Nicotinic Acid Upregulates Intestinal Antimicrobial Peptides via Manipulating the Histone Acetylation Modication to Enhance the Resistance of Escherichia Coli Infection in Weaned Piglets

Background: Nicotinic acid (NA) has been used to treat different inammatory disease with positive inuence, the mechanisms by which NA exerts its anti-inammatory effects remain largely undened. Here we proposed a new mechanism that NA manipulated endogenous antimicrobial peptides (AMPs) which contributed to the elimination of enterotoxigenic Escherichia coli (ETEC) K88, and thus affects the alleviation of inammation. Results: The results showed that NA alleviated the clinical symptoms of weaned piglets infected with ETEC K88. NA signicantly reduced the amount of ETEC K88 in the spleen and liver (P < 0.05). The intestinal morphological damage caused by ETEC K88 infection was alleviated by NA in weaned piglets. In addition, NA signicantly alleviated the expression of inammatory cytokine (IL-6, IL-8, and TNF-α) in the serum and intestines of weaned piglets infected with ETEC K88 (P < 0.05). and increased in intestinal mucosa. NA signicantly increased the content of SIgA and the expression of antimicrobial peptides (pBD2, PG1-5 and PR39) in intestines of weaned pigs. NA increased the diversity of microora in colonic contents, while NA signicantly reduced the relative abundance of Bacteroidetes, Bacteroidales and bacteroidia in weaned piglets infected with ETEC K88 (P < 0.05). Furthermore, the NA group signicantly reduced the level of HDAC7 in jejunum (P < 0.05) and increased the level of SIRT1 in the colon compared with the Control group. Moreover, NA signicantly increased the levels phosphorylation of histone H3 at Ser10 (pH3S10) in ileum and the levels of acetylation of lysine 9 on histone 3 (acH3K9) and acH3K27 in colon (P < 0.05) in weaned piglets infected with ETEC K88 (P < 0.05). acH3K27 and pH3S10 the these the expression levels of acH3K9, acH3K27 and pH3S10 in the promoter region by activating intestinal histone deacetylase SIRT1 or inhibiting HDAC7, the expression endogenous SIgA: Secretory immunoglobulin A; LDA: linear discriminant analysis; TP: total protein; ALB: albumin; GLOB: globulin; A/G: ALB/GLOB; AST: aspartate aminotransferase; ALP: alkaline phosphatase; LDH: lactic dehydrogenase; BUN: blood urea nitrogen; GLU: glucose.


Background
Infants and other mammalian neonates often suffer from diarrhea during the weaning period, which is the leading killer of children under ve years of age in developing countries all over the word [1,2].
Studies have shown that the piglets were usually faced with some problems such as physical or mental disorders, changes in small intestinal structure, disturbed intestinal microbiota and diminished immune responses during weaning [3,4], which will easily lead to diarrheal disease caused by the invasion of various pathogenic bacteria, especially enterotoxigenic Escherichia coli (ETEC). ETEC postweaning diarrhea, also named as postweaning enteric colibacillosis, is a crucial factor causing mortality of nursery pigs in the global swine production. The infection of ETEC in nursery pigs may induce diarrhea during the rst 1 or 2 weeks of postweaning periods usually resulting in dehydration, reduced weight gain, and death [5]. Therefore, it is extremely urgent to nd an effective way to improve the disease resistance of weaned piglets.
Nicotinic acid (NA), also known as Vitamin B3, is one of the most important and water-soluble B vitamins in mammals, and widely used as a feed additive in modern animal husbandry. Previous studies had shown that NA played an important role in anti-pellagra and regulation of cellular energy metabolism [6].
