冠心静及其单体藁本内酯抑制巨噬细胞活化与抗炎作用研究

Role of Guanxinjing and its Active Component Ligustilide in Inhibiting Macrophage Activation and Inflammatory Response

关键词:  冠心静  斑马鱼  巨噬细胞  抗炎  脂多糖

Key words:  Guanxinjing  zebrafish  RAW264.7 cells  anti-inflammatory  lipopolysaccharide

摘要:

目的 采用斑马鱼和RAW264.7巨噬细胞炎症模型，评价活血化瘀功效中成药冠心静的抗炎作用，并对其活性成分及作用机制进行探讨。方法 分别采用脂多糖 (lipopolysaccharide, LPS)诱导Tg(mpeg:eGFP)巨噬细胞荧光标记转基因斑马鱼与RAW264.7巨噬细胞构建炎症模型，评价冠心静对体内巨噬细胞聚集及体外巨噬细胞活化的调控作用。使用HPLC色谱分析鉴定冠心静中主要化学成分，并使用上述斑马鱼炎症模型和RAW264.7巨噬细胞NO释放水平筛选代表性活性成分。针对筛选出的关键成分藁本内酯在体内外模型中验证其抗炎作用，并采用Western blot法测定细胞中c-Jun氨基末端激酶 (c-Jun N-terminal kinase, JNK)、p-JNK蛋白表达水平，探讨分子机制。结果 与模型组相比，冠心静显著抑制LPS诱导的斑马鱼卵黄囊巨噬细胞聚集，并降低RAW264.7巨噬细胞中NO生成、iNOS基因表达及TNF-α分泌，提示有效抑制巨噬细胞活化。HPLC分析鉴定出7种主要成分，分别为丹参素、芍药内酯苷、芍药苷、丹酚酸B、丹酚酸A、洋川芎内酯A和藁本内酯。抗炎活性筛选显示，藁本内酯可显著减少斑马鱼巨噬细胞聚集和降低斑马鱼ROS水平。进一步发现藁本内酯浓度依赖性抑制LPS诱导巨噬细胞的NO释放、 iNOS表达、及炎症因子IL-6的转录及蛋白水平，并通过下调p-JNK蛋白表达抑制炎症应激相关JNK信号通路激活。结论 冠心静能够有效减轻炎症反应，其中藁本内酯是其关键的抗炎成分，可显著调控炎症介质释放、抑制巨噬细胞活化，其作用机制可能与JNK信号通路相关。

Abstract:

OBJECTIVE To evaluate the anti-inflammatory effects of Guanxinjing, a traditional Chinese medicine (TCM) with blood-activating and stasis-resolving effects, using zebrafish and RAW264.7 cell inflammation models, and to explore its active components and mechanisms of action. METHODS Lipopolysaccharide (LPS)-induced inflammation models were established in macrophage-labeled transgenic zebrafish line Tg(mpeg:eGFP) and RAW264.7 cells to assess the regulatory effects of Guanxinjing on macrophage aggregation in vivo and macrophage activation in vitro. High-performance liquid chromatography (HPLC) was employed to identify the main chemical components of Guanxinjing. Representative active components were screened using the zebrafish inflammation model and the NO release level of RAW264.7 cells. For the key identified component, ligustilide, its anti-inflammatory effects were validated in both in vivo and in vitro models. Western blot was performed to determine phosphorylated JNK (p-JNK)/total JNK protein levels, elucidating the molecular mechanism. RESULTS Compared with the model group, Guanxinjing significantly inhibited LPS-induced macrophage aggregation in the zebrafish yolk sac and reduced NO production, iNOS gene expression, and TNF-α secretion in RAW264.7 cells, indicating effective suppression of macrophage activation. HPLC identified seven major components in Guanxinjing: danshensu, albiflorin, paeoniflorin, salvianolic acid B, salvianolic acid A, senkyunolide A, and ligustilide. Anti-inflammatory screening revealed that ligustilide significantly reduced macrophage aggregation and ROS levels in zebrafish. Furthermore, ligustilide exhibited concentration-dependent inhibition of LPS-induced NO release, iNOS expression, and both transcriptional and protein levels of the inflammatory cytokine IL-6 in macrophages, while suppressing the activation of the inflammation-related JNK signaling pathway via downregulation of p-JNK protein expression. CONCLUSION Guanxinjing effectively alleviates inflammatory responses, with ligustilide serving as its key anti-inflammatory component. Ligustilide significantly regulates the release of inflammatory mediators and inhibits macrophage activation, likely through modulation of the JNK signaling pathway.