Rheumatoid arthritis (RA) is a chronic autoimmune disease causing significant quality-of-life impairment. Current treatments have potential side effects, prompting research into natural alternatives. Lycium barbarum polysaccharide (LBP), a bioactive component of Lycium barbarum with various beneficial properties, including anti-inflammation and gut microbiota regulation, has shown promise in RA treatment. Previous studies demonstrated LBP's effectiveness in improving RA symptoms in rat models, by maintaining bone integrity and reducing inflammatory mediators. However, the underlying mechanism, particularly its interaction with gut microbiota, remained unclear. Given the established link between gut microbiota and RA pathogenesis (enteric malnutrition and gut dysbiosis are common features), this study aimed to investigate whether LBP's RA-alleviating effects are mediated by gut microbiota modulation and subsequent changes in host gene expression within the intestinal epithelium. The study hypothesized that LBP alters the gut microbiome, leading to changes in epigenetic modification (DNA methylation) of host genes involved in RA pathogenesis, thereby reducing their expression and alleviating RA symptoms.

Existing literature establishes a strong link between rheumatoid arthritis and the gut microbiome. Studies have consistently shown that individuals with RA exhibit imbalances in their gut microbiota, often characterized by reduced beneficial bacteria like Bifidobacteria and Bacteroidetes, and increased abundance of harmful bacteria such as Prevotella. The dysbiosis can contribute to chronic low-grade inflammation, which is a key factor in RA pathogenesis. Furthermore, research suggests that restoring gut homeostasis is a potential therapeutic strategy for reducing RA severity. The gut-joint axis, a concept highlighting the interconnectedness between intestinal microecology and joint inflammation, has gained traction in the field. Several studies demonstrate that specific bacterial species can positively or negatively influence RA development and progression. While the exact mechanisms remain incompletely understood, modulation of the immune system, epigenetic modification, and metabolic changes are likely involved. Previous research has explored the therapeutic potential of various natural polysaccharides, demonstrating their efficacy in alleviating RA symptoms. However, the mechanisms of action, especially the role of gut microbiota, needs further investigation, especially with LBP.

This study employed a collagen-induced arthritis (CIA) rat model to evaluate the effects of LBP. Twenty-four SPF female Wistar rats were divided into three groups: Control, Model (CIA induced), and Lyc (CIA induced + LBP treatment). The CIA model was established via two subcutaneous injections of bovine type II collagen and incomplete Freund's adjuvant (IFA), seven days apart. The Lyc group received daily oral LBP administration (400 mg/kg body weight) for 42 days. Paw swelling and arthritis scores were measured to assess disease severity. Serum cytokine levels (IL-1α, IL-1β, IL-10, IL-12, IL-17) were measured using ELISA. Cecal content was analyzed via 16S rDNA sequencing to assess gut microbiota composition. Colonic epithelial tissue was analyzed using RNA sequencing (RNA-Seq) to identify differentially expressed genes (DEGs) and methylation sequencing to identify differentially methylated regions (DMRs). The correlation between gut microbiota composition, gene expression, methylation patterns, and RA symptoms was analyzed. Statistical analyses included t-tests, ANOVA, PLS-DA, and correlation analysis.

LBP significantly alleviated RA symptoms in the rat model, reducing paw swelling and arthritis scores compared to the Model group (P<0.01). LBP intervention also modulated serum cytokine levels, decreasing pro-inflammatory cytokines (IL-1α, IL-1β, IL-12, IL-17) and increasing anti-inflammatory cytokine IL-10 (P<0.05). 16S rDNA sequencing revealed that LBP significantly altered the gut microbiota composition. The relative abundance of Lachnospiraceae_NK4A136_group and uncultured_bacterium_f_Ruminococcaceae decreased, while the abundance of Romboutsia, Lactobacillus, Dubosiella, and Faecalibaculum significantly increased (P<0.05). RNA-seq identified 755 differentially expressed genes (DEGs) in the colonic epithelium of the Lyc group compared to the Model group. Methylation sequencing showed 697 differentially methylated regions (DMRs), annotating 375 genes. Ten genes (Dpep3, Gstm6, Slc27a2, Col11a2, Sycp2, SNORA22, Tnni1, Gpnmb, Mypn, and Acsl6) showed both hypermethylation and downregulation in the Lyc group. These genes are potentially associated with RA-related inflammation and cartilage degradation. The colonic epithelial SAM level was significantly increased in the Lyc group (P<0.05), positively correlating with the abundance of the four increased bacteria (Romboutsia, Lactobacillus, Dubosiella, and Faecalibaculum).

The findings demonstrate that LBP effectively alleviates RA symptoms in a rat model, likely through its modulation of gut microbiota. The observed changes in gut microbial composition, specifically the increase in beneficial bacteria and decrease in potentially harmful bacteria, support the gut-joint axis hypothesis. The reduction in pro-inflammatory cytokines and the improvement of joint pathology further solidify this correlation. The significant changes in gene expression, primarily due to DNA hypermethylation, indicate an epigenetic mechanism through which LBP exerts its effects. The elevation of SAM, a key methyl donor, as a result of the microbiota change is a plausible mechanism explaining this hypermethylation and subsequent suppression of RA-related genes. These results support the notion that LBP can modulate the gut microbiota, leading to epigenetic changes in host cells, resulting in downregulation of genes associated with RA pathogenesis. This mechanism could potentially explain LBP's beneficial effects in reducing inflammation and improving joint health in RA.

This study provides strong evidence that LBP effectively alleviates RA symptoms in a rat model, likely by modifying the gut microbiome, increasing SAM levels, inducing DNA hypermethylation of key genes involved in inflammation and cartilage destruction, and subsequently reducing their expression. The findings support the gut-joint axis and suggest that LBP could be a promising therapeutic agent for RA. Future studies should investigate the specific mechanisms through which individual bacteria mediate SAM production and the precise functions of the identified differentially expressed genes in RA pathogenesis. Further research is needed to confirm these findings in human clinical trials and to explore the optimal dose and administration route of LBP for RA treatment.

This study utilized a rat model, which may not perfectly reflect the complexity of human RA. The study focused on a single dose of LBP; further investigation is needed to determine the optimal dose-response relationship. While the correlation between gut microbiota changes, SAM levels, gene methylation, and RA alleviation is strong, direct causation remains to be fully established through additional experiments. The specific mechanisms by which individual bacteria influence SAM production and the functions of several genes warrant further exploration.