基于脂多糖诱导血管内皮细胞和斑马鱼研究白芍的血管保护作用及谱效关系

Protective effect of Radix paeoniae alba on angiopoiesis in lipopolysaccharide-induced vascular endothelial cell and zebrafish and its spectrum-effect relationship

关键词:  白芍  指纹图谱  血管内皮细胞  Tg(fli1:egfp)系斑马鱼  谱效关系  芍药苷

Key words:  Radix paeoniae alba  fingerprints  vein endothelial cells  Tg(fli1:egfp) zebrafish  spectral effect relationship  paeonin

摘要:

目的 建立白芍 (Radix paeoniae alba) HPLC 指纹图谱,研究白芍指纹图谱与保护血管内皮细胞活性之间的谱效关系。 方法 以HPLC 法建立白芍指纹图谱；采用脂多糖诱导人脐静脉内皮细胞(HUVEC)和转基因Tg(fli1:egfp)系斑马鱼模型,测定17批白芍提取物对血管内皮细胞的体内外药效作用,运用灰色关联度和偏最小二乘法分析白芍指纹图谱与药效作用的谱效关系。 结果 构建白芍的HPLC图谱,标出11个共有峰,有较高的关联度,表明白芍中的多个成分可能对保护血管内皮细胞活性具有协同作用；与体内活性的关联序分别为P6＞P7＞P10＞P11＞P9＞P5＞P3＞P2＞P8＞P1＞P4,与体外活性的关联序分别为P6＞P4＞P2＞P3＞P5＞P1＞P10＞P9＞P7＞P11＞P8,其中指认6号峰为芍药苷,经偏最小二乘标准方程回归系数为正值,与保护血管内皮细胞活性和Tg(fli1:egfp)系斑马鱼血管呈正相关,且VIP值均＞1.0；进一步检测活性成分药效作用发现芍药苷可提高脂多糖诱导内皮细胞模型增殖、迁移和成管能力,促进Tg(fli1:egfp)系斑马鱼血管生成,且存在浓度相关性,可认为是白芍保护血管内皮细胞的药效物质。 结论 通过白芍HPLC指纹图谱与保护脂多糖血管内皮细胞和斑马鱼模型药效作用的相关性分析,挖掘白芍保护血管炎症损伤的药效物质,为药材质量控制提供参考。

Abstract:

ABSTRACT: Objective: To establish the HPLC fingerprint of Radix paeoniae alba and investigate the spectral efficacy relationship between the fingerprint and the protection against vascular injury. Methods: The HPLC method was used to establish the fingerprint of Radix paeoniae alba. Lipopolysaccharide-induced human umbilical vein endothelial cells (HUVEC) and Tg(fli1:egfp) zebrafish models were used to evaluate in vivo and in vitro protective effects of 17 batches of Radix paeoniae alba extracts. Grey correlation degree and partial least squares method were used to analyze the spectral efficacy relationship between the fingerprint of Radix paeoniae alba and the protection effects. Results: The HPLC fingerprint of Radix paeoniae alba was constructed, and 11 common peaks were obtained, and the correlation was high, indicating that multiple components of Paeony may have synergistic effects on the protection of vascular endothelial cell activity. The correlation sequence with in vivo activity was P6＞P7＞P10＞P11＞P9＞P5＞P3＞P2＞P8＞P1＞P4, and in vitro activity was P6＞P4＞P2＞P3＞P5＞P1＞P10＞P9＞P7＞P11＞P8, respectively. Peak 6 was identified as paeoniflorin. The regression coefficient of the partial least squares standard equation was positive, positively correlated with the protection of vascular endothelial cell activity and the vascular development in Tg(fli1:egfp) zebrafish, with VIP values were ＞1.0. Furthermore, the verification experiments of active ingredient showed that paeoniflorin could improve cell viability, migration, and tube formation, as well as promote vascular development in Tg(fli1:egfp) zebrafish in a dose-dependent manner. The above results indicated that paeoniflorin might be the major active component of Radix paeoniae alba for the protection effect on vascular endothelial cell.