Korean J Physiol Pharmacol.  2016 Jan;20(1):15-23. 10.4196/kjpp.2016.20.1.15.

Carnosic acid protects against acetaminophen-induced hepatotoxicity by potentiating Nrf2-mediated antioxidant capacity in mice

Affiliations
  • 1Department of Ultrasound, The Second Hospital Affiliated of Zhengzhou University, Zhengzhou, Henan 450014, P. R. China.
  • 2Department of Gastroenterology, The Second Hospital Affiliated of Zhengzhou University, Zhengzhou, Henan 450014, P. R. China. zhengpeng-yuandr@126.com

Abstract

Acetaminophen (APAP) overdose is one of the most common causes of acute liver failure. The study aimed to investigate the protective effect of carnosic acid (CA) on APAP-induced acute hepatotoxicity and its underlying mechanism in mice. To induce hepatotoxicity, APAP solution (400 mg/kg) was administered into mice by intraperitoneal injection. Histological analysis revealed that CA treatment significantly ameliorated APAP-induced hepatic necrosis. The levels of both alanine aminotransferase (ALT) and aspartate transaminase (AST) in serum were reduced by CA treatment. Moreover, CA treatment significantly inhibited APAP-induced hepatocytes necrosis and lactate dehydrogenase (LDH) releasing. Western blot analysis showed that CA abrogated APAP-induced cleaved caspase-3, Bax and phosphorylated JNK protein expression. Further results showed that CA treatment markedly inhibited APAP-induced pro-inflammatory cytokines TNF-alpha, IL-1beta, IL-6 and MCP-1 mRNA expression and the levels of phosphorylated IkappaBalpha and p65 protein in the liver. In addition, CA treatment reduced APAP- induced hepatic malondialdehyde (MDA) contents and reactive oxygen species (ROS) accumulation. Conversely, hepatic glutathione (GSH) level was increased by administration of CA in APAP-treated mice. Mechanistically, CA facilitated Nrf2 translocation into nuclear through blocking the interaction between Nrf2 and Keap1, which, in turn, upregulated anti-oxidant genes mRNA expression. Taken together, our results indicate that CA facilitates Nrf2 nuclear translocation, causing induction of Nrf2-dependent genes, which contributes to protection from acetaminophen hepatotoxicity.

Keyword

Acetaminophen; Anti-oxidation; Carnosic acid; Hepatotoxicity; Nuclear factor erythroid 2-related factor 2

MeSH Terms

Acetaminophen
Alanine Transaminase
Animals
Aspartate Aminotransferases
Blotting, Western
Caspase 3
Cytokines
Glutathione
Hepatocytes
Injections, Intraperitoneal
Interleukin-6
L-Lactate Dehydrogenase
Liver
Liver Failure, Acute
Malondialdehyde
Mice*
Necrosis
Reactive Oxygen Species
RNA, Messenger
Tumor Necrosis Factor-alpha
Acetaminophen
Alanine Transaminase
Aspartate Aminotransferases
Caspase 3
Cytokines
Glutathione
Interleukin-6
L-Lactate Dehydrogenase
Malondialdehyde
RNA, Messenger
Reactive Oxygen Species
Tumor Necrosis Factor-alpha

Figure

  • Fig. 1 CA protected against APAP-induced liver injury. Male C57BL/6J mice were administered with either CA (100 mg/kg per day) or an equal volume of 0.5% CMC-Na for three days. At day 3, the mice were intraperitoneally injected with either 400 mg/kg APAP or an equal volume of PBS. (A) Representative images of H&E-staining in liver sections harvested at 6 hours post-APAP injection Magnification: 200×. (B, C) The serum levels of both ALT and AST were determined by a commercial kit. Data are shown as means±S.E.M. *p<0.05 versus APAP alone group (n=8).

  • Fig. 2 CA ameliorated APAP-induced hepatocytes DNA fragment. (A) Representative images of TUNEL staining in liver sections harvested at 6 hours post-APAP injection Magnification: 400×. (B) The serum levels of LDH were determined by a commercial kit. Data are shown as means±S.E.M. *p<0.05 versus APAP alone group (n=8).

  • Fig. 3 The effects of CA on APAPinduced cell death-related protein expression. Total cellular protein from liver was extracted and the protein levels were determined by western bolt. β-actin was used as the endogenous control. The bands were analyzed by densitometry. Data are shown as means±S.E.M. *p<0.05, **p<0.01 versus APAP alone group (n=8).

  • Fig. 4 CA inhibited APAP-induced hepatic inflammation. (A) Total RNA from liver was isolated and target genes mRNA levels were determined by real-time PCR. β-actin was used as the endogenous control. (B) Total cellular protein from liver was extracted and the indicated protein levels were determined by western bolt. β-actin was used as the endogenous control. The bands were analyzed by densitometry. Data are shown as means±S.E.M. *p<0.05, **p<0.01 versus APAP alone group (n=8).

  • Fig. 5 CA treatment reduced APAP-induced hepatic oxidative stress. Liver homogenate supernatants were used to determine the levels of ROS, MDA and GSH using a commercially available kit. Data are shown as means±S.E.M. *p<0.05 versus APAP alone group (n=8).

  • Fig. 6 CA promoted APAP-induced Nrf2 translocation into nuclear through blocking the interaction between Nrf2 and Keap1. (A) Total or nuclei Nrf2 protein levels were determined by western blot. β-actin and Lamin B1 were used as the endogenous control. The bands were analyzed by densitometry. Data are shown as means±S.E.M. *p<0.05 versus APAP alone group. (B) The interaction between Nrf2 and Keap1 in the liver was measured by co-immunoprecipitation assay. Representative data are shown.

  • Fig. 7 CA increased anti-oxidant genes expressions in APAP-treated mice. Total RNA from liver was isolated and target genes mRNA levels were determined by real-time PCR. β-actin was used as the endogenous control. Data are shown as means±S.E.M. *p<0.05 versus APAP alone group (n=8).


Cited by  1 articles

Loss of RAR-α and RXR-α and enhanced caspase-3-dependent apoptosis in N-acetyl-p-aminophenol-induced liver injury in mice is tissue factor dependent
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Korean J Physiol Pharmacol. 2021;25(5):385-393.    doi: 10.4196/kjpp.2021.25.5.385.


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