Cholecystitis is primarily an acute or chronic inflammatory reaction in the wall of the gallbladder, usually caused by gallstones or a bacterial infection of the gallbladder duct (1). Epidemiological data indicates that approximately 20 million people in the United States are affected by gallstones (2), with 1 to 4% of gallstone patients developing conditions such as chronic cholecystitis, acute cholecystitis, or gallstone-induced pancreatitis (3). Furthermore, delayed treatment of biliary infections can still lead to systemic inflammatory reactions and potentially fatal outcomes, with mortality rates ranging from 11 to 27% (4). Modern comprehensive studies on cholecystitis have revealed a significant association between its etiology and factors such as diet and lifestyle. Therefore, investigating early intervention and preventive strategies for cholecystitis remains crucial in mitigating the societal health burden (5).

Bile is a biofluid synthesized in the liver, stored, and concentrated in the gallbladder, where it is released into the intestine, specifically the duodenum, following meals. Alterations in the gallbladder due to disease can lead to changes in the gut microbiota composition as a result of bile influence on the gut ecosystem (6). Molinero et al. conducted 16S rRNA sequencing (16S rRNA sequencing is a method to study and identify bacteria and archaea by analyzing a specific genetic marker called the 16S ribosomal RNA gene. It helps reveal the composition and diversity of gut microbiota in various environments) and metagenomic sequencing (Metagenomic sequencing is a method to study all genetic material in gut microbiota, offering insights into diversity and functional potential) of bile samples from the gallbladder. Their findings revealed similarities in the distribution of major functional categories in the biliary metagenome to those described in the human gut microbiota, suggesting potential associations between the two (7). The human gut microbiota is composed of trillions of microorganisms. These microorganisms in the gut can undergo translocation through the duodenum via the Oddi sphincter or enter the liver through the bloodstream, subsequently being excreted into the bile. This process facilitates bacterial migration and colonization within the bile system (8). Recent research has indicated a significant association between the gut microbiota and the occurrence of acute cholecystitis. Likewise, Liu et al. utilized 16S rRNA and metagenomic sequencing techniques, obtaining a total of 185,713 high-quality sequence reads from fecal samples collected from 15 patients and 13 healthy controls. Their findings revealed a correlation between the gut microbial family Enterobacteriaceae and acute cholecystitis (9). Furthermore, several studies have proposed a significant association between Helicobacter pylori infection and cholecystitis. The underlying mechanism may involve Helicobacter pylori’s capability to infect the biliary system, leading to chronic inflammation of its mucosa. Consequently, this can impair bile acid secretion and reduce calcium salt solubility, thereby increasing the likelihood of gallstone formation (10–12).

It is well known that the human immune system relies on various micronutrients, such as vitamins A, D, C, E, B6, B12, and folate, as well as zinc, iron, copper, and selenium. These micronutrients play critical and synergistic roles in various stages of the immune response, ensuring the normal functioning of physical barriers and immune cells (13). Given this, there may exist potential regulatory mechanisms between circulating vitamin levels in the bloodstream and the occurrence of cholecystitis.

Existing research has also substantially demonstrated the interaction between the gut microbiota and circulating vitamin levels. The gut microbiota has the capacity to produce, consume, or compete for vitamin B, and supplementation or deficiency of vitamin B may impact the growth of specific bacteria, leading to changes in the composition of the gut microbiota (14). Li and Didier, for instance, found that antioxidant vitamins A, E, and β-carotene can improve gut barrier function and maintain normal immune system function by modulating the composition and metabolic activity of the gut microbiota, or by directly influencing the body’s immune response in response to oxidative stress (15, 16). Luthold and colleagues, in a study investigating vitamin D intake in 150 healthy adults, demonstrated that the role of vitamin D in maintaining immune homeostasis partly involves interactions with the gut microbiota (17). Interestingly, an observational study indicated that circulating vitamin C levels have a protective effect against cholesterol gallstones (18). Notably, vitamin D exerts potent regulatory effects on both innate and adaptive immunity (19), with research suggesting that maintaining serum 25(OH)D concentrations above 40 ng/mL can significantly reduce symptomatic diseases and decrease the prevalence of infections and chronic conditions (20).