As reported, nicotinamide treatment could ameliorate the course of bacterial and chemical induced colitis by enhancing neutrophil-speci c antibacterial clearance [7]. What's more, accumulating evidence from mouse has shown that NA alleviated intestinal mucosal in ammation and enhanced the expression of endogenous antimicrobial peptides in intestinal epithelium [8]. Endogenous antimicrobial peptides are an important part of innate immunity in animals. More and more evidences show that antimicrobial peptides play a key role in pathogen resistance and immune regulation [9,10]. However, there are few studies on the mechanism of NA regulating intestinal antimicrobial peptides to enhance resistance of ETEC infection in weaned piglets. Thus, a model of ETEC K88 infected early-weaned piglets was established, aiming to investigate the mechanism of NA regulating intestinal immunity to enhance resistance of weaned mammalian neonates, as assessed by analyzing intestinal morphology, intestinal immune responses, microbial community and metabolites, and the histone acetylation modi cation in this study.

Materials And Methods
Animals, experimental design, and sample collection The animal protocol was approved by the Animal Care Committee of the Institute of Animal Science, Guangdong Academy of Agricultural Sciences. Twenty-four weaned piglets (Duroc×Landrace×Yorkshire, age of 21d) were randomly assigned to 1 of 4 treatments based on BW and sex, each treatment with 6 piglets and 1 piglet per pen in a temperature-controlled room. The control (Control) and NA-treated (NA) groups were administered 20 mL normal saline or 20 mL NA solution (40 mg NA was dissolved in equal volume of normal saline). The K88 challenged (K88) and NA-treated plus K88 challenged group (K88+NA) groups were administered 20 mL normal saline or 20 ml nicotinic acid solution once daily for 3 consecutive days. On the fourth day, the K88 and K88+NA groups were treated with oral administration of 4*10 9 cfu /mL ETEC K88. All piglets were provided with access to water ad libitum. The piglets were checked daily for signs of diarrhea. At the end of experiment, the animals were individually weighed, weight loss of piglets was counted. Samples of the duodenum, jejunum, ileum, and colon were collected for analysis. Serum was obtained from the separation gel coagulation promoting tubes after centrifugation at 3000×g for 15 min at 4℃ and stored immediately at -20℃.
Bacterial plate counting analysis About 2.5 g liver and spleen tissues of pigs in 2.25 ml sterilized buffer liquid, and then homogenized.
Aliquots of 10 ml of the dilutions to be analyzed are placed into LB agar medium at plate, test three parallel plates for each sample. Plates are inverted and incubated for18 to 24 h at 37°C in a constant temperature incubator. Calculate the CFUs of bacterial transfer as the weighted mean from the successive dilutions, which contain between 30 and 300 colonies. The calculate result is the weighted means of the successive dilution multiply by dilution factor.

Intestinal morphology analysis
Formalin-xed duodenum, jejunum, ileum, and colon samples were embedded in para n wax. Segment cross sections were microtomed at approximately 5μm thick and stained with haematoxylin and eosin (H&E). In each section, villus height and associated crypt depth were measured using a DM3000 microscope (Leica Microsystems, Wetzlar, Germany). Images were obtained via using a DM3000 microscope (Leica Microsystems, Wetzlar, Germany). For each section, measurements of 6, wellorientated and intact villi were examined in each piglets' duodenum, jejunum, and ileum. In the end, the mean villus height was then calculated per piglet with Image-Pro software (Media Cybernetics, Rockville, MD). Histopathologic damage scores were determined according to the statement in Feng's publication [11].

Immunoglobulins, cytokines and biochemistry measurements
The concentrations of secretory IgA (SIgA) in the jejunal and ileal mucosa of piglets were determined using the commercially available enzyme-linked immunosorbent assay (ELISA) kits from TSZ ELISA (Framingham, MA) according to the manufacturer's instructions. The concentrations of IgM, IgA, IgG, IL-6, IL-8, TNF-α, and IFN-β in serum of piglets were determined using ELISA kits from Nuoyuan Co., Ltd.

