Cholestatic liver disease (CLD) refers to a group of disorders in which bile synthesis, secretion, and excretion are compromised for a variety of reasons (1). CLD dominantly includes primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC). PBC, a chronic cholestatic liver disease, is hallmarked by non-suppurative inflammation within the small intrahepatic bile duct (2). PSC, a rare cholestatic liver disease, could result in bile duct fibrosis and strictures. In contrast to PBC, which has a female predominance, the majority of PSC patients are male (3). Up to 80% of PSC patients also suffer from IBD, indicating the involvement of the gut-liver axis in PSC (4). Patients with PBC can effectively control the disease by taking medications such as Ursodeoxycholic Acid, Bezafibrate and Fenofibrate (5–7). However, there is currently no satisfactory treatment for PSC.

Though the exact mechanism underlying PBC and PSC is still not fully illustrated, it is reported that genetics, environment, immune factors, gut microbiota, and individual susceptibility may all contribute to the development of these diseases (4, 8, 9).

Environmental factors are thought to cause PBC and PSC in individuals who are genetically predisposed, resulting in a loss of tolerance to self-antigens. Molecules derived from microbiota can activate the immune system and lead to autoimmune inflammation (10). According to recent research, gut microbiota dysbiosis can affect the immune system leading to autoimmune diseases such as celiac disease, inflammatory bowel disease, and cholestatic liver diseases (11–13). In numerous studies focused on the gut-liver axis, a link has been established between gut microbiota dysbiosis and PBC and PSC pathophysiology (14–16). Research has indicated a significant reduction in the abundance of microbiota in individuals with PBC and PSC compared to healthy controls (17, 18). However, some bacterial genera, such as Haemophilus, Veillonella, Clostridium, and Bifidobacterium, are increased in PBC patients compared to healthy controls (19, 20), while Lactobacillus, Streptococcus, and Veillonella proportions are higher in PSC patients than healthy controls (21).

Nevertheless, it is important to note that observational studies are often influenced by confounding factors. Moreover, the aforementioned studies have examined different populations with varying dietary habits. Therefore, these cross-sectional studies do not allow for definitive conclusions to be drawn.

Mendelian randomization (MR) is an innovative approach to investigate the association between an exposure and a noteworthy outcome (22). Alleles are randomly allocated, according to Mendelian’s laws of inheritance, and genotypes are fixed at conception. Thus, confounders and reverse causality are unlikely to affect the causal relationship. MR analysis exploited common genetic variations to represent a modifiable environmental exposure, which has become widely used to investigate potential causal relationships between environmental exposures and outcomes. In two-sample MR analysis, single-nucleotide polymorphisms (SNPs) can serve as instrumental variables (IVs) to investigate casual associations between exposures and outcomes (23).

In summary, the causality of the associations between the gut microbiota and PBC and PSC remains inconclusive. In this study, we conducted a two-sample Mendelian randomization analysis using comprehensive summary statistics from large-scale genome-wide association studies (GWAS) of gut microbiota (GM), PBC and PSC to conduct this question.

In this two-sample Mendelian Randomization study, we found that 7 bacterial taxa were associated with PBC, and 9 bacterial taxa were associated with PSC.

Previous article reported (28) that Order Selenomonadales, Order Bifidobacteriales, Genus Lachnospiraceae_UCG_004, Family Peptostreptococcaceae, and Family Ruminococcaceae were related to PBC, which have been validated in our study. In addition, our results also found that order Bacillales and genus Anaerotruncu were potential risk factor for PBC. These results expand previous research and provide a foundation for further research on PBC, and we also compared the differences between PBC and PSC.

We identified order Selenomonadales as a protective factor in both PBC and PSC. Selenomonadales are anaerobic bacteria that typically have a curved or bent shape. They contribute to the formation and function of complex gut microbiota. These bacteria can utilize various carbon sources such as glucose, lactose, and cellulose to produce organic acids and gases through fermentation (29).

It was also found that the family Peptostreptococcaceae is a protective factor for PBC, while the genus Peptococcus has a similar protective effect on PSC. These results indicate that certain gut microbiota might play a common role in the occurrence of PBC and PSC.

The family Peptostreptococcaceae belongs to the phylum Firmicutes (30). This family includes the genera Peptostreptococcus, Finegoldia, and Anaerococcus. Bacteria in the family Peptostreptococcaceae are typically anaerobic organisms and can be found in the digestive tract, skin, and other body surfaces of humans and animals, as well as in soil and water environments.

There are also some bacteria that play different roles in PBC and PSC. For example, the family Ruminococcaceae plays a protective role in PBC, while the genus RuminococcaceaeUCG013 increases the risk of PSC. These results suggest that different bacteria within the same family may also have different effects.

