Cholelithiasis (also known as gallstones) is defined as a solid clot in the gallbladder or biliary system (Lammert et al., 2016). Approximately 90% of cholelithiasis occurrences are cholesterol gallstones, while the incidence of other stone types (including black and brown pigment stones) is below 10% (Sun et al., 2022). Approximately 10–20% of the global population has gallstones, and over 20% of cases develop gallstone diseases, such as acute cholecystitis, acute cholangitis, and obstructive jaundice (Cortés et al., 2020). Gallstone disease is one of the most expensive gastrointestinal conditions from a societal perspective (Grigor’eva and Romanova, 2020). Epidemiological studies identified numerous risk factors for cholesterol stones, which include type 2 diabetes, physical inactivity, and over-nutrition (Di Ciaula et al., 2019). This finding can be attributed to the risk factors that lead to excess cholesterol or disruption of cholesterol homeostasis (Rudling et al., 2019).

The gut microbiota has a non-negligible impact on metabolic disorders, including insulin resistance (Gomes et al., 2018), obesity (Abenavoli et al., 2019), and hyperlipidemia (Michels et al., 2022). These disorders are known risk factors for increased hepatic cholesterol synthesis, gallstone formation, and symptomatic gallstones (Di Ciaula et al., 2019). However, the role of intestinal microbiota in gallstone development remains unclear. Elevated levels of cholesterol and bilirubin in the bile and decreased levels of bile salts cause cholesterol gallstones (Sun et al., 2022). Decreased bile salt levels are observed in liver disease and in conditions such as Crohn’s disease or in individuals that have undergone colectomy or intestinal resection, where the enterohepatic circulation of bile salts is impaired (Molina-Molina et al., 2018). These findings led us to hypothesize that the gut microbiota and intestine may play key roles in influencing gallstone formation in the host. The diversity and taxonomy of gut microbiota are associated with bile acid levels in gallstone disease, and increased concentrations of taurodeoxycholic acid and taurocholic acid are associated with the presence of conditionally pathogenic bacteria (Petrov et al., 2020). However, intestinal flora is often influenced by confounding factors, including age, sex, environment, alcohol consumption, diet, and lifestyle (Zmora et al., 2019). Eliminating these confounders in observational studies can be challenging, thereby limiting research on the causal role of gut microbiota in gallstones. Fortunately, Mendelian randomization (MR) analysis can be employed to explore the causal role of intestinal microbiota in the etiology of human diseases independent of confounding factors (O’Donnell et al., 2023).

MR borrows economically inspired statistical techniques to enable researchers to examine the causal factors affecting human diseases (Birney, 2022). Numerous risk factors related to diseases have not established causation owing to the limitations of observational studies that cannot avoid the influence of confounders. Thus, MR has become one of the most effective methods for addressing issues in human biology and epidemiology, including the relationship between intestinal microbiota and disease (Georgakis and Gill, 2021). The correlation between genetic variation and outcome is independent of confounders in MR analysis (Bowden and Holmes, 2019). For instance, MR analysis has revealed that Bifidobacterium is causally linked to preeclampsia-eclampsia (Li et al., 2022), and the fecal abundance of Oscillibacter and Alistipes is causally associated with decreased triglyceride levels (Liu et al., 2022).

This study used summary-level statistics of the genome-wide association study (GWAS) from the MiBioGen and FinnGen consortia to perform a two-sample MR analysis to investigate the causal relationship between intestinal microbiota and cholelithiasis.

The two-sample MR study relied on three assumptions to draw conclusions regarding causation. Genetic variants strongly predict microbiome exposure independent of confounding factors and outcomes. Genetic variants influence outcomes through exposure (Emdin et al., 2017).

This study used the MiBioGen database and cholelithiasis data from FinnGen to investigate the causal relationship between gut microbes and cholelithiasis using MR analysis. Clostridium senegalense, Coprococcus3, and Lentisphaerae increase the risk of cholelithiasis. In contrast, Holdemania, Lachnospiraceae UCG010, and Ruminococcaceae NK4A214 showed protective effects. The causal relationship identified BSH as a potential link between bile salt metabolism and the gut microbiota.

The intestinal microbiota is a metabolic organ that produces numerous metabolites (Connell et al., 2022) (including BAs and indole derivatives) (Agus et al., 2018) that play crucial roles in regulating host metabolism (Cai et al., 2022b). Cholesterol oxidized by liver enzymes results in the production of BAs that are further metabolized by the intestinal microbiota (Collins et al., 2023). The gut microbiota regulates key enzymes, such as cholesterol-7α-hydroxylase (CYP7A1), involved in BA synthesis (Hartmann et al., 2018; Fukui, 2021), and BA synthesis is tightly controlled by negative feedback inhibition through the farnesoid X receptor (FXR) (Jia et al., 2018). Bile acid deconjugation is primarily mediated by bacteria with BSH activity (Smirnova et al., 2022). Thus, intestinal microbiota plays a significant role in the key enzymatic processes involved in cholesterol synthesis in the liver, secondary BA production, and enterohepatic circulation of BAs.

The balance between cholesterol and bile salts is a critical factor for gallstones (Ye et al., 2021). Previous studies have revealed direct associations between taurocholic acid, taurochenodeoxycholic acid, and alpha diversity of the microbiota, together with positive associations with the genera Chitinophagaceae, Microbacterium, Lutibacterium, and Prevotella intermedia (Petrov et al., 2020). Patients with gallstones exhibit an increased richness of 7a-dehydroxylating microbiota and decreased levels of Fimicutes and diversity of gut microbiota (Wang et al., 2020). Additionally, the abundance of Lactobacillus strains significantly reduced in lithogenic diet-induced gallstones through the mediation of FXR signaling (Ye et al., 2022). However, the causal correlation between the intestinal flora and cholelithiasis remains unclear. Our study predicted that presence of abundance of Clostridium senegalense and Coprococcus3 have a causal relationship with cholelithiasis.

