the human gut microbiota. Nature 2015;528:262–266.Article PubMed PMC PDF
16. Wang D, Dubois RN. The role of COX-2 in intestinal inflammation and colorectal cancer. Oncogene 2010;29:781–788.Article PubMed PMC PDF
17. Karin M, Greten FR. NF-kappaB: linking inflammation and immunity to cancer development and progression. Nat Rev Immunol 2005;5:749–759.Article PubMed PDF
18. Ghosh S, Hayden MS. New regulators of NF-kappaB in inflammation. Nat Rev Immunol 2008;8:837–848.Article PubMed PDF
19. Dejea CM, Fathi P, Craig JM, et al. Patients with familial adenomatous polyposis harbor colonic biofilms containing tumorigenic bacteria. Science 2018;359:592–597.Article PubMed PMC
20. Foretz M, Guigas B, Bertrand L, Pollak M, Viollet B. Metformin: from mechanisms of action to therapies. Cell Metab 2014;20:953–966.Article PubMed

Figure & Data

REFERENCES

Citations

Citations to this article as recorded by

PubReader
ePub Link

Cite

CITE: export Copy Download Format; Close

Download Citation

Download a citation file in RIS format that can be imported by all major citation management software, including EndNote, ProCite, RefWorks, and Reference Manager.

Format:

RIS — For EndNote, ProCite, RefWorks, and most other reference management software
BibTeX — For JabRef, BibDesk, and other BibTeX-specific software

Include:

Citation for the content below
Citation and abstract for the content below

Impact of stool transplantation and metformin on polyp reduction and inflammation in an APC Min mouse model

DOI: https://doi.org/10.5217/ir.2025.00011

XML Download

Figure

Impact of stool transplantation and metformin on polyp reduction and inflammation in an APC Min mouse model

Fig. 1. Changes in gut microbial abundance. Relative levels of total bacteria were assessed across experimental groups using 16S rRNA gene quantification by qPCR. Fecal samples were collected weekly throughout the experimental period, and DNA was extracted using a commercially available kit. qPCR, quantitative polymerase chain reaction.

Fig. 2. Reduction in polyp burden. (A) Total polyp counts in the small intestine and colon were determined under a stereomicroscope by 2 independent observers blinded to group assignments. (B) Representative images of the small intestine and colon were captured to visualize polyp distribution. aP<0.01. NS, not significant.

Fig. 3. Inflammatory cytokine profiles in serum and intestinal tissue. (A) Cytokine levels of IL-6, TNF-α, and IL-10 in serum were measured using ELISA, with concentrations expressed as pg/mL on the y-axis. (B) Cytokine levels of IL-6, TNF-α, and IL-10 in intestinal tissue lysates were assessed using ELISA, with concentrations expressed as pg/mg of tissue on the y-axis. aP<0.01. NS, not significant.

Fig. 4. Evaluation of tumorigenic markers in intestinal tissues. (A) The expression levels of NF-κB, Cox2, c-myc, and β-catenin mRNA in intestinal tissues were evaluated using real-time PCR. Total RNA was extracted with TRIzol, reverse-transcribed into cDNA, and amplified using gene-specific primers. (B) Protein levels of NF-κB, Cox2, c-myc, and β-catenin in intestinal tissues were assessed using ELISA to evaluate the effects of the interventions on tumorigenic pathways. aP<0.05. NS, not significant; mRNA, messenger RNA; cDNA, complementary DNA; PCR, polymerase chain reaction; ELISA, enzyme-linked immunosorbent assay.

Fig. 5. Analysis of tumorigenic markers in colorectal cancer cell lines. Colorectal cancer cell lines (HCT116 and HT29) were treated with stool (1:100 or 1:500 dilution) and metformin (40 mM) for 6 hours. Western blot analysis was performed to evaluate the expression of β-catenin, c-myc, NF-κB, and Cox-2. β-Actin was used as a loading control.

Fig. 1.

Fig. 2.

Fig. 3.

Fig. 4.

Fig. 5.

Impact of stool transplantation and metformin on polyp reduction and inflammation in an APC Min mouse model