柴胡皂苷d对LPS诱导的RAW264.7细胞炎症的抗炎作用机制

Anti-inflammatory Effect of Saikosaponin d on LPS-induced RAW264.7 Cell Inflammation

摘要:

该文以细菌脂多糖（Lipopolysaccharide, LPS）刺激的小鼠单核巨噬细胞RAW264.7为模型，对柴胡皂苷d（Saikosaponin d, SSd）的体外抗炎作用机制进行深入研究。结果显示，SSd在8 μmol/L的浓度下能够显著抑制RAW264.7炎症细胞一氧化氮（Nitric Oxide, NO）的释放（P<0.01），并以剂量依赖性（4 μmol/L、8 μmol/L）抑制细胞一氧化氮合酶（Inducible Nitric Oxide Synthase, iNOS）、环氧合酶2（Cyclooxygenase-2, COX-2）及白细胞介素-6（Interleukin-6, IL-6）、肿瘤坏死因子（Tumor Necrosis Factor-α，TNF-α）等炎症相关基因mRNA的表达（P<0.01）。蛋白印迹结果表明，SSd有效抑制了iNOS和COX-2蛋白的表达。扫描电镜观察分析发现SSd可以显著改善LPS诱导的RAW264.7细胞形态改变。此外，SSd能够抑制Toll样受体4（Toll-Like Receptor 4, TLR4）、IκB激酶（IκB Kinase-α, IκBα）、核因子κB（Nuclear Factor Kappa-B, NF-κB）、丝氨酸/ 苏氨酸激酶（Serine/Threonine Kinase Proteins, GSK3β）等炎症相关信号通路关键蛋白的表达或磷酸化水平。结果表明，SSd对LPS诱导的RAW264.7细胞炎症有较好的抑制作用，其作用机制与TLR4/NF-κB以及GSK3β等炎症相关信号通路相关，可为全面理解SSd的抗炎作用提供数据支撑。

Abstract:

A mouse model of lipopolysaccharide (LPS)-induced RAW264.7 cell inflammation was developed to investigate the in vitro anti-inflammatory mechanism of saikosaponin d (SSd). SSd demonstrated a significant inhibition of nitric oxide release from RAW264.7 cells at a concentration of 8 μmol/L (P<0.01) and suppressed mRNA level expression of inflammation-related genes, including inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2), interleukin-6, and tumor necrosis factor-α, in a dose-dependent manner at concentrations of 4 and 8 μmol/L (P<0.01). Western blot analysis revealed that SSd effectively reduced the protein level expression of iNOS and COX-2. SEM images confirmed that SSd alleviated LPS-induced morphological changes in RAW264.7 cells. Additionally, SSd inhibited the expression and phosphorylation levels of key proteins involved in inflammation-related signaling pathways, including toll-like receptor 4 (TLR4), IκB kinase, nuclear factor κB (NF-κB), and glycogen synthase kinase-3β (GSK3β). These findings indicate that SSd strongly inhibits LPS-induced RAW264.7 cell inflammation by targeting the TLR4/NF-κB and GSK3β signaling pathways. These findings contribute to our enhanced understanding of the anti-inflammatory effects of SSd.