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

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.


Introduction
Intestinal homeostasis seems to be a defining factor for poultry health that is affected by oxidative stress either produced by heat stress or feed stress (1). The poultry birds such as broilers, layers, geese, and turkeys are continuously exposed to lipopolysaccharides (LPS) via different routes such as feed, water, and fine dust particles in the house that always contain some amounts of LPS. However, the major natural source of LPS is the complex community of gram-negative bacteria in the intestines (2). LPS is an outer membrane component of gram-negative bacteria such as Enterobacteriaceae (3), Escherichia coli (E. coli) (4), Bacterodales (5), and Cyanobacteria (6) that are recognized by toll-like receptors (TLRs), particularly TLR4 and then invade the intestinal tissues and get access to the bloodstream thereby provoking reactive oxygen species (ROS)-induced systemic diseases. This leads to a leaky gut (7) that causes diarrhea, decreased nutrient absorption, and internal fluid loss in broilers (8).
From different studies, two supporting shreds of evidence suggest that nuclear factor kappa B (NF-kB) is activated either by ROS (9) or LPS (10). Further, LPS and ROS phosphorylate NF-kB inhibitor a (IkB-a) and let the NF-kB migrate to the nucleus and then exert their inflammatory and apoptotic impacts by activating NF-kB pathway (11). When NF-kB pathway is established, then it consistently contributes to inducing chronic low-grade inflammation (12) through upregulating oxidative stress (N. 13), pro-inflammatory mediators i.e. inducible nitric oxide (iNOS) and cyclooxygenase (COX)-2 and pro-inflammatory cytokines (i.e. IL-1b, IL-6, and TNF-a) (14, 15).
Gut microbial-induced LPS-mediated NF-kB activity orchestrates chronic low-grade inflammation that is a major disease risk factor in today's animals' life (16). Thus, modifiable factors that can reduce LPS-induced inflammation may potentially modulate disease risk. Different possible modifiable factors such as feed antibiotics (17), dietary fiber and threonine (18), pectin (19), Schisandra A (12), phytochemicals (20), and oral alkaline phosphatase (21) have been used in animals to detoxify LPS. Feed antibiotics increase food-born pathogenic bacterial-induced LPS resistance (22) while phytochemicals used as anticancer drugs show minimum side effects in animals (20). Albeit different dietary fibers detoxify LPS-induced chronic lowgrade inflammation (18), nobody did clearly explain the mechanisms of how dietary fibers ameliorate LPS-mediated systemic inflammation facilitated by gut microbiota in meat geese.
Different dietary fiber sources such as glucomannan, oligosaccharides, sialyl lactose, and galactooligosaccharides shape the gut microbiota to regulate intestinal ALP in rats and other animals (23, 24). Intestinal ALP is an endogenous antimicrobial peptide that is secreted from the apical enterocytes of small intestine and then moves toward the large intestine (25). Intestinal ALP has been shown to dephosphorylate LPS, CpG-DNA, and flagellin (26). In previous studies, lipid A moiety plays a role of bridge in activating myeloid differentiation factor 88 (MyD88) pathway by binding LPS with TLR4 and then activating oxidative stress (27) and NF-kB-induced systemic inflammation (12). In previous studies is not defined clearly to what extent and in which way the intestinal ALP dephosphorylates LPS by breaking TLR4/MyD88-induced ROS production and NF-kB-induced systemic inflammation. Furthermore, intestinal ALP promotes healthy homeostasis of gut microbiota (28) and gut barrier functions (29), which has been associated with lowering systemic inflammation in several studies of healthy adults (30) and their specific health conditions (e.g. obesity, diabetes, and cardiovascular disease) (31). The increasing evidence of the salutary functions of ALP emphasizes the significance of the naturally occurring brush border enzyme. Therefore, there is a need to find optional nutritional strategies that could naturally induce and regulate the endogenous growth of intestinal ALP in poultry birds. One of the optional possible nutritional strategies is the application of dietary fiber which has been used in different studies to induce intestinal ALP production (24).
Geese are herbivorous, and because of their unique ability to use high fiber feeds, pasture was suggested to be included to promote health (32). In China, the most commonly available pasture is ryegrass, which is rich in protein, dietary fiber, fatty acids, iron, zinc, magnesium, calcium, vitamins, essential amino acids, alkaloids, steroids, flavonoids, glycosides, phenols, and tannins (33). Recently, several reports suggest that ryegrass could regulate intestinal microflora of Beijing-you chickens (34) and improve ethnomedical properties like being antioxidant, antimicrobial, and antiinflammatory diseases in animals (35,36). However, up to now, formal mediation analysis experiments about the association between ryegrass (high dietary fiber) intake-induced endogenous ALP-regulated kelch-like ECH-Associating protein 1-nuclear factor erythroid 2 (Keap1-Nrf2) pathway and LPS-induced oxidative stress is lacking. Moreover, the Keap1-Nrf2 system plays a central role in the regulation of the oxidative stress response, and that Nrf2 coordinately regulates cytoprotective genes (37). Here we reported that the regulation of gut microbial-induced endogenous intestinal ALP by pasture intake preserves the normal homeostasis of gut microbiota, dephosphorylates LPS/TLR4/MyD88 pathwaymediated ROS insults, and activates Keap1-Nrf2 pathway to alleviate NF-kB-induced systemic inflammation in meat geese.

Ethical approval
According to animal care guidelines, the study was conducted using Wanfu geese. All used procedures were approved by the Research Bioethics Committee of the Henan Agricultural University (approval HENAU-2021).

Animals, diets, and housing
A total of 180, 25-day-old Wanfu mixed-sex geese from the commercial geese farm were purchased from Henan Daidai goose Agriculture and Animal husbandry development Co., Ltd (Zhumadian, China). The geese with an average weight of 693.6 g ( ± 3.32) were divided into two homogeneous groups: (1) in-house feeding group (IHF, n = 90) and (2) artificial pasture grazing group (AGF, n = 90), 12h artificial pasture grazing (06:00-18:00h) with once a day (19:00h) in-house feeding group. Each group consisted of six replicates with 15 geese per replicate. All the geese had free access to feed and freshwater ad-libitum. The IHF group meat geese were fed a commercial diet (Table 1). Two diets were used: a grower diet (25 to 45 days) and a finisher diet (46 to 90 days). The AGF system was established in form of grazing of meat geese at the expense of ryegrass. The nutritional composition of ryegrass was dry matter (90%), crude protein (15.47%), ash (8%), neutral detergent fiber (65%), acid detergent fiber (38%), ether extract (3.3%), calcium (0.90%), and phosphorous (0.47%). The experiment lasted for 66 days (Supplementary Figure 1).

