Aspirin eugenol ester affects ileal barrier function, inflammatory response and microbiota in broilers under lipopolysaccharide-induced immune stress conditions

Abstract

Aims: The aim of this study was to investigate the effects of aspirin eugenol ester (AEE) on ileal immune function in broilers under lipopolysaccharide (LPS)-induced immune stress.

Methods: Two hundred and forty one-day-old male Arbor Acres chicks were randomly divided into four groups (saline, LPS, saline + AEE and LPS + AEE) with six replicates of ten broilers each. The saline group and LPS group were fed the normal diet, while the other two groups received normal diet plus 0.1 g/kg AEE. Broilers in the LPS and LPS + AEE groups were injected intraperitoneally with 0.5 mg/kg B.W LPS in saline for seven consecutive days beginning at 14 days of age, while broilers in the saline and saline + AEE groups were injected with saline only.

Results: The results showed that AEE improved the ileal morphology and increased the ratio of villus height to crypt depth of immune-stressed broilers. LPS-induced immune stress significantly reduced the expression of the genes for the tight junction proteins occludin, zonula occludens-1 (ZO-1), claudin-1 and claudin-2, in the ileum, while AEE significantly up-regulated the expression of these genes. Compared with the saline group, the LPS-treated chickens showed significantly increased mRNA expression of the inflammatory factors tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10), cyclooxygenase-2 (COX-2), and microsomal Prostaglandin E Synthesase-1 (mPGES-1) in the ileum, while they were significantly decreased by AEE supplementation. In addition, analysis of the ileal bacterial composition showed that compared with saline and LPS + AEE groups, the proportion of Firmicutes and Lactobacillus in the LPS group was lower, while the proportion of Proteobacteria and Escherichia-Shigella was higher. Similarly, Line Discriminant Analysis Effect Size (LEfSe) analysis showed that compared with the LPS group, Brevibacillus was dominant in the saline group, while the LPS + AEE group was rich in Rhizobium, Lachnoclostridium, Ruminococcaceae, Faecalibacterium, Negativibacillus, Oscillospiraceae, and Flavonifractor.

Conclusion: These results indicate that dietary supplementation with 0.1 g/kg AEE could protect the intestinal health by improving the intestinal villus morphology, enhancing the expression of tight junction genes and alleviating inflammation to resist the immune stress caused by LPS stimulation in broilers, and the mechanism may involve COX-2-related signal transduction and improved intestinal microbiota composition.

Keywords: aspirin eugenol ester; broiler; ileal microbiota; immune stress; intestinal barrier function.

Figure 1

Effects of AEE on ileal morphology in immune-stressed broilers. (A) Morphological structure of ileal tissue. (B) Villus height. (C) Crypt depth. (D) V/C, ratio of villus height to crypt depth. Each vertical bar represents the mean ± SEM (n = 6). Bars with different letters differed significantly (p < 0.05). Scale bar, 200 μm.

Figure 2

Effects of AEE on relative expression of tight junction protein mRNA in the ileum of immune-stressed broilers. Relative expression of occludin (A), ZO-1 (B), claudin-1 (C), and claudin-2 (D) mRNA in ileum. The gene for GAPDH was used as a reference for normalization. Each vertical bar represents the mean ± SEM (n = 6). Bars with different letters differed significantly (p < 0.05).

Figure 3

Effects of AEE on relative mRNA expression of inflammatory cytokines in the ileum of immune-stressed broilers. Relative mRNA expression of TNF-α (A), IL-1β (B), IL-6 (C), IL-10 (D), COX-2 (E), and mPGES-1 (F) in ileum. The gene for GAPDH was used as a reference for normalization. Each vertical bar represents the mean ± SEM (n = 6). Bars with different letters differed significantly (p < 0.05).

Figure 4

Effects of AEE on ileal microbiota composition and diversity in immune-stressed broilers. Alpha-diversity indicated by observed Simpson (A) and Shannon (B) index. Venn diagram showing overlap of compositions of bacterial OTUs in different groups (C). Beta-diversity indicated by principal component analysis (PCA) on the phylum level (D). Average relative abundances of dominant bacterial phylum (E) and genus level (F) in ileum under different treatments.

Figure 5

Cladograms and bar plots obtained by LEfSe analysis showed differences in microbiota between the treatment groups. LEfSe analysis of the saline and LPS groups (A). LEfSe analysis of the LPS and LPS + AEE groups (B). LEfSe analysis of the saline and saline + AEE groups (C). The different color nodes in cladogram represent the microbial groups that are significantly enriched in the corresponding group and have a significant effect on the difference between the groups. Light yellow nodes represent microbial groups that do not differ significantly between groups, or have no significant effect on differences between groups. The LDA scores represent the effect size of each abundant species. The bar plots show the microbiota with LDA score > 2, which is a biomarker of statistical difference (LDA score threshold = 2).