Plasma proteins and irritable bowel syndrome

Abstract

Irritable bowel syndrome (IBS) is a complex disorder with unclear etiology and underlying mechanisms. This research aimed to identify possible plasma proteins linked to IBS using a proteome-wide Mendelian randomization (MR) analysis. We performed a bidirectional two-sample MR analysis to investigate the causal relationship between 4907 plasma proteins and IBS. Genetic data from 35,559 Icelanders and IBS genome-wide association study data from 329,381 individuals in the FinnGen consortium were used to select instrumental variables meeting stringent criteria. Several MR methods such as inverse variance weighted, MR-Egger, and Weighted Median, along with sensitivity analyses, were utilized to confirm the robustness of the findings. Enrichment analyses and gene interaction studies were also conducted to investigate biological pathways related to the significant findings. MR analysis identified 20 plasma proteins significantly associated with IBS. Of these, Scavenger Receptor Class A Member 5 and Fibroblast Growth Factor Binding Protein-1 were negatively associated with IBS, indicating a protective effect. The other 18 plasma proteins associated with IBS onset include but are not limited to Leucine-rich repeat kinase 2, Ras and Rab interactor 3, and Coiled-coil-helix-coiled-coil-helix domain-containing protein 10, all of which showed a positive correlation with increased IBS risk. Reverse MR analysis revealed no evidence that IBS influences plasma protein expression, suggesting that these proteins may play a causal role in the development of IBS. Enrichment analyses highlighted significant involvement in mitochondrial processes, synaptic vesicle recycling, and endosomal transport. In summary, this study identified 20 plasma proteins as potential causal factors in the development of IBS. The findings suggest that these proteins could serve as therapeutic targets, offering new insights into the underlying mechanisms of IBS.

1. Introduction

Irritable bowel syndrome (IBS) is a prevalent functional bowel disorder, characterized by a combination of chronic abdominal pain associated with a change in stool frequency without any underlying organic disease.^[1]In fact, the pathophysiological mechanisms of IBS are complex and not yet fully understood. Potential contributing factors include visceral hypersensitivity, gastrointestinal motility abnormalities, immune dysfunction, gut dysbiosis, small intestinal bacterial overgrowth, food sensitivities, genetic predisposition, and psychological factors.^[2–4] Traditionally, IBS is considered to have familial aggregation, with a higher prevalence in monozygotic twins compared with dizygotic twins.^[5] IBS is prevalent worldwide, affecting more than 10% of the population, with significant geographical variability in prevalence.^[6] The disorder mainly impacts young individuals and women, reducing the quality of life and work productivity of patients and imposing a substantial socioeconomic burden.^[7] Therefore, it is essential to thoroughly investigate the pathogenesis of IBS, identify its biomarkers, and find personalized therapeutic targets to develop effective prevention and treatment strategies. In recent years, besides the conventional use of antispasmodics and antidepressants, various diverse treatment approaches such as manipulating the gut microbiota^[8] and mind-body therapies^[9] have gained attention. For example, treating diarrhea-predominant IBS patients with multi-strain probiotics has significantly increased beneficial bacteria in the gut and effectively alleviated gastrointestinal symptoms.^[10] These methods align with the emerging concept of personalized medicine and can provide effective health management for IBS patients.

Blood plasma proteins, originating from either cellular leakage or active secretion, are primary sources for biomarkers and therapeutic targets, making their levels and functions essential in medical diagnosis and treatment for evaluating health and disease progression.^[11] A cross-sectional study found that serum levels of brain-derived neurotrophic factor were significantly elevated in IBS patients compared with healthy controls (IBS patients group mean 3.1, SD 4.3; control group mean 1.7, SD 2.7) (P = .02; Cohen’s d = 0.51).^[12] Additionally, another prospective cohort study showed that the serum levels of monocyte chemoattractant protein-1 in IBS patients were not significantly higher than those in the control group (204 ± 130 vs 174 ± 73 pg/mL; P = .311).^[13] However, the aforementioned literature mainly relies on observational studies to investigate the relationship between plasma proteins and IBS. These studies have several limitations, such as susceptibility to confounding factors, difficulty in distinguishing reverse causality, selection bias, and measurement errors. Thus, it is crucial to develop new approaches to examine the relationship between plasma proteins and IBS.