Sample collection
Body weight and feed intake were measured every week. The pasture feed intake was measured using the method described by Cartoni Mancinelli et al. (38). On days 45, 60, and 90, we selected six healthy meat geese per replicate with a body weight range of ± 1 std. from mean 1.63-2.31 kg, 3.33-4.28 kg, and 4.38-5.39 kg respectively. Blood samples were collected in non-anticoagulant sterile blood vessels from the jugular vein. Serum samples were then obtained by centrifuging the blood samples at 4,000 × g for 15 minutes at 4°C and stored at -80°C until analysis. After blood sampling, the geese were slaughtered and pH was determined from the proventriculus, gizzard, ileum, and cecum. Fresh cecal chyme was collected from the cecum using sterile 5 ml centrifuge tubes and then stored at -80°C for further analysis. The cecal tissues were immediately removed, thoroughly washed with phosphate-buffered saline (PBS), stored in liquid nitrogen, and then preserved at -80°C for further analysis. Cecal tissues were fixed by immersion in 4% and 10% neutral buffered formalin.

Measurement of LPS, ROS, and ALP levels
The serum and cecal tissues were sampled to measure LPS, ROS, and ALP activities. The kits were purchased from Shanghai Enzyme Link Biotechnology Co., Ltd (Shanghai, China) and all experimental procedures were performed according to the manufacturer's instructions.

Bacterial growth conditions
LPS is a key virulence factor of Escherichia coli (E. coli) that triggers innate immune responses via activation of the toll-like receptor 4 signaling pathway (39). To identify, whether E. coli contributes to activating the LPS, the batch cultivation for E. coli was carried out in Luria broth (LB) medium at 37°C with a 2-L working volume. LB medium was from recipe of Miller (5 g yeast extract, 10 g peptone tryptone, 10 g NaCl) (40). The pH was maintained at 6.95 automatically by titration with 5% H 2 SO 4 or 5% NaOH. Ampicillin was added to control the growth of other bacteria. Peptone tryptone and yeast extract were from OXOID, NaCl from Sigma, agar, and ampicillin from Solarbio (life sciences). The medium was made in distilled water and autoclaved under standard conditions. Dissolved oxygen in the culture was maintained at 40% saturation automatically by varying the speed of impeller rotation. Culture growth (OD600) was monitored with a DU640 Spectrophotometer (Beckman). Further, E. coli was cultured onto the Petri dishes for 24h at 37°C. The CFU/g stool for E. coli from the Petri dishes was counted.

Measurement of gut permeability
The concentration of tight junction proteins is a widely recognized indicator known as gut permeability. For this, the tight junction proteins ZO-1, Occludin, and Claudin concentrations were determined from the cecal tissues in the present study. The kits were purchased from Shanghai Enzyme Link Biotechnology Co., Ltd (Shanghai, China) and all experimental procedures were performed according to the manufacturer's instructions. Further, mRNA expression levels of ZO-1, Occludin, and Claudin and 2 genes encoding tight junction proteins dlg1 and E-cadherin were also measured from cecal tissues for gut barrier functions. Details related to gene expression are proposed to be harmonized in section 2.14.
Hematoxylin and eosin staining F i r s t , t h e c e c a l t i s s u e s w e r e fi x e d w i t h 1 0 % paraformaldehyde and stained with Harris' hematoxylin solution for 6h at a temperature of 60-70°C and were then rinsed in tap water until the water was colorless. Next, 10% acetic acid and 85% ethanol in water were used to distinguish the tissue 2 times for 2h and 10h, and again the tissues were rinsed with tap water. In the bluing step, we soaked the tissue in saturated lithium carbonate solution for 12h and then rinsed it with tap water. Finally, staining was achieved with eosin Y ethanol solution for 48h.

Paraffin embedding
The tissues were dehydrated with 95% ethanol twice for 0.5h, and then soaked in xylene for 1h at 60-70°C followed by paraffin for 12h. For the cecal tissues, we used 0.5 ml of 95% ethanol in dehydration.

Slicing and imaging
The stained tissues were cut into 3 sections of 7-µm slices with well-oriented parts using a Leica RM2235 microtome and then imaged using Nikon NIS-Elements microscopy.

Localization of NF-kB and Nrf2 by immunofluorescence analysis
The tissue sections were fixed in 4% paraformaldehyde for 10 minutes. Paraffin sections were dewaxed with xylene for 15 minutes, dehydrated with gradient alcohol, and rinsed in distilled water. Dewaxed and rehydrated slides of cecal tissue sections were boiled in citrate-EDTA antigen retrieval solution for 2 minutes and were blocked with 10% fetal bovine serum for 30 minutes. The tissue sections were incubated for NF-kB and Nrf2 with primary antibody Rabbit Anti-NF-kB (Affinity, P65-AF5006, 1:200; v/v) or Rabbit anti-Nrf2 (Bioss, bs-1074R, 1:500; v/v) respectively, at 4°C overnight followed by secondary antibody (HRP Goat Anti-Rabbit IgG (SeraCare, 5220-0336, 1:400; v/v) with incubation at room temperature in the dark for 50 minutes. Tyramine salt fluorescein was added dropwise to the tissues (configured with phosphate-buffered saline with Tween 20 (PBST) containing 0.0003% H 2 O 2 ) and incubated at room temperature for 20 minutes. 4',6-dia-midino-2-phenylindole (DAPI) was used to incubate the slides for 10 minutes at room temperature. Finally, the slides were sealed with anti-fluorescent quenching mounting medium, and the localization of Nrf2 and NF-kB was detected with a confocal fluorescence microscope (TCSSP8STED, Leica, Wetzlar, Germany). The nuclei stained by DAPI are blue under ultraviolet excitation and are positively expressed as the corresponding fluorescein-labeled green light.

RNA extraction and RT-qPCR
Total RNA from the cecal tissues (about 50 to 100 mg) was extracted by the addition of 1 mL of MagZol-reagent (#R4801-02; Magen Biotechnology, Guangzhou, Guangdong, China) according to the manufacturer's instructions. The concentration and purity of the total RNA were assessed using a NanoDrop 2000 UV-vis spectrophotometer (Thermo Scientific, Wilmington, USA). Subsequently, the RNA was reverse transcribed to cDNA using the ReverTra Ace ® qPCR RT Master Mix with gDNA Remover (TOYOBO, OSAKA, Japan) according to the manufacturer's instructions. The cDNA samples were amplified by real-time quantitative polymerase chain reaction with ChamQ Universal SYBR qPCR Master Mix from Vazyme Biotechnology (Nanjing, Jiangsu, P. R. China). Gene-specific primers for each gene were designed using Primer3web, version 4.1.0 (Supplementary Table 1). PCR was performed on the C1000 Touch PCR Thermal cycler (BIO-RAD Laboratories, Shanghai, China) using ChamQ Universal SYBR qPCR Master Mix from Vazyme Biotechnology (Nanjing, Jiangsu, P. R. China) and was as follows: 40 cycles of 95°C for 15 s and 60°C for 30 s. All measurements will be performed in triplicate. The messenger ribonucleic acid (mRNA) expression of target genes relative to beta-actin (b-actin) was calculated using 2 -DDCT method (41).

