Macleaya cordata isoquinoline alkaloids attenuate Escherichia coli lipopolysaccharide-induced intestinal epithelium injury in broiler chickens by co-regulating the TLR4/MyD88/NF-κB and Nrf2 signaling pathways

Abstract

This study sought to explore the effects and potential mechanisms of dietary supplementation with isoquinoline alkaloids (IA) from Macleaya cordata to alleviate lipopolysaccharide (LPS)-induced intestinal epithelium injury in broilers. A total of 486 1-day-old broilers were assigned at random to a control (CON) group, LPS group, and LPS+IA group in a 21-d study. The CON and LPS groups received a basal diet, while the LPS+IA group received a basal diet supplemented with 0.6 mg/kg IA. At 17, 19, and 21 days of age, the LPS and LPS+BP groups were injected intraperitoneally with LPS, and the CON group was intraperitoneally injected equivalent amount of saline solution. The results manifested that LPS injection caused intestinal inflammation and lipid peroxidation, disrupted intestinal barrier and function, and increased the abundance of harmful microorganisms. However, dietary IA supplementation alleviated LPS-induced adverse changes in intestinal morphology, apoptosis, mucosal barrier integrity, cecum microorganisms, and homeostasis disorder by decreasing inflammatory cytokines and enhancing antioxidant-related genes expressions; inhibited LPS-induced increases in TLR4 and NF-κB expressions and decreases in Nrf2 and GPX1 genes expressions. Our findings indicated that Macleaya cordata IA addition attenuated LPS-induced intestinal epithelium injury and disorder of intestinal homeostasis by enhancing the anti-inflammatory and antioxidant capacity of broiler chickens possibly via co-regulating TLR4/MyD88/NF-κB and Nrf2 signaling pathways.

Keywords: Macleaya cordata isoquinoline alkaloid; broiler; intestinal epithelium injury; intestinal inflammation; lipopolysaccharide; oxidative stress.

Figure 1

Effects of dietary supplementation with isoquinoline alkaloids on serum inflammatory in LPS-challenged broiler chickens. (A) Interleukin-1β (IL-1β); (B) Interleukin-6 (IL-6); (C) Interleukin-10 (IL-10); and (D) Tumor necrosis factor-α (TNF-α). CON group, broiler chickens fed a basal diet; LPS group, LPS-challenged broilers fed basal diets; and LPS+IA group, LPS-challenged broilers fed basal diets with isoquinoline alkaloids 0.6mg/kg. Values are presented as mean and standard error of mean (n = 6). #0.05 ≤ P < 0.10, **P < 0.01, ***P < 0.001.

Figure 2

Effects of dietary supplementation with isoquinoline alkaloids on serum D-lactate and DAO in LPS-challenged broiler chickens. (A) DAO; and (B) D-lactate. CON group, broiler chickens fed a basal diet; LPS group, LPS-challenged broilers fed basal diets; and LPS+IA group, LPS-challenged broilers fed basal diets with isoquinoline alkaloids 0.6mg/kg. Values are presented as mean and standard error of mean (n = 6). #0.05 ≤ P < 0.10, **P < 0.01, ***P < 0.001.

Figure 3

Effects of dietary supplementation with isoquinoline alkaloids on intestinal morphology and pathology in LPS-challenged broiler chickens. (A) Representative images (×100) were stained with hematoxylin and eosin; (B) Villus height; (C) Crypt depth; and (D) Villus height to crypt depth ratio. CON group, broiler chickens fed a basal diet; LPS group, LPS-challenged broilers fed basal diets; and LPS+IA group, LPS-challenged broilers fed basal diets with isoquinoline alkaloids 0.6mg/kg. Values are presented as mean and standard error of mean (n = 6). #0.05 ≤ P < 0.10, *P < 0.05, ***P < 0.001.

Figure 4

Effects of dietary supplementation with isoquinoline alkaloids on serum and intestinal antioxidant indexes in LPS-challenged broiler chickens. (A, F) Glutathione, (GSH); (B, G) Superoxide dismutase, (SOD); (C, H) Malondialdehyde, (MDA); (D, I) Total antioxidant capacity, (T-AOC); and (E, J) Hydrogen peroxide, (H₂O₂). CON group, broiler chickens fed a basal diet; LPS group, LPS-challenged broilers fed basal diets; and LPS+IA group, LPS-challenged broilers fed basal diets with isoquinoline alkaloids 0.6mg/kg. Values are presented as mean and standard error of mean (n = 6). #0.05 ≤ P < 0.10, *P < 0.05, ***P < 0.001.

Figure 5

Effects of dietary supplementation with isoquinoline alkaloids on intestinal barrier function in LPS-challenged broiler chickens. (A) Trefoil peptides (TFF); (B) Transforming growth factor alpha (TGF-α); (C) Mucin 2 (MUC2); and (D) Zonula occludens-1 (ZO-1). CON group, broiler chickens fed a basal diet; LPS group, LPS-challenged broilers fed basal diets; and LPS+IA group, LPS-challenged broilers fed basal diets with isoquinoline alkaloids 0.6mg/kg. Values are presented as mean and standard error of mean (n = 6). #0.05 ≤ P < 0.10, *P < 0.05, ***P < 0.001.

Figure 6

Effects of dietary supplementation with isoquinoline alkaloids on caspases levels in LPS-challenged broiler chickens. (A) caspase-3; (B) caspase-8; and (C) caspase-9. CON group, broiler chickens fed a basal diet; LPS group, LPS-challenged broilers fed basal diets; and LPS+IA group, LPS-challenged broilers fed basal diets with isoquinoline alkaloids 0.6mg/kg. Values are presented as mean and standard error of mean (n = 6). **P < 0.01, ***P < 0.001.

