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. 2026 Jun 3;26:489. doi: 10.1186/s12876-026-04981-5

Fusobacterium nucleatum exacerbates chronic atrophic gastritis in rats via mitochondrial dysfunction and inflammatory activation

Peixun Zhao 1,2, Yiyang Fan 1,2, Haohan Liu 1,2, Yue Jin 1,2, Yizinaer Abula 1,2, Adili Abudourousuli 3, Yangyang Zhang 1,4,✉
PMCID: PMC13449696  PMID: 42237224

Abstract

Background

Chronic atrophic gastritis (CAG) is a premalignant condition with limited therapeutic strategies. The role of the oral pathogen Fusobacterium nucleatum (F. nucleatum), associated with gastrointestinal carcinogenesis, in CAG remains unclear.

Methods

This study investigated whether F. nucleatum aggravates gastric mucosal injury through inflammation and mitochondrial dysfunction in CAG. A rat model of CAG was induced using N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) for 10 weeks, combined with oral gavage of F. nucleatum. Gastric pathology, bacterial colonization, serum cytokine levels, and mitochondrial membrane potential were evaluated. In vitro, human gastric epithelial cells (GES-1) were treated with MNNG and co-cultured with F. nucleatum at multiplicities of infection (MOI) of 50, 100, or 150. Cytokine concentrations in cell culture supernatants were quantified using corresponding human enzyme-linked immunosorbent assay kits. Mitochondrial membrane potential was assessed via flow cytometry.

Results

F. nucleatum colonized gastric tissues and intensified MNNG-induced injury, including rugal flattening, glandular atrophy, intestinal metaplasia, and inflammatory cell infiltration. It significantly increased pro-inflammatory cytokine levels and further decreased mitochondrial membrane potential. In GES-1 cells, F. nucleatum dose-dependently elevated secretion of interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) and impaired mitochondrial function.

Conclusions

F. nucleatum exacerbates CAG by promoting gastric inflammation and mitochondrial dysfunction. These findings may underscore the microbiota–mitochondria axis as a potential therapeutic target to prevent CAG progression.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12876-026-04981-5.

Keywords: Fusobacterium nucleatum (F. nucleatum), Chronic atrophic gastritis (CAG), Mitochondrial dysfunction, Inflammation

Introduction

Chronic atrophic gastritis (CAG) is a precancerous condition characterized by the progressive loss of gastric glands, intestinal metaplasia, or dysplasia. Its global prevalence reaches approximately 33%, with notably high rates in gastric cancer–endemic regions such as East Asia and Eastern Europe [1–3]. Epidemiological studies indicate that CAG confers an annual risk of progression to gastric cancer of 0.1%–0.3%, which is further increased by cofactors such as Helicobacter pylori (H. pylori) infection, autoimmune gastritis, and high-salt dietary intake [4–6]. Although H. pylori eradication reduces cancer risk, it is insufficient in reversing established gastric atrophy or intestinal metaplasia [7, 8], and certain individuals remain vulnerable to disease progression despite successful eradication [9]. Furthermore, long-term proton pump inhibitor–induced hypochlorhydria may alter the gastric microbiota and exacerbate mucosal injury [10, 11]. These limitations underscore the need to investigate additional microbial contributors, such as Fusobacterium nucleatum (F. nucleatum), to elucidate disease mechanisms and identify novel therapeutic targets.

Recent studies have identified mitochondrial dysfunction in gastric mucosal cells as a central pathological mechanism in CAG [12–14]. Damaged mitochondria, the primary source of reactive oxygen species (ROS), contribute to oxidative stress, activate the NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome, and sustain chronic inflammation via interleukin-1β (IL-1β) and interleukin-18 (IL-18) release [12–14]. The H. pylori virulence factor cytotoxin-associated gene A (CagA) impairs mitochondrial electron transport chain complexes, increases ROS production, and promotes epithelial apoptosis and malignant transformation [15–17]. Beyond H. pylori, other bacteria, such as the oral-derived F. nucleatum, may elicit similar mitochondrial injury. In colorectal cancer, for example, F. nucleatum increases mitochondrial ROS and mitochondrial DNA (mtDNA) leakage via its Fusobacterium adhesin A (FadA), thereby activating the cyclic GMP-AMP synthase–stimulator of interferon genes (cGAS–STING) pathway and intensifying inflammation [18–20]. Whether analogous mechanisms are involved in CAG remains to be determined.

