Pyrrolizidine alkaloids (PAs) are common secondary plant compounds with hepatotoxicity. The consumption of herbal medicines and herbal teas containing PAs is one of the main causes of hepatic sinusoidal obstruction syndrome (HSOS), a potentially life-threatening condition. The present study aimed to reveal the mechanism underlying the cytotoxicity of intermedine (Im), the main PA in Comfrey. We evaluated the toxicity of the retronecine-type PAs with different structures to cell lines derived from mammalian tissues, including primary mouse hepatocytes, human hepatocytes (HepD), mouse hepatoma-22 (H₂₂) and human hepatocellular carcinoma (HepG2) cells. The cytotoxicity of Im to hepatocyte was evaluated by using cell counting kit-8 assay, colony formation experiment, wound healing assay and dead/live fluorescence imaging. In vitro characterization showed that these PAs were cytotoxic and induced cell apoptosis in a dose-dependent manner. We also demonstrated that Im induced cell apoptosis by generating excessive reactive oxygen species (ROS), changing the mitochondrial membrane potential and releasing cytochrome c (Cyt c) before activating the caspase-3 pathway. Importantly, we directly observed the destruction of the cell mitochondrial structure after Im treatment through transmission electron microscopy (TEM). This study provided the first direct evidence of Im inducing hepatotoxicity through mitochondria-mediated apoptosis. These results supplemented the basic toxicity data of PAs and facilitated the comprehensive and systematic evaluation of the toxicity caused by PA compounds.

中文翻译：

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吡咯里西啶生物碱复合中间体的肝毒性：与其他吡咯里西啶生物碱的比较及其毒理学机制

吡咯里西啶生物碱 (PAs) 是常见的具有肝毒性的次生植物化合物。食用含有 PA 的草药和草药茶是肝窦阻塞综合征 (HSOS) 的主要原因之一，这是一种可能危及生命的疾病。本研究旨在揭示Comfrey中的主要 PA 中间体 (Im) 的细胞毒性机制。我们评估了不同结构的逆转录型 PAs 对哺乳动物组织细胞系的毒性，包括原代小鼠肝细胞、人肝细胞 (HepD)、小鼠肝细胞瘤 22 (H ₂₂) 和人肝细胞癌 (HepG2) 细胞。采用细胞计数kit-8测定、集落形成实验、伤口愈合测定和死/活荧光成像评估Im对肝细胞的细胞毒性。体外表征表明，这些 PA 具有细胞毒性，并以剂量​​依赖性方式诱导细胞凋亡。我们还证明了 Im 通过产生过量的活性氧 (ROS)、改变线粒体膜电位和在激活 caspase-3 途径之前释放细胞色素 c (Cyt c) 来诱导细胞凋亡。重要的是，我们通过透射电子显微镜 (TEM) 直接观察到 Im 处理后细胞线粒体结构的破坏。该研究提供了Im通过线粒体介导的细胞凋亡诱导肝毒性的第一个直接证据。