Music intervention mitigates LPS-induced gut barrier disruption and immune stress in broilers via TLR4/NF-κB regulation

Abstract

Immune stress induced by harsh environment in intensive farming can impair broiler intestinal health. Although music as an environmental intervention can alleviate short-term stress injury, its long-term regulatory mechanism on intestinal inflammation has not been clarified. In this study, we investigated the effects of a music-enriched environment on growth performance, intestinal barrier function, and inflammatory responses in lipopolysaccharide (LPS)-induced immunostressed broilers. AA broilers were randomly divided into four groups: control group (CON), music-enriched environment group (MUC), LPS-induced immune stress group (LPS) and music-enriched environment + LPS group (MUC+LPS). On the 14th, 16th and 18th days, the LPS and MUC+LPS groups were injected intraperitoneally with 500 μg of LPS to construct an immune stress model, and the CON and MUC groups were injected with an equal amount of saline. On day 28, the birds were sacrificed to detect the indicators associated with intestinal barrier and inflammation. The LPS group showed a significant decrease in performance from 14 to 28 days, with elevated serum levels of CORT, ACTH, DAO, and d-LA, and a decrease in the activity of intestinal mucosal SOD/GSH-Px, and impaired gut morphology. impaired; music remission significantly alleviated the decline in production performance, reduced the levels of stress hormones and markers of intestinal barrier damage, while elevating jejuno-ileal GSH-Px activity and improving intestinal morphology. Significant inflammatory gene expression characteristics were observed in jejunum and ileum tissues after LPS injection: upregulation of TLR4, NF-κB, TNF-α, IL-1β, and IL-6, and significant suppression of jejunal IL-10 expression. Notably, IL-10 and IFN-γ expression in the ileum did not show statistical differences. Inflammation-related gene expression showed an overall down-regulation trend after the music intervention, but was still significantly different from the control group. Music intervention on the regulation of jejunal MYD88 and ileal TNF-α - the LPS group did not show statistically significant differences in the expression of these two key inflammatory nodes with the LPS+MUS group. Mechanistic studies have shown that LPS triggers an oxidative stress cascade through activation of the TLR4/NF-κB signaling axis, leading to disruption of intestinal barrier integrity. In contrast, music exposure exerts a protective effect through a dual mechanism: on the one hand, it helps to enhance the expression of the tight junction protein ZO-1/Occludin to repair the physical barrier; on the other hand, it inhibits the activation of the TLR4/NF-κB pathway, which can effectively alleviate LPS-induced immunopathological damage.

Keywords: Inflammation; Intestinal barrier function; Lipopolysaccharide; Music-enriched environment; TLR4/NF-4 κB signaling pathway.

Fig. 1

Processing of experimental broilers and flowchart of sample collection times.

Fig. 2

Long-term musical mitigation of lipopolysaccharide attack on broiler growth performance. (A) 1d-14d, 15d-28d average daily gain (ADG) of broilers. (B) 1d-14d, 15d-28d average daily feed intake (ADFI) of broilers. Bars without the same letter differed significantly (P < 0.05).

Fig. 3

(A) Scanning electron microscopy (SEM) images of the jejunum and ileum (SU8010, 5.0 kV, 8.0 mm, ×120 LMlUL, 400 µm). In the LPS group for the jejunum, the injury sites are marked with red arrows. In the MUS+LPS group, the injury sites are indicated with green arrows. For the ileum, injury sites in the LPS group are marked with blue arrows, while in the MUC+LPS group, they are marked with purple arrows. (B) Transmission electron microscopy (TEM) results for the jejunum and ileum (magnification: ×15,000). In the LPS group for the jejunum, vacuoles are indicated by red arrows, and mitochondrial cristae disruption is marked with green arrows. In the LPS group for the ileum, mitochondrial damage is marked with blue arrows. (C) The serum levels of DAO and d-LA in broilers. Differences marked with lowercase letters denote a significant difference (P < 0.05), while the same letter indicates no significant difference (P > 0.05).

Fig. 4

The Effects of Long-term Music Exposure on Oxidative Stress, Intestinal Barrier, and Intestinal Permeability in Broiler Chickens Injected with Lipopolysaccharide (LPS). (A) SOD activity, GSH-Px activity, and MDA content. (B) Concentrations of CORT and ACTH. (C) Relative mRNA expression of tight junction proteins (Claudin 2, Occludin, ZO-1) and mucin protein Mucin 2 in the jejunal and ileal mucosa of broiler chickens. (D) Immunofluorescence images of tight junction proteins (Claudin 2, Occludin, ZO-1) and mucin protein Mucin 2 in the jejunal and ileal mucosa of broiler chickens. The statistical results of the mean optical density of tight junction proteins (Claudin 2, Occludin, ZO-1) and mucin protein Mucin 2 in the jejunal and ileal mucosa are shown below the immunofluorescence images. Differences marked with lowercase letters denote a significant difference (P < 0.05), while the same letter indicates no significant difference (P > 0.05).

Fig. 5

Music has potential anti-inflammatory effects and can effectively alleviate inflammation in the jejunum and ileum by inhibiting the LPS-triggered TLR4/NF-κB signaling pathway. (A) The figure shows the relative mRNA expression levels of the TLR4/NF-κB inflammatory signaling pathway and related inflammatory factors in the jejunum and ileum of broilers. (B) The figure presents the immunoblot images and corresponding statistical results of TLR4, IKB-α, NF-κB p65, and TNF-α proteins in the jejunum and ileum of broilers. (C) The figure shows the measurement results of IL-1β, IL-6, and IL-10 levels in the jejunum and ileum of broilers across different groups. Differences marked with lowercase letters denote a significant difference (P < 0.05), while the same letter indicates no significant difference (P > 0.05).

Fig. 6

Prolonged exposure to a music-enriched environment attenuates LPS-induced intestinal inflammation in chickens by modulating the oxidative-antioxidative response and the TLR4/NF-κB signaling pathway in the gut.