Pasture intake protects against commercial diet-induced lipopolysaccharide production facilitated by gut microbiota through activating intestinal alkaline phosphatase enzyme in meat geese

Abstract

Introduction: Diet strongly affects gut microbiota composition, and gut bacteria can influence the intestinal barrier functions and systemic inflammation through metabolic endotoxemia. In-house feeding system (IHF, a low dietary fiber source) may cause altered cecal microbiota composition and inflammatory responses in meat geese via increased endotoxemia (lipopolysaccharides) with reduced intestinal alkaline phosphatase (ALP) production. The effects of artificial pasture grazing system (AGF, a high dietary fiber source) on modulating gut microbiota architecture and gut barrier functions have not been investigated in meat geese. Therefore, this study aimed to investigate whether intestinal ALP could play a critical role in attenuating reactive oxygen species (ROS) generation and ROS facilitating NF-κB pathway-induced systemic inflammation in meat geese.

Methods: The impacts of IHF and AGF systems on gut microbial composition via 16 sRNA sequencing were assessed in meat geese. The host markers analysis through protein expression of serum and cecal tissues, hematoxylin and eosin (H&E) staining, localization of NF-қB and Nrf2 by immunofluorescence analysis, western blotting analysis of ALP, and quantitative PCR of cecal tissues was evaluated.

Results and discussion: In the gut microbiota analysis, meat geese supplemented with pasture showed a significant increase in commensal microbial richness and diversity compared to IHF meat geese demonstrating the antimicrobial, antioxidant, and anti-inflammatory ability of the AGF system. A significant increase in intestinal ALP-induced Nrf2 signaling pathway was confirmed representing LPS dephosphorylation mediated TLR4/MyD88 induced ROS reduction mechanisms in AGF meat geese. Further, the correlation analysis of top 44 host markers with gut microbiota showed that artificial pasture intake protected gut barrier functions via reducing ROS-mediated NF-κB pathway-induced gut permeability, systemic inflammation, and aging phenotypes. In conclusion, the intestinal ALP functions to regulate gut microbial homeostasis and barrier function appear to inhibit pro-inflammatory cytokines by reducing LPS-induced ROS production in AGF meat geese. The AGF system may represent a novel therapy to counteract the chronic inflammatory state leading to low dietary fiber-related diseases in animals.

Keywords: Keap1-Nrf2; gut microbiota; inflammation; intestinal alkaline phosphatase; lipopolysaccharides; meat geese; oxidative stress; pasture grazing.

Figure 1

Artificial pasture grazing system modulates gut microbiota to inhibit LPS synthesis induced by in-house feeding system. (A) Average abundance per sample of genes related to the four main LPS biosynthesis-related functions. Red indicates a positive correlation; green indicates a negative correlation, (B) Relative contributions of the different phyla to the total LPS-encoding capacity of the gut microbiome, and (C) Contribution of individual genera to LPS biosynthesis functions. The average abundances of genes related to any of the four LPS-related GO functions are shown for individual genera within each phylum. LP A biosyn. AC.; lipid A biosynthesis acyltransferase, LP A biosyn.; lipid A biosynthesis, LPS trans. peri. prot. lptA; lipopolysaccharide transport periplasmic protein lptA, and LPS biosyn. proc.; lipopolysaccharide biosynthesis process. Red indicates a positive correlation; blue indicates a negative correlation. In-house feeding system (IHF) and artificial pasture grazing system (AGF). The asterisks symbol indicates significant differences *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2

