Dietary resistant starch ameliorating lipopolysaccharide-induced in ammation associated with alteration in gut microbiome and glucagon-like peptide 1 signaling

Simeng Qin Sichuan Agricultural University Chengdu Campus Weiqiang Bai Sichuan Agricultural University Chengdu Campus Todd J Applegate University of Georgia Keying Zhang Sichuan Agricultural University Chengdu Campus Gang Tian Sichuan Agricultural University Chengdu Campus Xuemei Ding Sichuan Agricultural University Chengdu Campus Shiping Bai Sichuan Agricultural University Chengdu Campus Jianping Wang Sichuan Agricultural University Chengdu Campus Li Lv Sichuan Agricultural University Chengdu Campus Huanwei Peng Sichuan Agricultural University Chengdu Campus Yue Xuan Sichuan Agricultural University Chengdu Campus Qiufeng Zeng (  zqf@sicau.edu.cn ) Institute of animal nutrition, Sichuan Agricultural University


Introduction
The intestine of animals and humans serves as the bridge for connecting intraluminal and enteric external milieu, which mainly functioned to prevent pathogenic entities invasion and maintain intestinal homeostasis. During normal physiological activities, some endotoxins and toxins produced by colonized bacterial in intestine usually can be degraded and cleaved to non-toxic fragments by innate immune cells [1,2]. Nevertheless, once the organism stuck in pathological conditions, the interrupted intestinal wall allow the pathogenic bacteria to tissue, the speci c molecular patterns in microbial components could been recognized by some immune factors such as toll-like receptors (TLRs) and induce the transcription of speci c genes involved in pro-in ammatory and anti-bacterial responses [3]. These dysregulated immune responses due to intestinal disequilibrium may contribute to the occurrence of in ammatory related diseases including in ammatory bowel disease, sepsis, endotoxemia [4][5][6]. Modern meat ducks are no exception, various factors especially genetic selective pressure, environmental stress, bacterial infection, and immunological stress result in increasingly common incidence in the intestinal in ammation of birds [7]. The destroyed gut barrier integrity and dysbacteriosis could induce the systemic and intestinal in ammation [8]. In practically the early stage of growth and intestinal development, which is an important period relative to optimizing digestive e ciency and performance [7], the disorder of intestinal homeostasis in this phase has been noticed to associated with irreversibly weakened growth performance. Therefore, enhancing gut barrier integrity and reducing in ammation that are supposed to be an effective way for animal growth and physical health maintenance.
Currently, some functional feed ingredients targeting gut microbiota are acknowledged to counteract intestinal in ammation [9] and might be alternatives for antibiotics to product the antibiotic-free animal production in domestic birds. Resistant starch (RS), a kind of natural prebiotics exists in most common feed ingredients, is quite suitable for being supplemented in diets to maintain animal health. The most typical bene cial functions exerted by dietary RS is to enhance short chain fatty acids (SCFAs) production through optimizing gut microbiota in hindgut because of its relatively resistant to digestion in small intestine by starch degradation enzyme produced by host [10,11]. Our previous studies showed that diet with 12% raw potato starch (RPS, Type RS) could improve intestinal morphology and enhance intestinal barrier function through upregulating the transcription of tight junction proteins (TJPs) in both ileum and cecum, as well as it also increased the gut microbial diversity and the abundance of bene cial bacteria including Fecalibacterium and Subdoligranulum in cecum of meat ducks [12,13]. Accumulating evidence from rodent studies indicated that RS supplemented diet are likely to alleviate tissue damage, including but not limited to intestine, during in ammatory stress by modulating in ammatory cytokines and gut microbiota [14][15][16][17]. In addition to the metabolites such as SCFAs, glucagon-like peptide-1 (GLP-1) secretion might be a key contributor to the bene cial effects on the gut [18]. GLP-1 is a gut peptide secreted in enteroendocrine L cells located predominantly in the ileum and colon. It has been shown to against intestinal injury and promote gut growth via binding its receptor (GLP-1R) [19,20]. Activation of GLP-1R is also associated with anti-in ammatory effects [21,22]. It is reasonable to assume that dietary RS could alleviate intestinal in ammation from LPS challenge through GLP-1/GLP-1R signaling in meat ducks.
The objective of the current study, therefore, was to investigate the effects of dietary RS supplementation in the form of RPS on intestinal barrier integrity, gut microbiota, as well as systemic and intestinal in ammatory response in lipopolysaccharide (LPS)-challenged meat ducks. Of note, the SCFAs production and GLP-1/GLP-1R signaling as potential mechanism underlying the relationship between the improved intestinal status and dietary RS manipulation were also evaluated. Our hypothesis was that dietary RS supplementation would protect against LPS-induced intestinal damage and abnormal release of in ammatory cytokines of meat ducks through SCFAs production and GLP-1/GLP-1R signaling under immunological stress.