DNA extraction and PCR amplification
According to the manufacturer's instructions, the microbial community genomic DNA was extracted from cecal chyme samples using the E.Z.N.A. ® soil DNA Kit (Omega Bio-Tek, Norcross, GA, U.S.). The DNA extract was checked on 1% agarose gel, and DNA concentration and purity were determined with NanoDrop 2000 UV-vis spectrophotometer (Thermo Scientific, Wilmington, USA). The hypervariable region V3-V4 of the bacterial 16S rRNA gene was amplified with primer pairs 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3') by an ABI GeneAmp ® 9700 PCR thermocycler (ABI, CA, USA). The PCR amplification of the 16S rRNA gene was performed as follows: initial denaturation at 95°C for 3 minutes, followed by 27 cycles of denaturing at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 45 s, and single extension at 72°C for 10 minutes, and end at 4°C. The PCR mixtures contain 5 × TransStart FastPfu buffer 4 mL, 2.5 mM dNTPs 2 mL, forward primer (5 mM) 0.8 mL, reverse primer (5 mM) 0.8 mL, TransStart FastPfu DNA Polymerase 0.4 mL, template DNA 10 ng, and finally ddH 2 O up to 20 mL. PCR reactions were performed in triplicate. The PCR product was extracted from 2% agarose gel and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) according to the manufacturer's instructions and quantified using Quantus ™ Fluorometer (Promega, USA).

Processing of sequencing data
The raw 16S rRNA gene sequencing reads were demultiplexed, quality-filtered by Trimmomatic, and merged by FLASH with the following criteria: (i) the 300 bp reads were truncated at any site receiving an average quality score of <20 over a 50 bp sliding window, and the truncated reads shorter than 50 bp were discarded, reads containing ambiguous characters were also discarded; (ii) only overlapping sequences longer than 10 bp were assembled according to their overlapped sequence. The maximum mismatch ratio of overlap region is 0.2. Reads that could not be assembled were discarded; (iii) samples were distinguished according to the barcode and primers, and the sequence direction was adjusted, exact barcode matching, 2 nucleotides mismatches in primer matching. The taxonomy of each OTU representative sequence was analyzed by RDP Classifier (http://rdp.cme.msu.edu/) against the 16S rRNA database (e.g. Silva SSU128) using a confidence threshold of 0.7. The species composition and relative abundance of each sample were counted at the phylum level, and the composition of the dominant species of different groups was visualized by the package pie chart of R (version 3.3.1) software. PICRUSt is a software package for the functional prediction of 16S amplicon sequencing results which was used to determine the COG IDs related to LPS production. Spearman rank correlation coefficient was performed to construct a correlation heatmap among the highly abundant GO terms and cecal microbiota at phylum as well as genus levels mostly relevant to LPS production. Further, to determine the effect of microbiota interacting with apparent performance, redundancy analysis (RDA) was performed at the genus level using the R language vegan packet on Spearman correlation analysis (RDA 2014).

Statistical analysis
Data were expressed as mean ± SEM. Statistical analyses were performed using SPSS 20.0 software (=D3 SPSS, Inc., 2009, Chicago, IL, USA www.spss.com). Data from two groups were evaluated by unpaired two-tailed student T-Test. Significance was considered to be at P< 0.05. Spearman correlation analysis of the Euclidean distance was performed using GraphPad Prism version 8.3.0., and origin 2021. To compare host markers' relationships, Pearson's correlation analysis was performed by OECloud tools (https://cloud.oebiotech.cn).

