Phytomedicine ( IF 6.7 ) Pub Date : 2022-06-06 , DOI: 10.1016/j.phymed.2022.154242 Jie Chen 1 , Di Cao 2 , Shiqin Jiang 3 , Xia Liu 1 , Wencong Pan 1 , Hui Cui 1 , Weiqun Yang 1 , Zhongqiu Liu 1 , Jing Jin 3 , Zhongxiang Zhao 1
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Background
Blood stasis syndrome (BSS) is a severe disorder involving disturbances in glycerophosphocholine metabolism. Ilex pubescens (IP) can regulate the levels of lipids, such as lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE); however, the main active constituent of IP and its corresponding mechanism in BSS treatment are still unclear.
Purpose
To explore the mechanisms by which triterpenoid saponins of IP (IPTS) promote blood circulation using system pharmacology-based approaches.
Methods
Sprague-Dawley (SD) rat BSS model was prepared by oral administration of IPTS for 7 days followed by adrenaline hydrochloride injection before immersion in ice water. Coagulation parameters in plasma and thromboxane B2 (TXB2), endothelin (ET) and 6-keto-PGF1α in serum were measured. The possible influence on abdominal aortas was evaluated by histopathology assessment. Human vein endothelial cells (HUVECs) were incubated with ox-LDL, and the effects of IPTS on cell viability and LDH release were investigated. UPLC-QTOF-MS/MS was used for metabolic profile analysis of lipid-soluble components in rat plasma and intracellular metabolites in HUVECs. Network pharmacology was used to predict the relevant targets and model pathways of BSS and the main components of IPTS. Molecular docking, molecular dynamics (MD) simulation and biochemical assays were used to predict molecular interactions between the active components of IPTS and target proteins. RT-PCR was used to detect the mRNA level of target proteins. Western blotting and immunohistochemistry (IHC) were used to verify the mechanisms by which IPTS promotes blood circulation in BSS.
Results
IPTS improved blood biochemical function in the process of BSS and played a role in vascular protection and maintenance of the normal morphology of blood vessels. Furthermore, metabolite pathways involved in steroid biosynthesis and sphingolipid metabolism were significantly perturbed. Both metabolomics analysis and network pharmacology results showed that IPTS ameliorates vascular injury and that lipid accumulation may be mediated by PI3K/AKT signaling pathway activation. MD simulation and enzyme inhibitory activity results suggested that the main components of IPTS can form stable complexes with PI3K, AKT and eNOS and that the complexes have significant binding affinity. PI3K, AKT, p-AKT, and eNOS mRNA and protein levels were considerably elevated in the IPTS-treated group. Thus, IPTS protects the vasculature by regulating the PI3K/AKT signaling pathway, activating eNOS and increasing the release of NO.
Conclusion
A possible mechanism by which IPTS prevents BSS is proposed: IPTS can promote blood circulation by modulating sphingolipid metabolism and activating the PI3K/AKT/eNOS signaling pathways.
中文翻译：
冬青中的三萜皂苷通过调节鞘脂代谢和PI3K/AKT/eNOS信号通路促进血瘀证的血液循环
背景
血瘀综合征 (BSS) 是一种严重的疾病，涉及甘油磷酸胆碱代谢紊乱。冬青(IP) 可以调节溶血磷脂酰胆碱 (LPC) 和溶血磷脂酰乙醇胺 (LPE) 等脂质的水平；然而，IP的主要活性成分及其在BSS治疗中的相应机制仍不清楚。
目的
采用基于系统药理学的方法探索IP三萜皂苷（IPTS）促进血液循环的机制。
方法
Sprague-Dawley (SD) 大鼠 BSS 模型通过口服 IPTS 7 天，然后注射盐酸肾上腺素，然后浸入冰水中制备。血浆中的凝血参数和血栓素 B 2 (TXB 2)、内皮素 (ET) 和 6-keto-PGF1α 在血清中进行了测量。通过组织病理学评估评估对腹主动脉的可能影响。人静脉内皮细胞 (HUVEC) 与 ox-LDL 一起孵育，并研究了 IPTS 对细胞活力和 LDH 释放的影响。UPLC-QTOF-MS/MS 用于大鼠血浆中脂溶性成分和 HUVEC 中细胞内代谢物的代谢谱分析。利用网络药理学预测BSS的相关靶点和模型通路以及IPTS的主要成分。分子对接、分子动力学 (MD) 模拟和生化分析用于预测 IPTS 的活性成分与靶蛋白之间的分子相互作用。RT-PCR用于检测靶蛋白的mRNA水平。
结果
IPTS改善了BSS过程中的血液生化功能，起到了血管保护和维持血管正常形态的作用。此外，参与类固醇生物合成和鞘脂代谢的代谢物途径受到显着干扰。代谢组学分析和网络药理学结果均表明，IPTS 可改善血管损伤，并且脂质积累可能是由 PI3K/AKT 信号通路激活介导的。MD模拟和酶抑制活性结果表明，IPTS的主要成分可以与PI3K、AKT和eNOS形成稳定的复合物，且复合物具有显着的结合亲和力。IPTS 治疗组的 PI3K、AKT、p-AKT 和 eNOS mRNA 和蛋白质水平显着升高。因此，
结论
提出了IPTS预防BSS的可能机制：IPTS可以通过调节鞘脂代谢和激活PI3K/AKT/eNOS信号通路促进血液循环。
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