F. nucleatum, a commensal microorganism and opportunistic pathogen (a pathobiont), is a member of the human oral microbiota, but it can disseminate to other ecological niches within the body under certain conditions [21]. F. nucleatum is a gram-negative anaerobe implicated in periodontal disease and associated with systemic disorders such as colorectal cancer and inflammatory bowel disease [22–25]. Sorino et al. systematically described the role transition of F. nucleatum from a periodontal pathogen to a potential gastric cancer pathogen [26]. Kamali et al. examined gastric biopsy samples from 300 patients with gastroduodenal diseases and reported that 215 patients (72%) were positive for F. nucleatum. Notably, gastric biopsies from gastric cancer patients negative for Helicobacter pylori (H. pylori) still showed a significant prevalence of F. nucleatum infection [27]. Metagenomic analyses have further revealed its enrichment in the gastric mucosa of patients with CAG [28–30]. Mechanistically, its adhesin FadA binds to E-cadherin (E-cadherin), activates β-catenin (β-catenin), and promotes epithelial–mesenchymal transition (EMT), while also inducing mtDNA release, which subsequently amplifies inflammation through toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-κB) and cGAS–STING signaling pathways [31–33]. In animal models, F. nucleatum colonization worsens chemically induced colitis and promotes the infiltration of immunosuppressive cells in the tumor microenvironment [34, 35]. Zhang et al. used mouse models to demonstrate that infection with F. nucleatum could induce chronic gastritis and promote gastric mucosal dysplasia [36]. However, direct experimental evidence supporting a role for F. nucleatum in driving CAG via mitochondrial dysfunction remains lacking.

Based on this evidence, we hypothesize that F. nucleatum exacerbates N-methyl-N′-nitro-N-nitrosoguanidine (MNNG)-induced CAG by enhancing inflammation and inducing mitochondrial damage. To test this, we established an MNNG-induced CAG rat model colonized with F. nucleatum and systematically evaluated: (1) gastric mucosal pathology and bacterial colonization; (2) inflammatory cytokine expression; and (3) mitochondrial function, with a focus on membrane potential. These findings aim to provide experimental evidence for the role of F. nucleatum in CAG and support the concept of a “microbiota–mitochondria axis” as a potential therapeutic target.

Materials and methods

Ethical approval

All of the animal experiments were approved by the Animal Research Ethics Committee of The First Affiliated Hospital of Xinjiang Medical University (Approval No.: IACUC-JT-20241015-07) and conducted in accordance with the U.S. National Institutes of Health (NIH) guidelines for the care and use of laboratory animals (2011).

Bacterial culture

The F. nucleatum strain (American Type Culture Collection 25586, ATCC 25586) used in this study is a reference type strain originally isolated from the human oral cavity, specifically from a cervicofacial lesion. The reference strain F. nucleatum (ATCC® 25586™) was obtained from the American Type Culture Collection (ATCC, USA). Cultures were maintained on Columbia blood agar plates in an anaerobic workstation at 37 °C. After 48 h of incubation, colonies were inoculated into 40 mL of brain heart infusion (BHI) broth and cultured anaerobically at 37 °C until mid-exponential phase. Bacterial density was determined by measuring optical density at 660 nm (OD660) and converted to colony-forming units (CFU)/mL. For in vivo experiments, a suspension containing 1 × 109 CFU was used. For in vitro assays, bacterial counts were verified using a hemocytometer, and serial dilutions were prepared to achieve the required multiplicities of infection (MOI).

