Chlorogenic acid protects against intestinal inflammation and injury by inactivating the mtDNA-cGAS-STING signaling pathway in broilers under necrotic enteritis challenge

Abstract

This study aimed to determine the effects of chlorogenic acid (CGA) on the growth performance, intestinal health, immune response, and mitochondrial DNA (mtDNA)-cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling pathway in broilers under necrotic enteritis (NE) challenge. The 180 one-day-old male Cobb 500 broilers with similar body weight of 44.59 ± 1.39 g were randomly allocated into 3 groups. The groups were control diet (Control group), control diet + NE challenge (NE group), and control diet + 500 mg/kg CGA + NE challenge (NE + CGA group), with 6 replicates per treatment. All broilers except the Control group were given sporulated coccidian oocysts (d 14) and Clostridium perfringens (d 19-21) by oral gavage. Our findings showed that CGA improved the growth performance and intestinal morphology in broilers under NE challenge. CGA supplementation elevated the barrier function in broilers under NE challenge, which reflected in the decreased serum concentrations of D-lactate and diamine oxidase, and upregulated jejunal protein expression of occludin. CGA supplementation also improved the immune function, which reflected in the increased concentrations and gene expressions of anti-inflammatory factors, and decreased concentrations and gene expressions of proinflammatory factors. CGA supplementation further enhanced intestinal cell proliferation and differentiation, which manifested in the increased number of goblet cells and positive cells of proliferating cell nuclear antigen on d 28 and 42. Furthermore, CGA supplementation decreased the mtDNA (d 42) and mitochondrial reactive oxygen species levels (d 28 and 42), and increased the mitochondrial membrane potential (d 42) and mitochondrial complex I (d 28 and 42) or III (d 28) activity. Broilers challenged with NE had upregulated jejunal protein expressions of cGAS, phospho-TANK-binding kinase 1, and phospho-interferon regulatory factor 7 compared with the Control group, which were downregulated after CGA supplementation. In conclusion, dietary supplementation CGA could protect against intestinal inflammation and injury by reducing the leakage of mtDNA and inactivating the cGAS-STING signaling pathway in broilers under NE challenge.

Keywords: broiler; cGAS–STING signaling pathway; chlorogenic acid; mitochondrial dysfunction; necrotic enteritis.

Graphical abstract

Figure 1

Effects of chlorogenic acid (CGA) supplementation on the intestinal morphology in broilers under necrotic enteritis (NE) challenge. (A and B) Jejunum and ileum tissues stained with hematoxylin and eosin on d 28 and 42, scale bar: 200 μm. (C and D) Scanning electron microscopy images of jejunum and ileum tissues on d 28 and 42, scale bar: 1 μm. Red arrows pointing to the microvillus, and blue arrows pointing to the tight junction. Control, control diet; NE, control diet + NE challenge; NE + CGA, control diet + 500 mg/kg CGA + NE challenge (n = 6).

Figure 2

Effects of chlorogenic acid (CGA) supplementation on the intestinal barrier function in broilers under necrotic enteritis (NE) challenge. (A and B) Serum D-LA and DAO concentrations on d 28 and 42. (C and D) Protein expressions for jejunal ZO-1 and occludin on d 28 and 42. Control, control diet; NE, control diet + NE challenge; NE + CGA, control diet + 500 mg/kg CGA + NE challenge; D-LA, D-lactate acid; DAO, diamine oxidase; ZO-1, zonula-occludens-1. ^a,bDifferent superscripts indicate significant differences (P < 0.05) (n = 6).

Figure 3

Effects of chlorogenic acid (CGA) supplementation on the jejunal concentrations of sIgA and inflammatory cytokines in broilers under necrotic enteritis (NE) challenge on (A) d 28 and (B) d 42. Control, control diet; NE, control diet + NE challenge; NE + CGA, control diet + 500 mg/kg CGA + NE challenge; sIgA, secretory immunoglobulin A; IL, interleukin; TNF-α, tumor necrosis factor α; IFN, interferon. ^a,b,cDifferent superscripts indicate significant differences (P < 0.05) (n = 6).

Figure 4

Effects of chlorogenic acid (CGA) supplementation on the jejunal mRNA expressions of inflammatory cytokines in broilers under necrotic enteritis (NE) challenge on (A) d 28 and (B) d 42. Control, control diet; NE, control diet + NE challenge; NE + CGA, control diet + 500 mg/kg CGA + NE challenge; IL, interleukin, TNF-α, tumor necrosis factor α; IFN, interferon. ^a,bDifferent superscripts indicate significant differences (P < 0.05) (n = 6).

Figure 5

Effects of chlorogenic acid (CGA) supplementation on the intestinal proliferation and differentiation in broilers under necrotic enteritis (NE) challenge. (A and B) Number of goblet cells per unit length on d 28 and 42 (scale bar: 100 μm). (C and D) Immunohistochemistry of PCNA on d 28 and 42 (scale bar: 100 μm). Control, control diet; NE, control diet + NE challenge; NE + CGA, control diet + 500 mg/kg CGA + NE challenge. ^a,bDifferent superscripts indicate significant differences (P < 0.05) (n = 6).

Figure 6

Effects of chlorogenic acid (CGA) supplementation on the mitochondrial damage in broilers under necrotic enteritis (NE) challenge. (A and B) Transmission electron microscopy observation of mitochondrial ultrastructure on d 28 and 42, scale bar: 1 μm. Red arrow indicates mitochondrial damage. (C and D) mtDNA and mtROS levels in the jejunum on d 28 and 42. (E and F) MMP, complex I and Ⅲ activities on d 28 and 42. Control, control diet; NE, control diet + NE challenge; NE + CGA, control diet + 500 mg/kg CGA + NE challenge; mtDNA, mitochondrial DNA; mtROS, mitochondrial reactive oxygen species. ^a,b,cDifferent superscripts indicate significant differences (P < 0.05) (n = 6).

Figure 7

Effects of chlorogenic acid (CGA) supplementation on the cGAS–STING signaling pathway in broilers under necrotic enteritis (NE) challenge. (A and B) Protein expressions of jejunal cGAS, STING, TBK1, p-TBK1, IRF7, and p-IRF7 on d 28 and 42. (C and D) Jejunal mRNA expressions of cGAS, STING, TBK1, and IRF7 on d 28 and 42. Control, control diet; NE, control diet + NE challenge; NE + CGA, control diet + 500 mg/kg CGA + NE challenge; cGAS, cyclic GMP–AMP synthase; STING, stimulator of interferon genes; TBK1, TANK-binding kinase 1; IRF7, interferon regulatory factor 7. ^a,bDifferent superscripts indicate significant differences (P < 0.05) (n = 6).