Mendelian randomization (MR) is an analytical technique that leverages natural genetic variations (such as single nucleotide polymorphisms [SNPs]) as instrumental variables (IVs) to simulate randomized trials, offering more reliable causal inference.^[14] This method helps eliminate the influence of confounding factors, enabling a more precise investigation of the causal relationship between an exposure and an outcome. In recent years, MR has been used to examine causal relationships between various molecules and IBS. For instance, one study found no causal link between serum calcium and vitamin D levels and the risk of IBS.^[15] Another study suggested that higher serum copper levels were associated with a reduced risk of IBS (odds ratio [OR] = 0.89; 95% confidence interval: 0.80, 0.98).^[16] However, a proteome-wide MR analysis investigating biomarkers and potential therapeutic targets for IBS has not yet been conducted. Interestingly, a comprehensive proteome-wide MR analysis revealed that elevated levels of 3 circulating proteins: Macrophage Stimulating 1 (MST1), NAD Kinase, and hepatocyte growth factor activator were significantly associated with a reduced risk of inflammatory bowel disease (IBD), highlighting these proteins as potential therapeutic targets.^[17] Consequently, this study utilizes a proteome-wide MR analysis to clarify the potential causal relationships between plasma proteins and IBS. These insights may provide valuable directions for identifying diagnostic biomarkers and developing targeted therapies for this condition.

2. Methods

2.1. Study design

We performed a two-sample MR analysis to assess the causal link between 4907 plasma proteins and IBS. First, we considered the 4907 plasma proteins as exposures and IBS as the outcome to examine their possible inhibitory or promoting effects on the onset of IBS. Then, we reversed the analysis, treating IBS as the exposure and plasma proteins as the outcomes to investigate alterations in plasma protein levels following the onset of IBS. The goal of this MR study is to pinpoint potential therapeutic targets for IBS. All included original studies received ethical approval, and the data sets used were publicly accessible. This study utilizes R statistical software (version 4.4.1) for data analysis.

2.2. Proteomic data source

Whole-genome sequencing data were generated by deCODE Genetics using Illumina SNP chips for 49,708 Icelanders with both genotype and phenotype data. This information was then utilized to impute the genomes of 166,281 genotyped individuals from Iceland, leading to the identification of over 27 million imputed variants 18. Of these participants, 35,559 had protein measurements available. Genetic associations with 4907 plasma proteins were uncovered through a large-scale protein quantitative loci (pQTL) study involving these 35,559 individuals. Plasma samples were analyzed using the SomaScan version 4 assay (SomaLogic).

2.3. Irritable bowel syndrome data source

The genome-wide association study summarized data for IBS were provided by the FinnGen consortium (R10 release).^[18] This study included 10,329 controls and 329,381 clinical IBS individuals (ICD-10 K58). IBS, as defined by the database (https://r10.risteys.finngen.fi/endpoints/K11_IBS), includes individuals who have been diagnosed with gastrointestinal symptoms characterized by chronic abdominal pain and altered bowel habits in the absence of any organic cause. Controls are those individuals who reported not having IBS.

2.4. IV selection

Choosing suitable IVs depends on fulfilling 3 critical assumptions (Fig. 1).^[19] IVs should have a strong association with the exposure factors (in this case, plasma protein levels); IVs must be independent of any confounding factors that lie between the exposure and the outcome (in this case, IBS); IVs are associated with outcomes only by exposure. To ensure their relevance, we performed an association analysis on 4907 plasma proteins, applying a stringent significance threshold of P < 5e-8.^[20] We then removed SNPs showing linkage disequilibrium based on the criteria of R² < 0.001 and Kb = 10,000.^[20,21] We also calculated the F-statistic for the remaining SNPs to detect and remove weak IVs. An F-statistic >10 signifies that the IVs are sufficiently strong.^[21,22] This process ensured that the selected SNPs for the 4907 plasma proteins were relevant and robust, adhering to the assumptions required for IV analysis.

