Interstitial cystitis (IC), also termed bladder pain syndrome (BPS), presents as a chronic non-bacterial inflammation of the bladder. It is characterized by varying degrees of frequency, urgency, dysuria, and/or suprapubic pain among affected individuals, sometimes extending to the urethra, perineum, vagina, and during sexual intercourse (1). The condition predominantly affects women, with a male-to-female ratio of approximately 1:10 (2) and an incidence rate as high as 6.53% (3) among adult women. Despite extensive research, the etiology of IC remains elusive (4), complicating the search for specific treatment modalities. Prolonged medical interventions for IC render patients vulnerable to mental health disorders such as depression and anxiety, contributing to a substantial suicide risk rate of 38.10% (5).

The human gut harbors a diverse microbiota comprising bacteria, fungi, archaea, viruses, and protozoa. This gut microbiota exists in symbiosis with the intestinal mucosa, playing crucial roles in immunomodulation, metabolism, and gastrointestinal protection in individuals with good health (6). Chronic dysbiosis can disrupt multiple bodily homeostatic mechanisms, potentially precipitating local and systemic inflammatory reactions (7). Researchers are increasingly intrigued by the interplay between gut microbiota and chronic inflammation-related disorders, as it unveils novel therapeutic avenues for conditions like inflammatory bowel disease (8). The gut microbiota has emerged as a critical factor in the pathogenesis of various inflammatory diseases, including asthma, inflammatory bowel disease, non-alcoholic fatty liver disease, rheumatoid arthritis, and type II diabetes mellitus (9). Recent studies have emphasized the “gut-bladder axis” in the interaction between the gut microbiota and bladder (10, 11). Similarly, research has explored the role of gut microbiota in the development and progression of IC and its implications for diagnostic and therapeutic strategies (12, 13). Comparisons with healthy controls revealed significant reductions in the levels of Eggerthella sinensis, Collinsella aerofaciens, Faecalibacterium prausnitzii, Odoribacter splanchnicus, and Lactonifactor longoviformis. An animal experiment demonstrated that fecal transplantation from wild-type mice into IC model mice alleviated IC symptoms, while fecal transplantation from IC model mice exacerbated pain and anxiety behaviors, suggesting the potential involvement of gut microbiota in IC development. However, comprehensive causal analysis of the interactions between gut microbiota and IC is still lacking in current literature.

Mendelian randomization (MR) is an innovative epidemiological method that utilizes genome-wide association study (GWAS) data and employs single nucleotide polymorphisms (SNPs) as instrumental variables (IVs) to establish causality and discern the causal relationship between an exposure and an outcome. The MR approach operates on the principle that distinct genotypes determine varied phenotypes, and adheres to Mendel’s Law of Inheritance, which stipulates that “parental alleles are randomly allocated to offspring during gamete formation.” Furthermore, alleles are randomly assigned to individuals at conception, predating both exposure and outcome, thereby obviating concerns of reverse causation. Theoretically, genetic variation remains unaffected by common confounders, such as postnatal environment and economic status. Notably, the MiBioGen consortium and the Dutch Microbiome Project (DMP) have recently published numerous loci linked to microbiome abundance, presenting an unparalleled opportunity to investigate causal relationships between gut microbiota and IC. Accordingly, we conducted a bidirectional two-sample MR analysis to elucidate the causal link between gut microbiota and IC while further probing the host gene-microbiota associations.

Figure 1 presents an overview of the study. Since our study was based on a public database, no additional ethical approval or informed consent was required.

The investigation initiated with MR analysis of two samples to explore the potential causal link between gut microbiota and IC, employing summary statistics from GWAS. Subsequently, we delved into the molecular mechanisms through which gut microbiota influences IC development and elucidated potential host gene-microbiota associations in IC patients. To our knowledge, this represents the inaugural MR study probing the causal association between gut microbiota and IC. Our study not only holds promise for effective IC prevention and treatment strategies but also furnishes novel insights into IC pathogenesis from a gut microbiota perspective.

