Data Sheet 1_Emodin mitigates rheumatoid arthritis through direct binding to TNF-α.docx

Emodin has shown certain anti-rheumatoid arthritis (RA) activity in preliminary studies. However, the precise mechanisms of emodin’s anti-RA effects, particularly its direct targets, remain unclear. This study aimed to evaluate the anti-RA activity of emodin and elucidate its potential mechanisms, with a specific focus on identifying its molecular targets. Employing a collagen-induced arthritis (CIA) rat model, along with transcriptomic analysis, thermal proteome profiling (TPP) and TNF-α-induced L929 cell model, the anti-RA activity of emodin was confirmed, identifying TNF-α as a potential target. Techniques such as drug affinity responsive target stability (DARTS), cellular thermal shift assay (CETSA), Affinity ultrafiltration-liquid chromatography/mass spectrometry (AUF-LC/MS), surface plasmon resonance (SPR) and bio-layer interferometry (BLI) validated the direct binding of emodin to TNF-α. Molecular dynamics simulation, ELISA and BLI further revealed that emodin stabilizes the asymmetric trimeric structure of TNF-α, disrupting the TNF-α-TNFR1 interaction. In vitro assays, including luciferase reporter gene assay and TNF-α-induced MH7A cell model, demonstrated that this disruption inhibits TNF-α-induced NF-κB activation, leading to the downregulation of inflammatory mediators such as IL-6, IL-1β, and COX2. In conclusion, emodin directly targets TNF-α, stabilizing its structure and blocking TNF-α-TNFR1 interaction, which subsequently suppresses downstream NF-κB pathway activation and contributes to its potent anti-RA properties.

大黄素（Emodin）在前期研究中已展现出一定的抗类风湿关节炎（rheumatoid arthritis, RA）活性。然而，大黄素发挥抗RA作用的精确机制，尤其是其直接作用靶标，仍未明确。本研究旨在评估大黄素的抗RA活性并阐明其潜在作用机制，重点聚焦于其分子靶标的鉴定。本研究采用胶原诱导性关节炎（collagen-induced arthritis, CIA）大鼠模型，结合转录组学分析、热蛋白质组分析（thermal proteome profiling, TPP）以及肿瘤坏死因子α（TNF-α）诱导的L929细胞模型，验证了大黄素的抗RA活性，并鉴定出TNF-α为其潜在作用靶标。通过药物亲和力响应靶标稳定性测定（drug affinity responsive target stability, DARTS）、细胞热位移测定（cellular thermal shift assay, CETSA）、亲和超滤-液相色谱/质谱联用（Affinity ultrafiltration-liquid chromatography/mass spectrometry, AUF-LC/MS）、表面等离子体共振（surface plasmon resonance, SPR）以及生物层干涉术（bio-layer interferometry, BLI）等技术，证实了大黄素可直接结合TNF-α。分子动力学模拟、酶联免疫吸附测定（enzyme-linked immunosorbent assay, ELISA）及BLI进一步揭示，大黄素可稳定TNF-α的不对称三聚体结构，阻断TNF-α与TNFR1的相互作用。体外实验（包括荧光素酶报告基因测定以及TNF-α诱导的MH7A细胞模型）结果表明，该阻断作用可抑制TNF-α介导的核因子κB（NF-κB）通路激活，进而下调白细胞介素6（IL-6）、白细胞介素1β（IL-1β）及环氧合酶2（COX2）等炎症介质的表达。综上，大黄素可直接靶向TNF-α，通过稳定其结构并阻断TNF-α与TNFR1的相互作用，继而抑制下游NF-κB通路激活，最终发挥强效的抗RA活性。