Figure 1.

The figure reveals the principle of Mendelian randomization and 3 main assumptions. Solid lines indicate desired associations, while dashed lines indicate undesirable associations.

2.5. MR analysis

In this study, we employed the bidirectional two-sample MR method to evaluate causal relationships between plasma proteins and IBS. Initially, we used the “TwoSampleMR” package to format and harmonize the exposure and outcome data, ensuring consistency in effect alleles and other alleles between the 2 datasets. Subsequently, we performed the main MR analysis on the harmonized data using 5 different methods: inverse variance weighted (IVW), MR-Egger, Weighted Median, Simple Mode, and Weighted Mode, with IVW being the principal technique employed.^[21,23] These methods allowed us to calculate effect estimates and generate ORs. Among them, the minimum and maximum true causal effects when applying the IVW method with 80% power are 0.569 and 0.576, respectively.

To ensure the robustness of our results, we conducted several sensitivity analyses, including individual SNP analysis, Leave-one-out analysis, and funnel plot. These analyses were used to assess the contribution of each SNP to the causal relationship, detect significant effects of individual SNPs on the overall results, and evaluate publication bias. Additionally, we performed multiple comparison corrections and heterogeneity tests, and applied the MR-PRESSO method to detect and correct for pleiotropy.

For further analysis and result selection, we focused on results from the IVW method with P-values <.05 and calculated false discovery rate (FDR) adjusted P-values. We also selected data where all ORs were either >1 or <1. For data with pleiotropy test P-values >.05, we extracted the corresponding IDs and filtered the MR analysis results associated with these IDs. Finally, we saved the filtered results in multiple files for subsequent analysis and validation.

2.6. Other analysis

To further analyze and present the MR results, we generated volcano plots and forest plots. The volcano plot is a scatter plot that visualizes effect estimates (on the x-axis, Beta values) and significance levels (on the y-axis, −log10(P-value)), enabling a clear identification of significant causal relationships. The forest plot offers a comprehensive visualization of the causal links between each exposure factor and the outcome, displaying effect estimates along with their 95% confidence intervals. This allows researchers to visually compare the magnitude and statistical significance of different causal relationships, enhancing the interpretation of MR analysis results.

Additionally, we performed GO and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses on plasma protein genes with significant associations. These analyses aim to understand the biological functions and pathways involving these genes, providing deeper insights into their roles in cellular components, molecular functions, and biological processes. Finally, we utilized the GeneMANIA tool (https://genemania.org/) for functional prediction and interaction network analysis of the selected genes, identifying potential gene functions and interactions.

