DataSheet1_Anti-Inflammatory Effects of Ginsenoside Rb3 in LPS-Induced Macrophages Through Direct Inhibition of TLR4 Signaling Pathway.xlsx

Panax ginseng has therapeutic effects on various inflammation-related diseases. Ginsenoside Rb3 (GRb3), a natural compound with anti-inflammatory and immunomodulatory properties, is one of the main active panaxadiol extracted from Panax ginseng. We explored whether GRb3 inhibited LPS-mediated inflammation through TLR4/NF-κB/MAPK signaling in macrophages. GRb3 attenuated NO and PGE2 production by attenuating iNOS and COX2 expression. GRb3 also suppressed pro-inflammatory cytokines levels, including IL-1β, IL-6, and TNF-α. Moreover, GRb3 administration significantly suppressed NF-κB (p65) nuclear translocation and the phosphorylation levels of p65, IκBα, JNK, p38, and ERK dose-dependently. Molecular docking demonstrated that GRb3 could dock onto the hydrophobic binding site of TLR4/MD2 complex, with a binding energy of −8.79 kcal/mol. Molecular dynamics (MD) displayed stable TLR4-MD2-GRb3 modeling. GRb3 dose-dependently inhibited LPS binding to cell membranes and blocked TLR4 expression. Surface plasmon resonance imaging (SPRi) revealed that GRb3 had an excellent binding affinity to TLR4/MD2 complex. Notably, resatorvid (TAK242), a selective TLR4 inhibitor, did not increase the repressive influence of GRb3 in RAW264.7 macrophages. Moreover, TLR4 overexpression partially reversed the repressive roles of GRb3 on the NF-κB/MAPK pathway and inflammatory mediators. Collectively, our study strongly indicated that GRb3 attenuated LPS-mediated inflammation through direct inhibition of TLR4 signaling. A novel insight into the underlying mechanism of anti-inflammatory effects of GRb3 in macrophages was confirmed.

人参（Panax ginseng）对多种炎症相关疾病具有治疗作用。人参皂苷Rb3（Ginsenoside Rb3, GRb3）是从人参中提取的主要人参二醇类活性成分之一，是一种具有抗炎与免疫调节特性的天然化合物。本研究探讨了GRb3是否可通过巨噬细胞内的TLR4/NF-κB/MAPK信号通路，抑制脂多糖（Lipopolysaccharide, LPS）介导的炎症反应。GRb3可通过下调诱导型一氧化氮合酶（Inducible Nitric Oxide Synthase, iNOS）与环氧合酶2（Cyclooxygenase 2, COX2）的表达，减少一氧化氮（Nitric Oxide, NO）与前列腺素E2（Prostaglandin E2, PGE2）的生成。GRb3还可抑制促炎细胞因子的表达水平，包括白细胞介素1β（IL-1β）、白细胞介素6（IL-6）与肿瘤坏死因子α（Tumor Necrosis Factor α, TNF-α）。此外，GRb3给药可显著抑制核因子κB（NF-κB，p65亚基）的核转位，以及p65、IκBα、JNK、p38与ERK的磷酸化水平，且该抑制作用呈剂量依赖性。分子对接实验显示，GRb3可结合于TLR4/MD2复合物的疏水结合位点，结合能为-8.79千卡/摩尔（kcal/mol）。分子动力学（Molecular Dynamics, MD）模拟结果表明，TLR4-MD2-GRb3复合物模型结构稳定。GRb3可呈剂量依赖性地抑制LPS与细胞膜的结合，并阻断TLR4的表达。表面等离子体共振成像（Surface Plasmon Resonance Imaging, SPRi）实验证实，GRb3与TLR4/MD2复合物具有优异的结合亲和力。值得注意的是，选择性TLR4抑制剂瑞莎托维（resatorvid, TAK242）并不会增强GRb3在RAW264.7巨噬细胞中的抗炎抑制效果。此外，TLR4过表达可部分逆转GRb3对NF-κB/MAPK信号通路及炎症介质的抑制作用。综上，本研究明确证实，GRb3可通过直接抑制TLR4信号通路减轻LPS介导的炎症反应，为阐明GRb3在巨噬细胞中发挥抗炎作用的潜在分子机制提供了全新视角。