Kashin-Beck disease (KBD) is a debilitating endemic degenerative osteochondrosis characterized by shortened and deformed fingers, joint deformities, and limited mobility. While metabolic processes, apoptosis, immune responses, cytoskeletal changes, cell movement, and extracellular matrix turnover are implicated in KBD chondrocyte injury, the underlying mechanisms remain unclear, hindering effective treatment. Evidence suggests that the interaction between genetic predisposition and environmental factors drives cartilage damage in KBD patients. Recent research highlights the potential influence of the gut microbiome on immune responses and metabolite levels, suggesting a link between gut dysbiosis and low-grade inflammation that contributes to cartilage injury and frailty. The gut microbiome's role in cartilage health is increasingly recognized, and exploring the cartilage-gut-microbiome axis may lead to new therapeutic approaches. Many microbial metabolites can affect osteochondral disease development, suggesting a connection between gut microbiome diversity and host metabolic health. Metabolomics studies the metabolites produced by a biological system, offering insights into cellular processes. These metabolites, as intermediates and end products of cellular processes, are found in various bodily fluids, acting as the ultimate response of biological systems to genotype, phenotype, and environment. Gut microbiota translocation to subchondral bone marrow and deeper cartilage zones, along with microbial conversion of dietary and host nutrients, may produce metabolites impacting cartilage metabolism through low-grade inflammation. However, the interaction between gut microbiota, metabolites, and KBD development hasn't been thoroughly investigated.

Several studies have explored the gut microbiome's role in other joint diseases. Rogier et al. (2017) found decreased Bacteroidaceae and increased Firmicutes in a collagen-induced arthritis mouse model. Scher et al. (2013) reported a lack of gut Bacteroides in patients with new-onset rheumatoid arthritis. These findings suggest that gut dysbiosis is associated with inflammatory joint diseases. Other research has demonstrated the impact of short-chain fatty acids (SCFAs) on bone mass and protection against bone loss (Lucas et al., 2018). The role of specific microbial metabolites, like succinic acid produced by *Prevotella copri* (Kovatcheva-Datchary et al., 2015), has also been studied, highlighting the complex interplay between diet, gut microbiota, and joint health. Studies show that an imbalance of nutrients and the low-selenium diet with Fusarium mycotoxin contamination could be one of the most important factors affecting the gut microbiota composition in KBD patients, which leads to joint degradation through the cartilage-gut-microbiome axis.

This study employed a case-control design, recruiting 32 KBD patients and 35 healthy controls from Xunyi County, a KBD-endemic region in China. Stringent inclusion and exclusion criteria were used to minimize confounding factors. Fecal and serum samples were collected and analyzed. 16S rDNA gene sequencing was performed to characterize the gut microbiota composition. The V3-V4 region of the 16S rDNA gene was amplified, sequenced, and analyzed using bioinformatics tools like QIIME2 to assess alpha and beta diversity, determine dominant taxa, and identify differentially abundant genera and species between KBD patients and controls. Linear discriminant analysis (LDA) effect size (LEfSe) was used to identify specific bacteria associated with KBD. Metagenomic sequencing was also performed on fecal samples (16 grade I KBD, 16 grade II KBD, 35 controls) to identify differentially expressed genes and assess functional changes using databases like KEGG and COG. Serum samples underwent LC/MS-based metabolomic analysis to identify differentially regulated metabolites (DRMs) between groups. PLS-DA was used for data analysis, DRMs were mapped to KEGG pathways, and correlation analysis examined the relationship between gut microbiota changes and metabolites. Statistical analyses included t-tests, Fisher's exact test, Spearman's correlation, and random forest modeling.

16S rDNA sequencing revealed no significant differences in alpha diversity between KBD patients and controls. However, beta diversity analysis showed significant differences in gut microbiota composition. KBD patients exhibited higher levels of *Fusobacteria* and *Bacteroidetes* and lower levels of *Firmicutes*, indicating gut dysbiosis. Specifically, genera *Alloprevotella*, *Robinsoniella*, *Megamonas*, and *Escherichia-Shigella* were more abundant in KBD patients. Metagenomic sequencing confirmed these changes at the species level, with most differentially abundant species belonging to the *Prevotella* genus. *Prevotella copri* was notably enriched in grade II KBD. Functional analysis predicted alterations in L-glutamate and L-glutamine biosynthesis and polyisoprenoid biosynthesis. Serum metabolomic analysis showed significant differences in metabolites involved in lipid metabolism, including unsaturated fatty acids and glycerophospholipids. Correlation analysis revealed relationships between specific microbial species and metabolite levels. For instance, unsaturated fatty acids were negatively correlated with *Paenibacillus montanisoli* and positively correlated with *Buttiauxella brennerae*. Lysolecithins showed negative correlations with *Parvimonas* sp. and positive correlations with *Rothia* sp.

The study's findings of gut dysbiosis in KBD patients, characterized by elevated *Bacteroidetes* and reduced *Firmicutes*, align with observations in other inflammatory joint diseases. The increased abundance of specific genera like *Alloprevotella* and *Prevotella* suggests potential roles in KBD pathogenesis. The observed alterations in lipid metabolism, particularly unsaturated fatty acids and glycerophospholipids, support the hypothesis of a disturbed lipid homeostasis in KBD. The correlation between specific microbial species and metabolites highlights the complex interplay between the gut microbiome and metabolome in KBD development. Selenium deficiency and *Fusarium* mycotoxins, known environmental risk factors for KBD, may contribute to the observed gut microbiota dysbiosis, potentially influencing metabolite production and leading to cartilage damage. The depletion of anti-inflammatory species like *Faecalibacterium prauznitzii* further supports a role for gut dysbiosis in KBD pathogenesis. The study suggests a potential mechanism where gut dysbiosis influences systemic metabolism, potentially leading to cartilage damage via the cartilage-gut-microbiome axis.

This study provides evidence of gut microbiota dysbiosis and metabolic alterations in KBD patients. The observed changes in specific microbial species and metabolites, particularly those related to lipid metabolism, point toward a novel interaction between the gut microbiome and metabolome in KBD pathogenesis. Future research should explore the causal relationships between specific gut microbiota alterations, metabolite profiles, and KBD progression. Further investigation into the effects of dietary interventions, probiotics, or other targeted therapies aimed at modulating the gut microbiome could offer new avenues for KBD prevention and treatment.

The relatively small sample size is a limitation of this study. The declining incidence of KBD and the challenges in identifying classic KBD cases in older populations due to potential overlap with osteoarthritis made recruiting a larger cohort difficult, despite strict inclusion/exclusion criteria. The cross-sectional design prevents establishing causal relationships between gut microbiota alterations and KBD development. Further longitudinal studies with larger sample sizes are needed to confirm these findings and explore the long-term effects of gut dysbiosis on KBD progression.