Psoraleae Fructus (PF) is a widely-used herb with diverse pharmacological activities, while its related hepatic injuries have aroused public concerns. In this work, a systematic approach based on RNA sequencing (RNA-seq), high-content screening (HCS) and molecular docking was developed to investigate the potential mechanism and identify major phytochemicals contributed to PF-induced hepatotoxicity. Animal experiments proved oral administration of PF water extracts disturbed lipid metabolism and promoted hepatic injuries by suppressing fatty acid and cholesterol catabolism. RNA-seq combined with KEGG enrichment analysis identified mitochondrial oxidative phosphorylation (OXPHOS) as the potential key pathway. Further experiments validated PF caused mitochondrial structure damage, mtDNA depletion and inhibited expressions of genes engaged in OXPHOS. By detecting mitochondrial membrane potential and mitochondrial superoxide, HCS identified bavachin, isobavachalcone, bakuchiol and psoralidin as most potent mitotoxic compounds in PF. Moreover, molecular docking confirmed the potential binding patterns and strong binding affinity of the critical compounds with mitochondrial respiratory complex. This study unveiled the underlying mechanism and phytochemicals in PF-induced liver injuries from the view of mitochondrial dysfunction.

补骨脂（PF）是一种广泛使用的草药，具有多种药理活性，但其相关的肝损伤引起了公众的关注。在这项工作中，开发了一种基于 RNA 测序 (RNA-seq)、高内涵筛选 (HCS) 和分子对接的系统方法，以研究潜在机制并鉴定导致 PF 诱导肝毒性的主要植物化学物质。动物实验证明口服PF水提取物可通过抑制脂肪酸和胆固醇分解代谢扰乱脂质代谢并促进肝损伤。 RNA-seq结合KEGG富集分析确定线粒体氧化磷酸化（OXPHOS）是潜在的关键途径。进一步的实验验证了 PF 会导致线粒体结构损伤、mtDNA 耗竭并抑制参与 OXPHOS 的基因表达。通过检测线粒体膜电位和线粒体超氧化物，HCS 确定补骨脂素、异补骨脂查酮、补骨脂酚和补骨脂素是 PF 中最有效的线粒体毒性化合物。此外，分子对接证实了关键化合物与线粒体呼吸复合物的潜在结合模式和强结合亲和力。本研究从线粒体功能障碍的角度揭示了 PF 引起的肝损伤的潜在机制和植物化学物质。