Serum pharmacochemistry combined with network pharmacology reveals the hepatotoxicity mechanism of Alangium chinense (Lour.) Harms.

Ethnopharmacological Relevance: Alangium chinense (Lour.) Harms, commonly known as A. chinense, is a member of the Alangiaceae family. This plant is traditionally utilized by the Miao nationality of Guizhou as a medicinal remedy for rheumatic discomfort, employing a method that targets and eliminates toxins. Research has demonstrated its efficacy in dispelling wind, reducing dampness, breaking up blood stagnation, and alleviating pain. Nonetheless, there have been indications that A. chinense may possess toxic properties; however, the exact mechanism underlying its toxicity remains not fully understood.

Aim Of The Study: Employing an integrated strategy that combines serum metabolomics and network pharmacology, this work intends to elucidate the toxic elements and comprehensively explore the fundamental processes of toxicity connected to A. chinense.

Materials And Methods: Rats were divided into thirteen groups, including normal control group and low-medium-high dose groups of water extract and ethanol extract of fibrous root and root, over 14 consecutive days. Toxic effects were evaluated through serum biochemistry and pathohistological examinations. Serum metabolomics were analyzed using UPLC-MS/MS to identify the main blood-absorbed constituents of A. chinense. Additionally, hepatotoxicity-related targets were compiled from OMIM, CTD, GeneCards, and DisGeNET databases alongside primary blood-absorbed component targets sourced from TCMSP and Swiss Target Prediction databases. We elucidated the toxic mechanism of A. chinense through compound-target and target-pathway networks. Finally, we verified the mechanism of A. chinense-induced hepatotoxicity in rats by molecular docking, qRT-PCR, western blotting and in vitro experiments.

Results: In vivo experiments revealed that A. chinense increased the levels of AST and ALT in serum. The sectioning results indicated that different medicinal parts and different extracts of A. chinense caused varying degrees of liver damage in a dose-dependent manner, with the water extract of fibrous root resulting in the greatest damage. The UPLC-MS/MS analysis revealed 75 blood-absorbed components in A. chinense, with 18 significantly linked to liver injury factors, such as Anabasine, Brucine, Tricin, and quercetin-3,4'-dimethyl ether. Additionally, network pharmacology revealed 123 potential targets associated with A. chinense-induced hepatotoxicity. KEGG pathway analysis revealed 247 signaling pathways associated with these common targets, emphasizing key pathways such as the PI3K-Akt signaling pathway, lipid and atherosclerosis signaling pathway, and chemical carcinogenesis-receptor activation signaling pathway. Molecular docking studies demonstrated that Mansonone D, Sudachitin, Mansonone E, Tricin, Brucine, and (-)-Anabasine exhibit strong affinity and low binding energy with PIK3CA, AKT1, mTOR, MAP2K1, and MAPK1 among the identified blood-absorbed ingredients. In addition, qRT-PCR combined with Western blotting analysis showed that compared with the control group, A. chinense water extract significantly increased the mRNA expression of PIK3CA, AKT1, mTOR, MAP2K1, MAPK1 and rheb in rat liver tissue. Abnormal activation of pmTOR/mTOR, pPI3K/PI3K, pAKT/AKT protein expression levels and the enzyme activity levels of caspase 3/7 and caspase 1, thereby activating PI3K/AKT/mTOR pathway to play hepatotoxic role through autophagy or apoptosis. This suggests an intensive activation of this specific biochemical transformation channel. Finally, the in vitro experiment showed that Anabasine, the alkaloids of fibrous root and root could significantly increase AST and ALT levels in mouse AML-12 cells.

Conclusions: Through network pharmacology and serum metabolomics analysis, we investigated the possible mechanism of A. chinense's hepatotoxicity and confirmed that A. chinense may be caused by abnormal activation of PI3K/AKT/mTOR signaling pathway. This study elucidates the potential mechanism of A. chinense-related hepatotoxicity, provides a theoretical basis for in-depth exploration of its toxicity mechanism and mitigation strategies, and provides valuable insights and scientific support for understanding the intrinsic toxicity mechanism of ethnomedicine from a holistic perspective.