3. Result

3.1. Proteome-wide MR analyses of IBS

Through the analysis of exposure data, we identified 31,315 SNPs associated with plasma proteins after removing linkage disequilibrium and weak IVs. Finally, we selected 3598 plasma proteins for MR analysis. The F-statistics for the chosen IVs ranged from 29.71 to 57,932.93, indicating strong instruments. Additional details on the selected SNPs can be found in supplementary material (Table S1, Supplemental Digital Content 1, Supplemental Digital Content). Subsequently, we applied the IVW method to comprehensively assess the impact of these 3598 plasma proteins on IBS. Our results identified significant associations between 20 plasma proteins and the onset of IBS (P_FDR < 0.3; Fig. 2). As shown in Figure 3, 2 protective plasma proteins were negatively associated with the incidence of IBS (scavenger receptor class A member 5 [SCARA5], OR = 0.7917, (95% confidence interval (CI), 0.6844 to 0.9158), P_FDR = 0.2782; fibroblast growth factor binding protein 1(FGFBP1), OR = 0.6097, (95%CI, 0.5078 to 0.7321), P_FDR = 0.0004). The genetic susceptibility of the remaining 18 plasma proteins was positively correlated with the incidence of IBS, including but not limited to (leucine-rich repeat kinase 2 (LRRK2), OR = 1.3108, (95%CI, 1.1379 to 1.5099), P_FDR = 0.1401; Ras and Rab interactor 3 (RIC3), OR = 1.4358, (95%CI, 1.1495 to 1.7935, P_FDR = 0.2529; coiled-coil-helix-coiled-coil-helix domain-containing protein 10 (CHCHD10), OR = 1.6432, (95%CI, 1.2798 to 2.1098), P_FDR = 0.1401; HSPA1L, OR = 1.1924, (95%CI, 1.0818 to 1.3142), P_FDR = 0.2079; MYNN, OR = 1.4882, (95%CI, 1.1701 to 1.8929), P_FDR = 0.2529; G protein-coupled receptor 68 (GPR68), OR = 1.4414, (95% CI, 1.1453 to 1.8141), P_FDR = 0.2905). Further details on the remaining plasma proteins can be found in the supplementary material (Table S2, Supplemental Digital Content 2, Supplemental Digital Content).

Figure 2.

This volcano plot shows the causal relationship between plasma protein and risk of IBS in the MR analysis. PFDR < 0.3 was considered a significant association. IBS = irritable bowel syndrome, MR = Mendelian randomization.

Figure 3.

Forest plot of causality between 20 plasma proteins and IBS. CI = confidence interval, IBS = irritable bowel syndrome, ORs = odds ratios.

3.2. Reverse MR analyses of IBS

To investigate whether IBS affects the expression levels of proteins in circulation, we conducted a reverse MR analysis using genetically predicted IBS as the exposure factor and the 20 plasma proteins identified in the previous analysis as the outcome factors. The results showed that none of the plasma proteins exhibited a reverse causal relationship with IBS (P > .05), suggesting that these 20 plasma proteins are likely key causal factors in the development of IBS rather than consequences of the disease (Fig. S1, Supplemental Digital Content 3 and Table S3, Supplemental Digital Content 4, Supplemental Digital Content).

3.3. GO and KEGG enrichment analysis and co-expression network

In the GO enrichment analysis, we used 2 types of charts to present the results: a bubble chart and a circular network plot. The bubble chart illustrates the gene enrichment and significance levels for different terms. The size of the bubbles represents the number of genes in each term, while the gradient color of the bubbles indicates the P-value. As shown in Figure 4A, the enriched terms are mainly concentrated in biological processes such as endosomal transport, mitochondrial-related processes (e.g., regulation of protein targeting to the mitochondrion, regulation of mitochondrial gene expression), synaptic vesicle recycling, retrograde transport from the endoplasmic reticulum to the Golgi, molecular functions like protein folding chaperones, and cellular components related to the mitochondrial matrix. The circular network plot visually displays the relationships between GO terms, highlighting significance and enrichment levels through color intensity and area size. This plot further corroborates the findings of the bubble chart, particularly the significant enrichment in mitochondrial-related processes, synaptic vesicle-related processes, and signaling-related molecules (Fig. 4B). Similarly, we conducted a KEGG enrichment analysis. As shown in Figure 4C, the results revealed significant enrichment in 4 pathways: Endocytosis, Spliceosome, Nicotinate and nicotinamide metabolism, and Legionellosis. Lastly, we used the GeneMANIA tool to perform a co-expression network interaction analysis on the 20 plasma protein genes. This analysis revealed that the biological functions of these genes and their co-expression network genes are related to endosomal transport (Fig. 4D).

Figure 4.

(A) GO analysis diagram of genes for 20 plasma proteins (bubble chart); (B) GO analysis diagram of genes for 20 plasma proteins (circlize chart); (C) KEGG analysis diagram of genes for 20 plasma proteins(bubble chart); (D) Co-expression network and function diagram of genes for 20 plasma proteins. KEGG = Kyoto Encyclopedia of Genes and Genomes.