Materials And Methods
The RS was used from RPS with a 57% RS content (Windmill ® potato starch, The Netherlands). LPS was purchased from Sigma-Aldrich (From Escherichia coli serotype O55: B5, MO, USA). GLP-1R agonist, liraglutide (Selleck, S8256). Birds were raised in temperature-and humidity-controlled room, and allowed to access water and feed freely throughout experiments. The ducklings were vaccinated at 1 d of age against Newcastle Disease and Infectious Bronchitis at the hatchery facilities. The basal diet was formulated to meet the requirement of meat ducks according to National Research Council (NRC, 1994). RS diet contained 12% RPS based on our previous work [12]. Experimental diets shown in Table   S1&2.

Birds and study design
To evaluate the response of intestinal status to chronic or acute LPS challenge and dietary RS supplementation. In the chronic LPS challenge, 240 1-d-old meat ducks were randomly assigned to control, LPS challenge (LPS), and LPS challenge with fed 12% RPS diet (LPS + RS) group. After 12-h feed deprivation, the ducks were intraperitoneally injected with either 1 mg/kg body weight (BW) of LPS or sterile saline at 14, 16, and 18 d (Fig. S1A). The routes of LPS injection were identical to our previous study [23]. Four hours after injecting on 18 d, 1 duck with a weight closest to the pen average was selected for blood samples, then centrifuged at 3,000 g for 15 min at 4°C to gain serum or plasma, and subsequently the distal ileal, ileal mucosa and cecal digesta were collected for historical analysis, intestinal integrity evaluation, SCFA determination, and microbial analysis, respectively. For the acute LPS challenge, a total of 144 1-d-old Cherry Valley meat ducks were randomly assigned to control, LPS, LPS + RS, or LPS challenge with liraglutide (100 μg/kg BW, LPS + Liraglutide), respectively. At day 14, birds were injected intraperitoneally with either 2 mg/kg BW of LPS or sterile saline (Fig. S1B). At 4 h after injecting, 1 bird from each pen was selected for blood, distal ileal, and ileal mucosa were collected, then snap frozen in liquid nitrogen and stored at -80°C for further analysis.

Histological analysis
The xed distal ileum using 10% neutral-buffered formalin for 24 h was embedded in para n. Sections of 4-mm thickness were subjected to hematoxylin and eosin staining (H&E). Images were taken from a microscope (BA400Digital, Mike Audi Industrial Group Co., Ltd., China) and were analyzed with Image-Pro Plus 6.0 (Media Cybernetics, Silver Spring, MD, USA).

Immuno uorescence analysis
The GLP-1R protein of ileum was determined by immuno uorescence. The 4% paraformaldehyde-xed samples were rehydrated in PBS, subjected to antigen retrieval with ethylene diamine tetraacetic acid buffer, and then blocked with 3% bovine serum albumin prior to incubation with mouse monoclonal antibodies to GLP-1R (1:100, sc-390774, Santa Cruz Biotechnology Inc., Dallas, TX, USA) overnight at 4°C. Slides were then detected with Cy3 conjugated Goat Anti-mouse IgG (GB21301, Servicebio Technology Co., Ltd, Wuhan, China) for 1 h at room temperature in the dark, and the nuclei were stained with 4′-6diamidino-2-phenylindole (DAPI) for 10 min. All slides were nally examined for uorescence using a confocal scanning microscope (NIKON ECLIPSE TI-SR), and images were taken with the NIKON DS-U3 software. The proportion of GLP-1R positive cells was determined by counting cells in each of three sections from each of 6 ducks.

Intestinal permeability determination
Ducks from chronic LPS challenge trial were used to determinize the intestinal permeability using uorescein isothiocyanate dextran (FITC-d, 4 kDa, Sigma, USA), an indicator to examine barrier function.
On d 18, 1 bird each pen close to the pen average weight were selected, and all ducks received orally FITCd (4.16 mg/kg). The serum was collected at 2.5 h post FITC-d administration and the uorescence was measured at 485nm excitation and 528nm emission (BioTek Instruments, Winooski, VT). The FITCdextran concentrations were determined from standard curves generated by the serial dilution of FITC-d.
All assays were performed as described by the manufacturer's instructions and done in duplicate.

