[Mechanism of Huangqin Qingre Chubi Capsule in inhibiting inflammation and proliferation of fibroblast-like synoviocyte in rheumatoid arthritis].

OBJECTIVES: To clarify the role of the ALKBH5/circ_0091685/eIF4A3 axis in rheumatoid arthritis fibroblast-like synoviocyte (RA-FLS) and to investigate whether a traditional Chinese medicine preparation Huangqin Qingre Chubi Capsule (HQC) regulates inflammation and proliferation of RA-FLS through this axis.

METHODS: Differentially expressed genes (DEGs) between RA patients and healthy individuals 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 performed to identify related biological processes and pathways. A co-culture model of peripheral blood mononuclear cells and RA-FLS was established. Through overexpression or silencing of ALKBH5, circ_0091685, and EIF4A3, combined with site-directed mutation of the N⁶-methyladenosine (m⁶A) sites within circ_0091685, we verified whetherALKBH5 targets eIF4A3 by mediating m⁶A modification of circ_0091685, thereby affecting RA-FLS inflammation and proliferation. 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 involving the ALKBH5/circ_0091685/eIF4A3 axis. Cell proliferation was assessed by 5-ethynyl-2 ´-deoxyuridine (EdU) staining; inflammatory cytokine levels were measured by ELISA; expression of EIF4A3 and circ_0091685 was detected by RT-qPCR; protein expression of ALKBH5 and eIF4A3 was analyzed by Western blotting; the binding between circ_0091685andeIF4A3 was validated by RNA immunoprecipitation (RIP); total RNA m⁶A levels and m⁶A modification on circ_0091685 were detected by colorimetric assay and methylated RNA immunoprecipitation-qPCR, respectively; and histopathological changes in rat knee joints were observed using hematoxylin and eosin staining, safranin O-fast green staining, and toluidine blue staining.

RESULTS: ALKBH5 and EIF4A3 wereamong the top 20 DEGs between RA patients and healthy individuals. GO analysis indicated enrichment of methylation-related biological processes, and KEGG analysis identified enriched pathways including the IL-17 signaling pathway. Compared with the ALKBH5 silencing control group, the ALKBH5-silenced group showed decreased expression of ALKBH5, circ_0091685, eIF4A3, reduced levels of IL-6, IL-17A, IL-23 and TNF-α, and a lower relative number of EdU-positive cells (all P<0.01), whereas the ALKBH5 overexpression group exhibited the opposite effects. Compared with the ALKBH5 overexpression+circ_0091685 control group, the ALKBH5 overexpression+circ_0091685 mutant group had significantly reduced IL-6, IL-17A, IL-23, and TNF-α levels (all P<0.01). RIP assays confirmed thatcirc_0091685 specifically binds to eIF4A3. Silencing circ_0091685or EIF4A3 decreased the levels of IL-6, IL-17A, IL-23 and TNF-α, and reduced the relative number of EdU-positive cells (all P<0.01), while overexpressingcirc_0091685 or EIF4A3 produced opposite effects. After HQC intervention, the pathological changes in the knee joints of adjuvant-induced arthritis rats were ameliorated. After treatment with HQC‑containing serum, RA‑FLS showed decreased expression of IL‑6, IL‑17A, IL‑23, and TNF‑α, along with a reduced relative number of EdU‑positive cells (all P<0.05), as well as downregulated expression of ALKBH5, circ_0091685, and eIF4A3 (all P<0.05), while total RNA m⁶A and circ_0091685 m⁶A levels were elevated (all P<0.05). In the presence of HQC‑containing serum, overexpression of ALKBH5 significantly upregulated the expression of IL‑6, IL‑17A, IL‑23 and TNF‑α (all P<0.01). Similarly, in the presence of HQC‑containing serum, overexpression of circ_0091685 significantly upregulated the expression of IL‑6, IL‑23 and TNF‑α (all P<0.01). These findings indicated that overexpression of ALKBH5 or circ_0091685 reversed the inhibitory effects of HQC‑containing serum on RA‑FLS inflammation and proliferation. Furthermore, both HQC‑containing serum treatment and circ_0091685 silencing significantly reduced IL‑6, IL‑23, TNF‑α and eIF4A3 expression (all P<0.01), suggesting a synergistic interaction between HQC‑containing serum and circ_0091685 silencing.

CONCLUSIONS: HQC inhibits inflammation and proliferation of RA-FLS by targeting eIF4A3 through ALKBH5-mediated m⁶A modification of circ_0091685.