4. Discussion

To the best of our knowledge, this is the first study to explore potential causal links between plasma proteins and IBS through a proteome-wide MR analysis. We performed a bidirectional two-sample MR analysis to evaluate the causal effects of 4907 plasma proteins from the deCODE database on IBS development. The analysis identified 20 key plasma proteins as significant contributors to IBS development, with no evidence of bidirectional causality among them. Of these, 18 proteins showed a positive association between their elevated expression and increased IBS risk, while 2 proteins, SCARA5 and FGFBP1, were negatively associated with IBS risk, suggesting a protective effect. Although direct evidence linking these proteins to IBS is currently lacking, their known biological functions provide plausible mechanisms.

Proteins such as complement component 1 Q subcomponent-binding protein (C1QBP), Heat Shock Protein Family A Member 1 Like (HSPA1), and GPR68 are involved in inflammation and immune responses, indicating their potential role in the chronic low-grade inflammation associated with IBS. C1QBP is a multifunctional acidic protein found in various cellular compartments, including mitochondria, the cell surface, cytoplasm, and nucleus, with the highest expression in mitochondria.^[24] Research has shown that the binding of C1q to C1QBP can activate endothelial cells, leading to the production of several inflammatory mediators such as IL-6, IL-8 and MCP-1.^[25] Conversely, inflammatory mediators like Interferon Gamma (IFN-γ), Tumor Necrosis Factor Alpha (TNF-α), and Escherichia coli Lipopolysaccharide (E. coli LPS) can upregulate C1QBP expression.^[26] Another study suggests that C1QBP not only acts as a receptor for C1q but also binds to plasma proteins and proteins from pathogenic microorganisms. Many of the plasma proteins that bind to C1QBP are coagulation proteins, implying that C1QBP plays a crucial role in regulating fibrin formation at sites of local immune injury and/or inflammation.^[27] These mechanisms suggest that C1QBP may contribute to and exacerbate the inflammatory response, potentially serving as an underlying mechanism in the pathogenesis of IBS. Heat shock proteins are a class of molecular chaperones that are highly expressed in cells under heat stress or other stress conditions, such as oxidative stress, infection, and inflammation. Their primary function is to assist in protein folding, repair, and prevention of protein aggregation, thereby protecting cells from damage.^[28] HSPA1L, a member of the HSP70 family (HSPA), is highly expressed in the male germline. A study analyzing whole-exome sequencing data from 136 IBS patients and 106 healthy individuals found that de novo mutations in HSPA1L, along with 5 other rare mutations, are closely associated with the development of IBD.^[29] Although there is currently no direct evidence linking HSPA1L mutations to IBS, the possibility that misfolded proteins due to these mutations may trigger inflammatory responses could be a potential mechanism in IBS pathogenesis. GPR68, a proton-sensing G protein-coupled receptor, is activated in acidic environments and plays a significant role in inflammatory responses.^[30] Research has shown that using a novel GPR68 inhibitor^[31] or knocking out the GPR68 gene in mice^[32] can alleviate symptoms of colitis. Results indicate that compared with control groups, mice treated with the inhibitor exhibited reduced macrophage infiltration, lower levels of pro-inflammatory cytokines TNF and IL-6, decreased fibrosis markers like Transforming Growth Factor Beta 1 (TGF-β1), and normal vascular distribution without thickening under endoscopy.^[31] This suggests that GPR68 may influence IBS by modulating intestinal inflammation. IBS patients often exhibit chronic low-grade inflammation, and GPR68 might exacerbate IBS symptoms by enhancing these inflammatory responses and altering gut permeability. 5’-Nucleotidase Ecto (NT5E), also known as CD73, is an enzyme involved in the generation of extracellular adenosine and plays a role in regulating immune responses and inflammation.^[33] Dysregulation of its function may influence IBS through immunomodulatory effects. Recent studies have shown that NT5E expression is significantly downregulated in Tregs of patients with systemic lupus erythematosus, and the NT5E axis mediates the immunosuppressive activity of Tregs,^[34] suggesting its role in IBS may be related to immune regulation in the gut.