Gene expression assays
Total RNA was extracted using a Trizol reagent (TaKaRa, China) from frozen ileal mucosa samples following the manufacturer's instructions. RNA integrity was tested by a 1% agarose gel electrophoresis.
Real-time PCR was performed on ABI QuantStudio™ 6 Flex system (Applied Biosystems, CA, USA). The primer sequences for the target genes designed using Primer 3 (Table S3). Relative gene expression was quanti ed by normalizing to the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin.

Cecal SCFAs analysis
Approximately 0.5 g of cecal digesta was diluted with 2 mL of ultrapure water mixed with a uniform. The solution was left for 30 min and then centrifuged at 3,000 × g for 15 min. Supernatants (1 mL) were mixed with 0.2 mL ice-cold 25% (w/v) metaphosphoric acid solution, and incubated at 4℃ for 30 min, followed by 11,000 × g centrifugation for 10 min. The SCFA contents including acetate, propionate, and butyrate were separated and determined by gas chromatograph (Varian CP-3800, USA).

Gut microbiome sequencing
Total DNA was extracted from cecal content with a DNA stool mini kit (Qiagen, Valencia, CA, United States). After determining the DNA concentration and integrity, an amplicon sequencing library was constructed based on the PCR-ampli ed V4 variable regions of 16s rDNA, and paired-end sequenced on an Illumina HiSeq 2500 platform (BGI, Shenzhen, China). The obtained sequences were processed using FLASH (v1.2.11) and USEARCH (v7 .0.1090) for alignment and clustering. The nal reads were clustered as operational taxonomic units (OTUs) with a 97% similarity threshold. The OTUs were further assigned to species level using the RDP classifer (v2.2) based on the Greengene database (V201305) of full-length 16S rRNA sequences. Partial least-squares discrimination analysis (PLS-DA) was performed by R package mixOmics.

Statistical analysis
The data obtained were analyzed by the Shapiro-Wilk and Levene's test to assess normal distribution and homogeneity of variances. Analyses were done using unpaired Student's t-tests and the Mann-Whitney U test for normally or non-normally distributed datasets, respectively in Graphpad Prism (GraphPad Software, San Diego, CA, USA), as indicated. For data of microbiota, signi cant genera were determined by R (v3.4.1) based on either Kruskal-Wallis test or Wilcox-test. Spearman correlation analysis was used to explore the correlation between gut bacteria and gene expression of cytokines. Differences with P ≤ 0.05 were considered signi cant.

