Gastric cancer (GC) ranked as the fifth most common cancer and cause of cancer-related deaths globally, with 968,000 new cases and 660,000 deaths reported worldwide in 2022 (Bray et al., 2024). East Asia and Eastern Europe are the regions with the highest incidence rates globally. Extensive research suggests that gastric carcinogenesis is associated with genetic factors, Helicobacter pylori infection, dietary habits, and regional environmental factors, all interacting within a complex network (López et al., 2023; Noto et al., 2022; Smyth et al., 2020). The dynamic balance of human gut microbiota (GM) is closely linked to both physiological and pathological conditions and can directly or indirectly influence the carcinogenesis, treatment, and prognosis of GC (Wang et al., 2023b; Wang et al., 2023d). Increasing evidence indicates that GC patients experience changes in GM diversity and abundance, with alterations in specific microbial communities associated with GC risk (Yu et al., 2024). Additionally, some GM metabolites, such as short-chain fatty acids (SCFAs) and bile acid metabolites, have been associated with the formation and progression of GC (Noto et al., 2022; Yu et al., 2024). These metabolic products can enter systemic circulation via the gut-liver and gut-blood axes, exerting significant effects on systemic metabolism (Ahlawat et al., 2021; Hsu and Schnabl, 2023).

Blood metabolites, as critical indicators of the body’s metabolic status, serve as biomarkers for the diagnosis and prognosis of various diseases (Kurilshikov et al., 2019; Xu Z. et al., 2023). Previous studies have shown that changes in blood metabolites are closely linked to GM dysbiosis and its metabolic byproducts (Vojinovic et al., 2019). Consequently, the GM and its metabolites, along with blood metabolites, may play important roles in the carcinogenesis and development of GC (Cao et al., 2023; Lario et al., 2017). Further investigation into these relationships could not only clarify the pathogenesis of GC but also provide novel insights and methodologies for its early diagnosis and prevention. However, as current research is predominantly observational, it is prone to confounding factors such as dietary patterns, environmental influences, and reverse causality, often with small sample sizes. Additionally, ethical and practical limitations pose challenges to conducting randomized controlled studies that involve all bacterial strains.

Mendelian randomization (MR) is a powerful data analysis method for inferring causal relationships and has been widely applied in epidemiological research (Sekula et al., 2016). With advancements in sequencing technologies and the public availability of large-scale genome-wide association study (GWAS) data, numerous reliable genetic variations, such as single nucleotide polymorphisms (SNPs), are now accessible for MR studies. In this study, we used the latest GWAS datasets to conduct bidirectional MR and mediation analyses, aiming to explore the causal associations among GM, circulating metabolites, and GC risk. Additionally, we integrated metabolomics and microbiomics data to better elucidate the potential mediating role of circulating metabolites in the relationship between GM and GC.

In this large-scale MR study, we identified potential causal relationships between 19 GM taxa and 7 blood metabolic traits with GC and simultaneously explored their potential interactions. Using a two-step MR method for mediation analysis, we found that five blood metabolic traits may mediate the causal relationship between GM and GC. These findings suggest that the protective effects of the Bacteroidia class and Bacteroidales order in reducing GC risk may be partially attributed to their influence on the concentrations of five blood metabolites.

In our study, Bacteroides exhibited a negative correlation with GC across taxonomic levels from phylum to genus, suggesting a potential protective role for Bacteroides in GC patients. Its reduced abundance may be associated with gastric carcinogenesis. An observational study examining fecal samples from 22 healthy participants and 20 GC patients reported findings consistent with ours, revealing a significant decrease in Bacteroides abundance from phylum to genus in GC patients compared to healthy controls (Liang et al., 2019). Additionally, an MR study conducted in a different population further supports our findings (Xie et al., 2023). Bacteroides is a key commensal bacterium in the human gut and a major producer of SCFAs, which play a crucial role in maintaining immune homeostasis within the gastrointestinal tract (Raoul et al., 2024; Wexler, 2007). In gastrointestinal neoplasms, both SCFA levels and the abundance of SCFA-producing bacteria are significantly reduced (Dong et al., 2023; Yu et al., 2024). Previous studies have shown that direct supplementation of dietary fiber and probiotics or the transplantation of fecal microbiota to alter GM composition can enhance SCFA levels and suppress neoplasm development (Dong et al., 2023; Yu et al., 2024).

