Objective  To explore the mechanism of acute liver injury induced by Cortex dictamni through network pharmacology and in vivo experiment in animal.

Methods  The chemical constituents and targets of Cortex dictamni were retrieved from TCMSP, TCMIP and SwissTargetPrediction databases, and the related targets of liver injury diseases were identified through GeneCards and CTD databases. The protein interaction network of the intersection targets was analyzed by STRING database and the core targets were selected. The GO function and KEGG pathway enrichment analysis were completed by DAVID database, and the multi-level association network diagram of "drug-component-target" was constructed by Cytoscape software. In the animal study, Cortex dictamni was administered to mice at a dosage of 92.7  g/ (kg·d) via intragastric administration, and the biological samples were collected after 7 days. The pathological changes of liver were observed by hematoxylin-eosin (HE), Masson and Oil Red O staining. The expression levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH) in serum, as well as malondialdehyde (MDA), superoxide dismutase (SOD), tumor necrosis factor-α (TNF-α), and interleukin (IL)-1β in liver tissues, were quantified using enzyme-linked immunosorbent assay (ELISA). The expressions of protein kinase B1 (AKT1), IL-6, TNF-α, tumor protein p53 (TP53), cystatin 3 (CASP3), and IL^-1β mRNA in liver tissues were determined using real-time quantitative reverse transcription PCR (qRT-PCR). Molecular docking was employed to verify the binding affinity of potentially toxic components to their respective targets.

Results  A total of 14 chemical constituents, 244 predicted targets and 202 intersection targets with liver injury were obtained. The GO biological process analysis mainly involved positive regulation of gene expression, negative regulation of apoptosis process. KEGG pathway enrichment analysis mainly included cancer pathway and PI3K-Akt, TNF, IL-17 signaling pathways. The pathological sections revealed severe hemorrhage, a considerable amount of hepatocyte necrosis, nuclear fragmentation or dissolution in the liver tissues of mouse with HE staining after the administration of Cortex dictamni. Masson staining showed evident fibrosis in the liver tissues, while Oil Red O staining indicated a substantial production of lipid droplets. Compared with the control group, the ELISA results demonstrated a significant increase in serum AST, ALT, ALP, LDH levels, as well as hepatic MDA, TNF-α, and IL-1β levels (P<0.05), and a decrease in hepatic SOD levels (P<0.05) in the treated group. The qRT-PCR results indicated a significant elevation in the expression levels of relevant mRNAs in the liver tissues of the treated mice (P<0.05). Molecular docking showed that the potentially toxic components of obacunone, dictamnine and fraxinellon had good binding affinity to AKT1, IL-6, TNF-α, TP53, CASP3 and IL-1β.

Conclusion  Obacunone, dictamnine, fraxinellon, and limonin might be the potential toxic components of acute liver injury induced by Cortex dictamni in mice. Cortex dictamni could act on the liver by changing the expressions of AKT1, IL-6, TNF-α, TP53, CASP3, IL-1β and other proteins, affecting energy metabolism, cell differentiation, inflammation, oxidative stress and immunity, leading to liver injury.

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