Many observational studies report a correlation between Clostridium and gallstone disease. For instance, patients with gallstones have elevated levels of Clostridium in the stool (Grigor’eva and Romanova, 2020), and Clostridium species were isolated from gallbladder stones (Liu et al., 2000), consistent with the results of our study. We found a causal association between Clostridium senegalense and cholelithiasis. Clostridium-encoded protein analysis revealed that Clostridium had two genes of BSH consistent with a previous study (Song et al., 2019). The presence of greater amounts of bacteria with BSH leads to increased bile salt deconjugation, resulting in elevated biliary deoxycholate levels, positive regulation of hepatic cholesterol secretion, and cholesterol crystallization (Lammert et al., 2016). Furthermore, increased hydrolysis can lead to steatosis, and increased secondary bile acid levels are associated with colorectal cancer (Horáčková et al., 2018). Recent research showing that theabrownin can reduce hypercholesterolemia by inhibiting the intestinal microbiota related to BSH activity indirectly supports our hypothesis. The underlying mechanism may be that low BSH activity increased the concentration of conjugated BAs, which could inhibit the FXR-FGF15 signaling pathway, thereby led to decreased hepatic cholesterol synthesis (Huang et al., 2019). However, the effect of BSH on the microbiota in cholelithiasis causation requires verification and further exploration.

Coprococcus was also predicted to be a risk factor for gallstone disease, and encoded BSH proteins which was similar to the previous study (Song et al., 2019). Moreover, the relative abundance of BSH in the microbiota is significantly associated with mortality from diabetes and cardiovascular disease (Song et al., 2019). This finding further emphasizes the significance of BSH in cholelithiasis. Further research is required to understand how BSH in Clostridium and Coprococcus affect the formation of cholesterol stones. Recent studies show that Desulfovibrionales are enriched in cholelithiasis patients, increase the synthesis of secondary BAs and intestinal cholesterol uptake, stimulate biliary secretion, and affect FXR and CYP7A expression [15]. Further research is required to determine whether BSHs in Clostridium and Coprococcus promote cholesterol stone formation by increasing the secondary BA synthesis.

The present study revealed the protective effects of Lachnospiraceae UCG010 and Ruminococcaceae NK4A214 against cholelithiasis. These strains did not express BSH. This result suggests that BSH deficiency might be a protective factor against gallstone formation; however, this hypothesis requires verification. Microbial conversion of cholesterol to coprostanol is another mechanism that reduces cholesterol and decreases the formation of cholesterol stones (Kenny et al., 2020). Lactobacillus curvatus KFP419 strain reduce cholesterol levels by increasing the conversion of cholesterol to coprostanol (Park et al., 2018). Lachnospiraceae and Ruminococcaceae are reportedly associated with high coprostanol levels (Antharam et al., 2016). An elevation of sterol metabolites coincides with increases in the Lachnospiraceae family in vitro, implying that this family promotes sterol metabolism (Blanco-Morales et al., 2020). These findings suggest that the protective effects of Lachnospiraceae and Ruminococcaceae against cholelithiasis may be related to cholesterol conversion; however, the underlying mechanisms require further exploration.

To the best of our knowledge, this is the first MR investigation revealing a link between gut microbiota and cholelithiasis. Robust genetic instruments were employed with a test of horizontal pleiotropy and two-sample heterogeneity to obtain reliable results from the MR analysis. Additionally, a leave-one-out analysis was performed to examine potential biases introduced by individual SNPs. Five sets of genetic instruments were used for MR analysis. In addition, the concept of BSH was introduced as an innovative approach to investigate the underlying mechanisms of the causal association between microbiota and cholelithiasis.

Nevertheless, this study has limitations. First, most patients with cholelithiasis in the analysis were of European descent, whereas the gut flora database encompasses other populations. Second, the potential association between microbiota and cholelithiasis by BSH was only superficially speculated. Further research is required to elucidate the underlying mechanisms and causal relationship. Third, subgroup analyses such as distinguishing between symptomatic and asymptomatic cholelithiasis was not possible because summary data for cholelithiasis was used in our analysis. Fourth, our exploration was limited to the genus level owing to the lowest taxonomic level available in the gut microbiota dataset, thus impeding a more detailed investigation at the species level. Additionally, some bacteria were predicted to be present at the phylum level; we could not analyze their BSH proteins. Finally, the sample size of the exposure group was relatively small; therefore, the reverse MR analysis results could not completely exclude the possibility of reverse causality.

Our investigations demonstrated a causal relationship between cholelithiasis and Clostridium senegalense and Coprococcus3, whereas Holdemania, Lachnospiraceae UCG010, and Ruminococcaceae NK4A214 had a protective effect. The causal relationship between the gut microbiota and cholelithiasis may be mediated by BSH. In addition, reverse MR analysis supported a causal relationship between cholelithiasis and the intestinal microbiota. Moreover, these findings suggest that cholelithiasis may influence the gut flora. Further validation and mechanistic studies are required.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ Supplementary Material .

Ethical approval was not required for the study involving humans in accordance with the local legislation and institutional requirements. Written informed consent to participate in this study was not required from the participants or the participants’ legal guardians/next of kin in accordance with the national legislation and the institutional requirements.

XL: Data curation, Formal Analysis, Investigation, Methodology, Resources, Visualization, Writing – original draft, Writing – review & editing. XQ: Data curation, Formal Analysis, Methodology, Writing – review & editing. RH: Data curation, Validation, Writing – original draft. TM: Supervision, Writing – review & editing. ZT: Funding acquisition, Investigation, Project administration, Writing – review & editing.