Growing evidence suggests that SCFAs, as natural histone deacetylase (HDAC) inhibitors produced by GM, play a critical role across various immune cells. SCFAs can promote macrophage differentiation and enhance antimicrobial activity by inhibiting HDAC3 (Schulthess et al., 2019). They also induce IL-10 expression in T cells and enhance B cell activation, thus promoting immune tolerance and maintaining gut microbial balance (Kim, 2023). Recent experimental studies have found that butyrate regulates CD8+ T cell cytotoxicity through the G protein-coupled receptor 109A (GPR109A) and homologous domain protein homologous box (HOPX) pathway, resulting in enhanced anti-cancer activity against tumor cells in the GC microenvironment (Yu et al., 2024). Notably, butyrate and other SCFAs have direct effects on the immune system and also indirectly suppress the development of gastrointestinal tumors, including GC, by preserving gut barrier function and preventing microbial dysbiosis (Gou et al., 2024; Kaźmierczak-Siedlecka et al., 2022). These findings suggest that increasing SCFA levels through dietary or probiotic interventions may have significant potential in reducing GC risk (Gou et al., 2024; Yu et al., 2024).

In our study, Butyrivibrio and Phascolarctobacterium genera exhibited the most significant negative correlations with GC risk. Both genera belong to the Firmicutes phylum and are producers of SCFAs. Butyrivibrio, a beneficial gut bacterium, generates butyrate, a metabolite linked to numerous health-promoting effects, including the maintenance of the intestinal barrier, energy homeostasis, and anti-inflammatory and antioxidant activities (Hodgkinson et al., 2023; Zhang et al., 2021). A cross-sectional study reported a significant increase in Butyrivibrio abundance in patients’ intestines following subtotal gastrectomy for early-stage GC (Lin et al., 2018). However, research on the association between Butyrivibrio abundance and GC risk remains limited, warranting further clinical studies to elucidate their relationship. Phascolarctobacterium, primarily utilizing succinate produced by Bacteroides as a nutritional substrate, may interact with Bacteroides in the gut (Ikeyama et al., 2020; Watanabe et al., 2012). We therefore hypothesize that Phascolarctobacterium abundance may decrease along with the reduction of Bacteroides in the gut of GC patients. A recent study indeed found a significant reduction in Phascolarctobacterium abundance among GC patients (Yu et al., 2024), which indirectly supports its protective effect against GC.

The Ruminococcus genus includes both beneficial and harmful species, and its impact on GC risk may vary across populations and classification levels. Our findings indicate a positive correlation between the Ruminococcaceae family and GC risk. A fecal microbiota analysis of a European cohort similarly revealed a marked increase in Ruminococcus abundance among GC patients (Youssef et al., 2018). Conversely, studies analyzing fecal samples from East Asian populations reported conflicting results (Lee et al., 2024; Yu et al., 2024). Consequently, additional research with larger, ethnically diverse samples is necessary to clarify the relationship between Ruminococcus and GC risk.

Our MR study identified several blood metabolic traits with causal effects on GC risk, providing insights from a genetic perspective. For instance, elevated levels of phospholipids and free cholesterol in small and medium LDL were associated with a decreased GC risk. Similarly, higher levels of total lipids, cholesterol esters in IDL, and increased IDL particle concentrations correlated with reduced GC risk. Prior research has predominantly examined traditional blood markers linked to lipoproteins and lipids in relation to GC (Qiu et al., 2023). A meta-analysis summary has demonstrated that serum high-density lipoprotein cholesterol (HDL-C) and total cholesterol (TC) levels are negatively associated with GC risk, while low-density lipoprotein cholesterol (LDL-C) and triglycerides (TG) levels show no association with GC risk (Xu S. et al., 2023). Our study is the first to examine the causal relationship between circulating lipoprotein subclasses and GC risk, providing new insights into the intricate biochemical mechanisms underlying lipid metabolism dysregulation in GC patients.

