Abstract
Background: Toluene can trigger oxidative stress, cellular injury, and dysregulation of apoptosis and autophagy pathways in the liver. Vitex agnus-castus (VAC) fruit extract, known for its rich antioxidant, flavonoid, and phenolic contents, may counteract these effects through its free radical-scavenging and cytoprotective actions. This study aimed to evaluate the protective role of VAC fruit extract on key apoptotic and autophagy-related marker genes in toluene-exposed mice, supported by bioinformatics analysis. Methods: Flavonoid and phenolic contents of VAC fruit extract were measured colorimetrically, and antioxidant activity was assessed using the 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay. Adult male mice received toluene (100 mg/kg), VAC fruit extract (100 or 200 mg/kg), or their combination. The evaluation included gene expression analysis by quantitative real-time polymerase chain reaction (qRT-PCR), measurement of serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), and molecular docking analysis along with gene network construction using bioinformatics tools. Results: The flavonoid, total phenolic content, and antioxidant potential of the VAC fruit extract were 60.24, 126.35, and 455.23 mg equivalents per gram of extract, respectively, indicating its high antioxidant capacity. Toluene upregulated Bax (P<0.01), Lc3 (P<0.05), and Beclin1 (P<0.05) gene expression, which was significantly downregulated by VAC (100 or 200 mg/kg; P<0.05 to P<0.01), while Bcl2 gene expression, as well as AST and ALT levels, remained unchanged. Molecular docking confirmed the strong binding of VAC compounds to the target proteins, which may contribute to their functional alterations. Conclusion: The strong antioxidant and bioactive features of VAC fruit extract contributed to the modulation of oxidative stress-induced apoptotic and autophagy gene expression in the liver of toluene-exposed mice, highlighting its potential as a hepatoprotective and regulatory compound against toluene-induced toxicity.