Therefore, it is crucial to determine whether circulating vitamin levels (vitamins A, B12, C, D, and E) in the bloodstream interact with the gut microbiota in the development of cholecystitis. Unfortunately, these studies still face the risk of confounding factors and reverse causality, making the potential causal relationship between the gut microbiota, circulating vitamin levels, and cholecystitis unclear. Previously, a Mendelian randomization (MR) study involving Genome-wide association study (GWAS) of the gut microbiota from 18,340 individuals and GWAS data of vitamin D from 417,580 individuals showed a causal relationship between Erysipelotrichales and circulating vitamin D levels (The effect size of the main MR analysis method result:β = −0.06, The p-value after multiple correction using the False Discovery Rate (FDR) method: P_FDR = 1.93 × 10⁻⁴) (21). However, to ensure the accuracy of the results, this study will conduct further research using a larger-scale GWAS meta-analysis of circulating vitamin D levels.

MR is a robust methodological framework that employs genetic variation as instrumental variables. By conducting a comprehensive analysis that integrates data from GWAS and gut microbiota, we can identify genetic variants closely associated with gut microbiota traits. This approach allows us to overcome limitations inherent in traditional observational studies, including confounding factors and reverse causality, thus providing more reliable evidence for causal inference (22). Furthermore, this study employs a series of sensitivity analyses to ensure the stability and reliability of the results. It also employs the cML-MA method to eliminate potential pleiotropy, both coherent and incoherent. The primary objective of this research is to assess the causal impact of gut microbiota on cholecystitis and the mediating role of circulating vitamins. This study aims to advance our understanding of the mechanisms underlying cholecystitis and holds significance for the development of preventive and therapeutic interventions. Future research should involve large-scale clinical randomized controlled trials to validate the causal relationships between gut microbiota, circulating vitamin levels, and the risk of cholecystitis.

This study employed large-scale GWAS summary data and applied a two-sample MR approach to investigate the association between gut microbiota, serum vitamin levels, and cholecystitis. Our findings revealed a positive correlation between an increase in the abundance of gut microbiota RuminococcaceaeUCG003 and an elevated risk of cholecystitis. Additionally, we observed that the increased risk of cholecystitis might be mediated by a decline in circulating vitamin D levels (proportion mediated = 5.5%).

Firstly, we observed a significant correlation between gut microbiota dysbiosis and the risk of cholecystitis, consistent with prior research. Studies suggest that bacteria can colonize the gallbladder either through the duodenal-ampullary complex or by hematogenous spread to the liver, eventually leading to the formation of a microbial environment in the gallbladder (37). For instance, Hu et al., using ITS and 16S rRNA gene high-throughput sequencing, studied the fungal and bacterial spectra in bile and gallstone samples from cholelithiasis patients, identifying the presence of Ruminococcus genus and other gallbladder microbiota consistent with our findings (38). Gut microbiota dysbiosis may promote the development of cholecystitis through multiple mechanisms. Firstly, it may trigger an inflammatory response within the gallbladder, leading to cholecystitis (39). Furthermore, a meta-analysis has shown a positive association between Helicobacter pylori infection and chronic cholecystitis and cholelithiasis (OR = 3.022, 95% CI: 1.897–4.815, I² = 20.1%), this could be due to H. pylori infection causing chronic inflammation of the biliary mucosa, leading to impaired bile acid secretion and increased risk of gallstone formation (11). Secondly, gut microbiota dysbiosis may affect the metabolism of bile acids, leading to changes in bile acid composition and function, thus promoting the formation of gallstones and the development of cholecystitis (40).