In our present MR investigation, we identified a causal link between eight bacterial taxa and the risk of IC. Notably, Butyricimonas, Coprococcus, Lactobacillales, Lentisphaerae, and Bilophila wadsworthia exhibited positive associations with IC risk. Conversely, taxa like Desulfovibrio piger, Oscillibacter, and Ruminococcus lactaris demonstrated protective effects against IC.

Desulfovibrio piger, one of the most prevalent pairs of sulfate-reducing bacteria in feces (43), releases hydrogen sulfide (H2S), a third gas signaling molecule widely involved in various biochemical reactions in the animal body. It plays a physiological role in the gastrointestinal tract, regulating cellular and tissue functions (44, 45). It can downregulate the expression of cyclooxygenase-2 mRNA and prostaglandin synthesis, as well as reduce the expression of the pro-inflammatory cytokine tumor necrosis factor-alpha mRNA, thereby exerting either pro-inflammatory or anti-inflammatory effects. Its anti-inflammatory effects may be one of the mechanisms through which it benefits individuals with IC (46, 47). Additionally, H₂S may also function as a signaling molecule in the host, potentially providing beneficial effects such as cardioprotection (48). Recent research has revealed Desulfovibrio piger’s positive impact on the growth and function of Faecalibacterium prausnitzii (49). Faecalibacterium prausnitzii, a crucial bacterium in the human gut flora, is a major producer of butyric acid, known for its anti-inflammatory effects, enzyme activity maintenance, and protection of the digestive system against intestinal pathogens (50). However, observational studies indicate significantly reduced levels of Faecalibacterium prausnitzii in the feces of IC patients (13). Ruminococcus, one of the earliest gastric bacteria identified, significantly influences metabolism, digestion, and the metabolism of resistant starch. Patients with inflammatory bowel disease often exhibit varied Ruminococcus numbers and activities in their intestines, affecting gut health and inflammation levels (51). Anomalies in Ruminococcus may contribute to immune-related diseases like allergy, eczema, and asthma (52). Although the precise mechanism remains unclear, studies suggest that Ruminococcus may impact the pathogenesis of immune-mediated diseases by influencing intestinal immune system regulation and inflammatory responses. Bilophila, an opportunistic pathogen group, represented by Bilophila wadsworthia, was initially isolated from patients with gangrenous and perforated appendicitis (53). It synergizes with a high-fat diet, leading to increased inflammatory responses, intestinal barrier dysfunction, and bile acid metabolism abnormalities, consequently raising the risk of glucose metabolism abnormalities and hepatic steatosis (54). Bilophila wadsworthia eventually metabolizes 2,3-dihydroxypropane-1-sulfonate, producing H2S through a recently discovered metabolic pathway (55). Butyricimonas, a beneficial bacterium, produces short chain fatty acids (SCFAs), the primary metabolites of anaerobic bacterial fermentation in the gut (56). SCFAs serve as crucial fuel for intestinal epithelial cells and exhibit potential anti-inflammatory effects, along with immune cell modulation (57). For example, SCFAs influence epithelial barrier function and mucosal and systemic immunity through G protein-coupled receptor signaling or histone deacetylase activity. SCFAs can also directly or indirectly affect immune responses in extramural sites such as the liver, lungs, reproductive tract, and brain, and are associated with various diseases. Coprococcus, a significant member of the Firmicutes phylum and Lachnospiraceae family, resembles Faecalibacterium prausnitzii in terms of its beneficial role in intestinal health and immune system balance. Actively fermenting carbohydrates, Coprococcus is a key butyric acid producer and serves as a microbial biomarker for assessing gastrointestinal health (58). Butyric acid exerts anti-inflammatory effects by directly influencing the differentiation of intestinal epithelial cells, phagocytes, B cells, plasma cells, as well as regulatory and effector T cells (57). Interestingly, we observed deleterious effects of the Butyricimonas and Coprococcus on IC, offering a novel perspective for future investigations.

