[Huangqin Qingre Chubi Capsules inhibit inflammation and proliferation of fibroblast-like synoviocytes in rheumatoid arthritis via the ALKBH5/circ_0091685/EIF4A3 axis].
Source: PubMed, NCBI / U.S. National Library of Medicine
To clarify the role of the ALKBH5/circ_0091685/EIF4A3 axis in rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) and to investigate whether a traditional Chinese medicine preparation Huangqin Qingre Chubi Capsules (HQC) regulates inflammation and proliferation of RA-FLS through this axis. Differentially expressed genes (DEGs) 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 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 N-methyladenosine (mA) sites within circ_0091685, we verified whether ALKBH5 targets EIF4A3 by mediating mA 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. 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 EdU staining; inflammatory cytokine levels were measured by ELISA; mRNA expression of EIF4A3 and circ_0091685 was detected by RT-qPCR; protein expression of ALKBH5 and EIF4A3 was analyzed by Western bl
Abstract
To clarify the role of the ALKBH5/circ_0091685/EIF4A3 axis in rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) and to investigate whether a traditional Chinese medicine preparation Huangqin Qingre Chubi Capsules (HQC) regulates inflammation and proliferation of RA-FLS through this axis. Differentially expressed genes (DEGs) 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 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 N-methyladenosine (mA) sites within circ_0091685, we verified whether ALKBH5 targets EIF4A3 by mediating mA 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. 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 EdU staining; inflammatory cytokine levels were measured by ELISA; mRNA 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_0091685 and EIF4A3 was validated by RNA immunoprecipitation (RIP); total mA levels and mA 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-eosin, safranin O-fast green, and toluidine blue staining. Among the top 20 DEGs between RA patients and healthy controls were ALKBH5 and EIF4A3. 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 proportion of EdU positive cells (all<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<0.01). RIP assays confirmed that circ_0091685 specifically binds to EIF4A3 protein. Overexpression of circ_0091685 or EIF4A3 increased the levels of IL-6, IL-17A, IL-23, and TNF-α and elevated the EdU positive cell ratio (all<0.01), while silencing circ_0091685 or EIF4A3 produced opposite effects. After HQC intervention, the pathological changes in the knee joints of adjuvant-induced arthritis rats were ameliorated. In RA-FLS, HQC treatment reduced the expression of IL-6, IL-17A, IL-23, and TNF-α, decreased the EdU positive cell ratio (all<0.01), downregulated ALKBH5/circ_0091685/EIF4A3 expression (all<0.05), and increased total m⁶A levels as well as m⁶A levels on circ_0091685 (all<0.05). Overexpression of ALKBH5 or circ_0091685 abolished the inhibitory effects of HQC containing serum on RA-FLS inflammation and proliferation. Under HQC containing serum treatment, further overexpression of ALKBH5 or circ_0091685 upregulated IL-6, IL-17A, IL-23, and TNF-α, whereas silencing these molecules had the opposite effects (all<0.01). Both HQC containing serum treatment and circ_0091685 silencing significantly reduced EIF4A3 protein expression (all<0.01). HQC inhibits inflammation and proliferation of RA-FLS by targeting EIF4A3 through ALKBH5 mediated m⁶A modification of circ_0091685.