Mechanism of Hepatotoxicity Induced by Ethanol Extract of Emilia sonchifolia (L.) DC Revealed by Proteomics and Metabolomics

Objective: Hepatotoxicity has been reported for Emilia sonchifolia (L.) DC (Emilia sonchifolia). The plant material is typically prepared using two extraction methods for practical application: water extraction and ethanol extraction. However, our previous research only investigated its water extract. Therefore, this study aims to systematically evaluate the hepatotoxicity and underlying mechanisms of the ethanol extract of Emilia sonchifolia, thereby providing a more comprehensive scientific basis for its rational application and safety assessment.
Methods: An acute toxicity preliminary screening study was conducted by orally administering Emilia sonchifolia ethanol extract to mice at doses ranging from 0 to 33.6 g/kg/day. Based on the results of the acute toxicity test preliminary screening study, mice were divided into a control group and an Emilia sonchifolia ethanol extract group (8.6 g/kg/day) for a 14-day delayed hepatotoxicity experiment based on clinical treatment duration. At the end of the intervention, hepatic pathological changes were examined using hematoxylin-eosin staining. Enzyme-linked immunosorbent assay (ELISA) was employed to quantify the levels of alanine aminotransferase , aspartate aminotransferase, total bilirubin, direct bilirubin, total bile acids, alkaline phosphatase, and γ-glutamyl transferase in serum, as well as malondialdehyde, superoxide dismutase, and catalase in liver tissue. Proteomics and metabolomics analyses were performed to investigate the mechanisms of hepatotoxicity induced by the ethanol extract. Additionally, the mRNA expression levels of Cyp3a41a, Cyp2c29, Ugt2b1, and Hsd3b3 in mice liver tissue were determined using quantitative reverse transcription polymerase chain reaction (qRT-PCR).
Results: The acute toxicity preliminary screening study results showed that a dose of 12.0 g/kg or higher of the Emilia sonchifolia ethanol extract caused acute liver failure and death in mice. A dose of 8.6 g/kg or lower of the Emilia sonchifolia ethanol extract produced dose-dependent acute hepatotoxicity. Meanwhile, a dose of 8.6 g/kg of the Emilia sonchifolia ethanol extract induced delayed toxicities in mice. Proteomics and metabolomics results revealed that the hepatotoxicity induced by the ethanol extract of Emilia sonchifolia was associated with cholestasis and oxidative stress caused by disruptions in drug metabolism, steroid hormone biosynthesis, and primary bile acid biosynthesis. Validation experiments showed that the levels of Cyp2c29 were decreased, while the mRNA levels of Cyp3a41a, Ugt2b1, and Hsd3b3 were increased in the liver tissues of mice treated with the ethanol extract of Emilia sonchifolia. Additionally, serum levels of total bilirubin, direct bilirubin, total bile acids, alkaline phosphatase, and γ-glutamyl transferase were significantly elevated. Furthermore, in the livers of mice treated with the ethanol extract, malondialdehyde levels were increased, whereas superoxide dismutase and catalase levels were decreased.
Conclusion: In summary, the ethanol extract of Emilia sonchifolia can induce hepatotoxicity in mice, and its mechanism is associated with cholestasis and oxidative stress mediated by disruptions in drug metabolism, steroid hormone biosynthesis, and primary bile acid biosynthesis.

研究目的：已有研究报道，一点红（Emilia sonchifolia (L.) DC）具有肝毒性。实际应用中，该药材通常采用两种提取方式制备：水提取法与乙醇提取法。但本课题组前期研究仅考察了其水提取物。因此，本研究旨在系统评价一点红乙醇提取物的肝毒性及其潜在作用机制，为其合理应用与安全性评价提供更为全面的科学依据。
研究方法：本研究先开展急性毒性预筛实验，以0至33.6 g/kg/天的剂量对小鼠灌胃给予一点红乙醇提取物。根据急性毒性预筛结果，参照临床给药周期，将小鼠分为对照组与一点红乙醇提取物给药组（8.6 g/kg/天），开展为期14天的延迟性肝毒性实验。干预结束后，采用苏木精-伊红（hematoxylin-eosin, HE）染色法观察肝脏病理变化。通过酶联免疫吸附测定（enzyme-linked immunosorbent assay, ELISA）定量检测小鼠血清中丙氨酸氨基转移酶（alanine aminotransferase, ALT）、天冬氨酸氨基转移酶（aspartate aminotransferase, AST）、总胆红素（total bilirubin, TBIL）、直接胆红素（direct bilirubin, DBIL）、总胆汁酸（total bile acids, TBA）、碱性磷酸酶（alkaline phosphatase, ALP）及γ-谷氨酰转移酶（γ-glutamyl transferase, GGT）的水平，同时检测肝组织中丙二醛（malondialdehyde, MDA）、超氧化物歧化酶（superoxide dismutase, SOD）与过氧化氢酶（catalase, CAT）的水平。通过蛋白质组学与代谢组学分析，探究该乙醇提取物诱导肝毒性的潜在机制。此外，采用实时荧光定量聚合酶链反应（quantitative reverse transcription polymerase chain reaction, qRT-PCR）检测小鼠肝组织中Cyp3a41a、Cyp2c29、Ugt2b1及Hsd3b3的mRNA表达水平。
研究结果：急性毒性预筛实验结果显示，给予12.0 g/kg及以上剂量的一点红乙醇提取物可导致小鼠急性肝衰竭甚至死亡；给予8.6 g/kg及以下剂量的一点红乙醇提取物则会引发剂量依赖性急性肝毒性。同时，8.6 g/kg剂量的一点红乙醇提取物可诱导小鼠产生延迟性毒性反应。蛋白质组学与代谢组学分析结果显示，一点红乙醇提取物诱导的肝毒性与药物代谢、类固醇激素生物合成及初级胆汁酸生物合成通路紊乱引发的胆汁淤积和氧化应激密切相关。验证实验结果表明，一点红乙醇提取物给药组小鼠肝组织中Cyp2c29的表达水平下调，而Cyp3a41a、Ugt2b1及Hsd3b3的mRNA表达水平上调。此外，给药组小鼠血清中总胆红素、直接胆红素、总胆汁酸、碱性磷酸酶及γ-谷氨酰转移酶水平均显著升高。进一步检测发现，给药组小鼠肝组织中丙二醛水平升高，而超氧化物歧化酶与过氧化氢酶活性降低。
研究结论：综上，一点红乙醇提取物可诱导小鼠产生肝毒性，其作用机制与药物代谢、类固醇激素生物合成及初级胆汁酸生物合成通路紊乱介导的胆汁淤积和氧化应激密切相关。