Results
Artificial pasture grazing system modulates gut microbiota to inhibit LPS synthesis induced by in-house feeding system The metagenome predicted functions classified using clusters of orthologous genes (COG) database in phylogenetic reconstruction of unobserved states (PICRUSt) software are performed to investigate the functional differences in the gut microbiota between the two feeding meat geese groups (in-house feeding group (IHF) and artificial pasture grazing group (AGF) meat geese) at different time points 45d, 60d, and 90d. A total of 4060, 4069, 4082, 3959, 4030, and 4060 COG IDs were identified in samples IHF45, AGF45, IHF60, AGF60, IHF90, and AGF90 respectively (unpublished data). To identify which strains contribute to LPS production in the meat geese gut, we focused on the GO terms and cecal microbiota at the phylum level mainly relevant to LPS biosynthesis ( Figure 1A and Supplementary Figures 2A-F). At 45d, in the IHF meat geese, Firmicutes families dominated the four while Actinobacteiota dominated the two main COG terms related to LPS biosynthesis. At 60d, in the IHF meat geese, Actinobacteriota and Proteobacteria families dominated the four and two main COG terms related to LPS biosynthesis, respectively. At 90d, only Bacteroidota family dominated the three main COG terms related to LPS biosynthesis in IHF meat geese.
Overall, Firmicutes and Bacteroidota families dominate both in the IHF and AGF meat geese at 45d, 60d, and 90d, but Firmicutes and Bacteroidota individually contribute 68.45% and 32.62% of the LPS biosynthesis in the IHF meat geese at 45d and 90d, respectively ( Figure 1B). In contrast, Actinobacteriota family is the minor contributor to LPS biosynthesis, with an average of 8.89% and 9.90% of the total LPS biosynthesis in IHF meat geese at 45d and 60d, respectively. In contrast to other bacterial families, Proteobacteria family with a minute quantity with an average of 2.94% contributes to the total LPS biosynthesis in IHF meat geese at 60d.
The quality and types of diet make alterations in the gut microbiota in a time-dependent manner. We next examined whether the bacterial composition of the samples would correlate with the potency of activation or inhibition of these samples ( Figure 1C). At the genus level, we determined the Spearman correlation between the GO terms and cecal microbiota falling in different phyla mainly relevant to LPS biosynthesis and those have been individually expressed in a supporting file (Supplementary Figures 3A-E). We identify a strong correlation between GO functions related to LPS biosynthesis and microbial composition at the genus level and could only find a moderate-to-strong correlation between a few microbial individual genera and the stimulatory potency of individual cecal LPS production in IHF meat geese ( Figure 1C). However, we found that the abundance of very few bacterial genera Lactobacillus and Ruminococcus_torques_group following Firmicutes phylum show a moderate correlation with the activation of lipid A biosynthesis and LPS transportation periplasmic protein lptA at 45d in IHF meat geese ( Figure 1C). While only single bacterial genera norank_f:norank_o: Gastranaerophilales following the phylum Cyanobacteria show a strong correlation with the activation of LPS transportation periplasmic protein lptA at 45d in IHF meat geese ( Figure 1C). In contrast, we found a maximum of the bacterial genera Prevotellaceae_UCG-001, Bacteroides, and Alistipes following Bacteroidota phylum were contributing to the lipid A biosynthesis acyltransferase and lipid A biosynthesis at 60d in IHF meat geese ( Figure 1C). Similarly, Bacteroides and Rikenellaceae_RC9_gut_group following Bacteroidota phylum B C A 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 follow the same trend in activating the lipid A biosynthesis acyltransferase and lipid A biosynthesis at 90d in IHF meat geese ( Figure 1C). Notably, Bacteroides dominate activating lipid A biosynthesis acyltransferase at 60d and 90d in IHF meat geese ( Figure 1C). Our results revealed that Bacteroidota phylum is by far the most abundant contributor to the LPS biosynthesis functions in IHF meat geese intestinal microbiota, consistent with their high abundance relative to other Gram-negative genera in the gut.
Inhibitory effects of artificial pasture grazing system on in-house feeding system-induced ROS production via LPS/ TLR4/MyD88 pathway in meat geese We observed significantly increased in pH of proventriculus, gizzard, ileum, and ceca in the AGF meat geese compared with IHF meat geese (Supplementary Figures 4A-D and Supplementary  Table 2). Then we measured the protein levels of intestinal ALP activity and expression levels of intestinal alkaline phosphatase gene (ALPi) and 2 separate alkaline phosphatase genes (CG5150 and CG10827) from the meat geese. The serum ALP activity by enzyme-linked immunosorbent assay (ELISA) kit ( Figure 2A) and mRNA expression of ALPi ( Figure 2B) and alkaline phosphatase genes (CG5150 and CG10827) ( Figure 2C) increased significantly in AGF meat geese as compared to IHF meat geese at 45d, 60d, and 90d. Furthermore, intestinal ALP may contribute to maintaining the normal gut microbial homeostasis by suppressing the E. coli and as well as detoxifying the LPS (42, 43). To identify, whether E. coli contributes to activating the LPS and the suppression of intestinal ALP, we cultured the E. coli onto the Petri dishes for 24h at 37°C. For this, we counted the colony-forming units (CFU) for E. coli from the Petri dishes. We observed that the CFU/g stool was less in the AGF meat geese compared with the IHF meat geese (Supplementary Figure 5A). To further verify our results, we pick up one colony from the petri dish and incubated it in the LB medium for 48h at 37°C. Then we determined the E. coli cell cultures based on spectrophotometer readings at OD600 for 48h with an interval of 2h. We found a significant decline in the concentration of E. coli cell cultures from the AGF meat geese as compared to IHF meat geese (Supplementary Figure 5B).
The genes in rfa cluster such as rfaK and rfaL are involved in the synthesis and modification of LPS core. These two genes play a vital role in the attachment of 0 antigens to the core. This result was following the higher protein abundance of serum LPS ( Figure 2D) Inhibitory effects of artificial pasture grazing system on in-house feeding system-induced ROS production via LPS/TLR4/MyD88 pathway in meat geese. 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. and mRNA expression of LPS biosynthesizing genes rfaK and rfaL ( Figure 2E) in cecal tissues of IHF meat geese. Next, to identify whether lipid A moiety of LPS permits LPS to bind with TLR4 and then activate MyD88 pathway, first, we determined the genes related to lipid A biosynthesis. We found that the mRNA expression of lpxA, lpxB, lpxC, and lpxD was lesser in the AGF meat geese compared with IHF meat geese ( Figure 2F). Next, we found decreased mRNA expression of LBP ( Figure 2G) and soluble cluster of differentiation 14 (sCD14) ( Figure 2H) in AGF meat geese. This suggests that the higher mRNA expression of genes related to lipid A may able the LPS to attach with TLR4 ( Figure 2I) and then activate MyD88 dependent pathway in cecal tissues of IHF meat geese compared with AGF meat geese at 45d, 60d, and 90d ( Figure 2J). The activation of TLR4/MyD88 pathway may contribute to ROS production (44). As expectedly, the higher TLR4/MyD88 gene expression was observed to be increased in IHF meat geese concerning higher serum ROS production ( Figure 2K).
Inhibitory effects of artificial pasture grazing system on in-house feeding system deteriorated nutrient absorption In our study, we found that intestinal ALP activity (Majorbio i-Sanger cloud platform (http://rdp.cme.msu.edu/)) was decreased in IHF meat geese along with increased ROS abundances ( Figure 2K) compared with AGF meat geese. Based on this scenario, next, to identify whether the IHF-induced intestinal dysbiosis may impact intestinal morphology in meat geese, we performed H&E staining of cecal tissues. The effect of AGF system on the morphology of cecal tissues is presented in Supplementary Table 3. Irrespective of a commercial diet, the villus height, villus width, surface area, and distance between villi in cecum of AGF meat geese group were improved as opposed to crypt depth compared to those of IHF meat geese group at different time points 45d, 60d, and 90d. While the villus height to crypt depth ratio (V:C) values of the cecum were not different. Further, the morphology of the cecal tissues from different feeding systems was measured and compared to one another as shown in Supplementary Figure 6. These results illustrated the nutrient absorption in cecum under different feeding systems.
Beneficial effects of artificial pasture grazing system on in-house feeding system-dependent apoptosis-induced gut permeability in meat geese Alterations in mitochondrial membrane permeability could initiate the stimulation of cytochrome C into the cytoplasm, which activates caspases that, in turn, trigger apoptosis. Before starting apoptosis-related experiments, we confirmed that cytochrome C activity was increased with a commercial diet in the cecal tissues of IHF meat geese (Majorbio i-Sanger cloud platform (http://rdp.cme.msu.edu/)), then further we confirmed it from the mRNA expression of cytochrome C from cecal tissues ( Figure 3A). To discover that apoptosis production is affected by feed type in meat geese, we tested mRNA expression levels of caspase 3 and 8 in cecal tissues from IHF and AGF meat geese at different stages such as 45d, 60d, and 90d. We found a significant increase in caspase3 (CASP3) and 8 (CASP8) activity in IHF meat geese (Figures 3B, C). Next, we performed H&E staining of cecal tissues. The upper normal limit for the number of apoptotic cells per field (Mean ± SD) in the villus at 45d, 60d, and 90d from the cecal tissues of IHF and AGF meat geese has been shown in Figure 3D and Supplementary Table 4. From different studies, this accelerated apoptosis has been involved in inducing intestinal mucosa disruption and intestinal permeability (45). To evaluate whether the mucus phenotype can be explained by altered structural organization of the mucosal barrier, we stained cecal tissues with H&E. A well-defined mucus-producing goblet cell genes (mucin2 (MUC2) and Mucin 5, subtype AC (MUC5AC)), the number of goblet cells per 20µm, and inner muscular tonic/muscularis mucosa layer thickness were observed in AGF meat geese group (Figures 3E, F; Supplementary Figures 7A; 8A, B and Supplementary Table 5). In agreement with a mucosal barrier, the mucus-producing goblet cell genes and inner muscular tonic/muscularis mucosa layer appeared less organized upon a commercial diet feeding. Furthermore, intestinal ALP is known to promote gut barrier function, and the disruption of the intestinal barrier is thought to play a critical role in gut permeability development, hence, we measured the gut permeability in IHF and AGF meat geese at 45d, 60d, and 90d. The ELISA kit method showed an IHF-dependent increase in endotoxemia (LPS) ( Figure 2D), significantly influenced by intestinal ALP deficiency in IHF meat geese (previously explained in Figures 2A-C). Furthermore, expression levels of intestinal tight junction proteins were measured in cecal tissues of IHF and AGF meat geese. Again diet and loss of endogenous ALP were associated with a significant reduction in protein expression levels of zona occludin-1 (ZO-1), Occludin, and Claudin ( Figures 3G-I).