Dietary RS inclusion improved BW and intestinal barrier during chronic LPS challenge
Prior to LPS challenge, RS inclusion signi cantly increased (P < 0.05) ducks' BW at 14 d. Subsequently, the BW at 18 d was notably decreased by chronic LPS-challenged, which was reversed due to dietary RS treatment (both P < 0.01; Figure 1A). Regarding intestinal barrier, histologic examination showed that ileum form the control group presented a regularly arranged villi and intact epithelial structure (Fig. 1B).
LPS injection resulted to obviously exfoliated ileal mucosa, disorderly arranged and broken villi and in ltrated with in ammatory cells, whereas the diet with RS alleviated ileal injury, showed by basically intact epithelia and slightly in ammatory cell in ltration after chronic LPS-challenged (Fig. 1B).
The outcomes from FITC-d and endotoxins, both biomarkers used to re ect intestinal permeability, suggested that the concentrations of FITC-d and endotoxins in serum were respectively signi cantly (P < 0.01) or slightly (P = 0.089) elevated in chronic LPS-challenged group than the control group, but them were numerical decrease by RS supplementation (Fig. 1C, D). Moreover, the genes encoding intestinal barrier were determined and showed that chronic LPS challenge signi cantly decrease (P < 0.01) mRNA expression levels of Occludin and Claudin-1 in ileum as compare with the control group, and the altered mRNA abundance of Claudin-1 was remarkably reversed with dietary RS administration (P < 0.01; Fig. 1E).
Gut microbiota and cecal SCFA concentrations response to chronic LPS challenge and dietary RS supplementation As illustrated in Figure 2A, there were no signi cant changes in individual SCFAs by challenging LPS (P > 0.05). Dietary RS supplementation signi cantly increased the concentrations of propionate and butyrate during chronic LPS challenge (P < 0.05). With regarding to the gut microbiota, the composition was apparently affected by experiment manipulation, indicated by totally separated from treatments (Fig. 2B). More speci cally, except increased the proportion of Lachnospira and Mucispirillum, the chronic LPS injection failed to change the abundance of Firmicutes, Bacteroides, Bi dobacterium, and Ruminococcus compared to control group. However, diet with RS increased the proportion of Firmicutes, and thus increased the radio of Firmicutes to Bacteroides, along with higher abundance of Bi dobacterium, Mucispirillum, and Ruminococcus following the LPS-challenged (Fig. 2C-I).
Dietary RS supplementation alleviated the in ammatory response induced by chronic LPS challenge might associate GLP-1 and gut microbiota Concerned the ileal and systemic in ammatory response, LPS challenge induced higher concentration of serum TNF-α relative to control group, which was normalized by supplementing dietary RS (P < 0.01; Figure 3A). However, experiment treatments did not alter the content of IL-1β and IL-17 in serum (P > 0.05; Fig. 3B, C). Re ecting to ileum, the mRNA levels of TNF-α, Interferon-γ (IFN-γ), IL-1β, IL-4 and IL-17 were increased in chronic LPS administration (P < 0.05), and reversed their increased levels by dietary RS (Fig. 3D).
We further analyzed the potentially associations between in ammatory action and GLP-1 signaling and gut microbiota. The result of immuno uorescence analysis for GLP-1R showed that which was decreased by LPS challenged tended to decrease the GLP-1R expression (P = 0.08), and then diet supplemented of RS upregulated the abundance of GLP-1R in ileum (P = 0.089; Fig. 4A, B). In addition, alterations in bacteria were also associated with one or more in ammatory response factors based on Spearman's correlation coe cients (Fig. 4C). For instance, at genera levels, both IL-4 and TLR4 were negatively correlated with Megamonas. IL-4 was also signi cantly negatively correlated with some healthassociated gut bacteria, including Bi dobacterium, Collinsella and Ruminococcus. TNFα was negatively associated with the abundance of Odribacter. IL-1β and IL-17 are positively associated with Adlercreutzia and Sphingomonas, respectively. IFNγ associated positively with Anaerostipes, Bacteroides, Eggerthella and Phascolarctobacterium, and most notably Sutterella and Bilophila, while negatively with Prevotella.
Dietary RS improved ileal integrity and decreased in ammation response during acute LPS challenge To further explore the role of GLP1/GLP-1R signaling in the bene cial effect of dietary RS attenuating intestinal in ammation induced by LPS challenge. The model of acute LPS challenge and GLP-1R agonist liraglutide were used, and the results showed that dietary RS inclusion did not signi cant in uence the BW in acute LPS challenged ducks among treatments (Fig. S1C). Histologic examination revealed that ileum of ducks in control groups presented a regularly arranged villi and intact epithelial structure, However, heavily in ltrated with in ammatory cells and partially exfoliated ileal mucosa were observed in LPS group, which were mitigated by RS supplementation or liraglutide injection, evidenced by basically intact epithelia with slightly in ammatory cell in ltration after acute LPS-challenged (Fig. 5A). The higher serum endotoxins level suggested an increase in gut permeability due to acute LPS challenge group when compared with that in control (P < 0.05). Both liraglutide injection and dietary RS supplementation reduced the concentration of serum endotoxins to varying degrees (Fig. 5B).
Moreover, acute LPS challenge also elevated genes expression of TNF-α (P = 0.083) and TLR4 (P < 0.05) in ileum as compared to the control, and dietary RS supplementation markedly reversed TLR4 mRNA level (Fig. 5C). Meanwhile, dietary RS and liraglutide treatment were signi cantly lowered IL-6 or promoted IL-10 transcriptional levels (both P < 0.05) relative to LPS-challenged group (Fig. 5C). In serum, acute LPS challenge increased the concentration of TNF-α, IL-1β, and IL-6 as compared with control group, and subsequently were suppressed by the administration with liraglutide injection or dietary RS (Fig. 5D-F).

