4-Hydroxyphenylacetic Acid Prevents Acute APAP-Induced Liver Injury by Increasing Phase II and Antioxidant Enzymes in Mice

Abstract

Acetaminophen (APAP) overdose is the principal cause of drug-induced acute liver failure. 4-hydroxyphenylacetic acid (4-HPA), a major microbiota-derived metabolite of polyphenols, is involved in the antioxidative action. This study seeks to investigate the ability of 4-HPA to protect against APAP-induced hepatotoxicity, as well as the putative mechanisms involved. Mice were treated with 4-HPA (6, 12, or 25 mg/kg) for 3 days, 1 h after the last administration of 4-HPA, a single dose of APAP was intraperitoneally infused for mice. APAP caused a remarkable increase of oxidative stress markers, peroxynitrite formation, and fewer activated phase II enzymes. 4-HPA increased Nrf2 translocation to the nucleus and enhanced the activity of phase II and antioxidant enzymes, and could thereby ameliorate APAP-induced liver injury. Studies reveal that 4-HPA, as an active area of bioactive dietary constituents, could protect the liver against APAP-induced injury, implying that 4-HPA could be a new promising strategy and natural hepatoprotective drug.

Keywords: 4-hydroxyphenylacetic acid 4-HPA; Nrf2; acetaminophen APAP; hepatotoxicity; nuclear factor erythroid 2-related factor; oxidative stress; polyphenols.

FIGURE 1

Effects of 4-HPA on APAP-induced increase in serum transaminases levels. Each value represents the mean ± SD of eight animals per group. ^∗ indicates statistically different group with APAP group (p < 0.05; AST and ALT, respectively). (1) Control: control group; (2) NAC100+APAP: treated with 100 mg/kg per day NAC and 300 mg/kg APAP; (3) APAP: a single dose of 300 mg/kg APAP; (4) 4HPA6+APAP: treated with 6 mg/kg per day 4-HPA and 300 mg/kg APAP; (5) 4HPA12+APAP: treated with 12 mg/kg per day 4-HPA and 300 mg/kg APAP; (6) 4HPA25+APAP: treated with 25 mg/kg per day 4-HPA and 300 mg/kg APAP.

FIGURE 2

Effects of 4-HPA on hepatic histopathological alterations following APAP treatment. Typical images were chosen from each experimental group. Original magnification was 10 × 10 and 40 × 10. The arrows mean liver injury.

FIGURE 3

Effects of 4-HPA on oxidative stress induced by APAP. (A) GSH, (B) CAT, (C) GPx, (D) SOD (E) MDA. Each value represents the mean ± SD of eight animals per group. ^∗ Above the bars indicate a statistically different group with APAP group (p < 0.05).

FIGURE 4

Effects of 4-HPA on protein expression of 3-NT and GCLC following APAP overdose. (A) Representative blots of 3-NT, GCLC, and GAPDH expression. Quantification of 3-NT. (B) GCLC (C) protein levels. ^∗ Above the bars indicate statistically a different group with APAP group (p < 0.05). Each value represents the mean ± SD of eight animals per group.

FIGURE 5

Effects of 4-HPA on protein and mRNA expression of CYP2E1 following APAP overdose. (A) Representative blots of CYP2E1 and GAPDH expression. (B) Quantification of CYP2E1 protein levels. (C) Relative mRNA levels of CYP2E1. Each value represents the mean ± SD of eight animals per group. ^∗ Above the bars indicate a statistically different group with APAP group (p < 0.05).

FIGURE 6

Effects of 4-HPA on mRNA and levels of phase II enzymes following APAP overdose. The levels of GST (A), UGTs (B), and SULTs (C) were determined by ELISA, and the mRNA levels of GSTa1 and GSTm1 (D), UGT1A1, UGT1A6 and UGT1A9 (E), SULT1A1 and SULT2A1 (F) were determined by real time PCR. Each value represents the mean ± SD of eight animals per group. ^∗ Above the bars indicate a statistically different group with APAP group (p < 0.05).

FIGURE 7

4-HPA induces expression of Nrf2. (A) Western blots of Nrf2, Lamin B, and GAPDH expression. (B) Nuclear Nrf2/Cytosolic Nrf2 content ratios and the densities of nuclear and cytosolic Nrf2 normalized by Lamin B and GAPDH. (C) Relative mRNA levels of Nrf2. Each value represents the mean ± SD of eight animals per group. ^∗ Above the bars indicate a statistically different group with the APAP group (p < 0.05).

FIGURE 8

A summary of 4-HPA’s effects on the APAP-induced injury. 4-HPA may stimulate the Nrf2/Keap1 into phosphorylated Nrf2 by MAPK, P13, ERK or JNK. In the nuclear, the phosphorylated Nrf2 combines with one of small Maf proteins and binds to the antioxidant response element (ARE) in the upstream promoter region of many antioxidative genes and phase II enzymes, and initiates their transcription. 4-HPA promotes the APAP transformation into a non-toxic form. 4-HPA increased Nrf-2 translocation to the nucleus and enhanced the activity of phase II enzymes and anti-oxidant enzymes, and could thereby ameliorate APAP-induced liver injury. It can inhibit the activity of CYP2E1, alleviate the oxidative stress of the liver, and counter the liver toxicity induced by APAP. The red arrows marks indicated that the results of 4-HPA treatments.