Inhibitory Effects of artificial pasture grazing system on in-house feeding system-induced NF-kB pathway and its systemic inflammation In our study first, we explained that the activation of MyD88 dependent pathway could lead to the stimulation of ROS in IHF meat geese. Along with this dependent pathway, the cellular protein LC8 (8-kDa dynein light chain) plays a role in the redox regulation of NF-kB pathway. Actually, LC8 binds to IkB-a in a redoxdependent manner, thereby preventing its phosphorylation by IKK (46). Here, IHF system-induced intestinal ALP disruption and the resulting ROS production oxidized LC8 which leads to the dissociation from IkB-a and then causes NF-kB activation. This result was under reduced mRNA expression levels of LC8 and IkB-a and increased mRNA levels of NF-kB in IHF meat geese ( Figures 4A-C). Furthermore, we investigated the genes related to regulating NF-kB pathway. The mRNA expression of NF-kBregulated genes IL-8, CCL2, PLAU, and BIRC3 in IHF meat geese ( Figures 4D) showed that IL-8, PLAU, and BIRC3 were by far the most abundant contributor genes involved in regulating the NF-kB pathway in the cecal tissues of IHF meat geese. Further to confirm the NF-kB pathway activation in IHF meat geese, we performed the nuclear translocation of NF-kB by the immunofluorescence analysis. As observed using a confocal fluorescence microscope, most of the NF-kB protein was translocated into the cell nucleus in the cecal tissues of IHF meat geese compared with AGF meat geese ( Figure 4E). Next, a significant increase in mRNA expression of pro-inflammatory mediators iNOS and COX2 and cytokines IL-1b, IL-6, and TNF-a in cecal tissues of IHF meat geese were observed instead of IL-1b as a function of diet composition. Here we observed that intestinal ALP deficiency was associated with significantly increased mRNA expression levels of these five cytokines ( Figures 4F-J).
Effects of artificial pasture grazing system on activation of Nrf2 pathway in meat geese When NF-kB pathway is established under the insults of ROS, then a natural immune defense mechanism is activated underpinning the Keap1-Nrf2 pathway. Keap1 regulates the activity of Nrf2 and acts as a sensor for oxidative stress. Upon oxidative stress, Keap1 loses its ability to ubiquitinate Nrf2, allowing Nrf2 to move in the nucleus and activate its target genes Beneficial effects of artificial pasture grazing system on in-house feeding system-dependent apoptosis-induced gut permeability in meat geese. 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. (47). For these consensus, first, we found that the mRNA expression levels of Keap1 were declined and mRNA expression levels of Nrf2 were increased by a limited ROS production in AGF meat geese ( Figure 5A). This demonstrates that the production of ROS activates Nrf2 pathway. Because Nrf2 pathway is known to promote cellular redox homeostasis, and as the impairment of Nrf2 activity is considered to play a crucial role in cellular defense system, we measured Nrf2 and Nrf2-regulated genes and the antioxidant enzymes regulated by Nrf2 in IHF and AGF meat geese at threetime points i.e. 45d, 60d, and 90d. With AGF system, the mRNA expression levels of Nrf2 ( Figure 5B) and Nrf2-regulated genes NQ01, Gclc, Gclm, and GSTA4 were increased in AGF meat geese ( Figure 5C). Next, to evaluate the impacts of AGF system on Keap1-Nrf2 pathway, we determined nuclear translocation of Nrf2 by immunofluorescence analysis. The results revealed increased Nrf2 in AGF versus IHF meat geese ( Figure 5D), which suggested that artificial pasture intake may involve in the activation of the Nrf2 pathway. It is clearly understood that the improved Nrf2 regulation should contribute in antioxidant defense mechanisms. Again we observed that the pasture intake was involved in increased protein levels of antioxidants HO-1, GSR, T-SOD, GSH-PX, T-AOC, and CAT from serum samples of meat geese at 45d, 60d, and 90d ( Figures 5E-J). Further, whether these antioxidants are involved in attenuating the mediators that caused ROS insults in IHF and AGF meat geese, we measured oxidative mediator MDA from serum samples. We examined a severe increase in protein levels of MDA in IHF meat geese compared with AGF meat geese at three time points 45d, 60d, and 90d ( Figure 5K). Artificial pasture grazing system attenuates the in-house feeding systeminduced endotoxemia, gut permeability, and chronic systemic inflammation of meat geese To further explore the effects of long-term establishment of AGF system on intestinal alterations at three-time points 45d, Inhibitory effects of artificial pasture grazing system on in-house feeding system-induced NF-kB pathway and its systemic inflammation. 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).
60d, and 90d, we hypothesized whether intestinal ALP may involve in activating Nrf2 pathway, and we performed correlation analysis among host markers ( Figure 9B). The results obtained from this relationship showed that intestinal ALP was positively correlated with Nrf2 and Nrf2-regulated genes and as well as its antioxidant enzymes. Further Nrf2 pathway including its antioxidation immune system was positively correlated with IL-4, IL-10, and tight junction proteins including 2 genes encoding tight junction proteins Discs large 1 (dlg1) and E-cadherin. IL4 and IL-10 are known to be anti-inflammatory cytokines (48). Indeed, we also measured the protein levels of cecal ALP by western blot analysis ( Figure 6A) and by ELISA kit method ( Figure 6B) in meat geese. The endotoxin (LPS) and ROS measured by ELISA kit method were increased in cecal tissues of IHF meat geese compared with AGF meat geese ( Figures 6C, D). Further, we measured the mRNA expression levels of TJs proteins including ZO-1, Occludin, and Claudin ( Figures 6E-G), 2 genes encoding tight junction proteins dlg1 and E-cadherin ( Figures 6H, I), antiinflammatory cytokines (IL-4 and IL-10) ( Figures 6J, K), and pro-inflammatory cytokines (iNOS, COX2, IL-1b, IL-6, and TNF-a) ( Figures 4F-J) in meat geese that have received AGF and IHF environment from 45d to 90d. The results obtained from correlation analysis suggest that the activation of Nrf2 pathway by intestinal ALP enzyme was primarily involved in attenuating endotoxemia, gut permeability, and proinflammatory cytokines in AGF meat geese.
Long-term artificial pasture grazing system attenuates the manifestation of KEAP1-induced aging phenotypes in meat geese The investigation of impact of Nrf2 pathway activation on cecal tissues aging from the expression levels of aging marker genes showed elevated level of p19ARF, p16INK4a, and p21 in IHF meat geese compared with cecal tissues of AGF meat geese at 45d, 60d, and 90d of age ( Figures 7A-C). The results illustrated that Nrf2 pathway activation induced by ROS-directed Keap1 inhibition effectively suppressed the manifestation of aging phenotypes in cecal tissues of AGF meat geese. Long-term artificial pasture grazing system improved metabolic profile in meat geese The AGF system was effective in preventing metabolic syndrome in AGF meat geese with a significantly improved body weight ( Figures 8A) and lipid profile (Figures 8B-E), as well as lowering blood glucose and urea nitrogen levels ( Figures 8F, G).
Ali et al. 10.3389/fimmu.2022.1041070 Frontiers in Immunology frontiersin.org with serum T-AOC and cecal pH, Occludin, and IL-4 and negatively correlated with serum LPS and cecal iNOS, CASP3, Keap1, p19ARF, and p21. Alistipes and Lactobacillus were strongly positively correlated with intestinal ALP and Nrf2 pathway and suppress all those bacteria (Subdoligranulum, norank_f:norank_o: Clostridia_UCG_014, and Erysipelatoclostridium) that were the causative factors for pathogenesis in AGF meat geese. As diet has a major influence on gut microbiota composition, richness, and diversity (49). It has been known that the origin, type, and quality of diet modulate the gut microbiota in a time-dependent manner. Based on the previous studies, we hypothesized whether AGF system as a high dietary fiber source modulates the gut microbiota at different time points or not. We further analyzed the correlation between microbiota and metabolic indices in the gut tract of meat geese at 60d. The results of which showed that the norank_f:norank_o:RF39 was significantly positively correlated with the cecal pH, serum HO-1 and GSH-PX and cecal Nrf2, IL-4, IL-10, Zo-1, Occludin, claudin, dlg1, and Ecadherin and negatively correlated with serum LPS and TG and cecal MyD88, COX2, TNF-a, Keap1, p19ARF, and p16INK4a in AGF meat geese. Romboutsia was positively correlated with cecal Nrf2, IL-4, IL-10, dlg1, E-cadherin, and LC8 and negatively correlated with cecal MyD88, iNOS, TNF-a, and p16INK4a. Norank_f:norank_o:RF39 and Romboutsia were positively correlated with Nrf2 pathway that was strongly involved in attenuating the harmful impacts of Peptococcus and Ruminococcus_torques_group in AGF meat geese.

