Rao Chang, Hu Ruixue, Hu Yongxin, Jiang Yan, Zou Xu, Tang Huilan, Chen Xing, He Xiaoli, Hu Guang
School of Pharmacy and Bioengineering, Chongqing University of Technology, Chongqing, 400054, China.
Chongqing Institute for Food and Drug Control, Chongqing, 401121, China.
Chin Med. 2024 Jul 16;19(1):100. doi: 10.1186/s13020-024-00970-6.
Salvia miltiorrhiza (Danshen, DS) and Radix Paeoniae Rubra (Chishao, CS) herbal pair (DS-CS) is a famous traditional Chinese combination which has been used as antithrombotic formular for centuries. However, there is still lack of sufficient scientific evidence to illustrate its underlying mechanisms. The purpose of this study is to investigate the antithrombotic effects of DS-CS extract in zebrafish and explore its possible mechanism of action.
The quality of traditional Chinese medicines DS and CS granules was evaluated using High Performance Liquid Chromatography (HPLC). Subsequently, the therapeutic effect of the DS-CS combination and its components, Salvianolic Acid A (SAA) and Paeoniflorin (PF), in various concentrations on thrombosis was experimentally validated. Moreover, the interaction between DS-CS and the thrombosis disease targets was analyzed through network pharmacology, predicting the potential antithrombotic mechanism of DS-CS. Molecular docking and in vivo zebrafish experiments were conducted to validate the predicted targets, with qRT-PCR utilized for target validation.
DS-CS exhibited anti-thrombotic effect in zebrafish with concentrations ranging from 25 to 300 μg/mL. The co-administration of PF and SAA at 25 μg/mL each revealed a synergistic antithrombotic effect exceeding that of individual components when contrasted with PHZ treatment. Protein-protein interaction (PPI) analysis identified key genes, including Albumin (ALB), Proto-oncogene tyro-sine-protein kinase Src (SRC), Matrix metalloproteinase-9 (MMP9), Caspase-3 (CASP3), Epidermal growth factor receptor (EGFR), Fibroblast growth factor 2 (FGF2), Vascular endothelial growth factor receptor 2 (KDR), Matrix metalloprotein-ase-2(MMP2), Thrombin (F2), and Coagulation factor Xa (F10), associated with the antithrombotic action of PF and SAA. Furthermore, KEGG pathway analysis indicated involvement of lipid metabolism and atherosclerosis pathways. Molecular docking revealed strong binding of PF and SAA to pivotal hub genes, such as SRC, EGFR, and F10. The experimental findings demonstrated that DS-CS could upregulate the mRNA expression levels of EGFR while inhibiting F10 and SRC mRNA levels, thereby ameliorating thrombotic conditions.
This research provided valuable insights into the potential mechanisms underlying the antithrombotic activity of DS-CS. Our findings suggested that PF and SAA could be the key active ingredients responsible for this activity. The antithrombotic effects of DS-CS appeared to be mediated through the regulation of mRNA expression of SRC, EGFR, and F10. These results enhanced our understanding of DS-CS's therapeutic potential and lay the groundwork for future studies to further elucidate its mechanisms of action.
丹参（Danshen，DS）和赤芍（Chishao，CS）药对（DS-CS）是一种著名的传统中药组合，数百年来一直作为抗血栓配方使用。然而，仍缺乏足够的科学证据来说明其潜在机制。本研究的目的是研究DS-CS提取物在斑马鱼中的抗血栓作用，并探讨其可能的作用机制。
采用高效液相色谱法（HPLC）对中药DS和CS颗粒的质量进行评价。随后，通过实验验证了不同浓度的DS-CS组合及其成分丹酚酸A（SAA）和芍药苷（PF）对血栓形成的治疗效果。此外，通过网络药理学分析DS-CS与血栓形成疾病靶点之间的相互作用，预测DS-CS潜在的抗血栓机制。进行分子对接和体内斑马鱼实验以验证预测的靶点，并利用qRT-PCR进行靶点验证。
DS-CS在25至300μg/mL浓度范围内对斑马鱼表现出抗血栓作用。与PHZ处理相比，PF和SAA各以25μg/mL共同给药显示出协同抗血栓作用，超过了单个成分的作用。蛋白质-蛋白质相互作用（PPI）分析确定了关键基因，包括白蛋白（ALB）、原癌基因酪氨酸蛋白激酶Src（SRC）、基质金属蛋白酶-9（MMP9）、半胱天冬酶-3（CASP3）、表皮生长因子受体（EGFR）、成纤维细胞生长因子2（FGF2）、血管内皮生长因子受体2（KDR）、基质金属蛋白酶-2（MMP2）、凝血酶（F2）和凝血因子Xa（F10），这些基因与PF和SAA的抗血栓作用相关。此外，KEGG通路分析表明脂质代谢和动脉粥样硬化通路参与其中。分子对接显示PF和SAA与关键枢纽基因如SRC、EGFR和F10有很强的结合。实验结果表明，DS-CS可以上调EGFR的mRNA表达水平，同时抑制F10和SRC的mRNA水平，从而改善血栓形成状况。
本研究为DS-CS抗血栓活性的潜在机制提供了有价值的见解。我们的研究结果表明，PF和SAA可能是负责这种活性的关键活性成分。DS-CS的抗血栓作用似乎是通过调节SRC、EGFR和F10的mRNA表达来介导的。这些结果加深了我们对DS-CS治疗潜力的理解，并为未来进一步阐明其作用机制的研究奠定了基础。