Studies have demonstrated that gut microbiota can regulate host physiology and potentially induce disease by influencing gene expression (59–61). Thus, we utilized FUMA to map SNPs to genes and explore genes that might exhibit a causal relationship with IC. To gain insight into the biological functions of these genes in disease, we conducted GO enrichment analysis. Our findings suggest that gut microbiota with beneficial roles may impact channel regulator activity, protein binding, cadherin binding, and other pathways. Conversely, gut microbiota with detrimental roles may influence protein repair, paranodal junction, cell-cell junction, protein binding, and other pathways. Subsequently, we conducted a comparative analysis of hub gene expression between IC patients and healthy controls using bulk RNA analysis. The results revealed significant down-regulation of SPTBN1 and PSME4 in IC patients, while CHAC2, ERLEC1, ASB3, STAT5A, and STAT3 were significantly up-regulated. Therefore, SPTBN1, PSME4, CHAC2, ERLEC1, ASB3, STAT5A, and STAT3 could potentially represent novel genes implicated in the underlying pathogenesis of IC and serve as therapeutic targets.

Spectrin Beta, Non-Erythrocytic 1 (SPTBN1) is a protein-coding gene known for its role in maintaining neuronal morphological stability, cellular structure, and cell signaling. Additionally, it contributes to synapse formation and normal neuronal function (62). Several studies have indicated a correlation between IC and neurological disorders such as fibromyalgia, depression, and memory impairment (63, 64). Moreover, neurogenic pain is considered a potential etiological factor in IC, wherein pain may arise from dysfunction in peripheral, central, and/or endogenous inhibitory pathways (65). Proteasome Activator Subunit 4 (PSME4) encodes a protein that regulates proteasome activity, thereby influencing the rate and selectivity of protein degradation (66). However, its association with inflammation and IC remains unclear. One of the primary intracellular functions of the protein encoded by the ChaC Glutathione Specific Gamma-Glutamylcyclotransferase 2 (CHAC2) gene, is its involvement in glutathione metabolism. Glutathione, a crucial antioxidant, safeguards cells from oxidative stress (OS)-induced damage within the cell. Research indicates that the expression level of the CHAC2 protein is influenced by intracellular OS levels, thereby regulating intracellular glutathione levels and subsequently impacting the cell’s redox state and its ability to counter OS (67, 68). OS is recognized as a significant contributing factor in inflammatory diseases. Notably, one of the key features underlying the pathobiology of IC is the excessive production of reactive oxygen species and cytokines, leading to pronounced OS (69). Mitigating OS has emerged as a potential therapeutic approach for IC (70, 71). The Endoplasmic Reticulum Lectin 1(ERLEC1) gene encodes a protein pivotal in the endoplasmic reticulum, particularly in protein quality control and folding. It aids in identifying proteins folding abnormally or with specific sugar groups, thereby participating in their subsequent processing and modification, including their targeting to appropriate pathways for degradation or repair (72). However, its association with inflammation and IC remains unclear. Ankyrin Repeat and SOCS Box Containing 3 (ASB3) is a protein-coding gene containing the SOCS box, facilitating the binding and degradation of various proteins. Members of the SOCS protein family are known to impede cytokine signaling by directly interacting with JAK kinase or activated cytokine receptors (73). Although ASB3 genes have been extensively studied in oncological disorders, such as promoting colorectal cancer cell growth and metastasis when mutated or down-regulated (74), their connection with inflammation and IC remains unelucidated. Signal Transducer and Activator of Transcription 5A (STAT5A) and Signal Transducer and Activator of Transcription 3 (STAT3) are pivotal transcription factors involved in various biological processes. Both are integral components of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway, which plays a critical role in regulating cell growth, differentiation, survival, and immune response (75). Moreover, the JAK/STAT pathway modulates the production of numerous inflammatory mediators, thereby regulating inflammation development (76). There is now a degree of certainty that inflammation plays a key role in the pathophysiology of IC (77). A prior investigation observed significant enrichment of the JAK/STAT signaling pathway in a cyclophosphamide (CYP)-induced IC mouse model (78), where pSTAT3 expression increased post-CYP treatment in a rat cystitis model (79). Inhibition of the JAK/STAT signaling pathway demonstrated effectiveness in reducing CYP-induced inflammation in rat ulcerative cystitis (80). Hence, we hypothesize that gut microbiota may contribute to the onset and progression of IC inflammation by modulating the JAK/STAT signaling pathway, thereby influencing IC inflammation development. In conclusion, further studies are warranted to elucidate the mechanisms underlying the action of these genes in IC and their therapeutic potential.