Inhibitory effects of artificial pasture grazing system on in-house feeding system-induced ROS production via LPS/TLR4/MyD88 pathway in meat geese. (A) ALP protein level in serum, (B) ALPi mRNA level in cecal tissues, (C) mRNA levels of intestinal ALP genes (CG5150 and CG10827) in cecal tissues, (D) LPS protein level in serum, (E) mRNA levels of LPS biosynthesizing genes (rfaK and rfaL) in cecal tissues, (F) mRNA levels of lipid A biosynthesizing genes (lpxA, lpxB, lpxC, and lpxD) in cecal tissues, (G) LBP mRNA level in cecal tissues, (H) sCD14 mRNA level in cecal tissues, (I) TLR4 mRNA level in cecal tissues, (J) MyD88 mRNA level in cecal tissues, and (K) ROS protein level in serum, normalized by β-actin and measured by qPCR. In-house feeding system (IHF) and artificial pasture grazing system (AGF). Data with different superscript letters are significantly different (P < 0.05) according to the unpaired student T-Test. The asterisks symbol indicates significant differences *P < 0.05, **P < 0.01.

Figure 3

Beneficial effects of artificial pasture grazing system on in-house feeding system-dependent apoptosis-induced gut permeability in meat geese. (A) Cytochrome C mRNA level in cecal tissues, (B) CASP3 mRNA level in cecal tissues, and (C) CASP8 mRNA level in cecal tissues. (D) H&E staining of cecal tissues (magnification, 40×) for the number of apoptotic cells per field (Mean ± SD). (E) MUC2 mRNA level in cecal tissues, and (F) MUC5AC mRNA level in cecal tissues, normalized by β-actin and measured by qPCR. (G) ZO-1 protein level in cecal tissues, (H) Occludin protein level in cecal tissues, and (I) Claudin protein level in cecal tissues. In-house feeding system (IHF) and artificial pasture grazing system (AGF). Data with different superscript letters are significantly different (P < 0.05) according to the unpaired student T-Test. The asterisks symbol indicates significant differences **P < 0.01.

Figure 4

Inhibitory effects of artificial pasture grazing system on in-house feeding system-induced NF-κB pathway and its systemic inflammation. (A) LC8 mRNA level in cecal tissues, (B) IKB-a mRNA level in cecal tissues, (C) NF-κB mRNA level in cecal tissues, (D) NF-κB-regulated genes (IL-8, CCL2, PLAU, and BIRC3) mRNA levels in cecal tissues, (E) Immunofluorescence (IF) analysis using Rabbit Anti-NF-κB (P65-AF5006) (1:200; v/v) showing nuclear translocation of NF-κB in the cecal tissues of meat geese. Blue: nucleus (DAPI); Green: NF-κB-staining; Cerulean blue: merge of blue and green indicating nuclear localization of NF-κB, scale bar = 20 µm. (F) iNOS mRNA level in cecal tissues, (G) COX2 mRNA level in cecal tissues, (H) IL-1β mRNA level in cecal tissues, (I) IL-6 mRNA level in cecal tissues, and (J) TNF-a mRNA level in cecal tissues, normalized by β-actin and measured by qPCR. In-house feeding system (IHF) and artificial pasture grazing system (AGF). Data with different superscript letters are significantly different (P < 0.05) according to the unpaired student T-Test. The asterisks symbol indicates significant differences *P < 0.05, **P < 0.01. The lack of a superscript letter means that all differences were nonsignificant (ns).

Figure 5

Effects of Artificial Pasture Grazing System on Activation of Nrf2 Pathway in Meat Geese (A) Keap1 mRNA level in cecal tissues, (B) Nrf2 mRNA level in cecal tissues, and (C) Nrf2-regulated genes (NQO1, Gclc, Gclm, and GSTA4) mRNA levels in cecal tissues, normalized by β-actin and measured by qPCR. (D) Immunofluorescence (IF) analysis using Rabbit anti-Nrf2 (bs1074R) (1:500; v/v) showing nuclear translocation of Nrf2 in the cecal tissues of meat geese. Blue: nucleus (DAPI); Green: Nrf2-staining; Cerulean blue: merge of blue and green indicating nuclear localization of Nrf2, scale bar = 20 µm. (E) HO-1 protein level (F) GSR protein level (G) T-SOD protein level (H) GSH-PX protein level (I) T-AOC protein level (J) CAT protein level and (K) MDA protein level. In-house feeding system (IHF) and artificial pasture grazing system (AGF). Data with different superscript letters are significantly different (P < 0.05) according to the unpaired student T-Test. The asterisks symbol indicates significant differences *P < 0.05, **P < 0.01.