GLP-1/GLP-1R signaling responses to dietary RS addition during acute LPS challenge
As presented in Figure 6A, although acute LPS change did not signi cantly change the concentration of plasma GLP-1, administrated with dietary RS and liraglutide result in a 32% and 24% increase in terms of serum GLP-1 content compared with LPS group, respectively (Fig. 6A). The GLP-1 synthesis related genes expression including proprotein convertase subtilisin/kexin type (Pcsk1) and solute carrier family 5 member 1 (Slc5al), as well as GLP-1R were also determined, and the data suggested that acute LPS challenge not notably affect the GLP-1R and Slc5al genes expression, but tended to increase the abundance of Pcsk1 mRNA (Fig. 6B-D). When compared to LPS group, liraglutide injection signi cantly increased GLP-1R and Pcsk1 mRNA level (Fig. 6B, C). Also, the diet supplemented with RS upregulated that transcription of Pcsk1 and Slc5al in ileum (Fig. 6C, D)

Discussion
Intestinal in ammatory disease has been becoming a great threat to poultry production in non-obvious ways due to impaired gut and growth performance of birds [24,25]. Numerous researches that included our previous study have con rmed the release of pro-in ammatory cytokines and immunological stress triggered by LPS intraperitoneal injection could impair nutrients absorption and depress growth performance in domestic birds [23,26,27]. In the present study, the reduced BW by chronic LPS injection has yielded strong evidence for a harmful consequence on growth performance of meat ducks induced by LPS challenge. Furthermore, the compromised BW by chronic-LPS challenge at 18-d-old ducks were elevated by RS supplementation, indicating that RS exerted protective effects on performance of ducks suffered from immunological stress. Our previous study noticed that RS functioned as prebiotics to maintain intestinal health of ducks by improving barrier function and modulating microbial composition [12,13], which could explain why the dietary RS ameliorated growth performance in LPS-challenged ducks. For this, intestinal morphology and permeability were further determined in the currently study. In lined with previous ndings in ducks [27], the higher serum FITC-d concentration suggested that LPS challenge resulted in a drastic increase intestinal permeability. At the molecular level, chronic LPS challenge downregulated the expression of Claudin-1 and Occludin, both important composition of TJPs in intestinal barrier [28]. Evidence from our recently studies showed that dietary RS administration could enhanced gut barrier function of ducks in normal feeding via increasing mRNA expression of TJPs in ileum [12]. In addition, RS was also found to effective in protecting the TJPs expression in dextran sulfate sodium (DSS)-induced mice [15]. The present results are congruent with previous outcomes and indicated that dietary RS is bene cial to enhance intestinal barrier integrity under immunological tress.
It is well-known that compromised intestinal integrity might trigger aberrant in ammatory responses and in conjunction with accumulation of in ammatory mediators and further aggravating tissue damage [29]. Herein, we also compared the differences in alteration of systemic and intestinal in ammation response to LPS challenge and dietary RS supplementation. In this study, in ammatory lesions in ileum was consistently observed in LPS-challenged ducks, and dietary RS supplementation could effectively alleviate in ammatory injury in villus and crypt of ileum induced by LPS. RS provided also exerted a protective role against DSS-induced colonic in ammation as re ected by a decrease in colonic proin ammatory cytokine levels of IL-1β, IL-6, TNF-α, and IFN-γ in proximal or distal segments [15]. Analogously, our results in ileum showed that chronic LPS-challenged increased the expression of IL-1β, IL4, IL-17, and TNF-α, and acute LPS challenge upregulated the TNF-α mRNA levels. As an antiin ammatory effect, dietary RS supplementation alleviated in ammatory response in ileum both in acute and chronic LPS challenge, evidenced by a noticeable reduction in transcriptional levels of those increased in ammatory cytokines mentioned above. Of note, the lipid A fraction contained in LPS can act as ligand for immune receptor TLR4 that elicits robust in ammatory responses in immune and somatic cells [30]. TLR4 is required to recruit speci c adaptor proteins including MyD88 (myeloid differentiation primary response gene 88 etc., to initiate the downstream signaling to produce in ammatory cytokines [31] and the activation of TLR4 is preceded the release of in ammatory cytokines [32]. TLR4 is thus considered as a classical signaling pathway in triggering cytokine production, in ammation and adaptive immune response. Whereupon, the gene expression of TLR4 in ileum was examined to verify whether RS exerts an anti-in ammatory effect through this pathway. The present study with RS supplementation observed a remarkably reversion in LPS-induced increase in TLR4 mRNA level under acute LPS challenge. Taken together, our results support the ability of dietary RS to exhibited an excellent anti-in ammatory e cacy when suffered LPS challenge, which might involve in the suppression of TLR4 role.