Discussion
Intracellular ROS production by diet-induced gut microbiota facilitated LPS generation (50) may lead to chronic low-grade inflammation and modern chronic inflammatory diseases (51). Discovering a safe and novel means of limiting its development is urgently required for the prevention and treatment of these diseases. Diet is a primordial need for life and today, the modern poultry industry is based on grains with lower content of dietary fiber (52). Moreover, the worldwide trends of excessive low dietary fiber intake have been implicated in today's chronic inflammatory diseases including diabetes mellitus, autoimmune, cancer, cardiovascular, and chronic kidney disease (53,54). However, the connections between the shifts in dietary fiber contents and attenuating the incidence of chronic inflammatory diseases by activating the intestinal ALPdependent-redox signaling mechanism remain to be elucidated in meat geese. Therefore the present study demonstrates for the first time, that a long-term pasture grazing can alleviate commercial diet-induced gut microbial dysbiosis, gut barrier dysfunction and integrity, inflammatory diseases, aging phenotypes, and metabolic syndrome.
Though recent dietary supplementation studies have addressed some impacts of dietary fiber on gut microbiota (55, 56). However, despite these captivating findings, the mechanisms underlying these diverse associations and their outcomings have not been fully explored. In our study, we established artificial pasture grazing system for meat geese that supports concurrent processes to impede in-house feeding system-induced metabolic endotoxemia and systemic inflammation. Consequently, the innovative feature of the AGF system as a high dietary fiber source is to build a pathway-based mechanism by which it increased the abundance of intestinal ALP-producing bacteria, and prevented IHF system-induced LPS-producing bacteria. These changes improve the intestinal nutrient absorption, mucus layer, and mucus-producing goblet cell genes, resulting in reduced metabolic endotoxemia (LPS), LPS-induced ROS production, and gut permeability. The subsequent reduction of proinflammatory cytokines lead to the prevention of chronic inflammatory diseases and aging phenotypes. Spearman and Pearson's correlation analysis, including the above-mentioned findings, strongly supports the proposed mechanisms.
Metabolic endotoxemia can be determined by the abundance of bacteria affecting LPS production (57). In this study, we found that AGF intervention reduced the enrichment of genes involved in LPS biosynthesis based on the predicted function by 16S rRNA sequencing and PICRUSt analysis. Interestingly, our current findings have shown similar results with AGF intervention similar to dietary capsaicin in human subjects (58). This would indicate the possibility that a lower abundance of Gram-negative microbiota must be responsible for the low abundance of COG orthology belonging to LPS biosynthesis functions in the AGF meat geese. This could mainly be due to the prevention of members of the Gramp o s i t i v e p h y l a F i r m i c u t e s ( g e n e r a L a c t o b a c i l l u s , Ruminococcus_torques_group, Subdoligranulum, and Christensenellaceae_R-7_group) and Actinobacteriota (genera norank_f_norank_o_Gastranaerophilales) and gram-negative phyla Bacteroidota (genera Prevotellaceae_UCG_001, Bacteroides, Alistipes, and Rikenellaceae_RC9_gut_group) with AGF intervention because these were the key bacterial phyla and their respective genera that largely contribute to the IHF meat geese. These lipopolysaccharides are bacterium-associated molecular patterns, which act via TLR4/MyD88 pathway by promoting the inflammatory response (59).
The production of ROS by LPS-induced TLR4/MyD88 pathway activation (44) may depend on a diet rich in high fat, high calorie, high protein, and high carbohydrates (60,61). The correct cellular response to ROS production is critical to preventing oxidative damage and maintaining cell survival. However, when too much cellular damage has occurred, it is to the advantage of a multicellular organism to remove the cell for the benefit of the surrounding cells. ROS can therefore trigger apoptotic cell death based on the severity of the oxidative stress (62) and may contribute to inducing NF-kB pathway (63). ROSinduced severe apoptotic cell death may accelerate intestinal mucosa disruption and result in causing intestinal permeability (64). Based on the current studies, we hypothesized how intestinal ALP would incinerate in LPS-induced TLR4/MyD88 facilitates ROS production pathway.
Intestinal ALP is a major enzyme of interest for its gut microbiota-modifying properties (65,66). Endogenous ALP production has been shown to inhibit the overgrowth of E. coli by dephosphorylating LPS (28,57,67). It is well known that intestinal ALP capacity to dephosphorylate LPS was shown to be present in the colon and feces of mice (26) and reduces LPSinduced gut permeability and inflammation in Caco2 and T84 cell lines (68). LPS binds specifically to TLR4 and stimulates inflammation by activating two distinct pathways, namely LPSdependent release of TNF-a and NF-kB (through MyD88dependent and -independent pathways) (69). The data from our experiment support the notion that the AGF system as high dietary fiber source enhanced the abundance of intestinal ALP producing Alistipes, and Lactobacillus, (45d), Norank_f: norank_o:RF39 and Romboutsia (60d), and Faecalibacterium (90d) genera. This microbiota was further seen to involve in suppressing E. coli and inactivating the capacity of lipid A biosynthesizing genes (lpxA, lpxB, lpxC, and lpxD) to bind LPS with TLR4 and then inhibit the activation ability of MyD88 dependent pathway. Some reports, utilizing dietary fiber as a nutrient source in animals, support our results (24,70).
It is well-known that endogenous ALP production enhances the expression of proteins (Zo-1, Occludin, and Claudin) involved in tight junctions, thereby preventing the translocation of endotoxins (LPS) by intestinal gram-negative bacteria (E. coli) across the gut barrier (21,57) (72), in which microbiallyinduced endogenous intestinal ALP production was observed to decrease the ROS production and apoptosis-related genes CASP3 and CASP8, improve the mucus-producing goblet cell genes MUC2 and MUC5AC as well as inner muscular tonic/ muscularis mucosal layer thickness. Moreover, tight junction proteins Zo-1, Occludin, and Claudin and 2 genes encoding tight junction protein dlg1 and E-cadherin were observed to be increased in AGF meat geese which gave the fact that these proteins were strongly involved in inducing nutrient absorption and overall intestinal health ( Figure 10).
