Lycium barbarum polysaccharide modulates gut microbiota to alleviate rheumatoid arthritis in a rat model

The study addresses whether the RA-alleviating effects of Lycium barbarum polysaccharide (LBP), a non-starch polysaccharide, are mediated by modulation of the gut microbiota and downstream host epigenetic regulation. RA is a chronic autoimmune disease marked by synovial inflammation and joint destruction, for which current pharmacotherapies can have notable side effects. LBP has reported anti-oxidant, barrier-enhancing, anti-inflammatory, and microbiota-modulating activities, and prior work indicates LBP can improve clinical indices in collagen-induced arthritis (CIA) models. Given that NSPs reach the colon and are metabolized by gut microbes, and that intestinal dysbiosis is linked to RA and can influence host gene expression via epigenetic mechanisms, the study aims to elucidate if LBP reshapes gut microbiota to produce metabolites (e.g., SAM) that drive DNA methylation changes in colonic epithelial genes associated with RA, thereby alleviating disease.

Prior studies show LBP exerts multiple beneficial effects including anti-oxidation, barrier protection, anti-tumor, and anti-inflammatory actions, and improves RA in CIA models by maintaining bone integrity and reducing inflammatory mediators. LBP also affects chondrocyte proliferation and reduces arthritis. Intestinal dysbiosis is a hallmark of RA, with reports of decreased Bifidobacteria and Bacteroidetes and increased Prevotella (e.g., P. copri) in new-onset RA. Gut microbiota can modulate host epigenetics and intestinal epithelial gene expression, suggesting a gut-joint axis. Specific taxa such as Fusobacteria and Lachnospiraceae relate to inflammatory bowel disease, a risk factor for RA, while beneficial taxa like Romboutsia and Faecalibacterium support intestinal integrity. Lactobacillus strains (e.g., L. salivarius, L. plantarum, L. fermentum, L. helveticus) have shown RA amelioration or prevention in models. DNA methylation commonly suppresses gene expression; epigenetic regulation has been implicated in inflammatory diseases and is influenced by microbial metabolites like SAM.

Design: Collagen-induced arthritis (CIA) rat model with LBP intervention and multimodal analyses (microbiome 16S rRNA sequencing, colonic epithelial transcriptomics and methylomics, cytokine quantification, histopathology, and SAM measurement). Animals and housing: 24 SPF female Wistar rats (8 weeks old) housed under controlled conditions. Ad libitum AIN-93M diet and sterile water. Procedures approved by Jinan University IACUC (2019827-02). RA induction: Bovine type II collagen emulsified with incomplete Freund’s adjuvant (IFA). Subcutaneous primary immunization at day 7 (0.2 mL emulsion, ~1.5 cm from caudal end) and booster at day 14 (1.5 mL emulsion, ~3 cm from caudal end). Controls received saline. Groups and treatment: Randomized into 3 groups (n=8 each): Control, Model (CIA), and Lyc (CIA + LBP). Lyc group received LBP 400 mg·kg−1 by daily gavage for 42 days; Control and Model received sterile distilled water. The rat dose corresponds to ~63.49 mg·kg−1 for humans. Timeline: 42-day experiment with daily gavage throughout. Clinical assessments: Hind paw diameter measured weekly with vernier caliper; arthritis scores (0–4 per paw; max 16 per rat) recorded weekly from day 7 per predefined criteria. Histopathology: Hind feet collected post-euthanasia; joints fixed in 4% paraformaldehyde, decalcified in 10% EDTA for 30 days, paraffin-embedded, sectioned (5 µm), HE-stained; evaluated for inflammatory infiltration, synovial/fibrous hyperplasia, pannus. Cytokines: Serum IL-1α, IL-1β, IL-10, IL-12, and IL-17 quantified by ELISA kits. Microbiome: Cecal bacterial DNA extracted; V3–V4 16S rRNA amplicons constructed and sequenced on Illumina HiSeq platform (HiSeq 2500). Bioinformatics included read processing, OTU clustering, taxonomic assignment, alpha/beta diversity, PLS-DA, and differential abundance analyses. Transcriptomics (colonic epithelium): Total RNA extraction, quality control (Agilent Bioanalyzer). Library prep with mRNA isolation, fragmentation, cDNA synthesis, end-repair, size selection (200–300 bp), PCR amplification (15 cycles), sequencing on Illumina HiSeq 4000 (paired-end). Expression quantified as FPKM. Differentially expressed genes (DEGs) identified using EdgeR with P<0.05 and |log2FC|>2; KEGG classification and pathway enrichment performed. Methylomics (colonic epithelium): Genomic DNA extraction; library prep with end-repair, adenylation, ligation of methylated adapters; hybridization capture with biotinylated RNA baits and streptavidin purification; bisulfite conversion (EZ DNA Methylation-Gold kit); sequencing and analysis (including Bismark/Bison pipelines). Identification and annotation of differentially methylated regions (DMRs) and sites (DMS) across gene elements (promoter, exon, intron, intergenic, UTR, TTS). Integration with transcriptome to identify genes with promoter/genic hypermethylation and concomitant downregulation. SAM quantification: SAM levels measured in colonic epithelial tissue and intestinal contents; group comparisons performed. Correlation analyses linked SAM levels with bacterial abundances, cytokines, clinical indices, and gene expression. Statistics: Group differences assessed with significance thresholds indicated in figures (typically P<0.05, P<0.01, P<0.001). Correlation heatmaps generated to relate clinical, microbial, metabolite, and gene expression variables.

