Formononetin protects against acetaminophen-induced hepatotoxicity through enhanced NRF2 activity.

Fen Jin,Weifeng Huang,Dewei Peng,Chunpeng Wan,Weifang Li,Liangliang Yao,Yuan Zou,Hongqian Zhao

doi:10.1371/journal.pone.0170900

Abstract

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.

Highlights

Acetaminophen (APAP, known as paracetamol, N-acetyl-p-aminophenol and Tylenol1), is a nonsteroidal analgesic and antipyretic drug that is clinically widely used
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-pbenzoquinoneimine (NAPQI)[1,2,3]
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 detoxified by conjugation with GSH

Summary

Introduction

Acetaminophen (APAP, known as paracetamol, N-acetyl-p-aminophenol and Tylenol1), is a nonsteroidal analgesic and antipyretic drug that is clinically widely used. 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-pbenzoquinoneimine (NAPQI)[1,2,3]. 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 detoxified by conjugation with GSH. The transcription factor, nuclear factor erythroid 2 p45-related factor 2 (Nrf2), regulates the expression of a wide array of genes involved in GSH synthesis, antioxidative system, drug metabolism via binding to the antioxidant response element (ARE) for hepatoprotection[8]. In Nrf2-deficient mice, the higher susceptibility to APAP leads to greater severity in hepatic damage and increased lethality[9, 10]

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