ROS production regulates NF-kB activity in a bidirectional fashion, namely, ROS may trigger activation or repression of NF-kB activity (73). In many studies, NF-kB inhibition is LC8 dependent (46). But in our study, we have shown that the activation of NF-kB by LPS-induced TLR4/MyD88-accelerated ROS production is collectively LC8 and IkB-a dependent. Some reports, utilizing LPS as a ROS inducer, support our results (74,75). The results of our study with a little modification from those of Cario (76) and Fukata et al. (69), evinced that the triggering of LPS facilitated MyD88 pathway and the subsequent ROS production may altogether activate the NF-kB signaling cascades, which played an important role in the development of inflammation by synthesizing and stimulating proinflammatory cytokines (iNOS, COX2, IL-1b, IL-6, and TNFa) (29,77). Dysregulated inflammatory cytokines production plays a pivotal role in developing low-grade inflammation (78). The above-mentioned so-called studies were unable to describe whether the activation of NF-kB pathway and the resulted proinflammatory cytokines were owing to connections between the dietary components (dietary fiber) and gut microbiota. Our results were in accordance with the report of Kyung-Ah Kim et al. (79); Eva d'Hennezel et al. (5); and Sun et al. (51) in which the microbially-induced LPS production increased the mRNA expression of NF-kB, iNOS, COX2, IL-1b, IL-6, and TNF-a in the liver of mice, pigs, and humans as that of our IHF treatment meat geese. Conversely, in our study, we discovered from the spearman correlation analysis between microbiota and host markers that AGF intervention prevented the IHF-induced upregulation of gut microbiota interacting with LPS, ROS, and pro-inflammatory cytokines by activating gut microbiota those directly interacting with intestinal ALP and Nrf2 pathway. Further, following the mechanism of Bates et al. (80) and Estaki et al. (28), we developed combined Pearson's correlation analysis among host markers to evaluate whether intestinal ALP is involved in activating Nrf2 pathway. We observed that the intestinal ALP was significantly positively correlated with Nrf2 in all stages of sample collection suggesting that intestinal ALP may contribute to activating Nrf2 pathway in AGF treatment meat geese.
In response to oxidative challenge, a stress response is activated to control ROS overproduction and provide optimal conditions for effective ROS signaling to support redox homeostasis. The Nrf2/Keap1 and nuclear factor kappa-lightchain-enhancer of activated B cells/inhibitory kB protein (NF-kB/IkB) systems were considered to be two major "master regulators" of the stress response. One of the most important ways in which NF-kB activity influences ROS levels is via increased expression of antioxidant proteins has been explained elsewhere (81). The way by which Keap1-Nrf2 responds to ROS has not been elucidated clearly in meat geese. Upon oxidative stress, Keap1 acts as a sensor and regulates the activity of Nrf2 thereby, Keap1 loses its ability to ubiquitinate Nrf2, allowing Nrf2 to move in the nucleus and activate its target genes (47,82). The results of our study following this mechanism by which dietary fiber in AGF meat geese was able to activate intestinal ALP that was significantly positively correlated with Nrf2 and Nrf2-regulated genes including NQ01, Gclc, Gclm, and GSTA4, and the antioxidant defense network-related enzymes such as HO-1, GSR, T-SOD, GSH-PX, T-AOC, and CAT and significantly negatively correlated with oxidative related enzyme MDA.
Several studies revealed that Nrf2 activity is modulated with different dietary interventions such as a high-fat diet or dietary energy restriction (83,84). The results suggest that the progression of age-related phenotypes p19ARF, p16INK4a, and p21 detected in this study are primarily caused by the decline of protective function by Nrf2 pathway in IHF meat geese. This may be because of chronic smoldering inflammation which is considered one of the important factors associated with lowfiber diet-related diseases and aging phenotypes (53,85). We indeed observed that the expression of pro-inflammatory cytokine genes iNOS, COX2, IL-1b, IL-6, and TNF-a were increased in IHF meat geese. While epidemiological evidence shows that TNF-a and IL-6 are predictive of many aging phenotypes (86). Hence, we found it to be associated with more pronounced aging phenotypes p19ARF, p16INK4a, and p21 in geese lacking pasture intake, whereas long-term AGF system significantly induced the potent anti-inflammatory action of Nrf2 and it may evolve in reducing the proin fl ammatory cy tokines in AGF me at geese with concomitantly, aging phenotypes. Of note, our results suggest that AGF-induced intestinal ALP positively correlates with Nrf2 and negatively correlates with Keap1 and pro-inflammatory cytokines. This notion coincides with the fact that Nrf2mediated inhibition of iNOS, COX2, IL-1b, IL-6, and TNF-a induction contributes to the prevention of delayed aging phenotypes (87) and the development of geese' health.
In a previous study, intestinal ALP regulation prevents and reverses the changes associated with a high-fat diet-induced metabolic syndrome (21). Furthermore, regulation of intestinal FIGURE 10 Diagram illustrating a proposed mechanism by which artificial pasture grazing system as a high dietary fiber source up-regulates intestinal ALPproducing 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 LPSproducing 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-kB 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.
ALP by dietary fiber-rich diets improves the lipid profile during low dietary fiber and low-fat diets (88). In the current study, we found a low dietary fiber-related spontaneous increase in the serum lipid profile and glucose levels in meat geese, significantly more pronounced in geese lacking pasture intake, underscoring the potential beneficial role of AGF-induced intestinal ALP in the prevention of metabolic diseases.
To prove the hypothesis that intestinal ALP might directly contribute to the reduction of endotoxemia, gut permeability, pro-inflammatory cytokines, and metabolic syndrome, we applied a combined correlation analysis among them. We found that ROS production owing to microbially-induced LPS was seen to be increased with IHF system and further involved in inducing intestinal mucosa deterioration, apoptosis, gut permeability, oxidants, NF-kB pathway, pro-inflammatory cytokines, aging phenotypes, and metabolic syndrome. The establishment of AGF system as a high dietary fiber source can reverse this process. Specifically, AGF system increase the abundance of ALP-producing bacteria and that intestinal ALP negatively correlates with ROS. The low production of ROS in AGF meat geese interacts with Keap1 and diminishes its activity and then alternatively activates the Nrf2 pathway. Activation of intestinal ALP and Nrf2 pathway collectively positively correlates with LC8, IKB-a, antioxidants (HO-1, GSR, T-SOD, GSH-PX, CAT, and T-AOC), tight junction proteins ZO-1, Occludin, and Claudin, including 2 genes encoding tight junction proteins dlg1 and E-cadherin, and anti-inflammatory cytokines (IL-4 and IL-10). IL-4 is produced by Th2 cells (89) whereas IL-10 is involved in Th2 differentiation (90) and both are known to be anti-inflammatory cytokines (48). Several pieces of evidence from previous studies revealed that IL-4 and IL-10 depletion is associated with pronounced ulcerative colitis and Crohn's disease, type 2 diabetes, metabolic syndrome (91,92). In our study, the activation of intestinal ALP, Nrf2 pathway, antioxidants, IKB-a, and anti-inflammatory cytokines potentially evolve in reducing endotoxemia, gut permeability, pro-inflammatory cytokines, aging phenotypes, and metabolic syndrome in AGF meat geese.
In summary, our data suggest that intestinal ALPas a natural brush border enzymeplays a critical role in animal health development through maintaining intestinal microbiome homeostasis, reducing LPS-induced ROS production, activating Nrf2 pathway, inducing anti-inflammatory immune responses, and preserving gut barrier function, decreasing low-grade inflammation, and metabolic syndrome. Further studies will focus on elucidating the precise mechanisms of intestinal ALP and Nrf2 pathways' beneficial role in different dietary patterns and aging. Given that AGF system safely induces intestinal ALP and Nrf2 pathways, targeting specific dietary fiber sources that could induce endogenous intestinal ALP production could represent a novel approach to preventing a variety of dietinduced gut microbial-related diseases in animals.