Animal housing and experimental procedures

Male Sprague-Dawley rats (6 weeks old, 180–200 g) were procured from the Laboratory Animal Center of Xinjiang Medical University (Animal Production License No.: SCXK (Xin) 2020-0002). Thirty rats were housed under specific pathogen-free conditions and randomly divided into three groups (n = 10 each): normal control (NC), MNNG-induced CAG (CAG), and MNNG-induced CAG with F. nucleatum intervention (CAG + F. nucleatum). Animals were maintained in a controlled environment (temperature: 20–26 °C; humidity: 50–70%) under a 12-h light/dark cycle with ad libitum access to standard chow and water. Following one week of acclimatization, CAG was induced by administering MNNG (170 µg/mL) in drinking water every other day for 10 weeks [37, 38]. Rats in the CAG + F. nucleatum group additionally received oral gavage of F. nucleatum (1 × 109 CFU in 3 mL suspension) once weekly for 10 weeks.

Sample collection from rats

At the conclusion of the 10-week protocol, the rats were weighed and anesthetized with 2% pentobarbital sodium at a dose of 2 mg/kg. Samples were then collected from the rats as follows: (1) serum—blood was drawn from the abdominal aorta, and serum was separated and stored at ˗80 °C for subsequent analysis; and (2) gastric tissue—the stomach was excised following euthanasia. Fresh tissue was immediately processed for flow cytometric assessment of mitochondrial membrane potential. Additional tissue was snap-frozen in liquid nitrogen for protein analysis, while the remainder was fixed in 10% neutral buffered formalin for 24 h, dehydrated in graded ethanol, and paraffin-embedded for histological examination. Finally, the rats were sacrificed through the injection of excessive pentobarbital sodium.

Examination of gastric tissue changes

For histopathological analysis, gastric tissues from each group were photographed under standardized magnification and height settings. Formalin-fixed samples were cleared using xylene, embedded in paraffin, and sectioned at 5 μm thickness using a microtome. Sections were stained with hematoxylin and eosin (H&E), and histological evaluation of gastric mucosal damage was conducted at 40× and 400× magnification using an optical microscope.

Quantitative real-time PCR detection of F. nucleatum in rat gastric mucosa

Total DNA was extracted from rat gastric tissues using the Animal Tissue/Cell Genomic DNA Extraction Kit (D1700, Solarbio) according to the manufacturer’s protocol. Genomic DNA from F. nucleatum cultures was isolated using the Bacterial Genomic DNA Extraction Kit (DP302-02, Tiangen). DNA concentration and purity were assessed using a nucleic acid/protein quantifier, and integrity was confirmed by agarose gel electrophoresis. Samples meeting quality criteria were stored at ˗20 °C until analysis. Target gene amplification and cloning were performed using primers listed in Supplementary Table S1, with polymerase chain reaction (PCR) conditions detailed in Supplementary Tables S2 and S3. Amplified products were purified with the SanPrep Column DNA Gel Extraction Kit (B518131, Sangon Biotech) and ligated into the PEASY-T1 vector (CT101-01, TransGen Biotech) using the ligation protocol described in Supplementary Table S4. The ligation mixture was transformed into competent Escherichia coli (E. coli) DH5α cells, and individual colonies were screened to confirm correct gene insertion. Positive clones harboring recombinant plasmids were expanded, and plasmid DNA was extracted using the Endo-Free Plasmid Mini Kit (B518161-0100, Sangon Biotech). Purified plasmids were validated and used to construct a standard curve for absolute quantification. Specifically, plasmids containing the F. nucleatum-specific gene fragment were serially diluted and subjected to quantitative real-time PCR (qPCR) to generate a standard curve. The abundance of F. nucleatum in gastric tissue samples was quantified by detecting the 16 S rRNA gene via qPCR. Copy numbers were calculated from Ct values relative to the standard curve. Detailed reaction components and cycling parameters are provided in Supplementary Tables S5 and S6.