Analysis of intestinal microbiota via 16S rRNA gene sequencing
The contents in the colon of the piglets was aseptically collected, and the total DNA of the colonic contents was extracted using a DNA Kit (SimGEN, Hangzhou, China) according to the instructions provided by the manufacturer. Subsequently, the purity and yield of the DNA samples were quanti ed using a NanoDrop 1000 (Thermo Fisher Scienti c, Waltham, MA) spectrophotometer. Then, twenty-four samples (n= 6) were sequenced on an Illumina HiSeq PE250 platform provided by Novogene (Beijing, China). Paired-end reads from the original DNA fragments were merged by using FLASH. Clustering was performed using the UPARSE pipeline, and sequences were classi ed into different operational taxonomic units (OTUs) based on the sequence similarity cut-off value (i.e., 97 %). Lastly, the diversity and composition of the bacterial communities were determined by α and β diversity according to Novogene's recommendations. At the phylum, class and order levels, LEfSe was used to identify metagenomic biomarkers, while linear discriminant analysis (LDA) was used to estimate the effect of abundance of each species on the difference between groups.
Untargeted metabolomic analysis of colonic contents Metabolite extractions: equal volume of liquid samples was dried on a freeze-drier, then 0.5 mL cold extraction solvent methanol/acetonitrile/H 2 O (2:2:1, v/v/v) was added to the sample, and adequately vortexed. After vortexing, the samples were incubated on ice for 20 minutes, and then centrifuged at 14,000 g for 20 minutes at 4°C. The supernatant was dried in a vacuum centrifuge. For LC-MS analysis, the samples were re-dissolved in 100 μL acetonitrile/water (1:1, v/v) solvent and transferred to LC vials.
LC-MS analysis, data analysis and bioinformatics analysis were performed according to the method in our previous study [12].
Relative quantitative in real-time PCR Total RNA was extracted from the intestinal tissue samples using Trizol reagent (Invitrogen, Carlsbad, CA). The amount of RNA extracted was determined and its purity was veri ed using NanoDrop 1000 (Thermo Fisher Scienti c). Contaminant DNA was removed by gDNA Eraser (Takara, Dalian, China). The cDNA was generated using 1μg aliquot of total RNA with a PrimeScript RT Reagent Kit (Takara). Synthesized cDNA was stored at −20°C prior to real-time PCR analysis.
Real-time PCR was performed using a CFX Connect Detection system (Bio-Rad, Hercules, CA). The sequences of primers used in this study were listed in Table 1. Primers for speci c porcine genes were synthesized by Biotechnology Inc. The cDNA was ampli ed with SYBR® Premix DimerEraser™ (TakaRa Biotechnology Inc.) containing 4-μL 20-fold diluted cDNA, 0.5 μL primers F (10 μM), 0.5 μL primers R (10 μM), 5 μL iTaq Universal SYBR Green Supermix (Bio-Rad), The PCR ampli cation was performed using the following conditions: 95°C for 30 s, followed by 40 cycles at 95°C for 5 s, 60°C for 30 s, and 72℃ for 30 s. The melting curves were systematically analyzed to evaluate the speci city after each run. All reactions were conducted in triplicate. To evaluate the relative quanti cation of mRNA expression, the cycle threshold (CT) values of the target genes were normalized to the CT-values of the β-actin, and the results were presented as fold changes using the 2 -ΔΔCt method.

Western blot analysis
Total protein was extracted from intestinal tissue samples using lysis buffer (KeyGEN, Nanjing, China). The protein concentrations of each sample was calculated with the BCA protein assay kit. Protein was used for western blot analysis, after adding 6× concentrated sample buffer (0.5M Tris, 30% glycerol, 10% SDS, 0.6M DTT, 0.012% bromophenol blue) and heating the samples for 5 min at 95°C. Proteins in supernatants were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene uoride membranes (PVDF) membranes. Next, the membranes were incubated with the primary antibodies for β-actin (Abcam, MA, USA), SIRT1, HDAC7, acH3, acH3K9, acH3K27 and pH3S10 (Abcam) overnight at 4°C after blocked with Tris-buffered saline containing 0.1% Tween-20 (TBS-T) and 5% low-fat milk blocked for 1 h at RT. After washing for 1 h in TBS-T ve times, the membranes were incubated for 1 h at RT with horseradish peroxidase (HRP) secondary antibodies.