Figure 7

Effects of dietary supplementation with isoquinoline alkaloids on intestinal apoptotic index in LPS-challenged broiler chickens. (A) TUNEL assay. The blue color represents the total cells, and the green color represents the apoptosis cells. (B) Positive cell density. CON group, broiler chickens fed a basal diet; LPS group, LPS-challenged broilers fed basal diets; and LPS+IA group, LPS-challenged broilers fed basal diets with isoquinoline alkaloids 0.6mg/kg. Values are presented as mean and standard error of mean (n = 6). *P < 0.05.

Figure 8

Effects of dietary supplementation with isoquinoline alkaloids on intestinal inflammatory factors and intestinal SIgA concentrations in LPS-challenged broiler chickens. (A) Tumor necrosis factor-α (TNF-α); (B) Interleukin-1β (IL-1β); (C) Interleukin-6 (IL-6); (D) Interleukin-10 (IL-10); (E) Interferon-γ (IFN-γ); and (F) Secretory immunoglobulin A (SIgA). CON group, broiler chickens fed a basal diet; LPS group, LPS-challenged broilers fed basal diets; and LPS+IA group, LPS-challenged broilers fed basal diets with isoquinoline alkaloids 0.6mg/kg. Values are presented as mean and standard error of mean (n = 6). #0.05 ≤ P < 0.10, **P < 0.01, ***P < 0.001.

Figure 9

Effects of dietary supplementation with isoquinoline alkaloids on the expression of barrier genes in LPS-challenged broiler chickens. (A) Zonula occludens-1 (ZO-1); (B) Occludin (OCLN); (C) Claudin-2 (CLDN2); and (D) Claudin-3 (CLDN3). CON group, broiler chickens fed a basal diet; LPS group, LPS-challenged broilers fed basal diets; and LPS+IA group, LPS-challenged broilers fed basal diets with isoquinoline alkaloids 0.6mg/kg. Values are presented as mean and standard error of mean (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 10

Effects of dietary supplementation with isoquinoline alkaloids on the expression of nutrient transport genes in LPS-challenged broiler chickens. (A) Glucose transporter type 2 (GLUT2); (B) Sodium-glucose transporter 1 (SGLT1); (C) y + L amino acid transporter-1 (y + LAT1); and (E) Fatty acid binding protein-1 (FABP1). CON group, broiler chickens fed a basal diet; LPS group, LPS-challenged broilers fed basal diets; and LPS+IA group, LPS-challenged broilers fed basal diets with isoquinoline alkaloids 0.6mg/kg. Values are presented as mean and standard error of mean (n = 6). **P < 0.01.

Figure 11

Effects of dietary supplementation with isoquinoline alkaloids on the expression of inflammatory genes in LPS-challenged broiler chickens. (A) Toll-like receptor 4 (TLR4); (B) Myeloid differentiation primary response 88 (MyD88); and (C) Nuclear factor-kappa B (NF-κB). CON group, broiler chickens fed a basal diet; LPS group, LPS-challenged broilers fed basal diets; and LPS+IA group, LPS-challenged broilers fed basal diets with isoquinoline alkaloids 0.6mg/kg. Values are presented as mean and standard error of mean (n = 6). #0.05 ≤ P < 0.10, *P < 0.05, ***P < 0.001.

Figure 12

Effects of dietary supplementation with isoquinoline alkaloids on the expression of antioxidant genes in LPS-challenged broiler chickens. (A) Sirtuin1 (Sirt1); (B) Nuclear factor erythroid 2-related factor 2 (Nrf2); (C) Heme-oxygenase 1 (HO-1); (D) Catalase (CAT); (E) Superoxide dismutase 1 (SOD1); (F) Superoxide dismutase 2 (SOD2); (G) glutathione peroxidase-1 (GPX1); and (H) NAD(P)H quinone oxidoreductase 1 (NQO1). CON group, broiler chickens fed a basal diet; LPS group, LPS-challenged broilers fed basal diets; and LPS+IA group, LPS-challenged broilers fed basal diets with isoquinoline alkaloids 0.6mg/kg. Values are presented as mean and standard error of mean (n = 6). #0.05 ≤ P < 0.10, *P < 0.05, **P < 0.01.

Figure 13

Effects of dietary supplementation with isoquinoline alkaloids on the expression of apoptosis genes in LPS-challenged broiler chickens. (A) Bcl-2 associated X (Bax); (B) B-cell lymphoma-2 (Bcl-2); and (C) Bax/Bcl-2 ratio. CON group, broiler chickens fed a basal diet; LPS group, LPS-challenged broilers fed basal diets; and LPS+IA group, LPS-challenged broilers fed basal diets with isoquinoline alkaloids 0.6mg/kg. Values are presented as mean and standard error of mean (n = 6). *P < 0.05.

Figure 14

Alpha diversity and richness of the cecal microbiota. (A) The species accumulation curves; (B) The rarefaction curve of OTU; (C) Shannon index; (D) Simpson index; (E) ACE index; and (F) Chao 1 index. CON group, broiler chickens fed a basal diet; LPS group, LPS-challenged broilers fed basal diets; and LPS+IA group, LPS-challenged broilers fed basal diets with isoquinoline alkaloids 0.6mg/kg. Values are presented as mean and standard error of mean (n = 6).

Figure 15

Beta diversity analysis of the cecal microbiota. (A) The principal coordinate analysis (PCoA); (B) Unweighted pair-group method with arithmetic mean (UPGMA) phylogenetic tree; and (C) The analysis of ANOSIM. CON group, broiler chickens fed a basal diet; LPS group, LPS-challenged broilers fed basal diets; and LPS+IA group, LPS-challenged broilers fed basal diets with isoquinoline alkaloids 0.6mg/kg. n = 6. Differences between groups were considered significant at P < 0.05.