IC, an autoimmune disease, is characterized by the infiltration of immune cells such as lymphocytes, macrophages, and mast cells into the bladder tissue of affected individuals. The activation and proliferation of these immune cells can lead to inflammation and damage to the bladder mucosa, resulting in symptoms such as pain, frequent urination, and urgency (81). Previous studies have shown that the development of IC involves the infiltration of various immune cells, including T cells, B cells, plasma cells, macrophages, neutrophils, and mast cells (81, 82). Growing evidence suggests that the gut microbiota plays a crucial role in shaping the immune system. First, the gut microbiota influences the systemic immune response by regulating intestinal barrier function. A healthy microbiota maintains the integrity of the intestinal barrier, preventing the invasion of harmful substances and pathogens, thereby reducing systemic inflammation. Conversely, an imbalanced gut microbiota can lead to barrier disruption, loss of immune tolerance, and chronic inflammation, which can affect autoimmune responses (83). Second, the gut microbiota directly impacts immune cell function through the production of various metabolites, such as SCFAs. For example, SCFAs produced from the metabolism of dietary fibers stimulate oxidative phosphorylation and glycolytic activity in CD8⁺ T cells, enhancing their effector functions. SCFAs also activate glycolysis in B cells via mTOR signaling (84). These metabolites can influence the immune cells in the bladder through the bloodstream. Moreover, a key role of the gut microbiota in the immune system is the promotion of immune tolerance. The immune system must recognize and attack pathogenic microbes while tolerating the normal microbiota. The gut microbiota assists in distinguishing harmful microbes from benign ones by producing signaling molecules and regulating immune cell functions. This helps prevent autoimmune diseases, although dysbiosis can disrupt immune tolerance and trigger autoimmune reactions (85). Looking ahead, integrating GWAS summary statistics on interstitial cystitis with single-cell RNA sequencing data (86), or employing new analytical methods, such as ‘scPagwas’ (87), could provide deeper insights into the immune cell heterogeneity in the bladder tissue of IC patients and elucidate the specific roles of these cells in IC pathogenesis. In summary, the interaction between the gut microbiota and the immune system is pivotal in the pathogenesis of IC. Understanding how the gut microbiota affects immune cells and their roles in IC could offer new insights for the diagnosis and treatment of this condition.

This study has several strengths. By employing MR analysis, we established a causal relationship between gut microbiota and IC, thereby controlling for confounding factors and reverse causation. Additionally, we utilized the cML-MA method to address biases due to correlated and uncorrelated pleiotropy and applied a two-sample MR design with non-overlapping exposure and outcome summary data to mitigate bias. While most current research focuses on analyses at the genus level, our study is the first to identify bacteria closely related to IC at the species level, specifically Desulfovibrio piger, Oscillibacter unclassified, and Ruminococcus lactaris. Identifying these species provides new insights into the gut microbiota’s role in IC. Future research should explore the potential of regulating these key bacterial taxa through fecal microbiota transplantation or probiotic treatments to alleviate IC symptoms. Increasing the abundance of Desulfovibrio piger, Oscillibacter unclassified, and Ruminococcus lactaris could offer effective intervention targets. For instance, enhancing the abundance of Desulfovibrio piger has been shown to positively influence the growth and function of the gut probiotic Faecalibacterium prausnitzii, suggesting new possibilities for personalized IC treatment. These new findings not only enhance our understanding of the etiology of IC but also provide clear directions for developing new clinical treatment strategies, particularly through microbiota modulation for symptom management. Such strategies may involve targeted supplementation or suppression of specific bacterial species, offering new therapeutic hope for IC patients. Additionally, we analyzed the associations between host genes and the microbiota, further exploring potential therapeutic targets and mechanisms underlying IC pathogenesis.