Cell infection and viability assay

The human gastric epithelial cell line GES-1 (GES-1) was obtained from Shanghai Yaji Biotechnology and cultured in RPMI-1640 medium supplemented with 1% penicillin–streptomycin at 37 °C in a humidified 5% carbon dioxide (CO2) incubator. A growth curve was generated to identify the logarithmic growth phase for experimental use (Supplementary Fig. S1A). Following established protocols [37, 38], a CAG model was induced by treating cells with 40 µM MNNG for 24 h, while control cells were cultured under standard conditions. After treatment, the culture medium was replaced with 100 µL of 10% Cell Counting Kit-8 (CCK-8) solution per well. After 1 h of incubation, absorbance at 450 nm was measured using a microplate reader. Successful model establishment was indicated by a significant reduction in cell viability in the MNNG-treated group (Supplementary Fig. S1B). GES-1 cells at 90% confluence and in healthy condition were harvested, resuspended at 5 × 104 cells/mL, and seeded into 96-well plates (100 µL/well) for 24 h. The medium was then replaced, and cells were treated with 40 µM MNNG and co-cultured with F. nucleatum at various MOIs (0, 50, 100, 150, 200, 250, 300) for 24 h. After treatment, the medium was discarded, and 100 µL of 10% CCK-8 solution was added per well. After 1 h of incubation, OD450 was measured to determine the half-maximal inhibitory concentration (IC50) of F. nucleatum in MNNG-injured cells (Supplementary Table S7, Fig. 1C). Based on these results, MOI values of 50, 100, and 150 were selected for subsequent experiments. Accordingly, GES-1 cells were divided into five groups: (1) GES-1 (cultured under normal conditions for 24 h); (2) GES-1 + MNNG (treated with 40 µM MNNG for 24 h); and (3–5) low-, medium-, and high-dose F. nucleatum groups (MOI = 50, 100, and 150, respectively), each co-treated with 40 µM MNNG for 24 h.

Fig. 1.

Fig. 1

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Gastric tissue morphological changes and bacterial ectopic colonization in rats after F. nucleatum infection. A Comparison of gross gastric morphology in each group (n = 8 per group); (B) Comparison of gastric tissue histopathological structure in each group (H&E staining, n = 8 per group); (C) Comparison of colonization in the gastric mucosa of rats detected by qPCR (n = 6 per group). Data are presented as mean ± standard deviation. One-way ANOVA was used for comparisons among groups. P < 0.05, P < 0.01, P < 0.001

ELISA for inflammatory cytokines in rat serum and cell culture supernatant

Prior to analysis, all of the reagents and samples were equilibrated to room temperature. Serum levels of IL-1β, IL-6, and TNF-α in rats were measured using the Rat IL-1β enzyme-linked immunosorbent assay (ELISA) Kit (EK301B-48, Lianke Bio), Rat IL-6 ELISA Kit (EK306-48, Lianke Bio), and Rat TNF-α ELISA Kit (EK382-48, Lianke Bio), respectively, according to the manufacturers’ instructions. Similarly, cytokine concentrations in cell culture supernatants were quantified using corresponding human ELISA kits: IL-1β (EK101B-48), IL-6 (EK106/2–48), and TNF-α (EK182-48), following the protocols provided.

Flow cytometric analysis of mitochondrial membrane potential

For the in vivo experiments, fresh gastric tissues from each group were collected, minced, and digested with 200 U/mL collagenase type II for 45 min. Single-cell suspensions were prepared in phosphate-buffered saline (PBS) at a concentration of 1 × 106 cells/mL. For cell-based assays, GES-1 cells were harvested after treatment and similarly resuspended in PBS at 1 × 106 cells/mL. The suspensions were centrifuged at 2000 rpm for 5 min, and the supernatant was discarded. Cell pellets were incubated with 500 µL of 5 µM JC-1 staining solution at 37 °C for 30 min, washed twice with ice-cold PBS, and resuspended in 500 µL PBS. Mitochondrial membrane potential was assessed by flow cytometry based on the fluorescence intensities of JC-1 aggregates (red) and monomers (green), followed by statistical analysis.