Protein immunoreactive bands were photographed. Each special banding gray value was digitized using ImageJ software, and the gray value of target protein was divided by internal reference β-actin.

Statistical analysis
Statistical signi cance analysis of the experimental data was determined by the analysis of variance (ANOVA) with Duncan's multiple range test using SPSS 18.0 software (SPSS Inc., Chicago, IL). Values are given as means ± SEM. The difference was considered to be signi cant at P < 0.05.

Results
Effects of NA on body weight loss, clinical symptoms and intestinal morphology weaned piglets infected by ETEC.
We found that NA effectively attenuated the rate of weight loss (Fig. 1A) and diarrhea in weaned piglets.
To investigate whether the positive effects of nicotinic acid treatment on clinical symptoms were connected to a reduction of the Escherichia coli load in the gut, bacterial plate counting analysis of colony-forming units (CFUs) in the liver and spleen tissues was performed. Compared with the Control group, the CFU counts of Escherichia coli in the liver and spleen of piglets was signi cantly increased in the K88 group (P < 0.05). The K88+NA group signi cantly decreased the CFU counts of Escherichia coli in liver and spleen tissues compared with the K88 group (P < 0.05) (Fig. 1B, 1C).
Then, we investigated the effects of nicotinic acid on intestinal morphology of piglets, the results showed that nicotinic acid not only improved intestinal morphology, but also improved intestinal integrity compared with the Control group (P < 0.05) (Fig. 1D, 1E, 1F, 1G).
Effects of NA on the in ammation in intestinal tissues and serum of weaned piglets infected by ETEC.
To determine the anti-in ammatory effect of NA on the resistance of weaned piglets to ETEC K88 infection, the expression and secretion of the in ammatory cytokines (IL-6, IL-8, TNF-a and IFN-β) were evaluated in intestinal tissues and serum. Compared with the Control group, there were higher levels of serum IL-6, IL-8 and TNF-a in the K88 group (p < 0.05). However, the levels of serum IL-6, IL-8 and TNF-a were signi cantly lower in K88+NA group compared with the K88 group (p < 0.05) ( Fig. 2A, 2B, 2C). Compared with the Control group, the K88 group signi cantly increased the expression of ileal in ammatory cytokines IL-6 and colonic in ammatory cytokines IL-8 and TNF-α (p < 0.05). However, the expression of in ammatory cytokines IL-6 and IL-8 in the ileum and colon and the expression of TNF-a in jejunum and colon were signi cantly lower in the K88+NA group compared with the K88 group (p < 0.05) (Fig. 2E, 2F, 2G). Taken together, these data indicated that with NA treatment ameliorated the in ammation caused by ETEC K88 infection.
Effects of NA on serum biochemistry and immunoglobulins in weaned piglets infected by ETEC.
The effects of NA on serum biochemistry and immunoglobulins caused by Escherichia coli infection of weaned piglets were respectively shown in Table 2 and Fig. 3. Compared with the K88 group, the level of serum lactate dehydrogenase (LDH) in the K88+NA group was signi cantly increased (p < 0.05). The K88 group signi cantly reduced blood glucose (GLU) content in serum piglets compared with the normal control group (p < 0.05). However, the K88+NA group can signi cantly improve serum glucose (GLU) content compared with the K88 group of piglets (p < 0.05). In the aspect of immunoglobulins (Fig. 3A, 3B,   3C), the K88 group had no signi cant effect on serum IgM, but signi cantly increased serum IgG and IgA compared with the Control group (P < 0.05). Compared with K88 group, the serum IgM in the K88+NA group was signi cantly increased (P < 0.05). The content of SIgA in jejunum and ileum mucosa of piglets was measured by ELISA (Fig. 3D, 3E). The results showed that the K88 group signi cantly increased the content of SIgA in jejunum mucosa of piglets compared with the Control group. Compared with the K88 group, the K88+NA group signi cantly ameliorated the increase of SIgA in jejunum mucosa of piglets (P < 0.05).
Effects of NA on the microbial community in colonic contents of weaned piglets infected by ETEC.
