5-O-Demethylnobiletin Alleviates CCl4-Induced Acute Liver Injury by Equilibrating ROS-Mediated Apoptosis and Autophagy Induction

Polymethoxyflavanoids (PMFs) have exhibited a vast array of therapeutic biological properties. 5-O-Demethylnobiletin (5-DN) is one such PMF having anti-inflammatory activity, yet its role in hepatoprotection has not been studied before. Results from in vitro study revealed that 5-DN did not exert a high level of cytotoxicity on HepG2 cells at 40 μM, and it was able to rescue HepG2 cell death induced by carbon tetrachloride (CCl₄). Subsequently, we investigated acute liver injury on BALB/c mice induced by CCl₄ through the intraperitoneal injection of 1 mL/kg CCl₄ and co-administration of 5-DN at (1 and 2 mg/kg) by oral gavage for 15 days. The results illustrated that treatment with 5-DN attenuated CCl₄-induced elevated serum aminotransferase (AST)/alanine aminotransferase (ALT) ratio and significantly ameliorated severe hepatic damage such as inflammation and fibrosis evidenced through lesser aberrations in the liver histology of 5-DN dose groups. Additionally, 5-DN efficiently counteracted and equilibrated the production of ROS accelerated by CCl₄ and dramatically downregulated the expression of CYP2E1 vitally involved in converting CCl₄ to toxic free radicals and also enhanced the antioxidant Enzymes. 5-DN treatment also inhibited cell proliferation and inflammatory pathway abnormally regulated by CCl₄ treatment. Furthermore, the apoptotic response induced by CCl₄ treatment was remarkably reduced by enhanced Bcl-2 expression and noticeable reduction in Bax, Bid, cleaved Caspase 3, Caspase 9, and apaf-1 expression. 5-DN treatment also induced the conversion of LC3 and promoted the autophagic flux. Conclusively, 5-DN exhibited hepatoprotective effects in vitro and in vivo and prevented liver fibrosis induced by CCl₄.