- Clinical improvement: LBP reduced paw swelling and arthritis scores versus Model; histology showed reduced inflammatory infiltration, synovial hyperplasia, and pannus in Lyc group. - Cytokines: CIA increased serum IL-1α, IL-1β, IL-12, IL-17 and decreased IL-10 relative to Control. LBP significantly lowered IL-1α, IL-1β, IL-12, IL-17 and restored IL-10 toward normal (P<0.05–0.01). - Microbiome restructuring: 1,840,370 valid reads; Lyc group exhibited 6 unique OTUs and distinct beta-diversity (PLS-DA). Phylum level: Fusobacteria reduced with LBP. Genus level (Model vs. Lyc): decreases in Lachnospiraceae_NK4A136_group (P=0.0073) and uncultured_bacterium_f_Ruminococcaceae (P=0.0237); increases in Faecalibaculum (P=0.0155), Romboutsia (P=0.0237), Lactobacillus (P=0.0101), Dubosiella (P=0.0029). Pro-inflammatory cytokines, paw swelling, and arthritis scores positively correlated with Lachnospiraceae_NK4A136_group and uncultured_bacterium_f_Ruminococcaceae, but negatively with Romboutsia, Lactobacillus, Dubosiella, and Faecalibaculum; IL-10 showed opposite correlations. - Transcriptome changes: 462 DEGs upregulated and 293 downregulated in Lyc vs. Model. Enriched KEGG pathways (32 significant) included immune-related and inflammatory pathways (e.g., TNF signaling, PPAR signaling, cytokine–cytokine receptor interaction). - Methylome reprogramming: 697 DMRs annotating 375 genes (6.9% in promoters). 51,298 methylation-specific sites identified (5.6% promoter, 8.1% exon; majority intergenic/intronic). Integration identified 89 genes with hypermethylation and suppressed expression in Lyc vs. Model; 10 prioritized genes with notable promoter/genic methylation and downregulation: Dpep3, Gstm6, Slc27a2, Col11a2, Sycp2, SNORA22, Tnni1, Gpnmb, Mypn, Acsl6. - Gene–phenotype correlations: Expressions of several genes positively correlated with IL-1α/β, IL-12, IL-17A and with paw diameter/arthritis score (e.g., Dpep3 and Mypn with IL-1α/β, IL-17A; Col11a2 and Gpnmb with IL-1β, IL-17A; SNORA22 with IL-1α, IL-12; Acsl6 with IL-1β, IL-12 and negatively with IL-10; Tnni1 and Gpnmb, Mypn, Acsl6 with paw swelling/arthritis scores). - SAM increase: SAM content significantly increased in colonic epithelium and intestinal contents in Lyc vs. Model (P<0.05). Epithelial SAM positively correlated with abundances of Romboutsia, Lactobacillus, Dubosiella, and Faecalibaculum. Bacterial SAM increase suggested microbial contribution (notably Lactobacillus) to methyl donor availability. - Mechanistic model: LBP reshapes gut microbiota, increases microbial/epithelial SAM, induces DNA hypermethylation and downregulation of RA-associated epithelial genes, reduces inflammatory cytokines and joint pathology, improving RA indices.

The findings support the hypothesis that LBP alleviates RA via modulation of the gut microbiota and consequent epigenetic regulation in the host intestinal epithelium. LBP administration reversed CIA-associated dysbiosis by increasing taxa associated with intestinal health (Romboutsia, Lactobacillus, Dubosiella, Faecalibaculum) and decreasing taxa linked to inflammation (Lachnospiraceae_NK4A136_group, uncultured Ruminococcaceae, Fusobacteria). These microbial shifts correlated with reduced pro-inflammatory cytokines, improved clinical scores, and elevated IL-10. Concomitantly, LBP increased SAM levels in the gut and epithelium, aligning with the observed DNA hypermethylation and downregulation of genes implicated in cartilage degradation, angiogenesis, lipid metabolism/inflammation, and cellular invasion (e.g., Col11a2, Acsl6, Gpnmb, SNORA22). The integrated multi-omics evidence points to a gut–joint axis wherein microbial metabolites modulate host epigenetics to attenuate RA pathology.

This study demonstrates that Lycium barbarum polysaccharide effectively alleviates CIA in rats by reshaping the gut microbiota and increasing SAM availability, which drives DNA hypermethylation and suppressed expression of RA-associated genes in colonic epithelium. Key microbial changes included increased Romboutsia, Lactobacillus, Dubosiella, and Faecalibaculum and decreased Lachnospiraceae_NK4A136_group and uncultured Ruminococcaceae. Epigenetic suppression of genes such as Dpep3, Gstm6, Slc27a2, Col11a2, Sycp2, SNORA22, Tnni1, Gpnmb, Mypn, and Acsl6 correlated with reduced inflammatory cytokines and improved joint outcomes. Future work should directly validate SAM production by the enriched bacterial taxa and clarify the specific roles of several identified genes in RA pathophysiology, as well as assess translatability to human disease.

- The study did not directly confirm SAM production capacity by the specific bacteria enriched after LBP intervention, despite correlative increases in bacterial and epithelial SAM. - The functional roles of several hypermethylated, downregulated genes (e.g., Dpep3, Sycp2, Tnni1, Mypn) in RA remain undefined and require further investigation. - Findings are based on a rat CIA model; extrapolation to human RA requires additional validation.