药源性肝损伤（Drug-induced liver injury，DILI）是药物安全性评价重点关注的药物不良反应，是急性肝衰竭的主要原因。中草药由于人类用药的悠久历史和对疾病具有独特的疗效而被公众广泛接受。但伴随着中草药和膳食补充剂的普及，中草药所引起 DILI 也开始被世界各地文献报道。中草药引起的肝损伤的几个因素包括中草药在体内的代谢、滥用、质量控制不明确以及提取工艺改变导致毒性部位的富集等。此外，个体及环境差异也是中草药造成DILI的主要原因之一。中草药引起的细胞毒性往往具有隐匿性，对易感个体可能更容易造成肝脏损伤。这些危险因素可能会导致细胞的氧化失衡、免疫应激和胆汁淤积，引起肝脏的内源代谢的阻滞和病理损伤，直接或间接的导致肝毒性。目前为止，大多数中草药的肝毒性物质基础和机制尚未阐明，且对于中草药及其提取物引起的DILI仍然缺乏有效的诊断标志物。因此，识别中草药中的毒性成分对认知和规避用药风险具有重要作用。

一、基于斑马鱼肝毒性模型的白鲜皮提取物FRA的代谢毒性机制研究

白鲜皮是一种具有抗炎和抗过敏作用的传统草药，但一些研究表明，以白蜡树酮（Fraxinellone, FRA）为代表的几种呋喃环化合物是从 CD 中提取的主要活性成分，它们自身拥有的呋喃环官能团可通过细胞色素 P450（cytochromeP450, CYP450）代谢生成顺丁烯结构而产生肝毒性，但潜在的毒性机制尚未阐明。因此，本实验室利用斑马鱼受精后 72 小时的斑马鱼幼鱼研究 FRA 的代谢性肝毒性，并探讨其毒性作用的分子机制。我们采用流式细胞术、实时荧光定量 PCR 技术（real-time fluorescent quantitative PCR, qRT-PCR）和 western blot 研究 FRA 的体内肝毒性机制，并通过靶向氨基酸代谢组学进一步进行系统验证。

结果表明，FRA（10-30 μM）可引起斑马鱼的肝损伤和氨基酸代谢组学的明显改变。FRA 可上调 JNK/p53/Bax 通路，促进线粒体膜电位失衡，导致活性氧（Reactive oxygen species, ROS）增加，并通过下调 P-糖蛋白（P-glycoprotein, P-gp）、胆酸盐外排泵（bile salt export pump, Bsep）和牛磺胆酸钠共转运蛋白（sodium taurocholate cotransporting polypeptide, Ntcp）的表达，介导胆汁淤积性肝损伤。此外，添加CYP3A抑制剂酮康唑（Ketoconazole，KCZ）（1 μM）可显著降低 FRA 的肝毒性。靶向代谢组学分析表明，氨基酸水平的变化可以与分子生物学相结合，阐明 FRA 诱导肝毒性的机制，氨基酸代谢监测可能为 FRA 的药物性肝损伤防治提供新的途径。同时，我们发现斑马鱼具有相对丰富的代谢酶和转运体表达，适合于胆汁淤积型肝毒性药物的筛选和机制探索，为评价和发现药物潜在的肝毒性提供一定的方法学参考。

二、何首乌提取物 HY-W-26 的体外肝毒性及机制研究

HY-W-26 是从蓼科植物何首乌 Polygonum multiflorum Thunb 中提取分离得到的一种二蒽酮类化合物。前期本实验室通过斑马鱼肝毒性筛选模型确认了 HY-W-26 具有较高的肝毒性潜力。现在已经报道的何首乌肝毒性物质主要是围绕蒽醌、酚类等化合物，对二蒽酮类化合物诱导的细胞损伤还未有明确的机制报道。为更好得了解二蒽酮介导的 DILI，我们使用 HepG2 细胞评估 HY-W-26 的肝毒性。

结果表明，HY-W-26 具有较强的肝毒性，能显著降低 HepG2 细胞的存活率，诱导细胞凋亡。生化指标检测检测显示，HY-W-26 增加细胞培养上清液中谷丙转氨酶（alanine aminotransferase, ALT）、谷草转氨酶（aspartate aminotransferase, AST）和乳酸脱氢酶（lactate dehydrogenase, LDH）的水平和细胞中碱性磷酸酶（alkaline phosphatase, AKP）的水平。与对照组相比，不同浓度的HY-W-26可以导致HepG2细胞中谷胱甘肽（glutathione, GSH）和超氧化物歧化酶（superoxide dismutase, SOD）水平的降低，脂质氧化产物丙二醛（malondialdehyde, MDA）和ROS含量的升高。HY-W-26 还可以降低线粒体膜电位（mitochondrial membrane potential, MMP），促进细胞色素 C（Cytochrome c, Cyt c）的释放，剂量依赖性的增加 JNK、Bax、cleaved Caspase-3和-9 的表达水平以及和 PARP 的裂解，并降低Bcl-2 和 DFF45 的水平。此外，我们还发现 HY-W-26 可以抑制 PI3K 和 mTOR 的磷酸化，增加活化 AKT 的水平。我们认为 AKT 的活化可以增加细胞对 ROS 的敏感性而加速细胞凋亡。HY-W-26 还可以抑制参与抗氧化相关基因的表达水平，这与 ROS 的升高和氧化还原失衡有着直接关系。

本研究证明，HY-W-26 的肝毒性基制与线粒体损伤和 ROS 的增加有关。HY-W-26 可以通过抑制抗氧化系统诱导细胞的氧化应激和凋亡效应。此外，JNK 和 AKT 的激活会启动 ROS 驱动的凋亡级联反应，增加 ROS 介导的细胞损伤。这项研究强调了 ROS 应激参与何首乌诱导肝毒性的重要性。同时，现在有关何首乌肝毒性报道主要是围绕以大黄素为主的单蒽醌类化合物，我们希望通过对二蒽酮化合物的毒性研究来丰富何首乌的潜在肝毒性物质基础。