We evaluated the effects of NA on the composition of microbiota caused by Escherichia coli infection in the colonic contents of weaned piglets using Illumina sequencing of the 16S rRNA ( Fig. 4-1, 4-2). The common and special OTUs distribution among the four groups was presented by Venn diagram (Fig. 4-1A). As shown in NMDS plot Fig. 4-1B , the K88+NA group formed a distinct cluster clearly separated from the K88 group. Bacteroidetes and Firmicutes were the two most abundance bacterial phyla in all samples ( Fig. 4-1C). By LDA score analysis, we identi ed a total of 8 discriminative species among the four groups ( Fig. 4-1D). Only oscillibacter was abundant in the K88 group. However, in the NA group, bacteroidetes and bacteroidia were abundant. Enterobacteriales, proteobacteria and clostridium were relatively abundant in the K88+NA group.
The relative abundance of top 10 in phylum, order and class were provided. Meanwhile, as shown in LEfSe, bacteroidetes, bacteroidia and bacteroidales were abundant among the four groups ( Fig. 4-2A, B, C). Results indicated that the relative abundance of Bacteroidetes, Bacteroidales and bacteroidia in the K88+NA group was signi cantly reduced compared with the K88 group (P < 0.05).
Effects of NA on the metabolomics in colonic contents of weaned piglets infected by ETEC.
Metabolomics analysis combined with enrichment analysis of colonic contents were shown in Table 3 and Fig. 5. These results revealed that the K88 group altered the concentrations of metabolites (e.g., Isobutyric acid, Oleic acid, Succinate, Heptadecanoic acid, Cholic acid and so on) compared with Control group, and these metabolites were involved in ABC transporters and citrate cycle. In addition, the K88+NA group altered the concentrations of metabolites (e.g., Propionic acid, Succinate, Hydroxy-isocaproic acid, Heptadecanoic acid, 2-Methyl-3-hydroxybutyric acid and so on) compared with the K88 group, and these metabolites were involved in ABC transporters and citrate cycle.
Effects of nicotinic acid on the expression of intestinal antibacterial peptides in weaned piglets.
Further research is required to explore the effect of niacin on the expression of antimicrobial peptides in intestinal mucosa (Fig. 6A, 6B, 6C). The NA group signi cantly improved the expression of antimicrobial peptide PG1-5 in jejunum, pBD2, PG1-5 and PR39 in ileum of weaned piglets compared with the Control group (P < 0.05). In addition, the NA group also signi cantly improved the expression of antimicrobial peptide PG1-5 and PR39 in colon of weaned piglets (P < 0.05).
Effects of NA on the histone acetylation modi cation in intestinal mucosa in weaned piglets.
The effect of nicotinic acid on intestinal histone acetylation modi cation of ETEC infected piglets was further studied by Western blot (Fig. 7A, 7B, 7C, 7D). The results showed that the NA group signi cantly reduced the level of HDAC7 in jejunum (P < 0.05) compared with the Control group. In addition, the K88+NA group signi cantly increased the level of SIRT1 in jejunum (P < 0.05) compared with the Control group; Compared with the K88 group, the levels of histone SIRT1 and pH3S10 in ileum were signi cantly increased in the K88+NA group (P < 0.05); The NA group signi cantly increased the level of SIRT1 in the colon (P < 0.05) compared with the Control group. Moreover, compared with the K88 group, the K88+NA group signi cantly increased the levels of histone acH3K9 and acH3K27 in colon (P < 0.05).

