目的 探討三葉青藤醇提物（IREE）通過(guò)抑制成纖維樣滑膜細(xì)胞（FLS）增殖、遷移，抑制炎癥通路活化及炎癥因子釋放，發(fā)揮抗類風(fēng)濕關(guān)節(jié)炎的作用機(jī)制。方法 將SD大鼠隨機(jī)分為對(duì)照組、模型組、青藤堿（50 mg·kg^-1）組和IREE低、高劑量（50、100 mg·kg^-1）組，除對(duì)照組外，采用弗氏完全佐劑尾根部sc建立佐劑性關(guān)節(jié)炎（AA）大鼠模型，給藥始于造模2周后，共30 d，每天ig給藥1次，對(duì)照組與模型組給予0.9%氯化鈉溶液；進(jìn)行關(guān)節(jié)炎指數(shù)（AI）評(píng)分；通過(guò)HE染色觀察大鼠踝關(guān)節(jié)滑膜組織的病理特征；實(shí)時(shí)熒光定量PCR（qRT-PCR）檢測(cè)滑膜組織中腫瘤壞死因子（TNF）-α、白細(xì)胞介素（IL）-1β和IL-6 mRNA的表達(dá)水平； Western blotting檢測(cè)Toll樣受體8（TLR8）/核因子κB（NF-κB）信號(hào)通路蛋白在AA大鼠踝關(guān)節(jié)滑膜中的表達(dá)情況。分離AA大鼠關(guān)節(jié)滑膜組織中的FLS，采用CCK-8法檢測(cè)不同質(zhì)量濃度IREE對(duì)FLS活力的影響，篩選最佳藥物濃度；將FLS分為對(duì)照組、青藤堿（200 nmol·mL^-1）組及IREE低、中、高質(zhì)量濃度（1、2、3 μg·mL^-1）組，通過(guò)細(xì)胞克隆形成實(shí)驗(yàn)、劃痕實(shí)驗(yàn)和Transwell遷移實(shí)驗(yàn)分別檢測(cè)FLS的增殖和遷移能力。結(jié)果 與對(duì)照組相比，模型組大鼠踝關(guān)節(jié)滑膜層顯著增厚，滑膜細(xì)胞多層堆積，炎癥細(xì)胞大量浸潤(rùn)，關(guān)節(jié)間隙狹窄，血管翳生成增多； AI評(píng)分顯著增加，滑膜組織中TNF-α、IL-1β和IL-6 mRNA水平顯著升高，TLR8、Myd88、P65、p-P65蛋白表達(dá)量顯著增多，差異均具有統(tǒng)計(jì)學(xué)意義（P＜0.05、0.01、0.001）；與模型組相比，IREE低、高劑量組明顯減輕滑膜組織增生和炎癥細(xì)胞浸潤(rùn)，顯著降低AI評(píng)分，并顯著降低TNF-α、IL-1β和IL-6 mRNA水平及TLR8、Myd88、P65、p-P65蛋白表達(dá)，差異均具有統(tǒng)計(jì)學(xué)意義（P＜0.05、0.01、0.001）。成功分離AA大鼠原代FLS，與對(duì)照組相比，IREE低、中、高質(zhì)量濃度組均顯著抑制FLS的增殖和遷移能力（P＜0.05、0.01）。結(jié)論 IREE通過(guò)抑制FLS的增殖和遷移能力，抑制TLR8/NF-κB信號(hào)通路活化，降低TNF-α、IL-1β和IL-6等促炎細(xì)胞因子的表達(dá)，從而發(fā)揮抗類風(fēng)濕關(guān)節(jié)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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廣西科技廳自然基金面上項(xiàng)目（2023JJA140940）；廣西中醫(yī)藥大學(xué)高層次人才培育創(chuàng)新團(tuán)隊(duì)-醫(yī)學(xué)免疫學(xué)創(chuàng)新團(tuán)隊(duì)（2022B006）；研究生創(chuàng)新創(chuàng)業(yè)訓(xùn)練項(xiàng)目（YCSW2023403）