Statistical analysis

All of the statistical analyses were conducted using Statistical Product and Service Solutions (SPSS) 26.0. Normally distributed continuous variables were expressed as mean ± standard deviation (x̄ ± s). Comparisons among multiple groups were performed using one-way analysis of variance (ANOVA) when the assumption of homogeneity of variance was satisfied, or Welch’s ANOVA when variances were unequal. Post-hoc pairwise comparisons were carried out using the least significant difference test (equal variances) or Dunnett’s T3 test (unequal variances). Non-normally distributed continuous variables were presented as median (interquartile range) (M [P25, P75]) and compared using the Kruskal–Wallis H test, followed by pairwise multiple comparisons using the Kruskal–Wallis one-way ANOVA (k samples) method. Graphs were generated with GraphPad Prism 9.0. Statistical significance was defined as p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***).

Results

F. nucleatum infection induces gastric morphological changes and bacterial translocation in rats

Gastric tissues were collected from each group for analysis. Gross examination revealed reduced gastric mucosal folds in the CAG group compared to the NC group, while the CAG + F. nucleatum group exhibited near-complete loss of mucosal folds relative to both controls (Fig. 1A).

Representative H&E-stained sections are shown in Fig. 1B. Gastric tissue from the NC group exhibited preserved architecture, clearly defined histological layers, no evidence of mucosal congestion or hemorrhage, abundant glandular cytoplasm, and normal staining patterns. In contrast, the CAG group displayed marked mucosal thinning, glandular atrophy with structural disorganization, and focal goblet cell increases indicative of intestinal metaplasia. Compared with the CAG group, the CAG + F. nucleatum group demonstrated more severe mucosal thinning, reduced pit length, diminished glandular cell populations, and pronounced inflammatory infiltration—predominantly eosinophils and lymphocytes—in the lamina propria and submucosa.

Bacterial colonization in the gastric mucosa was quantified using qPCR. The copy number of the F. nucleatum 16 S rRNA gene in the CAG + F. nucleatum group was 9493.36 ± 4610.73 copies/ng, whereas minimal colonization was observed in the NC and CAG groups (Fig. 1C). Group differences were statistically significant (one-way ANOVA, p < 0.05).

F. nucleatum elevates serum inflammatory cytokine levels and reduces mitochondrial membrane potential in gastric tissues of rats

Serum samples were collected from each rat group, and concentrations of IL-1β, IL-6, and TNF-α were quantified by ELISA (Fig. 2A–C). In the NC group, cytokine levels were 20.63 ± 3.56 pg/mL (IL-1β), 112.91 ± 37.93 pg/mL (IL-6), and 2.68 ± 0.23 pg/mL (TNF-α). These values increased significantly in the CAG group to 28.96 ± 3.57 pg/mL, 391.95 ± 155.86 pg/mL, and 3.33 ± 0.21 pg/mL, respectively (p < 0.05, one-way ANOVA). In the CAG + F. nucleatum group, further elevations were observed: 37.01 ± 5.97 pg/mL, 665.10 ± 131.59 pg/mL, and 4.45 ± 0.30 pg/mL, respectively—all significantly higher than in the CAG group (p < 0.05).

Fig. 2.

Fig. 2

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Serum inflammatory cytokine levels and mitochondrial membrane potential in the gastric tissues of rats following F. nucleatum infection. A–C Serum concentrations of IL-1β, IL-6, and TNF-α in each group as detected by ELISA (n = 6 per group); (D) Bar graph showing JC-1 monomer levels in gastric tissues measured by flow cytometry (n = 6 per group; JC-1 monomer levels are inversely correlated with MMP); (E) Representative flow cytometry plots. Q2: cells with high MMP; Q3: cells with low MMP. All quantitative data are presented as mean ± standard deviation. One-way ANOVA was used for intergroup comparison. P < 0.05, P < 0.01, P < 0.001