Discussion
Colibacillosis of weaned piglets caused by pathogenic Escherichia coli, with severe diarrhea as the main clinical symptom, is one of the most serious diseases in China's livestock industry [13]. At present, ETEC is the most popular pathogenic bacteria in actual production of pigs. It colonized and proliferated in intestinal epithelial cells of weaned piglets through adhesion factors, thus destroying the normal balance of intestinal ora and causing secondary infections. Meanwhile, it could also release some enterotoxins to induce intestinal in ammation. These factors eventually resulted in diarrhea because of the unbalance of intestinal water and electrolyte metabolism [14]. Whether the morphological structure of intestinal epithelium is intact or not will affect the normal intestinal mucosal immune response and barrier function [15]. It is vital for the body to resist the infection of external pathogenic microorganisms. Villus height and crypt depth is an important index of the intestinal health of piglet. Previous studies found that villus height and crypt depth of duodenum and jejunum of weaned piglets were signi cantly increased after caused by Escherichia coli infection [16]. The present study focused on studying the effects of nicotinic acid on intestinal morphology and clinical symptoms caused by Escherichia coli infection. We demonstrated that administration of nicotinic acid effectively improved mental state, attenuated the intestinal tissue injury and the weight loss. Moreover, the number of bacterial translocations in the liver and spleen of piglets was signi cantly reduced. Research has shown that nicotinic acid supplements in the diet not only help improve the performance of weaned piglets, but also could reduce the occurrence of diarrhea piglets [17]. It was found that supplementation with tryptophan could alleviated the reduction in average daily gain (ADG) of piglets caused by Escherichia coli infection [18]. In addition, nicotinic acid supplements in the diet could increase the villus height of the small intestine in piglets caused by Escherichia coli infection [19].
Due to the immature development of the intestinal immune system of weaned piglets, the intestinal mucosa of the weaned piglets is often susceptible to the invasion of some pathogenic bacteria, which causes intestinal in ammation [20]. The main characteristic of intestinal in ammation is that in ammatory mediators are enriched in the intestinal mucosa. Cytokines play an important role in mediating intestinal tissue damage and coordinating in ammatory response, and are considered as the cornerstone of the body's intracellular monitoring system. Cytokines are small molecule peptides or glycoproteins secreted by antigen-presenting cells (APCs) and have biological functions such as participating in in ammatory response and regulating immune response. Interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interferon-β (IFN-β) are important pro-in ammatory cytokines. These cytokines may be upregulated in response to in ammation in the body [21]. Studies have con rmed that the mRNA expression of TNF-α, IL-6, IL-8 and other cytokine genes was increased in intestinal tissues of weaned piglets caused by Escherichia coli infection [22,23]. The results of the current study showed that nicotinic acid supplements in the diet could ameliorate in ammation by down-regulated the expression of TNF-α, IL-6 and IL-8 in piglets caused by Escherichia coli infection. This nding was partly consistent with the study by Kwon et al., who demonstrated that niacin alleviated pulmonary in ammation by reducing the expression of IL-6 and TNF-α in serum [24]. Another study showed that niacin reduced the release of TNF-α IL-6 and IL-8 by inhibiting the nuclear factor kappa-β (NF-κβ) signaling pathway in mouse alveolar in ammatory cells induced by lipopolysaccharide (LPS) [25]. In addition, it is reported that niacin can alleviate intestinal in ammation by inhibiting the expression of in ammatory cytokines in mouse macrophages or reducing intestinal epithelial cell apoptosis [26].
Immunoglobulin is produced when the body is stimulated by antigens such as bacteria and viruses. It could make pathogens lose their pathogenic effect by reacting with antigens and blocking the harm of pathogens to the body [27]. Secretory immunoglobulin A (SIgA), as mucosal humoral immune antibody, plays an important role in the local in ammatory response in the aspect of resisting external pathogenic microorganisms on mucous membrane of the host [28]. Research has shown that pathogens will be easier to will more easily adhere and invade to the intestinal mucosa epithelium, causing enteritis and enterogenous systemic infection when the level of intestinal mucosa secretion SIgA is reduced [29]. Our results demonstrated that nicotinic acid can signi cantly alleviate the increase of SIgA content in jejunal mucosa caused by challenge.
