Huangqin Qingre Chubi Capsules inhibit inflammation and proliferation of fibroblast-like synoviocytes in rheumatoid arthritis via the ALKBH5/circ_0091685/EIF4A3 axis

ObjectiveTo clarify the role of the a-ketoglutarate-dependent dioxygenase alk B homolog 5 (ALKBH5)/circ_0091685/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, circ_0091685 and EIF4A3, combined with mutation of the N⁶‑methyladenosine (m⁶A) sites in circ_0091685, we verified whether ALKBH5 targets EIF4A3 by mediating m⁶A modification of circ_0091685, 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/circ_0091685/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 circ_0091685 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 circ_0091685 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/circ_0091685/EIF4A3 axis, IL‑6, IL‑17A, IL‑23, and TNF‑α, along with a reduced proportion of EdU‑positive cells (all P<0.01); the opposite trends were observed in the ALKBH5 overexpression group. Compared with the ALKBH5 overexpression + circ_0091685 control group, the ALKBH5 overexpression + circ_0091685 mutation group showed markedly downregulated levels of IL‑6, IL‑17A, IL‑23 and TNF‑α (all P<0.01). RNA immunoprecipitation assays confirmed that circ_0091685 specifically bound to the EIF4A3 protein. Overexpression of circ_0091685 or EIF4A3 increased the cellular expression of the pro‑inflammatory cytokines IL‑6, IL‑17A, IL‑23 and TNF‑α, and elevated the proportion of EdU‑positive cells (all P<0.01), whereas silencing circ_0091685 or EIF4A3 yielded the opposite effects. After intervention with HQC, the histopathological changes in knee joint tissues of adjuvant‑induced arthritis rats were ameliorated. In RA‑FLS, the expression of IL‑6, IL‑17A, IL‑23 and TNF‑α was decreased, the proportion of EdU‑positive cells was reduced (all P<0.01), and the levels of ALKBH5/circ_0091685/EIF4A3 were downregulated (all P<0.05); meanwhile, global m⁶A modification and m⁶A levels on circ_0091685 were increased (all P<0.05). Overexpression of ALKBH5 or circ_0091685 abolished the inhibitory effects of HQC‑containing serum on inflammation and proliferation in RA‑FLSs. Under treatment with HQC‑containing serum, further overexpression of ALKBH5 or circ_0091685 upregulated IL‑6, IL‑17A, IL‑23 and TNF‑α expression, while their silencing exerted the opposite effect (all P<0.01). Either treatment with HQC‑containing serum or circ_0091685 silencing significantly reduced EIF4A3 protein expression (all P<0.01).ConclusionHQC inhibits RA inflammation and proliferation by ALKBH5-mediated m⁶A modification of circ_0091685 to target EIF4A3.