Research Article: Formononetin protects against acetaminophen-induced hepatotoxicity through enhanced NRF2 activity

Date Published: February 24, 2017

Publisher: Public Library of Science

Author(s): Fen Jin, Chunpeng Wan, Weifang Li, Liangliang Yao, Hongqian Zhao, Yuan Zou, Dewei Peng, Weifeng Huang, Aamir Ahmad.


To examine the effects of formononetin (FMN) on Acetaminophen (APAP)-induced liver injury in vitro and in vivo. Human non-tumor hepatic cells LO2 were pretreated with either vehicle or FMN (20, 40 μM), for 6 h, followed by incubation with or without APAP (10 mM) for 24 h. In an in vivo assay, male BALB/c mice were randomly divided into four groups: (1) control group; (2) APAP group; (3) APAP + FMN (50 mg/Kg); (4) APAP + FMN (100 mg/Kg). The mice in the control and APAP groups were pre-treated with vehicle; the other two groups were pretreated daily with FMN (50, 100 mg/Kg) orally for 7 consecutive days. After the final treatment, acute liver injury was induced in all groups, except the control group, by intraperitoneal (i.p.) injection of 300 mg/Kg APAP. In LO2 cells, APAP exposure decreased the cell viability and glutathione (GSH) content, which were both greatly restored by FMN pretreatment. Overdose of APAP increased hepatic malondialdehyde (MDA) content, serum alanine aminotransferase (ALT), and aspartate aminotransferase (AST) activity in experimental mice. Supplementation with 100 mg/Kg FMN significantly reduced APAP-induced elevated levels of MDA (1.97 ± 0.27 vs 0.55 ± 0.14 nmol/mg protein, p < 0.001), ALT (955.80 ± 209.40 vs 46.90 ± 20.40 IU/L, p < 0.001) and AST (1533.80 ± 244.80 vs 56.70 ± 28.80 IU/L, p < 0.001), and hepatic GSH level (5.54 ± 0.93 vs 8.91 ± 1.11 μmol/mg protein, p < 0.001) was significantly increased. These results were further validated by histopathology and TdT-mediated biotin-dUTP nick-endlabeling (TUNEL) staining, pretreatment with 100 mg/Kg FMN significant decreased APAP-induced hepatocellular damage and cell apoptosis (36.55 ± 3.82 vs 2.58 ± 1.80%, p < 0.001). Concomitantly, FMN stimulated the expression of Nrf2 and antioxidant gene expression in the presence of APAP. These data provide an experimental basis for the use of FMN in the treatment of patients with APAP-induced hepatotoxicity.

Partial Text

Acetaminophen (APAP, also known as paracetamol, N-acetyl-p-aminophenol and Tylenol®), is a nonsteroidal analgesic and antipyretic drug that is clinically widely used. At therapeutic doses, APAP is metabolized to water-soluble metabolites in the liver by UDP-glucuronosyltransferases (UTGs) and sulfotransferase (SULTs), with only a small amount being converted by cytochrome P450 (CYP450) 2E1 into the highly reactive, cytotoxic intermediate N-acetyl-p-benzoquinoneimine (NAPQI)[1–3]. The small amounts of NAPQI generated are normally effectively detoxified by glutathione (GSH) conjugation and cleared from the liver[4]. However, excessive intake of APAP saturates the glucuronidation and sulfation routes, resulting in the formation of large a mounts of NAPQI converted by CYP450, which is then detoxified by conjugation with GSH. Once GSH is depleted, NAPQI covalently binds to possibly critical cellular proteins, with formation of reactive oxygen species (ROS), causing hepatocellular necrosis and apoptotic cell death, eventually resulting in severe centrilobular hepatotoxicity and acute liver failure[5–7].

In summary, our present study revealed that FMN has a protective effect on APAP-induced hepatocellular toxicity in LO2 and in experimental mice. The hepatic cell protection effect by a decrease of lipid peroxidation, reduced liver injury, and restore hepatocellular GSH, at least in part through enhancing Nrf2 and antioxidant genes expression.




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