Mitochondrial membrane potential (MMP) in rat gastric tissues was assessed using JC-1 staining combined with flow cytometry (Fig. 2D–E). JC-1 is a cationic fluorescent dye whose aggregation state is directly correlated with MMP: when MMP is high, JC-1 accumulates in the mitochondrial matrix as aggregates, emitting red fluorescence (corresponding to the Q2 quadrant in flow cytometry plots); when MMP decreases, JC-1 remains in the cytoplasm as monomers, emitting green fluorescence (corresponding to the Q3 quadrant). Thus, the level of JC-1 monomers is inversely correlated with MMP. The results showed that, compared with the NC group, the level of JC-1 monomers in gastric tissues of rats in the CAG group was significantly increased (P < 0.001), indicating that gastric mucosal cells exhibited decreased MMP and mitochondrial dysfunction in the CAG state. Furthermore, the JC-1 monomer level in the CAG + F. nucleatum (F.n) group was further significantly elevated compared with that in the CAG group (P < 0.001), which was consistent with the flow cytometry data: the proportion of cells in the Q3 quadrant (low MMP cells) increased from 16.2% in the CAG group to 24.6% in the CAG + F.n group, while the proportion of cells in the Q2 quadrant (high MMP cells) decreased from 83.8% to 75.2%. These findings confirm that F. nucleatum infection further exacerbates MMP reduction and mitochondrial dysfunction in the gastric mucosal cells of CAG rats.

F. nucleatum increases inflammatory cytokine levels in culture supernatant and reduces mitochondrial membrane potential in GES-1 cells

Culture supernatants from GES-1 cells were analyzed for IL-1β, IL-6, and TNF-α levels (Fig. 3A–C). In untreated cells, concentrations were 2.74 ± 0.94 pg/mL (IL-1β), 2.31 ± 0.30 pg/mL (IL-6), and 18.64 ± 3.27 pg/mL (TNF-α). Exposure to MNNG significantly increased levels to 5.31 ± 0.35 pg/mL, 6.21 ± 1.32 pg/mL, and 102.41 ± 9.60 pg/mL, respectively (p < 0.05). Co-treatment with F. nucleatum further elevated cytokine secretion in a dose-dependent manner: Low dose: 8.01 ± 0.37 pg/mL (IL-1β), 11.50 ± 3.08 pg/mL (IL-6), 139.06 ± 13.92 pg/mL (TNF-α); Medium dose: 10.71 ± 1.61 pg/mL, 14.14 ± 1.61 pg/mL, 169.15 ± 12.84 pg/mL; High dose: 14.92 ± 0.81 pg/mL, 18.05 ± 1.86 pg/mL, 215.07 ± 18.38 pg/mL All of the values were significantly higher than in the MNNG-only group (p < 0.05).

Fig. 3.

Fig. 3

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Changes in inflammatory cytokine levels in cell culture supernatant and mitochondrial membrane potential in cells following F. nucleatum intervention. A–C Levels of IL-1β, IL-6, and TNF-α in the culture supernatant of cells in each group detected by ELISA (n = 3 per group); (D) Bar graph showing JC-1 monomer levels in cells detected by flow cytometry (n = 3 per group; JC-1 monomer levels are inversely correlated with MMP); (E) Representative flow cytometry plots of cells. Q2: cells with high MMP; Q3: cells with low MMP. All quantitative data are presented as mean ± standard deviation. One-way ANOVA was used for intergroup comparisons. P < 0.05, P < 0.01, P < 0.001

The MMP in GES-1 cells was assessed using JC-1 staining combined with flow cytometry (Fig. 3D–E). The results showed that, compared with the GES-1 control group, the level of JC-1 monomers in the GES-1 + MNNG group was significantly increased (P < 0.01), indicating that MNNG could induce a decrease in MMP and mitochondrial dysfunction in GES-1 cells. Furthermore, with increasing F. nucleatum (F.n) infection dose, the JC-1 monomer level was further elevated in a dose-dependent manner, with extremely significant differences observed in both the medium- and high-dose F.n groups compared with the GES-1 + MNNG group (P < 0.001). Consistent with the flow cytometry plots, the proportion of cells in the Q3 quadrant (low MMP cells) increased from 3.57% in the GES-1 group to 8.35% in the GES-1 + MNNG group, and further increased to 13.4% (low-dose), 17.8% (medium-dose), and 21.9% (high-dose) with escalating F.n doses; meanwhile, the proportion of cells in the Q2 quadrant (high MMP cells) decreased gradually from 96.4% to 91.6%, 86.5%, 82.2%, and 78.0%. These findings confirm that F.n can exacerbate the reduction in MMP and mitochondrial dysfunction in GES-1 cells in a dose-dependent manner under MNNG-induced injury.

