School of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine, Guangzhou 510006, China.
School of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine, Guangzhou 510006, China; School of Pharmacy, Wannan Medical College, Anhui, 241002, China.
Phytomedicine. 2022 Sep;104:154242. doi: 10.1016/j.phymed.2022.154242. Epub 2022 Jun 6.
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.
To explore the mechanisms by which triterpenoid saponins of IP (IPTS) promote blood circulation using system pharmacology-based approaches.
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 B (TXB), 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.
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.
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.
血淤证（BSS）是一种严重的代谢紊乱，涉及甘油磷酰胆碱代谢的紊乱。毛冬青（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。
采用基于系统药理学的方法探讨 IP 三萜皂苷（IPTS）促进血液循环的机制。
SD 大鼠 BSS 模型通过连续 7 天灌胃 IPTS，再用盐酸肾上腺素注射，然后浸入冰水中制备。检测血浆凝血参数和血清血栓素 B（TXB）、内皮素（ET）和 6-酮-PGF1α。通过组织病理学评估评估对腹主动脉的可能影响。孵育人脐静脉内皮细胞（HUVEC）用 ox-LDL，研究 IPTS 对细胞活力和 LDH 释放的影响。采用 UPLC-QTOF-MS/MS 对大鼠血浆脂溶性成分和 HUVEC 细胞内代谢物进行代谢谱分析。采用网络药理学预测 BSS 相关靶点和模型途径以及 IPTS 的主要成分。分子对接、分子动力学（MD）模拟和生化测定用于预测 IPTS 活性成分与靶蛋白之间的分子相互作用。采用 RT-PCR 检测靶蛋白的 mRNA 水平。采用 Western blot 和免疫组化（IHC）验证 IPTS 在 BSS 中促进血液循环的机制。
IPTS 在 BSS 过程中改善了血液生化功能，在血管保护和维持血管正常形态方面发挥了作用。此外，类固醇生物合成和鞘脂代谢途径的代谢物明显受到干扰。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，脂质积累可能通过激活 PI3K/AKT 信号通路来介导。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，并且复合物具有显著的结合亲和力。IPTS 治疗组的 PI3K、AKT、p-AKT 和 eNOS mRNA 和蛋白水平显著升高。因此，IPTS 通过调节 PI3K/AKT 信号通路、激活 eNOS 和增加 NO 释放来保护血管。
提出了 IPTS 预防 BSS 的可能机制：IPTS 通过调节鞘脂代谢和激活 PI3K/AKT/eNOS 信号通路促进血液循环。
