钩吻叶三萜皂苷 A 通过激活细胞坏死诱导肝损伤。
Pterocephin A, a novel Triterpenoid Saponin from Pterocephalus hookeri induced liver injury by activation of necroptosis.
机构信息
College of Pharmaceutical Sciences, Southwest University, Chongqing 400715, PR China.
TAAHC-SWU Medicinal Plant R&D Center, XiZang Agriculture and Animal Husbandry College, Nyingchi, Tibet 860000, PR China.
出版信息
Phytomedicine. 2021 May;85:153548. doi: 10.1016/j.phymed.2021.153548. Epub 2021 Mar 19.
BACKGROUND
Pterocephalus hookeri (C. B. Clarke) Höeck, a Tibetan medicine widely used for treatment of rheumatoid arthritis, was recorded in Chinese Pharmacopoeia (2020 version) with slight toxicity. The liver injury was observed in mice with administration of n-butanol extract (BUE) in our previously study. However, the toxic components and the mechanism were still unrevealed.
PURPOSE
The present study was aimed to isolate and structural elucidate of the toxic compound pterocephin A (PA), as well as evaluate its liver toxicity and investigate its mechanism.
METHODS
PA was isolated from the BUE of P. hookeri. Its structure was determined by analysis of HRMS, NMR and ECD data. L-02 cellular viability, LDH, ALT, AST, ROS, intracellular Ca and the fluidity of cell membrane were assessed by multifunctional microplate reader. The PI staining, cell membrane permeability assessment, and mitochondrial fluorescence staining analysis were determined through the fluorescence microscope. Liver samples for mice were assessed by pathological and immunohistochemistry analysis. Expression levels of indicated proteins were measured by western blotting assays.
RESULTS
PA was determined as a previously undescribed oleanolane-type triterpenoid saponin. In vitro study revealed PA significantly induced hepatotoxicity by inhibition of L-02 cell growth, abnormally elevation of ALT and AST. Mechanically, PA induced the damage of cell membrane, fragmentation of mitochondria, and subsequently increase of intracellular Ca and ROS levels, which trigged by necroptosis with the activation of RIP1 and NF-κB signaling pathways. In vivo study confirmed PA could induce liver injury in mice with observation of the body weight loss, increasing of serum ALT and AST, and the histopathological changes in liver tissues.
CONCLUSION
Our present study indicated that PA was an undescribed toxic constituent in P. hookeri to induce liver injury in mice by activation of necroptosis and inflammation. And the findings are of great significance for the clinical use safely of this herb.
背景
翼首草（C. B. Clarke）是藏药的一种，广泛用于治疗类风湿关节炎，在 2020 年版《中国药典》中有记载，具有轻微的毒性。在之前的研究中，我们观察到翼首草正丁醇提取物（BUE）在小鼠体内可引起肝损伤。然而，其毒性成分和机制仍未被揭示。
目的
本研究旨在从翼首草的 BUE 中分离并结构鉴定出有毒化合物翼首草素 A（PA），评估其肝毒性并探讨其作用机制。
方法
PA 从翼首草的 BUE 中分离得到。通过高分辨质谱（HRMS）、核磁共振（NMR）和圆二色（ECD）数据分析确定其结构。多功能微孔板读数仪评估 PA 对 L-02 细胞活力、乳酸脱氢酶（LDH）、丙氨酸转氨酶（ALT）、天冬氨酸转氨酶（AST）、活性氧（ROS）、细胞内 Ca 水平和细胞膜流动性的影响。荧光显微镜检测碘化丙啶（PI）染色、细胞膜通透性评估和线粒体荧光染色分析。对小鼠肝组织进行病理和免疫组织化学分析。通过 Western blot 测定法检测各指示蛋白的表达水平。
结果
PA 被确定为一种以前未被描述的齐墩果烷型三萜皂苷。体外研究表明，PA 通过抑制 L-02 细胞生长，异常升高 ALT 和 AST，显著诱导肝毒性。机制上，PA 诱导细胞膜损伤、线粒体碎片化，随后细胞内 Ca 和 ROS 水平升高，这是由 necroptosis 触发的，涉及 RIP1 和 NF-κB 信号通路的激活。体内研究证实，PA 可引起小鼠肝损伤，观察到体重减轻、血清 ALT 和 AST 升高以及肝组织的组织病理学变化。
结论
本研究表明，PA 是翼首草中的一种未被描述的有毒成分，可通过激活 necroptosis 和炎症诱导小鼠肝损伤。这些发现对于该草药的临床安全应用具有重要意义。
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