Discussion

CAG, a key precancerous lesion of gastric cancer, remains incompletely understood in terms of its pathogenesis. Although H. pylori infection is recognized as the primary etiological factor [4–6], eradication therapy demonstrates limited efficacy in reversing established atrophy and intestinal metaplasia [7, 8]. Moreover, some patients remain at risk of disease progression even after successful eradication [9], suggesting that additional microbial factors beyond H. pylori contribute to CAG pathogenesis [10, 11]. Recent advances in microbiome research have identified the oral-derived bacterium F. nucleatum as a potential contributor to gastrointestinal diseases [22, 24]. However, its specific role in CAG remains undefined. In this study, we employed an MNNG-induced CAG rat model with F. nucleatum intervention to systematically assess its impact on gastric pathology, inflammatory responses, and mitochondrial function.

Our findings demonstrated that F. nucleatum infection markedly exacerbated gastric mucosal injury in the MNNG-induced CAG rat model. Supporting evidence included: (1) macroscopic loss of gastric mucosal folds in the CAG + F. nucleatum group (Fig. 1A); (2) histopathological aggravation, including enhanced glandular atrophy, increased goblet cell metaplasia, and intensified inflammatory infiltration (Fig. 1B); and (3) quantitative PCR (qPCR) confirmation of robust F. nucleatum colonization in gastric mucosa (9493.36 ± 4610.73 copies/ng) (Fig. 1C). These observations are consistent with Chen et al. [31], who reported epithelial barrier disruption and inflammation in a colorectal cancer model. In contrast, Koga [6] proposed that F. nucleatum colonization of the stomach requires a hypochlorhydric environment induced by long-term proton pump inhibitor use. This discrepancy may be attributed to (i) our use of direct intragastric inoculation versus natural colonization, (ii) MNNG-induced mucosal injury creating a permissive niche, and (iii) interspecies differences (Sprague-Dawley rats vs. humans). Notably, we identified a positive correlation between F. nucleatum colonization and the severity of intestinal metaplasia, paralleling the “F. nucleatum–β-catenin–EMT” axis previously reported in colonic tissues by Rubinstein et al. [29], here demonstrated for the first time in gastric mucosa. These findings support the role of F. nucleatum as a potential driver of precancerous progression in CAG.

Mechanistically, this study is the first to elucidate how F. nucleatum exacerbates CAG through an microbiota–mitochondria axis. In rats, infection significantly elevated serum levels of pro-inflammatory cytokines (IL-1β: 37.01 ± 5.97 pg/mL; IL-6: 665.10 ± 131.59 pg/mL; TNF-α: 4.45 ± 0.30 pg/mL; all p < 0.05 vs. CAG) (Fig. 2A–C) and reduced MMP in gastric tissue (JC-1 monomers: 26.52 ± 2.98% vs. 16.13 ± 1.29% in CAG; p < 0.05) (Fig. 2D–E). These effects were further validated in GES-1 gastric epithelial cells, where F. nucleatum induced up to a 10.5-fold increase in TNF-α (Fig. 3A–C) and a 4.5-fold increase in JC-1 monomers in a dose-dependent manner (Fig. 3D–E). These findings align with those of Zhou et al. [12], who reported mitochondrial injury via the mtDNA–cGAS–STING pathway. An in-depth analysis of the mechanisms underlying the inflammation and mitochondrial damage induced by F. nucleatum in CAG requires considering factors at the level of microbe–host interactions. Studies have shown that F. nucleatum can promote IL-1β secretion by activating the NLRP3 inflammasome, thereby exacerbating inflammatory responses [39]. At the same time, this bacterium can also trigger mitochondrial damage by inducing the production of ROS, which further amplifies inflammatory effects through a cascade while disrupting homeostasis in the intestinal and oral mucosa [40]. The findings of the present study preliminarily confirm that F. nucleatum not only aggravates chronic inflammatory responses, but that it also induces mitochondrial damage, ultimately promoting the progression of CAG. However, the precise mechanism by which F. nucleatum contributes to the initiation and development of CAG remains to be further elucidated.

