目的 探讨三叶青藤醇提物（IREE）通过抑制成纤维样滑膜细胞（FLS）增殖、迁移，抑制炎症通路活化及炎症因子释放，发挥抗类风湿关节炎的作用机制。方法 将SD大鼠随机分为对照组、模型组、青藤碱（50 mg·kg^-1）组和IREE低、高剂量（50、100 mg·kg^-1）组，除对照组外，采用弗氏完全佐剂尾根部sc建立佐剂性关节炎（AA）大鼠模型，给药始于造模2周后，共30 d，每天ig给药1次，对照组与模型组给予0.9%氯化钠溶液；进行关节炎指数（AI）评分；通过HE染色观察大鼠踝关节滑膜组织的病理特征；实时荧光定量PCR（qRT-PCR）检测滑膜组织中肿瘤坏死因子（TNF）-α、白细胞介素（IL）-1β和IL-6 mRNA的表达水平； Western blotting检测Toll样受体8（TLR8）/核因子κB（NF-κB）信号通路蛋白在AA大鼠踝关节滑膜中的表达情况。分离AA大鼠关节滑膜组织中的FLS，采用CCK-8法检测不同质量浓度IREE对FLS活力的影响，筛选最佳药物浓度；将FLS分为对照组、青藤碱（200 nmol·mL^-1）组及IREE低、中、高质量浓度（1、2、3 μg·mL^-1）组，通过细胞克隆形成实验、划痕实验和Transwell迁移实验分别检测FLS的增殖和迁移能力。结果 与对照组相比，模型组大鼠踝关节滑膜层显著增厚，滑膜细胞多层堆积，炎症细胞大量浸润，关节间隙狭窄，血管翳生成增多； AI评分显著增加，滑膜组织中TNF-α、IL-1β和IL-6 mRNA水平显著升高，TLR8、Myd88、P65、p-P65蛋白表达量显著增多，差异均具有统计学意义（P＜0.05、0.01、0.001）；与模型组相比，IREE低、高剂量组明显减轻滑膜组织增生和炎症细胞浸润，显著降低AI评分，并显著降低TNF-α、IL-1β和IL-6 mRNA水平及TLR8、Myd88、P65、p-P65蛋白表达，差异均具有统计学意义（P＜0.05、0.01、0.001）。成功分离AA大鼠原代FLS，与对照组相比，IREE低、中、高质量浓度组均显著抑制FLS的增殖和迁移能力（P＜0.05、0.01）。结论 IREE通过抑制FLS的增殖和迁移能力，抑制TLR8/NF-κB信号通路活化，降低TNF-α、IL-1β和IL-6等促炎细胞因子的表达，从而发挥抗类风湿关节炎的作用。

Objective To investigate the mechanism of IREE in treating rheumatoid arthritis (RA) by inhibiting fibroblast-like synoviocyte (FLS) proliferation, migration, and inflammatory cytokine release. Methods SD rats were randomly divided into the control group, the model group, the sinomenine (50 mg·kg^-1) group and the IREE low-dose (50 mg·kg^-1) and high-dose (100 mg·kg^-1) groups. Except for the control group, the adjuvant arthritis (AA) rat model was established by subcutaneous injection of Freund's complete adjuvant at the tail base. Administration began two weeks after modeling and lasted for 30 days, with one oral administration per day. The control and model groups were given 0.9% sodium chloride solution. The arthritis index (AI) score was evaluated. Hematoxylin-eosin (HE) staining was used to observe pathological features of ankle synovial tissues. Real-time fluorescence quantitative PCR (qRT-PCR) was performed to detect mRNA levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 in synovial tissues, Western blotting detection of Toll like receptor 8 (TLR8)/nuclear factor kappa B (NF-κB) signaling pathway proteins in the synovial tissue of AA rats' ankles. FLS were isolated from AA rat synovial tissues, and the CCK-8 assay was used to evaluate IREE’s effects on FLS viability to determine the optimal concentration. FLS were divided into blank, sinomenine (200 nmol·mL^-1), and low-, medium-, and high-dose IREE (1, 2, 3 μg·mL^-1) groups. Cell colony formation, scratch, and Transwell migration assays were conducted to assess FLS Proliferation and migration. Results Compared with the control group, the ankle joint synovial layer of the model group rats was significantly thickened, with multiple layers of synovial cells accumulated and a large number of inflammatory cells infiltrated. The joint space was narrowed, and the pannus formation was increased. The AI score was significantly increased, and the mRNA levels of TNF-α, IL-1β and IL-6 in the synovial tissue were significantly elevated, as well as the protein expression levels of TLR8, Myd88, P65 and p-P65, with all differences being statistically significant (P < 0.05, 0.01, 0.001). Compared with the model group, the low and high dose groups of IREE significantly alleviated the synovial tissue hyperplasia and inflammatory cell infiltration, significantly reduced the AI score, and significantly decreased the mRNA levels of TNF-α, IL-1β and IL-6 and the protein expression of TLR8, Myd88, P65 and p-P65, with all differences being statistically significant (P < 0.05, 0.01, 0.001). Primary FLS from AA rats were successfully isolated. Compared with the control group, the low, medium and high concentration groups of IREE significantly inhibited the proliferation and migration ability of FLS (P < 0.05, 0.01). Conclusion IREE exerts anti-rheumatoid arthritis effects by suppressing FLS proliferation and migration, as well as reducing the expression of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6).

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广西科技厅自然基金面上项目（2023JJA140940）；广西中医药大学高层次人才培育创新团队-医学免疫学创新团队（2022B006）；研究生创新创业训练项目（YCSW2023403）