Aspirin Eugenol Ester Alleviates Energy Metabolism Disorders by Reducing Oxidative Damage and Inflammation in the Livers of Broilers Under High-Stocking-Density Stress

Abstract

This study aimed to evaluate the effects of aspirin eugenol ester (AEE) on growth performance, oxidative liver damage, inflammation, and liver metabolomics in broilers under high-stocking-density (HSD) stress. A total of 360 broilers were divided into four groups: normal density (ND, 14/m²), high density (HD, 22/m²), ND-AEE (ND + 0.01% AEE), and HD-AEE (HD + 0.01% AEE). HSD decreased total antioxidant capacity, increased malondialdehyde (MDA) levels, and elevated the expression of cyclooxygenase-2 (COX-2) and microsomal prostaglandin E synthase-1 (mPGES-1) mRNA, which contributed to the reduced performance of broilers. Specifically, HSD caused abnormalities in linoleic acid metabolism, leading to elevated levels of Prostaglandin E2 (PGE2) and Leukotriene B4 (LTB4) synthesis, which aggravated inflammation, increased liver lipid levels, and impaired ATP production. AEE counteracted the decline in broiler production performance induced by HSD by enhancing total antioxidant capacity, reducing MDA levels, protecting the liver from oxidative damage, and maintaining mitochondrial oxidative phosphorylation. AEE positively regulated the linoleic acid metabolism by promoting the synthesis of γ-linolenic acid and phosphatidylcholine, which reduced the synthesis of COX-2 and mPGES-1. AEE alleviated the metabolic imbalance caused by HSD stress and enhanced the efficiency of mitochondrial fatty acid oxidation, which reduced excess lipid accumulation in the liver and promoted ATP production. In summary, this study provides strong support for the dietary addition of AEE to alleviate liver oxidative damage, inflammation, and energy metabolism disorders caused by HSD stress.

Keywords: AEE; broilers; energy metabolism; high stocking density; inflammation; oxidative damage.

Figure 1

Effect of AEE on the relative expression levels of inflammatory factor mRNA in the livers of broilers across four treatment groups. ND, broilers at normal stocking density fed basal diet; HD, broilers at high stocking density fed basal diet; ND-AEE, normal-stocking-density group fed basal diet supplemented with 0.01% AEE; HD-AEE, high-stocking-density group fed basal diet supplemented with 0.01% AEE. (A–F) mRNA levels of COX-2, mPGES-1, IL-1β, TNF-α, IL-6, and IL-10 at 21, 28, 35, and 42 days of age. The gene for GAPDH was used as a reference for normalization. Bars labeled with different letters (a, b, c) indicate significant differences across all groups (p < 0.05), with data presented as mean ± SEM (n = 6).

Figure 2

Effect of AEE on liver pathology and lipid accumulation in broilers under HSD stress as shown by H&E and Oil Red O staining of liver sections at 21, 28, 35, and 42 days of age (A–D). In H&E staining, pink represents the cytoplasm and blue the nucleus; red arrows indicate inflammatory cells, and blue arrows point to lipid vacuoles. In Oil Red O staining, the nucleus appears blue, and lipid droplets are stained red (400× magnification). Scale bar: 20 μm. Representative histopathological section of the liver from each group (n = 4).

Figure 3

(A−F) Volcano plot screening for differentially expressed metabolites in the ND vs. HD and the HD vs. HD-AEE groups at 28, 35, and 42 days of age. Those with FC > 2 and p < 0.05 are marked in red, while those with FC < 0.5 and p < 0.05 are shown in blue. Differential metabolites of non-significance are marked in gray. For each group, n = 4.

Figure 4

Analysis of significantly different metabolites in the ND vs. HD and HD vs. HD-AEE groups. (A,B) Metabolite profile scores from orthogonal partial least squares discriminant analysis (OPLS-DA) for each group at 28 days of age. (C,D) OPLS-DA for each group at 35 days of age. (E,F) OPLS-DA for each group at 42 days of age. PC1 is principal component 1, PC2 is principal component 2, and different colored points and ellipses represent samples and confidence intervals for different groupings. (G) Histogram of differential metabolites (n = 4).

Figure 5

Comparative analysis of differential metabolic pathways in the liver of broilers from different treatment groups. (A–C) Factor diagram of KEGG enrichment analysis for the comparison of ND vs. HD at 28, 35, and 42 days of age. (D–F) Factor diagram of KEGG enrichment analysis for the comparison of HD vs. HDAEE at 28, 35, and 42 days of age (n = 4). The degree of enrichment of each pathway was determined by p-value and the number of metabolites, with significance assessed using the hypergeometric test (p < 0.05). The diameter of the circle indicates the number of metabolites. The varying colors, ranging from brown to green, symbolize the magnitude of the p-value. A lower p-value means a higher degree of significance of the enrichment level.

Figure 6

Diagram illustrating the proposed mechanism of how AEE alleviates oxidative damage, inflammation, and energy metabolism disorders in the livers of broilers under HSD stress. AEE restored normal liver morphology and mitochondrial function by upregulating the expression of key antioxidants, reducing inflammatory mediator expression, and inhibiting lipid peroxidation. This ensures normal lipid metabolism, decreases inflammatory responses and excessive lipid accumulation, and restores the energy metabolism balance in broilers under HSD stress. Red arrows indicate effects of HSD; green arrows represent effects of AEE. Upward and downward arrows denote increase and decrease, respectively.