Similarly, proteins like LRRK2 and Syndecan 3 (SDC3), which are involved in neural signaling, may influence the gut-brain axis, a key pathway in the pathophysiology of IBS. LRRK2 is a serine/threonine kinase rich in leucine repeat sequences and is primarily expressed in the brain, where it plays a role in cellular signaling, microtubule dynamics, and autophagy. Mutations in LRRK2 are closely linked to neurodegenerative diseases such as Parkinson’s disease.^[35] Recent studies suggest that LRRK2 is not only a pathogenic gene for Parkinson’s disease but also associated with IBD, highlighting a potential connection between gastrointestinal inflammation and neurodegeneration that may involve the gut-brain axis.^[36] Given that the pathogenesis of IBS is also linked to the gut-brain axis, LRRK2 likely represents a potential factor in IBS development. SDC3 is a cell surface protein belonging to the heparan sulfate proteoglycan family and plays a crucial role in cell signaling, adhesion, and tissue repair.^[37] MYNN, a transcription factor, is involved in the regulation of gene expression, influencing various cellular functions, including cell growth, differentiation, and stress response.^[38] SDC3 and MYNN have been shown to play significant roles in the nervous system, particularly as biomarkers for the early detection of Alzheimer’s disease.^[39,40] Therefore, it is hypothesized that they may impact the development of IBS by modulating the function of the enteric nervous system and the gut-brain axis. Resistance to Inhibitors of Cholinesterase 3 (RIC3) is an intracellular protein primarily involved in neural signaling by regulating the folding and expression of nicotinic acetylcholine receptors (nAChRs).^[41] It plays a crucial role in neuroprotection and cell survival by ensuring the proper folding and surface expression of nAChRs on neurons, thereby facilitating neural transmission. Several studies have shown that RIC3 expression is upregulated in lymphocytes of patients with multiple sclerosis,^[42,43] suggesting a potential role in neuroinflammation. Although there is currently no direct evidence linking RIC3 to the gut-brain axis, its critical role in the nervous system and its upregulation in neuroinflammatory diseases like multiple sclerosis suggest that RIC3 may influence IBS by modulating neuroinflammation and neuroimmune interactions. Specifically, RIC3 may affect neural regulation and immune responses in the gut, contributing to the pathogenesis of IBS.