Intestinal micro ora plays an important role in preventing pathogen adhesion and colonization [30], promoting digestion and metabolism, enhancing autoimmunity and maintaining health [31]. If the intestinal micro ora is out of balance, it may destroy the normal physiological function of the body, which will greatly increase the incidence of diseases, and thus lead to intestinal stress syndrome, in ammatory enteritis and diarrhea [32]. Bacteroidetes and Firmutes are the dominant ora in the intestinal tract of piglets, accounting for more than 90% [33]. With the change of growing environment, the dominant ora in the intestinal tract will change accordingly. Earlier report has shown that the increase of intestinal microbial diversity may enhance the stability of intestinal micro ora, and thus enhance its ability to resist the invasion of pathogenic bacteria [34]. It is shown that ETEC infection not only cause the imbalance of intestinal micro-ecological environment, but also signi cantly reduce the diversity of intestinal micro ora [35]. It is reported that the relative abundance of bacteroides is closely relative to the diarrhea rate of weaned piglets [36]. In this study, the results show that nicotinic acid could signi cantly increase the diversity of microbial species in colon contents of weaned piglets. Meanwhile, compared with the K88 group, the K88+NA group was signi cantly reduced the relative abundance of bacteroidetes in phylum, class and order levels. There is accumulating evidence indicating that the metabolite of the intestinal micro ora-butyrate could upregulate endogenous host defense peptides to enhance disease resistance in piglets [37,38].
Antimicrobial peptides that were secreted by intestinal epithelial and immune cells of piglets not only play an important role in killing bacteria directly, regulating the immune system and immune regulation, but also improve the body's resistance to pathogen infection by enhancing intestinal epithelial barrier function [39,40]. It has always been the focus of researchers that nutrition regulates the expression of intestinal antimicrobial peptides to improve resistance to disease. As far as we know, NA could alleviate intestinal in ammation and promote the expression of endogenous antimicrobial peptides. Some studies reported that NA could improve their ability of resistance to pathogen infection by improving the mice expression of antimicrobial peptide (CAMP and LF) [41]. In addition, there is research has revealed that NA supplements in the diet could signi cantly improve the expression of antimicrobial peptides in the intestinal epithelial cells of mice, which could alleviate intestinal in ammation and diarrhea caused by the lack of tryptophan [42]. In this study, we evaluated the effect of NA on the expression of antimicrobial peptides in intestinal mucosa. The results found that NA could signi cantly improve the expression of antimicrobial peptide PG1-5 in jejunum, pBD2, PG1-5 and PR39 in ileum, PG1-5 and PR39 in colon of piglets. However, the regulatory mechanism that nicotinic acid improved the expression of intestinal endogenous antimicrobial peptides in weaned piglets is unclear, and further research is still needed to do.
Studies showed that histone modi cation could accurately regulate the expression of the innate immune response and the corresponding defense genes [43]. Histone acetylation is of great importance for the transcriptional regulation of intestinal epithelial antimicrobial peptides [44]. Already there is evidence that butyric acid could upregulated endogenous host defense peptides by inhibiting histone deacetylase to strengthen disease resistance of piglets [37]. It has also been shown that some HDAC inhibitors can regulate the expression of antimicrobial peptide LL-37 in gastrointestinal cells by inhibiting histone deacetylase [45]. Other study has shown that when HDAC7 is overexpressed in mice, it causes in ammation in macrophages in mice [46]. SIRT-1 is an NAD-dependent deacetylase, and the changes of NAD counts in vivo will affect the activity of SIRT1 [47]. Studies have found that NAM can activate the NAD-sirtuins pathway to improve the expression of SIRT1 gene in the liver [48], and in some cases, NAM could act as an activator of SIRT1 [49]. Another study found that SIRT1 activator (resveratrol) increased the expression of cathelicidin antimicrobial peptide (CRAMP) in mouse cells [50]. Furthermore, transcription factors enter chromatin to bind promoters of innate immune genes, requiring acetylation of histone H3 lysine residues and phosphorylation of histone H3S10 [51]. In addition, some studies showed that HDAC inhibitors (TSA or SAHA) could signi cantly improve the expression of antimicrobial peptides (HBD2 and LL-37) in intestinal epithelial cells via increasing the acetylation level of histone H3K9 lysine residues [44]. It was found that HDAC inhibitor (butyric acid) increased the expression of histone H3K9 in the antimicrobial peptide promoter region, thereby promoting the expression of antimicrobial peptides (PBD2 and PR39) in porcine macrophages [37]. These results suggest that the elevated level of histone H3K9 in the promoter region may be a typical marker of transcriptional activation of antimicrobial peptide genes. In our study, we found that nicotinic acid activated the histone deacetylase SIRT1 and inhibited HDAC7, associated with improving the histone modi cation sites H3K9, acH3K27 and pH3S10 in the promoter region. Taken together, these results suggest that nicotinic acid may increase the expression levels of acH3K9, acH3K27 and pH3S10 in the promoter region by activating intestinal histone deacetylase SIRT1 or inhibiting HDAC7, and then further up-regulated the expression of endogenous AMPs in weaned piglets.