Family Ruminococcaceae family are usually anaerobic organisms. These bacteria are able to utilize cellulose and other components of plant cell walls to produce organic acids and gases through fermentation, providing energy and nutrients to the host. These bacteria in the human gut are associated with intestinal health and metabolism (31).

Due to Mendelian randomization analysis using GM GWAS data, different levels of bacteria, such as genus, family, and order, may extract the same SNPs, which results in different levels of bacteria having the same effect. For example, order Bifidobacteriales and family Bifidobacteriaceae both extract 13 SNPs, and the results indicated the same protective effect on PBC. What’s more, although the number of extracted SNPs differed, order Actinomycetales, family Actinomycetaceae, and genus Actinomyces are all having protective effects on PSC.

Bifidobacterium is generally regarded as probiotics and possesses numerous advantages, such as facilitating food digestion, synthesizing vitamins, and augmenting immune system functionality (32). However, our findings provide evidence that the family Bifidobacteriaceae may elevate the risk of PBC, which is consistence with previous research which identified Bifidobacterium is increased in PBC patients (20).

Actinomycetales are widely present in natural environments, including soil, water bodies, and plant surfaces. They can also survive in the bodies of humans and other animals, such as in the oral cavity, intestines, and skin. Actinomycetales have the ability to produce some important enzymes and bioactive substances (33). For example, they can produce cellulases, proteases, and acid phosphatases, as well as antioxidants and anti-tumor substances.

We found that specific bacterial features are causally related to the risk of PBC and PSC. The underlying mechanism of the influence of bacteria features on PBC and PSC has been extensively studied. Metabolites, especially short-chain fatty acids (SCFAs), are one of the most crucial factors (34, 35), with butyric acid, propionic acid, and acetic acid being the predominant constituents. A critical function of SCFAs is to act as signaling molecules that regulate the immune system, cellular growth, and metabolic activity of the host (36).

Butyrate, an essential metabolite derived from the GM, contributes to maintaining the integrity gut barrier by supplying energy to colonic epithelial cells. Additionally, it modulates genes associated with the circadian clock, thereby performing its anti-inflammatory function (37). Moreover, it can modulate T cell proliferation and regulate the activation of B cells that produce IL-10 and/or IL-17 (38). These cytokines will aggravate the inflammation of bile duct cells in patients with PBC and PSC and worsen bile stasis. There is also evidence that bacterial-derived peptides induce CD8⁺ T cell clonal expansion (39, 40), which are the main effector cells causing bile duct damage in patients with PBC and PSC (41).

Bile acids also play an important role in the pathogenesis of PBC and PSC. Bile acids not only play a role in digesting food, but also serve as important messengers for liver and intestinal communication.

Firstly, bile acids have a direct bactericidal effect and have an inhibitory effect on the growth of gut microbiota. In addition, bile acids regulate the composition of gut microbiota by regulating farnesoid X receptors. Finally, bile acids can also serve as raw materials for gut microbiota to promote the proliferation of some bacteria (42, 43). Dysbiosis of the GM, characterized by a decrease in microbial diversity and changes in specific bacterial species, may be linked to an elevated risk of developing PSC and PBC (44–46).

Based on our Mendelian randomization study, we have found a reasonable correlation between the gut microbiota taxa, PBC, and PSC. Some probiotics and their metabolites can restore the ecological balance of gut microbiota, repair the intestinal mucosal barrier and regulate systemic immune function. Our research can provide a foundation for further research (47). Future studies are required to better understand the mechanisms behind this prevalent disease and identify potential therapeutic targets.

Our study has some limitations: (a) Since the normally used threshold (p < 5 × 10⁻⁸) did not yield enough IVs, we set a lenient threshold (p < 1 × 10⁻⁵). (b) It’s difficult to determine whether some specific species are related to the outcome since most GM studies using 16S rRNA permit resolution at the genus level. (c) We refrained from conducting multiple corrections in our study. However, it is worth mentioning that the rigorous application of a multiple-testing correction may be excessively conservative and could potentially miss out on partially potential GM taxa that are causally correlated to CLD. Hence, we made the decision not to incorporate multiple correlations. Furthermore, it is important to note that the Bonferroni correction has the possibility of generating false negative results.

Our findings offer novel evidence that supports the causal influence of particular bacterial abundance on the risk of PBC and PSC. The GM is anticipated to be a promising treatment and prevention target for PBC and PSC.

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding authors.

JY: Data curation, Investigation, Writing – original draft. GM: Investigation, Methodology, Writing – original draft. KW: Software, Writing – original draft. HY: Software, Writing – original draft. SJ: Data curation, Methodology, Writing – original draft. QF: Methodology, Validation, Writing – original draft. XZ: Supervision, Writing – review & editing. GG: Supervision, Visualization, Writing – review & editing. YH: Funding acquisition, Project administration, Writing – review & editing.