Differential toxic phenotypes and liver injury induced by Atractylenolides (I, II, and III): Insights from zebrafish (Danio rerio) models and network toxicology

Abstract

Atractylenolides (I, II, and III), active sesquiterpene lactones from Atractylodes macrocephala Koidz, exhibit diverse pharmacological activities but have been reported to impair drug-metabolizing enzymes and hepatocellular function. However, a comprehensive safety assessment of these compounds remains lacking. In this study, we investigated the developmental toxicity profile of Atractylenolides (I, II, and III) in zebrafish embryos, with a particular focus on hepatotoxicity and its underlying mechanisms. Exposure to Atractylenolides (I, II, and III) resulted in concentration-dependent mortality, with 96-h median lethal concentrations (LC₅₀) of 81.64 μM, 138.40 μM, and 151.90 μM, respectively. Atractylenolides (I, II) induced multiple developmental abnormalities, among which Atractylenolide-I uniquely led to neuronal developmental arrest and diminished locomotor activity. Importantly, Atractylenolides (I, II) exhibited marked hepatotoxicity, evidenced by liver shrinkage, reduced liver-specific fluorescence intensity, and elevated levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT). In contrast, exposure to Atractylenolide-III did not induce significant toxic effects. Network toxicology analysis revealed that cytochrome P450 (CYP450) metabolism and apoptosis were closely associated with Atractylenolides (I, II) induced hepatotoxicity. qRT-PCR analysis revealed that Atractylenolides (I, II) suppressed mRNA expression of key drug-metabolizing genes, including cyp3c1 and cyp3a65. Simultaneously, Atractylenolides (I, II) downregulated genes associated with cell proliferation (top2α, uhrf1). Co-treatment with the hepatoprotective agent silybin partially reversed the liver injury and the alterations in drug metabolism gene expression induced by Atractylenolides (I, II). Collectively, our results provide important insights into the safety evaluation of Atractylenolides (I, II, and III).