To date, no studies have directly linked the Bacteroidia class or the Bacteroidales order to circulating lipoprotein subfractions. Previous observational studies have confirmed associations between specific GM taxa and subfractions of very-low-density lipoprotein (VLDL) and HDL, offering some understanding of the GM’s influence on host systemic metabolism (Vojinovic et al., 2019). The mechanisms by which GM may influence circulating metabolites remain incompletely understood, but they likely involve GM’s role in bile acid and SCFA metabolism (Ghazalpour et al., 2016). Prior studies suggest that bacteria-derived bile acids entering the bloodstream can participate in systemic lipid metabolism (Allayee and Hazen, 2015), while SCFAs such as butyrate, acetate, and propionate may also influence lipid biosynthesis (Allayee and Hazen, 2015). Through mediation analysis, we further explored the potential mediating effects of circulating metabolites on the association between GM and GC. Overall, the mediation proportions of these blood metabolite categories in the GM-GC relationship were relatively low. These modest mediation percentages likely reflect the complex and multifactorial nature of GC etiology, suggesting that blood metabolites play a limited mediating role in the relationship between GM and GC. Furthermore, the similarity in mediation proportions observed among certain metabolite categories may be attributed to shared lipid metabolism pathways and the overlapping biological functions of these lipid components, leading to parallel mediating effects on GC risk. From the funnel plots in our study, we observed that when GM was used as the exposure, the SNP distribution for certain taxa was uneven, indicating potential heterogeneity in the results. As with most MR studies on GM (Chen et al., 2023; Li et al., 2023; Zhou et al., 2023), this heterogeneity may be due to the limited number of available IVs. Consequently, the observed associations between these taxa and GC cannot entirely rule out the influence of chance or unmeasured confounding factors. Further research is therefore needed to confirm the definitive causal relationships between these taxa and GC.

This study has several strengths. We utilized the most recent and largest GWAS summary data available for blood metabolites and GC, with over 136,000 participants for metabolite data, 476,116 European participants, and 167,122 East Asian participants for GC data. Furthermore, we validated associations between blood metabolites and GC in both European and East Asian populations, ensuring robust statistical power. This study is also the first to investigate causal relationships among human GM, blood metabolites, and GC from a genetic standpoint, minimizing the confounding effects commonly encountered in observational studies.

Conversely, several potential limitations of this study should be acknowledged. First, our analysis relied exclusively on GM data from European populations. Given the substantial variability in GM composition across populations and ethnic groups, the generalizability of our findings may be restricted. Second, the GM data were generated using 16S rRNA sequencing, which inherently limits taxonomic resolution and hampers differentiation between closely related bacterial species (Rühlemann et al., 2021). Additionally, our study was restricted to bacterial taxa, while other non-bacterial microbiome constituents, such as fungi, viruses, archaea, and protozoa, are increasingly recognized for their roles in gastrointestinal health and disease (Jaswal et al., 2023). Expanding future analyses to encompass these groups could provide a more comprehensive understanding of the microbiome’s role in GC. Third, although we used a bidirectional MR approach to evaluate causal relationships, the power of the reverse MR analysis may be limited by the relatively low number of GC cases, potentially impacting the robustness of these reverse causal findings. Thus, we recommend cautious interpretation of these results. Additionally, although our MR analysis detected no pleiotropy, it cannot be entirely ruled out, especially as GM influences luminal metabolites that may not persist in the bloodstream or be detectable at all sampling points. Lastly, we identified GM taxa with nominally significant associations with GC, though these associations lost significance after applying multiple correction tests. Given the complex interdependencies among GM taxa (Faust and Raes, 2012), multiple testing corrections may be overly conservative in high-dimensional datasets (Wang et al., 2023c). Therefore, while not meeting the threshold for strict significance, the nominal associations identified should not be dismissed entirely, particularly as prior studies have corroborated that some of these taxa are associated with GC risk. Future studies with larger sample sizes and refined correction techniques may help to confirm these associations more robustly.

In summary, our MR analysis using genetic instruments identified several blood metabolites with potential causal associations to GC risk. Additionally, it provided suggestive evidence for a causal relationship between GM and GC, as well as the potential mediating role of blood metabolites in the GM-GC association. While this exploratory study offers valuable insights into the complex interactions between GM, blood metabolites, and GC, further research is essential to validate these causal relationships and elucidate the underlying mechanisms. Such efforts could ultimately inform novel GC treatment strategies centered on GM modulation and targeted metabolite interventions.