The novelty of this work lies in two key contributions. First, at the conceptual level, we propose the “microbiota–mitochondria axis” as a central mechanism in CAG progression, providing a novel explanation for disease persistence despite H. pylori eradication. Second, from a methodological perspective, we established a robust F. nucleatum-associated CAG animal model and complementary in vitro system. However, his study still has several limitations. The mechanistic exploration was limited to the detection of inflammatory factors and analysis of mitochondrial membrane potential, so the key signaling pathways and molecular targets through which F. nucleatum regulates the microbiota–mitochondria axis were not elucidated. Moreover, as a basic experimental study, this research lacks validation using clinical samples. The colonization rate, load distribution, and correlation with disease severity of F. nucleatum in patients with different stages of CAG remain to be confirmed by large‑sample, multicenter clinical studies. Future research directions include: (1) multicenter clinical studies to assess F. nucleatum colonization across CAG stages; (2) development of therapies targeting F. nucleatum or its virulence factors; and (3) investigation of mitochondrial protective agents as potential interventions for CAG. Such interdisciplinary approaches may offer innovative strategies for the prevention and treatment of CAG.

Conclusions

In summary, this study systematically elucidates the pivotal role and underlying mechanisms of F. nucleatum in the development and progression of CAG, utilizing both in vivo and in vitro models. The major findings are as follows: (1) F. nucleatum successfully colonizes the gastric mucosa and exacerbates MNNG-induced CAG pathology, characterized by increased glandular atrophy, enhanced intestinal metaplasia, and extensive inflammatory infiltration; (2) F. nucleatum mediates its pathogenic effects via an “microbiota–mitochondria axis”, significantly increasing pro-inflammatory cytokine levels and inducing mitochondrial dysfunction; and (3) F. nucleatum promotes inflammatory responses and mitochondrial depolarization in GES-1 cells in a dose-dependent manner. Collectively, these findings provide new mechanistic insights and identify potential therapeutic targets for the early diagnosis and precise treatment of CAG.

Supplementary Information

Supplementary Material 1. (223KB, docx)

Acknowledgements

Not applicable.

Abbreviations

CAG

Chronic atrophic gastritis

F. nucleatum

Fusobacterium nucleatum

H. pylori

Helicobacter pylori

MNNG

N-methyl-N′-nitro-N-nitrosoguanidine

ROS

Reactive oxygen species

NIH

National Institutes of Health

BHI

Brain heart infusion

CFU

Colony-forming units

MOI

Multiplicities of infection

H&E

Hematoxylin and eosin

Authors’ contributions

YZ, AA, and PZ conceptualized the study. YZ, AA, PZ, YF, and HL performed the investigation involving animal experiments and sample collection. YZ, PZ, YJ, and YA performed the investigation involving cell experiments and laboratory assays. YZ, PZ, YF, and HL conducted the formal analysis. PZ, YF, and HL wrote the original draft. All authors contributed to the writing—review and editing. YZ supervised the study. All authors read and approved the final version of the manuscript.

Funding

This work was funded by the Undergraduate Innovation and Entrepreneurship Training Program of Xinjiang Medical University (grant no. X202410760047).

Data availability

The datasets used or analyzed during the current study are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

The animal study protocol was approved by the Animal Research Ethics Committee of the First Affiliated Hospital of Xinjiang Medical University (Approval No.: IACUC-JT-20241015-07).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1. (223KB, docx)

Data Availability Statement

The datasets used or analyzed during the current study are available from the corresponding author upon reasonable request.

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