Huangqin Qingre Chubi Capsules inhibit inflammation and proliferation of fibroblast-like synoviocytes in rheumatoid arthritis via the ALKBH5/circ₀091685/EIF4A3 axis

ObjectiveTo clarify the role of the a-ketoglutarate-dependent dioxygenase alk B homolog 5 (ALKBH5) /circ₀091685/eukaryotic initiation factor 4A-Ⅲ (EIF4A3) axis in rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS), and on this basis, to explore whether Huangqin Qingre Chubi Capsules (HQC) regulates the inflammation and proliferation of RA-FLS through this axis. MethodsDifferentially expressed genes between RA patients and healthy controls were screened based on microarray datasets from the Gene Expression Omnibus (GEO) database. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were subsequently performed to identify the biological processes and functional pathways associated with these differentially expressed genes. A co‑culture model of peripheral blood mononuclear cells and RA‑FLS was established. Through overexpression/silencing of ALKBH5, circ₀091685 and EIF4A3, combined with mutation of the N⁶‑methyladenosine (m⁶A) sites in circ₀091685, we verified whether ALKBH5 targets EIF4A3 by mediating m⁶A modification of circ₀091685, thereby affecting the inflammation and proliferation of RA‑FLS. Meanwhile, an adjuvant‑induced arthritis rat model was established to observe the therapeutic effects of HQC in vivo. Furthermore, HQC‑containing serum was applied to RA‑FLS to explore the underlying mechanism of the ALKBH5/circ₀091685/EIF4A3 axis. In this study, cell proliferation was detected by 5‑ethynyl‑2′‑deoxyuridine (EdU) staining; the levels of inflammatory cytokines were determined by enzyme‑linked immunosorbent assay (ELISA) ; the mRNA expression levels of EIF4A3 and circ₀091685 were measured by reverse transcription quantitative polymerase chain reaction (RT‑qPCR) ; the protein expression of ALKBH5 and EIF4A3 was analyzed by Western blotting; the binding interaction between circ₀091685 and EIF4A3 was validated by RNA immunoprecipitation (RIP) ; the m⁶A modification level was detected by colorimetric assay and methylated RNA immunoprecipitation combined with quantitative polymerase chain reaction; and the histopathological changes in rat knee joint tissues were observed using hematoxylin‑eosin, safranin O‑fast green and toluidine blue staining. ResultsThe top 20 differentially expressed genes in samples from RA patients versus healthy controls included ALKBH5 and EIF4A3. GO analysis revealed enrichment of biological processes related to methylation, while KEGG analysis identified enriched pathways such as the IL‑17 signaling pathway. Compared with the ALKBH5 silencing control group, the ALKBH5 silenced group exhibited decreased expression of the ALKBH5/circ₀091685/EIF4A3 axis, IL‑6, IL‑17A, IL‑23, and TNF‑α, along with a reduced proportion of EdU‑positive cells (all PPPPPPPPConclusionHQC inhibits RA inflammation and proliferation by ALKBH5-mediated m6A modification of circ₀091685 to target EIF4A3.