Eukaryotic Translation Initiation Factor 4 Gamma 3 (EIF4G3), a member of the eukaryotic translation initiation factor 4G family, plays a crucial role in regulating protein synthesis and serves as an important node in cellular stress response and metabolic regulation.^[44] THO Complex Subunit 1 (THOC1), as part of the transcription-export (TREX) complex, is involved in mRNA processing and export.^[45] These functions highlight the critical roles of EIF4G3 and THOC1 in maintaining cellular function and gene expression regulation. Although the exact mechanisms are not yet fully understood, it is hypothesized that these proteins may influence IBS development by affecting protein synthesis, gene expression, and cellular function in intestinal epithelial cells, thus impacting gut barrier function and inflammatory responses. CHCHD10 is a mitochondria-associated protein that is primarily involved in maintaining mitochondrial structure and function, playing an essential role in cellular energy metabolism and stress response.^[46] Studies have shown that dominant mutations in CHCHD10 can lead to amyotrophic lateral sclerosis and frontotemporal dementia by disrupting mitophagy through the CHCHD10-PARL-PINK1 pathway.^[47] Family With Sequence Similarity 210 Member A (FAM210A) is another mitochondrial protein involved in regulating mitochondrial function and energy metabolism, and it plays a key role in maintaining cellular energy balance and responding to cellular stress.^[48] We speculate that mitochondrial dysfunction might lead to insufficient energy supply to intestinal epithelial cells, increased oxidative stress, and inflammation, thus impairing gut barrier function and contributing to IBS pathogenesis. Furthermore, Ras-related protein Rab-35 (RAB-35) and Intersectin 1 (ITSN1) have also been positively correlated with IBS development in our analysis, suggesting their potential roles in the pathophysiology of IBS. RAB-35 is a small GTPase primarily involved in membrane transport and endocytosis, regulating the formation and sorting of endosomes.^[49] Its role in regulating intracellular membrane transport may affect the absorption and barrier function of intestinal epithelial cells, thus influencing gut health. ITSN1, a multi-domain scaffold protein, is involved in signal transduction, vesicle transport, and synaptic plasticity.^[50] Although there is currently no direct literature linking RAB-35 and ITSN1 to IBS, their roles in cellular transport, signal transduction, and cellular morphology regulation suggest that they might influence IBS pathogenesis by modulating the function of the enteric nervous system or interactions between intestinal epithelial cells and their environment. COP9 Signalosome Subunit 2 (COPS2) is part of the COP9 signalosome complex, which is involved in regulating protein degradation, DNA repair, and gene expression, playing a crucial role in maintaining cellular homeostasis and responding to stress.^[51] A study on IBS-D patients identified COPS2, regulated by hsa-miR-19b1–5p, as a key protein in visceral hypersensitivity through miRNA-mRNA-protein integrated analysis.^[52] Calcium Binding Protein 7 (CABP7), a calcium binding protein with an EF-hand domain, primarily regulates the generation of vesicular carriers in the trans-Golgi network in neurons and may also be involved in regulating cytokinesis in mammalian cells, a process essential for normal cell growth and development.^[53] Notably, CABP7 is related to calcium signaling, which plays a crucial role in regulating gut motility and sensation. Transmembrane Protein 87B, a transmembrane protein, may influence IBS by regulating cell signaling and membrane transport,^[54] affecting intestinal epithelial cell function and their interactions with the enteric nervous system. N-Acetylated Alpha-Linked Acidic Dipeptidase-Like 1 (NAALADL1) is a zinc-dependent metallopeptidase with aminopeptidase activity involved in the final digestion and absorption of proteins/peptides, primarily expressed in the small intestine.^[55] Given its role in amino acid metabolism, abnormal expression of NAALADL1 in the gut may alter the homeostasis of the intestinal microenvironment, thereby affecting gut function. Taken together, these plasma proteins may contribute to IBS pathogenesis by influencing intestinal cell function and metabolism.

Finally, we discuss 2 protective plasma proteins, FGFBP1 and SCARA5, whose expression levels are negatively correlated with IBS onset. FGFBP1 is a regulatory protein that interacts with FGFs, enhancing their biological activity and promoting angiogenesis and wound healing,^[56] which may play a crucial role in the gut. Additionally, FGFBP1’s ability to promote angiogenesis may optimize intestinal microcirculation, facilitating the removal of metabolic waste and inflammatory mediators, thus reducing the inflammatory burden in the gut. Notably, FGFBP1 expression is significantly positively correlated with the infiltration of Tregs.^[57] By promoting Treg infiltration, FGFBP1 may exert anti-inflammatory effects in the gut of IBS patients, inhibiting excessive inflammatory responses and protecting the gut from inflammation-induced damage. This mechanism may explain the protective effect of FGFBP1 against IBS. On the other hand, SCARA5 is a member of the class A scavenger receptors, primarily expressed in macrophages and dendritic cells, and is involved in clearing various pathogens and particulate debris.^[58] Recent insights suggest that SCARA5, as a receptor for HMGB1, can inhibit the synergistic effect of CpG-ODN with HMGB1 in Toll-like receptor 9 (TLR9)-mediated inflammatory responses.^[59] HMGB1 is a key mediator in inflammatory responses, while TLR9 is a pattern recognition receptor involved in innate immunity. By inhibiting HMGB1 and TLR9-mediated inflammatory signaling, SCARA5 may play a crucial role in reducing IBS-related gut inflammation. Moreover, the absence of SCARA5 in aged mice leads to symptoms of autoimmune disease, including the accumulation of lymphocytes in the stromal connective tissues of multiple organs and the production of antinuclear antibodies, indicating that SCARA5 is vital in maintaining immune homeostasis and preventing autoimmune reactions.^[58] Therefore, the presence of SCARA5 may help prevent abnormal immune activation in IBS patients, reducing IBS-associated autoimmune responses. Although direct evidence is lacking, it is reasonable to infer from the mechanisms described above that SCARA5 and FGFBP1 may protect the gut from inflammation and immune abnormalities through multiple pathways, thereby lowering the risk of IBS development.