Re ecting to systemic in ammation. Our outcomes from serum in ammatory cytokines showed that both acute and chronic LPS challenge increased concentrations of serum endotoxins. In addition to as indictor of intestinal permeability, endotoxin is also a sort of LPS that constituting much of the outer membrane of Gram-negative bacteria. When suffered bacterial infection, high concentrations of endotoxin in gut and many other tissues would be present [33]. According to results of researches in human subjects, endotoxin levels in plasma or serum are normally low, but elevated during infections or gut in ammation [30]. Therefore, systemic in ammation caused by LPS injection in this study may be evidenced by the elevated endotoxins in serum of ducks. Meanwhile, LPS induces in ammation indirectly via the pro-in ammatory cytokines such as TNF-α, IL-6 and IL-1β was related to increased endotoxin level [33]. We therefore examined a variety of serum in ammatory cytokines concentrations in the current study. The concentration of TNF-α signi cantly increased after chronic LPS-challenge and both the content of TNF-α and IL-6 in serum were also stimulated by acute LPS-challenge, which were reduced by dietary RS supplementation to varying degrees, implying that dietary RS supplementation contributes to normalized the release of pro-in ammatory cytokines during in ammatory infection induced by LPS challenge.
Gut microbiota is considered to be a crucial part of intestinal homeostasis and paly key role in in ammatory response. There are accumulating evidence have con rmed that RS is bene cial for modulating microbiota in hindgut [10]. As we described in our previous study, diet with 12% RPS had a higher relative abundance of Firmicutes [13]. In the present study, we also con rmed that RS supplementation resulted in a shift in the microbial structure, and there was a consistent increase in the abundance of Firmicutes and the ratio of Firmicutes and Bacteroidetes following RS diet with LPSchallenged compared to basal diet with LPS-challenged, suggesting that members of the Firmicutes had more selective advantage than members of the Bacteroides when supplementing diet with RS, as similar results were also observed in study on human with high levels of RS diet [11]. R.brommi that belongs to Ruminococcus has been shown to degrade RS, and numerous studies in human and mice have reported RS supplementation could increase the amounts of Ruminococcus and Lachnospira [11,17,34,35]. Besides, following these changes, the relative abundance of Ruminococcus and Lachnospira were enriched in RS supplemented diet. These two genera are known as butyrate-producing bacteria, which was in line with the increase of butyrate in the cecal contents of ducks consuming RS diet. In addition, SCFAs is known as the microbiota-derived metabolites produced in cecum and colon, which are the signatures of the gut microbiota and contribute to modulate intestinal immune activity and in ammatory responses [36]. Our results of SCFAs showed that dietary RS supplementation elevated the concentration of propionate and butyrate, which was in agreement with our previous study [13], in which diet supplemented with 12% RPS could increase SCFAs contents in normal feeding experiment. To link the gut microbiota with the in ammatory markers, a correlation analysis was integrated and suggested that there were various bacteria signi cantly associated with these in ammatory cytokines and mediators. For This study raises three new question need to be elaborated. The rst is the response of gut microbiota to chronic LPS challenge. It was established that in ammation is triggered by LPS derived from the gut microbiota. However, in this study, except increased the proportion of Lachnospira and Mucispirillum, the chronic LPS injection failed to change the abundance of Firmicutes, Bacteroides, Bi dobacterium, and Ruminococcus. The second is the difference of GLP-1R expression between acute and chronic LPS challenge. Herein, chronic LPS challenge decreased the expression of GLP-1R using histometric method, whereas no apparently change was observed in acute LPS injection via RT-PCR determination. The third is LPS challenge model. The assessment for the link of dietary RS, in ammation, and GLP1/GLP-1R signaling was conducted using acute but not chronic LPS challenge, it is possible that the responses of GLP1/GLP-1R signaling to acute and chronic LPS challenge are complicated and changeable. Therefore, some of inaccurate conclusion highlighted the roles of dietary RS addition in modulating LPS-induced in ammatory response might be included in this study. Further researches would be essential to exclude this possibility.

Conclusion
In conclusion, the research has revealed a substantial e cacy of dietary RS supplementation in ameliorating in ammatory severity of LPS-induced in meat ducks, which were associated with improved intestinal integrity and gut microbiota, as well as SCFAs production. Moreover, this anti-in ammatory effect exerted by dietary RS in LPS challenge possibly also involves the TLR4 and GLP-1/GLP-1R signaling pathway. These results will bring a valuable strategy of nutritional immunity for protecting host by exploiting dietary RS supplementation ways to against immunological stress for domestic birds and human.

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Availability of data and material
All data generated or analysed during this study are included in this manuscript and its supplementary information les.

Competing interests
We declare that we have no nancial and personal relationships with other people or organizations that can inappropriately in uence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as in uencing the content of this paper.

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