Recently, research on the relationship between gut microbiota and circulating vitamin levels has become a hot topic. Gut microbiota metabolism can influence the synthesis, absorption, and metabolism of vitamins. For example, LeBlanc et al. proposed that B vitamins can be synthesized through lactate and potentially by bifidobacteria present in the gut (41). On the other hand, Traber et al. suggested that dietary overnutrition could lead to an increased abundance of gram-negative bacteria in the gut, causing increased inflammation and compromised gut function, subsequently affecting the status of antioxidant vitamins (such as vitamin C and vitamin E) (42). Additionally, some studies have indicated a high expression of vitamin D receptors in the gastrointestinal tract, but the relationship between gut microbiota and vitamin D is not yet well-established (43). Hence, we aimed to elucidate the mediating role of different circulating vitamin levels in the association between gut microbiota and cholecystitis. The causal relationship between serum vitamin levels and gut microbiota, influencing the occurrence of cholecystitis, may involve various mechanisms. Firstly, dysregulated gut microbiota may reduce vitamin A levels, and the metabolite all-trans retinoic acid plays a crucial role in mucosal immune response, with immune dysregulation contributing to the development of cholecystitis (44). Secondly, the reduced levels of vitamins such as vitamin C and vitamin E may impair the antioxidant defense system, increase oxidative stress in the gallbladder, and thus promote the development of cholecystitis (45). Our primary finding was that circulating vitamin D levels mediated the causative relationship between RuminococcaceaeUCG003 and cholecystitis. Research suggests that circulating vitamin D can affect the normal contraction of the gallbladder and bile excretion, with a decline in circulating vitamin D levels leading to bile stasis and consequent cholecystitis (46). For instance, Fangji Yang et al. using NTCP-deficient mice (exhibiting common phenotypes of high choline blood levels, vitamin D deficiency, bone loss, and gallbladder anomalies), observed significant changes in serum bile acid levels. Therefore, there is a close relationship between circulating vitamin D levels and bile acid status. Moreover, as cholecystitis is often associated with bacterial infections, and vitamin D regulates the innate immune system, enhancing the cellular killing of pathogenic microorganisms, some studies have suggested an inverse correlation between serum 25(OH)D levels and infection risk (47). In conclusion, our study revealed complex interactions and potential mechanisms between gut microbiota, serum vitamin levels, and cholecystitis. Important mechanisms may include gut microbiota dysbiosis-induced inflammation, alterations in bile acid metabolism, immune imbalance, compromised antioxidant defense systems, impaired gut barrier function, and gallstone formation. These findings are of significant importance for understanding the potential relationships between gut microbiota, serum vitamin levels, and cholecystitis. Future research should further explore potential therapeutic strategies for preventing and managing cholecystitis by modulating gut microbiota and supplementing vitamin levels.

Moreover, it is essential to acknowledge the limitations of this study. Firstly, MR relies on the fundamental assumption that genetic variations serving as instrumental variables influence the outcome solely through the exposure factor. However, despite our efforts to minimize confounding factors and use the cML-MA method to address pleiotropy, there may still be unmeasured confounding factors. Secondly, our study primarily focused on European populations, and the generalizability of our findings to other populations may be limited. We acknowledge that other potential mediators, aside from circulating vitamin levels, could contribute to the observed relationship between gut microbiota and cholecystitis. Future studies could explore these additional mediating factors to provide a more comprehensive understanding of the complex interactions involved. In addition, future research should consider utilizing larger samples of genetic variations in the gut microbiota to conduct a more comprehensive evaluation of our study.

In summary, our two-sample MR analysis unveiled the causal relationship between circulating vitamin D levels mediating gut microbiota and cholecystitis, along with the underlying mechanisms. Future research should further explore potential therapeutic strategies by modulating gut microbiota and supplementing vitamin levels to prevent and mitigate the risk of cholecystitis.

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.

CM: Writing – original draft, Writing – review & editing. LX: Writing – original draft, Writing – review & editing. XX: Software, Writing – original draft, Writing – review & editing. SH: Writing – original draft. JL: Investigation, Writing – review & editing. KC: Writing – original draft, Writing – review & editing.