Effects of Ferulic Acid on Lipopolysaccharide-Induced Oxidative Stress and Gut Microbiota Imbalance in Linwu Ducks

Abstract

Oxidative stress is a major factor that limits the development of the poultry industry. Ferulic acid (FA) has an antioxidant effect in birds, but the mechanism is not fully understood. In this study, we stimulated oxidative stress in 28-day-old female Linwu ducks by lipopolysaccharide (LPS) and fed them a diet supplemented with FA for 28 days. Results showed that FA alleviated LPS-induced growth performance regression, oxidative stress, and microbiota imbalance in ducks. An integrated metagenomics and metabolomics analysis revealed that s_Blautia_obeum, s_Faecalibacterium_prausnitzii, s_gemmiger_formicilis, and s_Ruminococcaceae_bacterium could be the biomarkers in the antioxidant effect of FA, which interacted with dihydro-3-coumaric acid, L-phenylalanine, and 13(S)-HODE, and regulated the phenylalanine metabolism and PPAR signaling pathway. This study revealed the mechanism of the antioxidant effect of FA, which provided evidence of applying FA as a new antioxidant in commercial duck production.

Keywords: Linwu duck; PPAR signaling pathway; antioxidant; ferulic acid; intestinal microbiota; metabolomics; metagenomics; phenylalanine metabolism.

Figure 1

Effects of LPS and FA on the growth performance of Linwu ducks. Data are presented as mean ±SD (n = 9) and analyzed by one-way ANOVA. “*” indicates p < 0.05. BW, body weight; ADG, average daily weight gain; FI, average daily feed intake; F:G, feed-to-gain ratio.

Figure 2

Evaluation of oxidative stress in Linwu ducks subjected to LPS and FA treatments. (A) Antioxidant enzymes and oxidative cytokines; (B) inflammatory substances. Data are presented as mean ± SD (n = 9) and analyzed by one-way ANOVA. “***” indicates p ≤ 0.001. SOD, superoxide dismutase; GSH-Px, glutathione peroxidase; CAT, catalase; T-AOC, total antioxidant capacity; MDA, malondialdehyde; GSH, glutathione; Ig, immunoglobulin; NF-κb, nuclear factor κB; IL-2, interleukin 2; IFN-γ, interferon γ; TNF-α, tumor necrosis factor α.

Figure 3

Comparison of bacterial composition and function in ducks’ cecal digesta. (A) Gut microbial composition at phylum level. (B) Gut microbial composition at species level. (C) α-diversity of gut microbiota in ducks. “*” indicates p ≤ 0.05. (D) Nonmetric multidimensional scaling (NMDS) plots of bacterial communities based on Bray–Curtis distance metric. (E) Difference in gut microbiota between CON and LPS. (F) Difference in gut microbiota between LPS and LPS-FA. Comparison was conducted by linear discriminant analysis effect size (LEfSe) analysis, with linear discriminant analysis (LDA) score > 2 and p < 0.05 being considered significant. (G) Comparison of KEGG pathway associated with differential bacteria between CON and LPS. (H) Comparison of KEGG pathway associated with differential bacteria between LPS and LPS-FA.

Figure 4

Comparison of metabolite composition in ducks’ cecal digesta. (A) OPLS-DA score plots of ESI+ and ESI−. (B) Volcano plot of the different metabolites (DMs) between CON and LPS. (C) Classification of the DMs between CON and LPS at the superclass level against the HMDB. (D) Enrichment analysis of KEGG pathway based on the DM between CON and LPS. (E) Volcano plot of the DMs between LPS and LPS-FA. (F) Classification of the DMs between LPS and LPS-FA at the superclass level against the HMDB. (G) Enrichment analysis of KEGG pathway based on the DMs between LPS and LPS-FA.

Figure 5

Integrated analysis of metagenome and metabolome in cecal digesta of ducks. (A) Venn diagram of KEGG pathways enriched from differential bacteria and differential metabolites in cecal digesta of ducks between CON and LPS. (B) Differential metabolites involved in pathways of glutathione metabolism and steroid hormone biosynthesis between CON and LPS. (C) Correlation of differential bacteria with metabolites and antioxidant-related cytokines between CON and LPS. (D) Venn diagram of KEGG pathways enriched from differential bacteria and differential metabolites in cecal digesta of ducks between CON and LPS. (E) Differential metabolites involved in pathways of phenylalanine metabolism and PPAR signaling pathway between CON and LPS. (F) Correlation of differential bacteria with metabolites and antioxidant-related cytokines between LPS and LPS-FA.