Elucidating Causal Relationships Among Gut Microbiota, Human Blood Metabolites, and Knee Osteoarthritis: Evidence from a Two-Stage Mendelian Randomization Analysis

<b><i>Background:</i></b> Although previous observational studies suggest a potential association between gut microbiota (GM) and knee osteoarthritis (KOA), the causal relationships remain unclear, particularly concerning the role of blood metabolites (BMs) as potential mediators. Elucidating these interactions is crucial for understanding the mechanisms underlying KOA progression and may inform the development of novel therapeutic strategies. <b><i>Objective:</i></b> This study aimed to determine the causal relationship between GM and KOA and to quantify the potential mediating role of BMs. <b><i>Methods:</i></b> Instrumental variables (IVs) for GM and BMs were retrieved from the MiBioGen consortium and metabolomics genome-wide association studies (GWAS) databases. KOA-associated single-nucleotide polymorphisms were sourced from the FinnGen consortium. Inverse-variance weighted approach was utilized as the main analytical method for Mendelian randomization (MR) analysis, complemented by MR-Egger, simple mode, weighted mode, and weighted median methods. The causal relationships between GM, BMs, and KOA were sequentially analyzed by multivariate MR. False discovery rate correction was applied to account for multiple comparisons in the MR results. Sensitivity analyses and reverse MR analysis were also conducted to verify the reliability of the findings. Finally, a two-step approach was employed to determine the proportion of BMs mediating the effects of GM on KOA. <b><i>Results:</i></b> MR analysis identified seven gut microbial species that are causally associated with KOA. Additionally, MR analysis of 1091 BMs and 309 metabolite ratios revealed 13 metabolites that influence the risk of KOA. Through two-step analysis, three BMs were identified as mediators of the effects of two GMs on KOA. Among them, 6-hydroxyindole sulfate exhibited the highest mediation percentage (10.26%), followed by <i>N</i>-formylanthranilic acid (6.55%). Sensitivity and reverse causality analyses further supported the robustness of these findings. <b><i>Conclusion:</i></b> This research identified specific GMs and BMs that have a causal association with KOA. These findings provide critical insights into how GM may influence KOA risk by modulating specific metabolites, which could be valuable for the targeted treatment and prevention of KOA.