目的

探究粪菌移植（FMT）联合甲氨蝶呤（MTX）治疗难治性类风湿关节炎（RA）患者的疗效、安全性及“菌群-代谢-免疫”三级通路的调控机制。

方法

本研究纳入2例对标准方案甲氨蝶呤治疗应答不良的难治性RA患者，从健康供体分离粪菌制备胶囊，予患者行粪菌移植共7次，联合MTX治疗，纵向随访24周。研究主要终点为16周ACR20应答情况，并收集基线（BP）、粪菌移植期（FP）、粪菌移植后（PP）三阶段的粪便、血清、PBMC标本，分别进行16S rRNA测序、LC-MS代谢组和RNA-seq转录组检测。利用多组学整合分析、功能富集（GO/KEGG/Reactome/DO）、蛋白互作网络（PPI）、相关性网络等，系统评估FMT干预的临床效应和分子机制。

结果

两例患者在16周均达到主要终点（ACR20应答），1例患者达到ACR50应答，1例患者达到ACR70缓解。DAS28、CRP、ESR等疾病活动指标显著下降，肿痛关节数、VAS、HAQ评分改善，FMT耐受性良好。16S rRNA 组学显示 FMT 后肠道菌群多样性显著提升，Escherichia-Shigella 丰度下降；Bifidobacterium 与 Faecalibacterium 低水平定植后趋于下降。代谢组发现Glu-Gln、N6-Succinyl Adenosine等抗炎小分子上调，富集于能量与氨基酸代谢通路。PBMC转录组提示FP期促炎因子下调，PP期免疫与代谢基因上调。多组学整合揭示菌群-代谢-免疫网络与疾病活动改善相关。

结论

FMT联合MTX可有效改善难治性RA患者炎症活动和关节功能，安全性良好。其机制可能与抗炎菌群低水平定植、关键代谢物上调及免疫相关基因表达调节有关，体现“菌群-代谢-免疫”协同调控趋势。尽管例数有限，本研究为FMT治疗RA和多组学揭示分子机制提供了初步的理论与实践依据，支持进一步扩大样本量深入研究。

Abstract

Objective

To evaluate the efficacy and safety of fecal microbiota transplantation (FMT) combined with methotrexate (MTX) in refractory rheumatoid arthritis (RA)who were irresponsive to standard MTX treatment, and to explore the potential mechanisms substantiating  the “microbiota–metabolite–immunity”modulation axis by multi-omics.

Methods

In combination with consecutive MTX treatment，two patients with refractory RA received a total of seven sessions of fecal microbiota capsule transplantation prepared from healthy donors. Longitudinal follow-up with several visits were arranged till  24 weeks and ACR20 response at 16 weeks was set as the primary clinical endpoint. Fecal, serum, and PBMC samples were collected at baseline (BP), during treatment (FP), and at post-treatment (PP) phases. 16S rRNA sequencing, LC-MS-based metabolomics, and RNA-seq transcriptomics were performed. Multi-omics integrative analyses, including GO/KEGG/Reactome/DO enrichment, protein–protein interaction (PPI) networks, and correlation analysis, were conducted to systematically assess the clinical effects and molecular mechanisms of FMT intervention.

Results

Both patients achieved the primary endpoint (ACR20 response) at week 16, with one achieving ACR50 and the other achieving ACR70 remission. Disease activity indicators including DAS28, CRP, and ESR showed significant reduction, along with improvements in swollen/tender joint counts, VAS, and HAQ scores. FMT was well tolerated. 16S rRNA sequencing revealed a significant increase in gut microbial diversity after FMT, with a marked decrease in Escherichia-Shigella abundance; Bifidobacterium and Faecalibacterium showed low-level colonization followed by a declining trend. Metabolomic analysis identified upregulation of anti-inflammatory small molecules such as Glu-Gln and N6-Succinyl Adenosine, enriched in energy and amino acid metabolic pathways. PBMC transcriptome data indicated downregulation of pro-inflammatory factors during the FP phase and upregulation of immune and metabolic genes in the PP phase. Multi-omics integration revealed that the microbiota–metabolite–immune gene network was associated with clinical improvement.

Conclusion

FMT combined with MTX effectively alleviated inflammation and improved joint function in patients with refractory RA, with a favorable safety profile. The underlying mechanism may involve low-level colonization of anti-inflammatory microbiota, upregulation of key metabolites, and modulation of immune-related gene expression, reflecting a coordinated regulation across the microbiota–metabolite–immune axis. Despite the limited sample size, this study provides preliminary theoretical and practical evidence supporting the use of FMT in RA treatment and the application of multi-omics to elucidate its molecular mechanisms, warranting further investigation in larger cohorts.