School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai, China.
School of Biosciences, Faculty of Health and Medical Sciences, Taylor's University, Subang Jaya, Selangor, Malaysia.
J Ethnopharmacol. 2025 Jan 10;336:118722. doi: 10.1016/j.jep.2024.118722. Epub 2024 Aug 23.
Triptolide is a major bioactive and toxic ingredient isolated from the traditional Chinese herb Tripterygium wilfordii (T. wilfordii) Hook F. It exhibits potent antitumor, immunosuppressive, and anti-inflammatory biological activities; however, its clinical application is hindered by severe systemic toxicity. Two preparations of T. wilfordii, including T. wilfordii glycoside tablets and T. wilfordii tablets, containing triptolide, are commonly used in clinical practice. However, their adverse side effects, particularly hepatotoxicity, limit their safe use. Therefore, it is crucial to discover potent and specific detoxification medicines for triptolide.
This study aimed to investigate the detoxification effects and potential mechanism of action of spironolactone on triptolide-induced hepatotoxicity to provide a potential detoxifying strategy for triptolide, thereby promoting the safe applications of T. wilfordii preparations in clinical settings.
Cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and crystal violet staining. Nuclear fragmentation was visualized using 4',6-diamidino-2-phenylindole (DAPI) staining, and protein expression was analyzed by Western blotting. The inhibitory effect of spironolactone on triptolide-induced hepatotoxicity was evaluated by examining the effects of spironolactone on serum alanine aminotransferase and aspartate aminotransferase levels, as well as liver pathology in a mouse model of triptolide-induced acute hepatotoxicity. Furthermore, a survival assay was performed to investigate the effects of spironolactone on the survival rate of mice exposed to a lethal dose of triptolide. The effect of spironolactone on triptolide-induced global transcriptional repression was assessed through 5-ethynyl uridine staining.
Triptolide treatment decreased the cell viability, increased the nuclear fragmentation and the cleaved caspase-3 levels in both hepatoma cells and hepatocytes. It also increased the alanine aminotransferase and aspartate aminotransferase levels, induced the hepatocyte swelling and necrosis, and led to seven deaths out of 11 mice. The above effects could be mitigated by pretreatment with spironolactone. Additionally, molecular mechanism exploration unveiled that spironolactone inhibited triptolide-induced DNA-directed RNA polymerase II subunit RPB1 degradation, consequently increased the fluorescence intensity of 5-ethynyl uridine staining for nascent RNA.
This study shows that spironolactone exhibits a potent detoxification role against triptolide hepatotoxicity, through inhibition of RPB1 degradation induced by triptolide and, in turn, retardation of global transcriptional inhibition in affected cells. These findings suggest a potential detoxification strategy for triptolide that may contribute to the safe use of T. wilfordii preparations.
ETHNOPHARMACOLOGICAL 相关性：雷公藤内酯醇是从传统中药雷公藤（Tripterygium wilfordii）（T. wilfordii） Hook F 中分离出的主要生物活性和有毒成分。它具有很强的抗肿瘤、免疫抑制和抗炎生物学活性；然而，其临床应用受到严重的全身毒性的阻碍。雷公藤的两种制剂，包括雷公藤苷片和雷公藤片，都含有雷公藤内酯醇，在临床上广泛应用。然而，它们的不良反应，特别是肝毒性，限制了它们的安全使用。因此，发现有效且特异的雷公藤内酯醇解毒药物至关重要。
本研究旨在探讨螺内酯对雷公藤内酯醇诱导的肝毒性的解毒作用及其潜在作用机制，为雷公藤内酯醇提供一种潜在的解毒策略，从而促进雷公藤制剂在临床中的安全应用。
使用 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴化物（MTT）测定法和结晶紫染色评估细胞活力。使用 4'，6-二脒基-2-苯基吲哚（DAPI）染色观察核片段化，并用 Western blot 分析蛋白表达。通过检测螺内酯对雷公藤内酯醇诱导的急性肝毒性小鼠模型中血清丙氨酸氨基转移酶和天冬氨酸氨基转移酶水平以及肝病理学的影响，评估螺内酯对雷公藤内酯醇诱导的肝毒性的抑制作用。此外，进行了生存实验，以研究螺内酯对暴露于致死剂量雷公藤内酯醇的小鼠的存活率的影响。通过 5-乙炔基尿苷染色评估螺内酯对雷公藤内酯醇诱导的全局转录抑制的影响。
雷公藤内酯醇处理降低了肝癌细胞和肝细胞的细胞活力，增加了核片段化和裂解的半胱天冬酶-3 水平。它还增加了丙氨酸氨基转移酶和天冬氨酸氨基转移酶水平，诱导肝细胞肿胀和坏死，并导致 11 只小鼠中的 7 只死亡。螺内酯预处理可以减轻上述影响。此外，分子机制研究表明，螺内酯抑制了雷公藤内酯醇诱导的 DNA 指导的 RNA 聚合酶 II 亚基 RPB1 降解，从而增加了新生 RNA 的 5-乙炔基尿苷染色的荧光强度。
本研究表明，螺内酯通过抑制雷公藤内酯醇诱导的 RPB1 降解，并进而延迟受影响细胞的全局转录抑制，发挥对雷公藤内酯醇肝毒性的有效解毒作用。这些发现为雷公藤内酯醇提供了一种潜在的解毒策略，可能有助于雷公藤制剂的安全使用。
