Kaurenoic acid extracted from <i>Sphagneticola trilobata</i> reduces acetaminophen-induced hepatotoxicity through inhibition of oxidative stress and pro-inflammatory cytokine production in mice

<p>Acetaminophen (paracetamol) is a widely used analgesic and antipyretic drug that is safe at therapeutic doses. However, acetaminophen overdose can be fatal. Currently, the only treatment available is the N-acetyl cysteine. The diterpene kaurenoic acid (<i>ent</i>-kaur-16-en-19-oic acid, KA) is the major constituent of <i>Sphagneticola trilobata</i> (L.) Pruski. KA presents anti-inflammatory, anti-nociceptive and antioxidant properties. In this study, we evaluated the efficacy of KA in a model of acetaminophen-induced hepatotoxicity. KA increased, in a dose-dependent manner, the survival rate after acetaminophen overdose. KA reduced acetaminophen-induced hepatic necrosis and ALT and AST levels. KA decreased acetaminophen-induced neutrophil and macrophage recruitment, oxidative stress and the production of IL-33, TNF-α and IL-1β, alongside with normalisation of IL-10 levels in the liver. Therefore, KA showed preclinical efficacy in acetaminophen-induced hepatotoxicity and lethality.</p

Pimaradienoic acid (PA) inhibits carrageenan-induced nitric oxide (NO) production.

<p>Mice were treated per oral (p.o.) with PA (10 mg/kg) or vehicle (DMSO 2% diluted in saline) 30 minutes before the intraperitoneal (i.p.) injection of carrageenan (300 μg/paw). Nitrite production in peritoneal exudates was determined 3 hours after carrageenan injection. Results are means ± SEM of six mice per group per experiment, and are representative of two separate experiments. [*p<0.05 compared with the saline group, and #p< 0.05 compared to the vehicle group (One-way ANOVA followed by Tukey’s test)].</p

Pimaradienoic acid (PA) inhibits carrageenan-induced oxidative stress.

<p>Mice were treated per oral (p.o.) with PA (10 mg/kg) or vehicle (DMSO 2% diluted in saline) 30 minutes before the intraplantar (i.pl.) injection of carrageenan (300 μg/paw). Paw skin (A) GSH levels, (B) ABTS⁺ scavenging activity, (C) the ability to reduce iron (FRAP), and (D) superoxide anion production were determined 3 hours after carrageenan injection. Results are means ± SEM of six mice per group per experiment, and are representative of two separate experiments. [*p<0.05 compared to the saline group; #p<0.05 compared to inflammatory stimulus group. (One-way ANOVA followed by Tukey’s test)].</p

Pimaradienoic acid (PA) inhibits carrageenan-induced paw edema and myeloperoxidase (MPO) activity.

<p>Mice were treated per oral (p.o.) with PA (10 mg/kg) or vehicle (DMSO 2% diluted in saline) 30 minutes before the intraplantar (i.pl.) injection of carrageenan (300 μg/paw). The evaluation of (A) paw edema was at 1–5 hours and (B) MPO activity at 5 hours after carrageenan injection. Results are means ± SEM of six mice per group per experiment, and are representative of two separate experiments. [*p< 0.05 compared with the saline group, #p <0.05 compared to the vehicle group (One-way ANOVA followed by Tukey’s test)].</p

Pimaradienoic acid (PA) inhibits carrageenan-induced peritoneal cytokine production.

<p>Mice were treated per oral (p.o.) with PA (10 mg/kg) or vehicle (DMSO 2% diluted in saline) 30 minutes before the intraperitoneal (i.p.) injection of carrageenan (500 μg/cavity). Peritoneal exudate samples were harvested 6 hours after carrageenan injection for (A) TNF-α and (B) IL-1β determination by ELISA. Results are means ± SEM of six mice per group per experiment, and are representative of two separate experiments. [*p<0.05 compared with the saline group, #p<0.05 compared to vehicle group (One-way ANOVA followed by Tukey’s test)].</p

Pimaradienoic acid (PA) structure.

<p>Chemical structure of PA[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149656#pone.0149656.ref029" target="_blank">29</a>].</p

Pimaradienoic acid (PA) inhibits carrageenan-induced total leukocyte and neutrophil recruitment in the peritoneal cavity.

<p>Mice were treated per oral (p.o.) with PA (0.1–10 mg/kg) or vehicle (DMSO 2% diluted in saline) 30 minutes before carrageenan (500 μg/ cavity) intraperitoneal (i.p.) injection. The (A) total number of leukocytes, (B) neutrophils and (C) mononuclear cells was evaluated 6 hours after carrageenan injection. Results are means ± SEM of six mice per group per experiment and are representative of two separate experiments. [*p < 0.05 compared to the saline group; #p < 0.05 compared to the vehicle group (One-way ANOVA followed by Tukey’s test)].</p

Pimaradienoic acid (PA) inhibited carrageenan-induced superoxide anion production.

<p>Mice were treated per oral (p.o.) with PA (10 mg/kg) or vehicle (DMSO 2% diluted in saline) 30 minutes before the intraperitoneal (i.p.) injection of carrageenan (500 μg/cavity). (A) Superoxide anion production and (B) NBT reaction positive cells (presence of formazan crystals) were determined in peritoneal cavity exudates 3 hours after carrageenan injection. Results are means ± SEM of six mice per group per experiment, and are representative of two separate experiments. [*p< 0.05 compared with the saline group, and #p< 0.05 compared to the vehicle group (One-way ANOVA followed by Tukey’s test)].</p