Ershiwuwei Lvxue Pill alleviates rheumatoid arthritis by different pathways and produces changes in the gut microbiota

Rheumatoid arthritis (RA) is a systemic autoimmune disease that often results in joint destruction. Ershiwuwei Lvxue Pill (ELP), a prescription of Tibetan medicine, has been used for centuries for the clinical treatment of RA in Tibet, China. In a previous study, we reported that ELP could ameliorate RA symptoms in CIA rats by inhibiting the inflammatory response and inducing apoptosis in synovial tissues. It is still needed further to clarify the mechanisms of action of ELP in mitigating RA.In this study, we aim to elucidate the mechanism of action of ELP to improve RA joint damage and explore the changes in the intestinal flora and host metabolites.Firstly, we analyzed the main absorbed constituents of ELP in the serum of rats by ultra-performance liquid chromatography quadrupole-time-flight mass spectrometry (UPLC-Q-TOF/MS). Then, we verified the alleviating effects of ELP on cartilage injury and bone erosion as well as the inflammatory response in CIA rats by microCT, H&E staining, safranin-O staining, and ELISA. Moreover, we investigated the main factors that mediate joint damage, including the production of matrix metalloproteinases (MMPs) and osteoclast activity in the ankle of rats by immunohistochemistry and tartrate-resistant acid phosphatase (TRAP) staining. Further, we explored the molecular mechanisms of the MMPs production and osteoclast activity in CIA rats treated with ELP through various experiments such as ELISA, qRT-PCR, western blotting, and immunofluorescence assay. Besides, we investigated gut microbiota composition by 16S rDNA sequencing and serum metabolites through untargeted metabolomics. In addition, we analyzed the correlation between gut microbiota and metabolites by Spearman correlation analysis.In this study, we identified 20 compounds from rat serum samples, which could be the ELP components that improve RA. Moreover, we found that ELP could alleviate cartilage and bone injury by reducing MMP-1, MMP-3, and MMP-13 expression and osteoclast activity in CIA rats. Further studies demonstrated that ELP could reduce joint damage by inhibiting osteoprotegerin (OPG)/receptor activator for nuclear factor-κB ligand (RANKL) /nuclear factor-κB (NF-κB) and extracellular signal-regulated kinase (ERK)/c-Jun N-terminal kinases (JNK) signal pathways. The 16S rDNA sequencing analysis indicated that there was a significant difference in the gut microbiota composition between the normal and CIA rats, and these differences were changed after ELP administration. ELP could alter the gut microbiota by increasing the abundance of the genus Lactobacillus and decreasing the abundance of Dorea, [Eubacterium]_ventriosum_group, Anaerostipes, Collinsella, Coprococcus_1, Ruminiclostridium_5, Ruminococcus_1, Family_XIII_UCG-001, Butyricicoccus, Erysipelotrichaceae_UCG-003, Lachnoclostridium, Faecalibacterium, Lachnospiraceae_UCG-010, Roseburia, Rs-E47_termite_group_norank, Treponema_2 genera. Non-targeted metabolomics analysis showed that ELP reduced arachidonic acid levels. The serum arachidonic acid level was significantly correlated with the abundance of 41 genera, particularly Collinsella and Lactobacillus.Our study shows that ELP can improve RA joint damage by inhibiting MMPs production and osteoclast activity, and regulating intestinal flora and host metabolites, which provides a novel insight into the ELP in alleviating RA.