Conclusions
In conclusion, microbially-induced ALP production by AGF system appears to preserve intestinal microbial homeostasis by targeting crucial intestinal alterations, including LPS-induced ROS, gut barrier dysfunction, systemic chronic low-grade inflammation, and metabolic syndrome. By targeting specific dietary fiber sources that could induce endogenous intestinal ALP production may represent a novel therapy to counteract the chronic inflammatory state leading to low dietary fiber-related diseases in animals. If confirmed in humans, these findings may help to better understand diseases with an affected gut barrier functions, such as obesity, ulcerative colitis, cardiovascular, and Crohn's disease.

Data availability statement
Sequence data for cecal microbiome has been uploaded in Sequence Read Achrive of NCBI under accession code: SRP395138.

Ethics statement
The animal study was reviewed and approved by Henan Agricultural University (approval HENAU-2021).

Author contributions
QA and SM designed research, conducted experiments, acquired data, analyzed data, performed statistical analysis, and wrote the manuscript. JN, FL, DL, ZW, HS, and YC acquired data and conducted experiments. YS, SM, and UF designed research, analyzed data, and critically revised the manuscript for intellectual content. All authors contributed to the article and approved the submitted version. providing Wanfu meat geese stocks used in this study. We thank Shi yinghua and San Ma for assistance with microbiome analysis.

Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher's note
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Overview of feeding and sampling strategies.

SUPPLEMENTAL FIGURE 2
Contributions of bacteria at phylum level to LPS biosynthesis functions. (A-F) Relative abundances (%) of the six most dominant phyla in the cecal chyme of the IHF and AGF meat geese. Data with different superscript letters are significantly different (P < 0.05) according to the unpaired student T-Test. *P < 0.05, **P < 0.01.

SUPPLEMENTAL FIGURE 4
Effect of different feeding systems on pH values of meat geese gastrointestinal tract. (A) pH of proventriculus, (B) pH of gizzard, (C) pH of ileum, and (D) pH of cecum. 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.

SUPPLEMENTAL FIGURE 5
Effects of different feeding systems on E. coli production in cecal tissues of meat geese. (A) Representative culture plate photos showing the difference between IHF and AGF meat geese in the growth of LPSproducing gram-negative E. coli. CFU/g stool. (B) E. coli cell cultures based on spectrophotometer readings at OD600 for 48h. Data with different superscript letters are significantly different (P < 0.05) according to the unpaired student T-Test. *P < 0.05, **P < 0.01.

SUPPLEMENTAL FIGURE 7
(A) Comparison of the goblet cell number (per 20µm) of meat geese with different feeding systems. H&E staining of cecal tissues (magnification, 40×). Goblet cell (GC), 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. SUPPLEMENTAL FIGURE 8 (A) Light micrograph of the wall of cecum tissues of meat geese, hematoxylin and eosin (40µm): 1outer layer of muscular tonic; 2inner layer of muscular tonic; 3outer layer of lamina muscularis mucosa (LMM); 4submucosal nerve node; and 5inner layer of lamina muscularis mucosa (LMM). (B) Comparison of the cecal membrane thickness of meat geese with different feeding systems (50µm). Inhouse feeding system (IHF) and artificial pasture grazing system (AGF).