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 N6-methyladenosine (m6A) sites within circ_0091685, we verified whether ALKBH5 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 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 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-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 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 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 P<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 P<0.01), downregulated ALKBH5/circ_0091685/EIF4A3 expression (all P<0.05), and increased total m⁶A levels as well as m⁶A levels on circ_0091685 (all P<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 P<0.01). Both HQC containing serum treatment and circ_0091685 silencing significantly reduced EIF4A3 protein expression (all P<0.01). HQC inhibits inflammation and proliferation of RA-FLS by targeting EIF4A3 through ALKBH5 mediated m⁶A modification of circ_0091685. 目的: 明确烷基化修复同源蛋白5（ALKBH5）/circ_0091685/真核起始因子4A-Ⅲ（EIF4A3）轴在类风湿关节炎成纤维样滑膜细胞（RA-FLS）中的作用，并在此基础上探讨黄芩清热除痹胶囊（HQC）是否通过该轴调控RA-FLS炎症和增殖。方法: 基于基因表达综合数据库（GEO）中的微阵列数据集筛选RA患者与健康对照者的差异表达基因，并进行基因本体（GO）和京都基因和基因组百科全书（KEGG）分析筛选差异表达基因相关的生物学过程和作用通路。建立外周血单个核细胞与RA-FLS共培养模型，通过过表达/沉默ALKBH5、circ_0091685、EIF4A3，以及circ_00916856-甲基腺嘌呤（m6A）位点突变，验证ALKBH5是否通过介导circ_00916856 m6A修饰靶向EIF4A3影响RA-FLS的炎症和增殖。同时建立佐剂性关节炎大鼠模型，观察HQC对大鼠的影响，并进一步通过HQC含药血清作用于RA-FLS探索ALKBH5/circ_0091685/EIF4A3轴在其中的作用机制。其中，细胞增殖能力采用5-乙炔基-2'-脱氧尿苷（EdU）染色检测，炎症因子水平采用酶联免疫吸附试验检测、EIF4A3、circ_0091685 mRNA表达水平采用RT-qPCR检测、ALKBH5和EIF4A3蛋白表达水平采用蛋白质印迹法检测、circ_0091685与EIF4A3结合采用RNA免疫共沉淀法验证，m6A修饰水平采用比色法和RNA甲基化免疫共沉淀-定量PCR检测，大鼠膝关节组织病理变化采用苏木精-伊红、番红O-固绿和甲苯胺蓝染色观察。结果: RA患者样本与健康对照样本的前20个差异表达基因包括ALKBH5和EIF4A3。GO分析显示生物学过程存在甲基化，KEGG分析的富集通路包括IL-17信号通路等。与ALKBH5沉默对照组比较，ALKBH5沉默组ALKBH5/circ_0091685/EIF4A3、IL-6、IL-17A、IL-23、TNF-α表达均减少，EdU阳性细胞比例减小（均P<0.01），而ALKBH5过表达组则相反。与ALKBH5过表达+circ_0091685对照组比较，ALKBH5过表达+circ_0091685突变组IL-6、IL-17A、IL-23、TNF-α表达显著减少（均P<0.01）。RNA免疫共沉淀检测结果显示，circ_0091685可以与EIF4A3蛋白特异性结合。过表达circ_0091685或过表达EIF4A3能升高细胞中IL-6、IL-17A、IL-23、TNF-α炎症因子表达，EdU阳性细胞比例增大（均P<0.01），沉默circ_0091685或EIF4A3则相反。HQC干预后，佐剂性关节炎模型大鼠膝关节组织病理有所改善，RA-FLS中IL-6、IL-17A、IL-23、TNF-α表达减少，EdU阳性细胞比例减小（均P<0.01），ALKBH5/circ_0091685/EIF4A3表达减少（均P<0.05），而m6A和circ_0091685 m6A表达增加（均P<0.05）。过表达ALKBH5或circ_0091685后，HQC含药血清对RA-FLS炎症和增殖的影响受到抑制。在加入HQC含药血清基础上过表达ALKBH5或circ_0091685能升高IL-6、IL-17A、IL-23、TNF-α的表达，反之则降低（均P<0.01）;加入HQC含药血清或沉默circ_0091685均能降低EIF4A3蛋白表达（均P<0.01）。结论: HQC通过调控ALKBH5介导的circ_0091685 m6A修饰靶向EIF4A3抑制RA-FLS炎症和增殖。.
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