Drug-induced liver injury (DILI) is one of the most important adverse drug reactions in drug safety evaluation and is the main cause of acute liver failure. Chinese herbal medicine and its extracts have been widely accepted by the public because of their long history of human use and their unique efficacy in the treatment of some metabolic diseases. However, with the popularity of Chinese herbal medicine and dietary supplements, the use of Chinese herbal medicine has gradually become a common factor causing DILI. Due to individual and environmental differences, the cytotoxicity caused by Chinese herbal medicine is often occult, which may cause liver damage more easily to susceptible individuals. These risk factors may lead to oxidative imbalance, immune stress and cholestasis, block the endogenous metabolism of the liver and pathological damage, and directly or indirectly lead to hepatotoxicity. At present, the hepatotoxic substance basis and mechanism of most Chinese herbal medicines have not been clarified, and there is still a lack of effective diagnostic markers for DILI caused by Chinese herbal medicines and their extracts. Therefore, the identification of toxic components in Chinese herbal medicine plays an important role in recognizing and avoiding the risk of drug use.

1. Study on the metabolic toxicity mechanism of fraxinellone extracted from Cortex Dictamni based on zebrafish hepatotoxicity model

Cortex Dictamni (CD) is a traditional herbal medicine with anti-inflammatory and anti allergic effects, but some studies have shown that several furan ring compounds, represented by fraxinellone (FRA), are the main active components extracted in CD, and their own possessed furan ring functional groups can produce hepatotoxicity via metabolizing by CYP450 to generate a cisbutene structure, but the underlying toxic mechanism has not been clarified. Therefore, 72 hpf (72 hours post fertilization) zebrafish larvae were used to evaluate the metabolic hepatotoxicity of the CD extract FRA and to explore the molecular mechanism of its toxic effects. We used flow cytometry, qRT-PCR and Western blot to investigate the mechanism of in vivo hepatotoxicity of FRA and performed further systematic confirmation by targeted metabolomics.

The results showed that FRA (10-30 μM) induced liver injury and obvious alterations in the metabolomics of zebrafish larvae. FRA can upregulate JNK/p53/Bax pathway to promote mitochondrial membrane potential imbalance leading to increased reactive oxygen species (ROS) and mediate cholestatic liver injury by downregulating the expression of bile acid transporters P-gp, Bsep, and Ntcp. In addition, the addition of ketoconazole (1 μM), a CYP3A inhibitor, significantly reduced the hepatotoxicity of FRA.

Targeted metabolomics analysis indicate the changes of amino acid levels can be combined with molecular biology to clarify the mechanism of hepatotoxicity induced by FRA, and amino acid metabolism monitoring may provide a new way for the prevention and treatment of DILI of FRA. At the same time, we found that zebrafish has relatively abundant expression of metabolic enzymes and transporters, which is suitable for the screening and mechanism exploration of cholestasis type hepatotoxic drugs, and may provide a certain methodological reference for evaluating and discovering the potential hepatotoxicity of drugs.

2. Study on the metabolic toxicity mechanism of fraxinellone extracted from Cortex Dictamni based on zebrafish hepatotoxicity model

HY-W-26 is a dianthrone compound extracted from Polygonum multiflorum Thunb. In the early stage, our laboratory confirmed that HY-W-26 has high hepatotoxicity potential through zebrafish hepatotoxicity screening model. At present, the hepatotoxic substances of Polygonum multiflorum have been reported mainly around anthraquinone, phenols and other compounds. There is no clear mechanism of cell damage induced by dianthrone compounds. To better understand dianthrone-mediated DILI, we used HepG2 cells to evaluate the hepatotoxicity of HY-W-26.

The results showed that HY-W-26 had strong hepatotoxicity and could significantly reduce the proliferation of HepG2 cells and induce apoptosis. The detection of biochemical indexes showed that HY-W-26 increased the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) in the supernatant of cell culture, and increased the level of alkaline phosphatase (AKP) in cells. Compared with the control group, different concentrations of HY-W-26 could reduce the levels of glutathione (GSH) and superoxide dismutase (SOD) and increase the contents of malondialdehyde (MDA) and ROS in HepG2 cells. HY-W-26 also decreased the mitochondrial membrane potential (mitochondrial membrane potential, MMP), promoted the release of Cytochrome c (Cyt c), increased the expression of JNK, Bax, cleaved Caspase-3 and-9 and the cleavage of PARP in a dose-dependent manner, and decreased the levels of Bcl-2 and DFF45. In addition, we also found that HY-W-26 could inhibit the phosphorylation of PI3K and mTOR and increase the level of activated AKT. We believe that the activation of AKT can increase the sensitivity of cells to ROS, which accelerates apoptosis. HY-W-26 can also inhibit the expression of genes involved in antioxidation, which is directly related to the increase of ROS and redox imbalance.

This study shows that the hepatotoxicity of HY-W-26 is related to mitochondrial damage and the increase of ROS. HY-W-26 can induce oxidative stress and apoptosis by inhibiting antioxidant system. In addition, the activation of JNK and AKT can initiate ROS-driven apoptosis cascade and increase ROS-mediated cell injury. This study emphasizes the importance of ROS stress in the hepatotoxicity induced by Polygonum multiflorum. At the same time, the current reports on hepatotoxicity of Polygonum multiflorum mainly focus on single anthraquinones based on emodin. We hope to enrich the potential hepatotoxic substance basis of Polygonum multiflorum by studying the toxicity of dianthrone compounds.