This research employed MR to systematically investigate the potential causal links between plasma proteins and IBS. One of the significant advantages of the MR method is its use of genetic variants as IVs, which effectively reduces the impact of confounding factors and reverse causation commonly seen in traditional observational studies, thereby providing more reliable causal inferences. Additionally, the large sample size in this study, encompassing data from over 320,000 individuals, not only increased the statistical power but also enhanced the generalizability of the findings. To ensure the robustness of our findings, we performed several sensitivity analyses, including individual SNP analysis, leave-one-out analysis, funnel plot analysis, and adjustments for multiple testing and heterogeneity. These methods reinforced the stability and reliability of our results. Additionally, MR-PRESSO was used to identify and correct potential pleiotropy, enhancing the credibility of our conclusions. Importantly, this study is the first to systematically assess the causal relationships between specific plasma proteins and IBS, identifying several protein biomarkers significantly associated with IBS. These proteins represent candidate causal mediators in IBS pathogenesis, but their translational potential should be evaluated in a subtype‑specific manner. Replication in subtype‑stratified cohorts and functional investigations are mandatory before any therapeutic inference can be drawn. Therefore, this study holds significant methodological, data-driven, and clinical relevance, making an important contribution to the field of IBS research.

Despite the comprehensive nature of our study, there are certain limitations. First, the genome-wide association study and proteomic data were exclusively from individuals of European descent, which may limit the applicability of the findings to other ethnic groups and affect the external validity of gene-phenotype associations globally. Future research should include a more diverse range of ethnic backgrounds to improve the generalizability of the results. Second, the MR approach itself has inherent limitations, such as the potential inability to completely eliminate all gene-environment interactions, which could introduce bias into causal inferences. Third, we only assessed the role of plasma proteins in IBS, lacking information on protein levels in other tissues. Future research should integrate proteomic data from various tissues to more thoroughly explore the pathogenesis of IBS. Finally, we acknowledge that our study is exploratory and that further research is needed to validate these associations. Future studies should focus on experimental validation of these proteins’ roles in IBS and explore the underlying mechanisms. Clinical studies are also necessary to confirm these findings and evaluate their potential for guiding new therapeutic approaches.

5. Conclusion

In summary, this study explored the bidirectional causal relationship between plasma proteins and IBS. Our forward MR analysis identified 20 plasma proteins as potential factors influencing the onset of IBS, while the reverse MR analysis found no substantial evidence suggesting that these proteins are a consequence of IBS. Moreover, we performed enrichment and co-expression network analyses to explore the molecular functions and biological significance related to these proteins. We hope that this study provides new insights into the underlying mechanisms of IBS and offers novel perspectives for identifying therapeutic targets.

Author contributions

Data curation: Yue Lei, Yiming Liu.

Formal analysis: Yue Lei, Yiming Liu.

Methodology: Yue Lei.

Project administration: Yue Lei.

Validation: Yue Lei, Yiming Liu.

Visualization: Yue Lei, Yiming Liu.

Writing – original draft: Yue Lei, Yiming Liu.

Writing – review & editing: Yue Lei, Yiming Liu.

Conceptualization: Yiming Liu.

Funding acquisition: Yiming Liu.

Resources: Yiming Liu.