Conclusions
In conclusion, NA could alleviate the clinical symptoms, the damage of intestinal morphology, and intestinal in ammation in weaned piglets infected ETEC K88, and NA may improve intestinal antimicrobial peptides to enhance resistance of Escherichia coli infection by regulating intestinal micro ora and its metabolites, histone deacetylase SIRT1 and HDAC7, histone modi cation sites (acH3K9, acH3K27 and pH3S10) in the promoter region.     Figure 1 Effects of niacin on body weight loss rate and intestinal morphology caused by Escherichia coli infection of weaned piglets. the weight loss rate of piglets was showed (A), the E. coli counts in liver (B) and spleen (C) tissues by bacterial plate counting analysis. Stained with H&E (bars, 500 μm) (Fig. D). Histological scores were determined as described in the Materials and Methods (Fig. E). Villous height in the jejunum, duodenum, and ileum (Fig. F). Crypt depth in the jejunum, duodenum, and ileum (Fig. G). All data are expressed as the mean ± SEM. * P < 0.05.  Effects of nicotinic acid on serum levels of immunoglobulins caused by Escherichia coli infection of weaned piglets. The serum levels of immunoglobulins IgM (Fig. A), IgG (Fig. B), IgA (Fig. C) and SIgA in jejunum (Fig. D) and ileum (Fig. E) mucosa were determined by ELISA. All data are expressed as the mean ± SEM. *P < 0.05.

Figure 4
Nicotinic acid improved the bacterial community caused by Escherichia coli infection in colonic contents of weaned piglets. The bacterial communities in the colonic contents of weaned piglets were investigated using Illumina sequencing of the 16S rRNA gene. Venn diagram shows the common and special OTUs distribution among the four groups ( Fig. 4-1A), nonmetric multidimensional scaling (NMDS) based on operational taxonomic unit levels (Fig. B), the LDA score analysis (LDA score ≥ 4) in four groups (Fig. C), UPGMA Clustering was conducted based on Unweighted Unifrac distance (Fig. D). The relative abundance of top 10 in phylum, order and class ( Fig. 4-2A), the LEfSe analysis identi ed the biomarker bacterial species (Fig. B), the signi cantly different species at each level (Fig. C). *P < 0.05.

Figure 5
Metabolomic analysis of the colonic contents in weaned pigs caused by Escherichia coli infection. Volcano plot in HILIC negative between K88 and Control (Fig. A) and negative between K88+NA and K88 (Fig. B); Enriched KEGG pathways analysis between K88 and Control (Fig. C), between K88+NA and K88 (Fig. D); Hierarchical clustering of the differentiated metabolites in HILIC negative between K88 and Control (Fig. E), between K88+NA and K88 (Fig. F).  Effects of nicotinic acid on the histone deacetylase modi cation in intestinal mucosa caused by Escherichia coli infection of weaned piglets. The expression of histone deacetylase SIRT1, HDAC7 and modi cation sites such as acH3K9, acH3K27 and pH3S10 in the promoter region in the jejunum, ileum, and colon mucosa were determined by Western blot (Fig. A), and the intensity of the bands was detected using ImageJ (Fig. B, C). * P < 0.05.