National and Local Collaborative Engineering Center of Chinese Medicinal Resources Industrialization and Formulae Innovative Medicine, Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, Jiangsu Key Laboratory for High Technology Research of TCM Formulae, Nanjing University of Chinese Medicine, Nanjing, 210023, China; Department of Pharmacy, School of Life and Health Sciences, Anhui Science and Technology University, Bengbu, 233100, China.
National and Local Collaborative Engineering Center of Chinese Medicinal Resources Industrialization and Formulae Innovative Medicine, Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, Jiangsu Key Laboratory for High Technology Research of TCM Formulae, Nanjing University of Chinese Medicine, Nanjing, 210023, China.
J Ethnopharmacol. 2025 Jan 30;337(Pt 2):118888. doi: 10.1016/j.jep.2024.118888. Epub 2024 Oct 3.
Tripterygii wilfordii Radix, (TW) as a toxic herbal medicine, is the root of Tripterygium wilfordii Hook. F. , which commonly used in China for the treatment of rheumatoid arthritis and autoimmune diseases, but its severe toxicity, particularly hepatotoxicity, significantly impacts its clinical application.
The hepatotoxicity and its molecular mechanism of 70% TW ethanol extract (TWE) on male mice were demonstrated based on metabolomics, network pharmacological analysis and experimental validation.
The toxic and bioactive ingredients in TWE were quantitative analyzed by Triple quadrupole (TQ) mass spectrometry method. The liver organ index, as well as the liver function indexes AST and ALT were evaluated after administering different doses of TWE for 24 h, and a pathological change was analyzed in liver tissue. Non-targeted metabolomics using UPLC-QTOF/MS was performed on both the plasma and liver tissue samples in combination with network toxicology to screen for key targets related to TWE toxicity in the liver. These key targets including caspase 3, NF-κB, TLR4, TNF-α, NQO1, and Bcl2 were subsequently verified through Western blotting experiments.
The six toxic and active ingredients of raphenolactone, ranolactone, triptolide tripterine, wilforlide A, demethylzeylasterain in TWE for the contents of 0.709, 1.408, 0.353, 0.354, 0.882, 0.227 mg g, respectively. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels increased and liver index decreased after administration of TWE for 24 h. Pathological analysis showed that TWE could produce toxicity to mouse liver, and its toxicity was dose-dependent. In the high-dose group, TW-D (11.23 g/kg) and TW-E (22.46 g/kg) caused a large amount of rupture in mouse liver nucleus and a large amount of inflammatory infiltration at the same time. Furthermore, 64 metabolites in plasma and 59 metabolites in the liver tissue were identified. The main metabolic pathways involved glycerol phospholipid metabolism, glycosylphosphatidylinositol-ether lipid metabolism, fatty acid metabolism, sphingomyelin metabolism, and ether lipid metabolism in plasma and liver tissue. Through analysis of the top 10 correlated targets, 6 out of the top 10 selected target proteins exhibited consistent expression patterns with liver injury. The levels of Bcl2 and NQO1 decreased with increasing exposure dose. The expression of Caspase 3, NF-κB, TLR4, and TNF-α increased with increasing dose. These findings suggest that protein expression has a regulatory effect at different doses groups compared to the control group.These findings suggest a regulatory effect of protein expression in different dose groups compared to the control group.
The hepatotoxic effects of TWE can increase ALT and AST levels in plasma, leading to hepatic oxidative damage and inflammatory response. The toxic mechanisms that produce are closely related to the regulating of the abnormal metabolites in plasma and liver tissue. Furthermore, the regulating the expression levels of targeted proteins of TNF-α, NF-κB, Caspase 3, NQO1, and Bcl2 were confirmed by examining the liver tissue. These data clearly elucidate the toxicity mechanism of TW, laying the foundation for ensuring the quality and safety of drugs.
雷公藤根（TW）作为一种有毒草药，是雷公藤钩藤的根，在中国常用于治疗类风湿性关节炎和自身免疫性疾病，但它的严重毒性，特别是肝毒性，显著影响了其临床应用。
本研究旨在基于代谢组学、网络药理学分析和实验验证，展示 70%TW 乙醇提取物（TWE）对雄性小鼠的肝毒性及其分子机制。
采用三重四极杆（TQ）质谱法定量分析 TWE 中的有毒和生物活性成分。给予不同剂量的 TWE 24 小时后，评估肝器官指数以及肝功能指标 AST 和 ALT，并分析肝组织的病理变化。使用 UPLC-QTOF/MS 进行非靶向代谢组学研究，结合网络毒理学筛选与 TWE 肝毒性相关的关键靶标。这些关键靶标包括 caspase 3、NF-κB、TLR4、TNF-α、NQO1 和 Bcl2，并通过 Western blot 实验进行验证。
TWE 中含有六种有毒和活性成分，分别为莱酚内酯、雷酚内酯、雷公藤红素、雷公藤内酯 A、去甲基泽拉木醛和 demethylzeylasterain，其含量分别为 0.709、1.408、0.353、0.354、0.882 和 0.227mg/g。给予 TWE 24 小时后，丙氨酸氨基转移酶（ALT）和天冬氨酸氨基转移酶（AST）水平升高，肝指数降低。病理分析表明，TWE 可对小鼠肝脏产生毒性，且其毒性呈剂量依赖性。在高剂量组中，TW-D（11.23g/kg）和 TW-E（22.46g/kg）导致大量小鼠肝细胞核破裂，同时大量炎症浸润。此外，在血浆中鉴定出 64 种代谢物，在肝组织中鉴定出 59 种代谢物。主要代谢途径涉及甘油磷脂代谢、糖基磷脂酰肌醇醚脂代谢、脂肪酸代谢、鞘脂代谢和醚脂代谢。通过分析前 10 个相关靶点，选择的前 10 个目标蛋白中有 6 个表现出与肝损伤一致的表达模式。Bcl2 和 NQO1 的水平随暴露剂量的增加而降低。Caspase 3、NF-κB、TLR4 和 TNF-α 的表达随剂量增加而增加。这表明与对照组相比，不同剂量组的蛋白表达具有调节作用。
TWE 的肝毒性作用可使血浆中 ALT 和 AST 水平升高，导致肝氧化损伤和炎症反应。产生的毒性机制与血浆和肝组织中异常代谢物的调节密切相关。此外，还通过检测肝组织证实了 TNF-α、NF-κB、Caspase 3、NQO1 和 Bcl2 靶向蛋白表达水平的调节。这些数据清楚地阐明了 TW 的毒性机制，为确保药物的质量和安全性奠定了基础。