Figure 6

Artificial pasture grazing system attenuates the in-house feeding system-induced endotoxemia, gut permeability, and chronic systemic inflammation of meat geese. (A) Western blot analysis was performed to detect the protein levels of ALP in the cecal tissues, normalized by GAPDH. (B) ALP protein level in cecal tissues, (C) LPS protein level in cecal tissues, and (D) ROS protein level in cecal tissues. (E) ZO-1 mRNA level in cecal tissues, (F) Occludin mRNA level in cecal tissues, (G) Claudin mRNA level in cecal tissues, (H) dlg1 mRNA level in cecal tissues, (I) E-cadherin mRNA level in cecal tissues, (J) IL-4 mRNA level in cecal tissues, (K) IL-10 mRNA level in cecal tissues. In-house feeding system (IH) and artificial pasture grazing system (AGF). Data with different superscript letters are significantly different (P < 0.05) according to the unpaired student T-Test. The asterisks symbol indicates significant differences *P < 0.05, **P < 0.01. The lack of a superscript letter means that all differences were nonsignificant (ns).

Figure 7

Effect of different feeding systems on KEAP1-induced aging phenotypes in meat geese. (A) p19ARF mRNA level in cecal tissues, (B) p16INK4α mRNA level in cecal tissues, and (C) p21 mRNA level in cecal tissues, normalized by β-actin and measured by qPCR. In-house feeding system (IHF) and Artificial pasture grazing system (AGF). Data with different superscript letters are significantly different (P < 0.05) according to the unpaired student T-Test. The asterisks symbol indicates significant differences **P < 0.01.

Figure 8

Effect of different feeding systems on metabolic profile of meat geese. (A) Body weight (kg), (B) LDL-C protein level in serum, (C) HDL-C protein level in serum, (D) TG protein level in serum, (E) T-CHO protein level in serum, (F) Blood glucose levels, and (G) BUN protein level in serum. In-house feeding system (IHF) and artificial pasture grazing system (AGF). Data with different superscript letters are significantly different (P < 0.05) according to the unpaired student T-Test. The asterisks symbol indicates significant differences **P < 0.01.

Figure 9

Association and model predictive analysis of the top 44 host markers with the highest correlation scores. (A) Correlation between gut microbiota and host markers by Spearman correlation analysis. Red squares indicate a positive correlation; whereas blue squares indicate a negative correlation. (B) The labels on the abscissa and the longitudinal axis represent Pearson’s correlation heatmap among host markers. Red squares indicate a positive correlation and blue squares indicate a negative correlation. Deeper colors indicate stronger correlation scores. The asterisks symbol indicates significant differences *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 10

Diagram illustrating a proposed mechanism by which artificial pasture grazing system as a high dietary fiber source up-regulates intestinal ALP-producing bacteria and Nrf2 signaling pathway while downregulating LPS-producing bacteria and ROS in meat geese. The increase in intestinal ALP-producing bacteria and the activation of Nrf2 signaling pathway maintain antioxidant and anti-inflammatory mechanisms that lower LPS-producing bacteria and LPS-induced ROS generation, intestinal mucosal deterioration, gut permeability, and metabolic endotoxemia. In the first step, the intestinal ALP attack on TLR4 and let the lipid A moiety not to allow LPS to bind with TLR4 and dephosphorylate LPS by breaking TLR4/MyD88-induced ROS production. In the second step, intestinal ALP activates Nrf2 pathway which reduces oxidative stress, so that ROS could not oxidize LC8 protein and deteriorate IKB-a to activate NF-κB pathway. In this way, intestinal ALP activates anti-inflammatory cytokines and then attenuates chronic low-grade inflammation, aging phenotypes, and metabolic syndrome. BG, blood glucose; TG, triglyceride; BW, body weight; BUN, blood urea nitrogen.