Hepatoprotection of Apocynum venetum flavonoids: Targeting oxidative stress via coordinated NRF2/BCHE induction and AR inhibition

Acute liver injury (ALI) represents a serious clinical condition characterized by rapid progression toward hepatic functional decompensation, potentially culminating in liver failure. Despite proposed therapeutic interventions, effective management strategies remain limited. Total flavonoids from Apocynum venetum leaves (TFAP), recognized for their antioxidant and anti-inflammatory properties, have demonstrated multi-organ protective effects in prior studies. However, the precise mechanisms underlying TFAP's role in attenuating ALI pathogenesis remain poorly elucidated.

Utilizing the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database and analysis platform, in conjunction with the drug mapping server, the active constituents of TFAP and their therapeutic targets for acute liver injury were identified. Specific targets pertinent to the treatment of acute liver injury were screened and compiled via the GEO database. Molecular docking techniques were employed to elucidate the interactions between the active compounds and their respective targets. Furthermore, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted to provide a comprehensive analysis of the targets associated with acute liver injury. To validate the network pharmacology findings, CCl4-induced acute liver injury cell and animal models were established. Cell viability was assessed using the Cell Counting Kit-8 (CCK8) solution, while flow cytometry, RT-qPCR, and Western blot assays were conducted to evaluate indicators related to cell apoptosis and the Nrf2 signaling pathway. Biochemical assays were employed to quantify the enzymatic activities of aspartate transaminase (AST), alanine transaminase (ALT), and superoxide dismutase (SOD), as well as the concentration of malondialdehyde (MDA) in both cellular and plasma samples. Hematoxylin-Eosin (HE) staining was conducted to examine the pathological alterations in hepatic tissues of mice across different experimental groups. Additionally, immunohistochemical methods were utilized to assess the expression levels of the proteins Nrf2, Keap1, HO-1, and GCLC in liver tissues.

A comprehensive transcriptome analysis combined with network pharmacology has indicated that TFAP may exert therapeutic effects on alcoholic hepatitis (ALD) by modulating the AR and BCHE signaling pathways. In vitro studies demonstrated that TFAP extended the survival of hepatocytes subjected to injury, significantly decreased the levels of AST, ALT, LDH, ROS, and MDA, and enhanced SOD activity in CCl₄-induced LO2 cells. Furthermore, treatment with these flavonoids resulted in a reduction of AR protein expression while increasing the expression of BCHE and Nrf2 proteins. In vivo experiments revealed that, in comparison to the blank control group, the model group exhibited significantly elevated levels of liver injury biomarkers ALT and AST (p < 0.01), increased MDA and SOD activities (p < 0.01), and a significant reduction in the expression of Nrf2, Keap1, HO-1, and GCLC proteins in liver tissue (p < 0.01). Histopathological analysis showed extensive steatosis, nuclear pyknosis, and cytoplasmic eosinophilic alterations in the liver tissue. In comparison to the model group, the groups receiving middle and high doses of TFAP demonstrated normal levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), malondialdehyde (MDA), and superoxide dismutase (SOD), alongside normal expression of nuclear factor erythroid 2-related factor 2 (Nrf2), Kelch-like ECH-associated protein 1 (Keap1), heme oxygenase 1 (HO-1), and glutamate-cysteine ligase catalytic subunit (GCLC) proteins within liver tissue. Additionally, hepatocyte morphology appeared normal. Conversely, in the low-dose group, hepatocytes continued to display karyolysis and steatosis; however, the extent of necrosis was significantly diminished (p < 0.01).

TFAP ameliorates carbon tetrachloride (CCl₄)-induced acute liver injury by counteracting oxidative stress through modulation of the Nrf2-Keap1 signaling pathway.