The Protective Effect of Aspirin Eugenol Ester on Paraquat-Induced Acute Liver Injury Rats

Abstract

Aspirin eugenol ester (AEE) possesses anti-inflammatory and anti-oxidative effects. The study was conducted to evaluate the protective effect of AEE on paraquat-induced acute liver injury (ALI) in rats. AEE was against ALI by decreasing alanine transaminase and aspartate transaminase levels in blood, increasing superoxide dismutase, catalase, and glutathione peroxidase levels, and decreasing malondialdehyde levels in blood and liver. A total of 32 metabolites were identified as biomarkers by using metabolite analysis of liver homogenate based on ultra-performance liquid chromatography-tandem mass spectrometry, which belonged to purine metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, glycerophospholipid metabolism, primary bile acid biosynthesis, aminoacyl-tRNA biosynthesis, phenylalanine metabolism, histidine metabolism, pantothenate, and CoA biosynthesis, ether lipid metabolism, beta-Alanine metabolism, lysine degradation, cysteine, and methionine metabolism. Western blotting analyses showed that Bax, cytochrome C, caspase-3, caspase-9, and apoptosis-inducing factor expression levels were obviously decreased, whereas Bcl-2 expression levels obviously increased after AEE treatment. AEE exhibited protective effects on PQ-induced ALI, and the underlying mechanism is correlated with antioxidants that regulate amino acid, phospholipid and energy metabolism metabolic pathway disorders and alleviate liver mitochondria apoptosis.

Keywords: antioxidation; aspirin eugnol ester; hepatotoxicity; metabolites; paraquat.

Figure 1

AEE reduces PQ-induced liver injury in rat. (A) Histopathological H&E staining of rat liver tissue (scale bar = 100 μm). Values are presented as the means ± SD where applicable (n = 6).

Figure 2

AEE attenuates PQ-induced oxidative stress in the liver of rat. (A) The activity of malondialdehyde (MDA) in serum of different treatment groups was detected. (B) The activity of superoxide dismutase (SOD) in serum of different treatment groups was detected. (C) The ratio of reduced glutathione/oxidized glutathione disulfide (GSH/GSSH) in serum different treatment groups was detected. (D) The activity of catalase (CAT) in serum of different treatment groups was detected. Values are presented as the means ± SD where applicable (n = 6). *p < 0.05 compared with the control group; ^#p < 0.05 compared with the PQ group.

Figure 3

Metabolomics analysis of the effect of AEE on PQ-induced ALI in rats. (A,G) PCA score plots based on supernatant of rat liver tissue of the control, PQ and AEE groups in positive and negative modes, ESI+: R² = 0.614, ESI–: R² = 0.589. (B,H) The loading plot of AEE and PQ groups in positive and negative modes. (C,I) OPLS-DA score plots of the AEE and PQ groups in positive and negative modes, ESI+: R²X = 0.566, R²Y = 0.994, Q² = 0.878; ESI–: R²X = 0.502, R²Y = 0.969, Q²= 0.752. (D,J) Permutation test of the OPLS-DA model, ESI+: the intercepts of R² = 0.926 and Q² = −0.387, ESI–: the intercepts of R² = 0.726 and Q² = −0.640. (E,K) OPLS-DA score plots of the control and PQ groups in positive and negative modes, ESI+: R²X = 0.592, R²Y = 0.993, Q² = 0.927; ESI–: R²X = 0.405, R²Y = 0.952, Q² = 0.820. (F,L) Permutation test of the OPLS-DA model, ESI+: the intercepts of R² = 0.915 and Q² = −0.641, ESI–: the intercepts of R² = 0.727 and Q² = −0.744. Values are presented as the means ± SD where applicable (n = 6).

Figure 4

The results of fold enrichment and path analysis of potential metabolites in the supernatant of liver tissue.

Figure 5

AEE decreased the level of apoptosis-related proteins in rat liver tissue induced by PQ. (A,B) The expression of AIF protein in liver tissue of different treatment groups was detected. (A,C) The expression of Bax protein in liver tissue of different treatment groups was detected. (A,D) The expression of Bcl-2 protein in liver tissue of different treatment groups was detected. (A,E) The expression of Caspase-3 protein in liver tissue of different treatment groups was detected. (A,F) The expression of Caspase-9 protein in liver tissue of different treatment groups was detected. (A,G) The expression of Cyt C protein in liver tissue of different treatment groups was detected. Values are presented as the means ± SD where applicable (n = 6). *p < 0.05 compared with the control group; ^#p < 0.05 compared with the PQ group.

Figure 6

The results of hepatoprotective effect of AEE on ALI rats.