However, our study has several limitations. Firstly, the majority of patients in the GWAS summary data used were of European descent, with limited representation from other ethnicities. This imbalance may introduce estimation bias and limit the generalizability of our findings. Consequently, the results may not be fully applicable to individuals of non-European origin. Future MR studies exploring the causal relationship between gut microbiota and IC should include diverse European and non-European populations to enhance generalizability. Secondly, subgroup analyses, such as with Hunner-type and non-Hunner-type, were not possible since summary data, rather than raw data, were used in the analyses, whereas symptoms are usually more severe in the former (88). Moreover, IC predominantly affects the female population, and gender-specific differences exist in gut microbiota composition (89). Unfortunately, our study did not conduct separate analyses for both genders. Future investigations should incorporate gender-specific MR analysis to provide comprehensive insights into the relationship between gut microbiota and IC across different populations. Then, although relaxing the P-value threshold increased the number of IVs, it may have introduced additional false positives. Employing a stringent LD threshold (r² < 0.001) to ensure SNP independence might have excluded some effective SNPs. Excluding SNPs with a MAF≤ 0.01 improved the stability of effect estimates but may have omitted biologically relevant low-frequency variants. While removing palindromic SNPs reduced data errors, it might have overlooked critical genetic signals. Future research should focus on optimizing SNP selection criteria, exploring different P-value and LD thresholds, using updated reference panels, expanding sample sizes, and employing advanced techniques to manage palindromic SNPs, thereby enhancing the reliability and comprehensiveness of research findings. Finally, our study has a relatively small sample size, with only 240 cases of IC, which is indeed a limitation and may affect statistical power. Although we conducted rigorous analyses and used the largest available dataset, we recognize that the small sample size might impact the robustness of our conclusions. To address this, we employed F-statistic calculations to assess statistical power, with instrumental variables yielding results greater than 10, indicating statistical significance. Additionally, we performed sensitivity analyses, all of which support the reliability of our findings. Nevertheless, we understand that using larger datasets in future studies is essential for improving statistical power and validating our results. Despite the limited sample size, we believe our study provides a valuable foundation for exploring the potential associations between gut microbiota and interstitial cystitis, guiding future research in this field. We once again thank the reviewer for their valuable comments, and we look forward to addressing the sample size issue in future research to further enhance our contribution to this field.

Our study suggests a potential causal relationship between gut microbiota and IC based on MR analysis, biological annotation, and bulk RNA data analysis. However, these conclusions are primarily derived from predictive analysis tools and lack experimental validation. Future research should integrate in vivo and in vitro experiments to confirm the actual impact of relevant gut microbiota species and their metabolites on IC. Additionally, experimental validation is required to verify whether the key genes and bacteria identified in the analysis are genuinely involved in the pathogenesis of IC. Such validation will contribute to a more comprehensive understanding of the role of gut microbiota in IC and further substantiate the proposed mechanisms, providing a more reliable scientific foundation for the prevention and treatment of IC.

In this study, we employed a MR design to investigate the association between gut microbiota and IC, marking the first exploration of its kind. Through this approach, we successfully pinpointed gut microbiota linked to IC, comprising both beneficial and harmful microbial communities, thus serving as potential biomarkers. Furthermore, leveraging FUMA, we annotated SNPs to discern the genes harboring these genetic variants, indicating a potential role of gut microbiota in IC pathogenesis through gene regulation. Additionally, by comparing the expression profiles of hub genes in healthy individuals and IC patients using bulk RNA sequencing data, we potentially identified diagnostic and prognostic biomarkers associated with IC. Overall, this study offers a novel avenue for delving into the underlying pathogenesis of IC, providing insights for the development of personalized therapeutic strategies and identifying preliminary targets for future drug development.