Butyrate Improves Porcine Endometrial Epithelial Cell Receptivity via Enhancing Acetylation of Histone H3K9.

SCOPE: Uterine receptivity is a major restriction of embryo implantation and survival, and the endometrial luminal epithelium serves as the transient gateway for uterine receptivity and embryo implantation. Butyrate is reported to promote the success of embryo implantation, but the effects and mechanism of butyrate on uterine receptivity are still unknown. METHODS AND RESULTS: Porcine endometrial epithelial cells (PEECs) are used as a model, and the cellular receptivity changes, metabolism, and gene expression profiles influenced by butyrate are analyzed. The study finds that butyrate improves receptive changes in PEECs, including inhibiting proliferation, exhibiting more pinocytosis on the cell surface, and increasing adhesiveness to porcine trophoblast cells. In addition, butyrate increases prostaglandin synthesis and markedly impacts purine metabolism, pyrimidine metabolism, and the FoxO signaling pathway. siRNA to inhibit the expression of FoxO1 and chromatin immunoprecipitation-sequencing (ChIP-seq) of H3K9ac are used to demonstrate that the H3K9ac/FoxO1/PCNA pathway can contribute to the effects of cell proliferation inhibition and uterine receptivity improvement induced by butyrate. CONCLUSION: The findings reveal that butyrate improves endometrial epithelial cell receptivity by enhancing the acetylation of histone H3K9, which shows nutritional regulation and therapeutic potential for poor uterine receptivity and difficulty in embryo implantation.

Rice bran oil supplementation protects swine weanlings against diarrhea and lipopolysaccharide challenge. / æ—¥ç²®æ·»åŠ ç±³ç³ æ²¹å¯å¢žå¼ºæ–­å¥¶ä»”çŒªæŠµæŠ—è ¹æ³»å’Œè„‚å¤šç³–åº”æ¿€.

Early weaned piglets suffer from oxidative stress and enteral infection, which usually results in gut microbial dysbiosis, serve diarrhea, and even death. Rice bran oil (RBO), a polyphenol-enriched by-product of rice processing, has been shown to have antioxidant and anti-inflammatory properties both in vivo and in vitro. Here, we ascertained the proper RBO supplementation level, and subsequently determined its effects on lipopolysaccharide (LPS)-induced intestinal dysfunction in weaned piglets. A total of 168 piglets were randomly allocated into four groups of seven replicates (42 piglets each group, (21±1) d of age, body weight (7.60±0.04) kg, and half males and half females) and were given basal diet (Ctrl) or basal diet supplemented with 0.01% (mass fraction) RBO (RBO1), 0.02% RBO (RBO2), or 0.03% RBO (RBO3) for 21 d. Then, seven piglets from the Ctrl and the RBO were treated with LPS (100 µg/kg body weight (BW)) as LPS group and RBO+LPS group, respectively. Meanwhile, seven piglets from the Ctrl were treated with the saline vehicle (Ctrl group). Four hours later, all treated piglets were sacrificed for taking samples of plasma, jejunum tissues, and feces. The results showed that 0.02% was the optimal dose of dietary RBO supplementation based on diarrhea, average daily gain, and average daily feed intake indices in early weaning piglets. Furthermore, RBO protected piglets against LPS-induced jejunal epithelium damage, which was indicated by the increases in villus height, villus height/crypt depth ratio, and Claudin-1 levels, as well as a decreased level of jejunal epithelium apoptosis. RBO also improved the antioxidant ability of LPS-challenged piglets, which was indicated by the elevated concentrations of catalase and superoxide dismutase, and increased total antioxidant capacity, as well as the decreased concentrations of diamine oxidase and malondialdehyde in plasma. Meanwhile, RBO improved the immune function of LPS-challenged weaned piglets, which was indicated by elevated immunoglobulin A (IgA), IgM, ß||-defensin-1, and lysozyme levels in the plasma. In addition, RBO supplementation improved the LPS challenge-induced dysbiosis of gut microbiota. Particularly, the indices of antioxidant capacity, intestinal damage, and immunity were significantly associated with the RBO-regulated gut microbiota. These findings suggested that 0.02% RBO is a suitable dose to protect against LPS-induced intestinal damage, oxidative stress, and jejunal microbiota dysbiosis in early weaned piglets.

Multi-omics analysis reveals gut microbiota-ovary axis contributed to the follicular development difference between Meishan and Landrace × Yorkshire sows.

BACKGROUND: The mechanism by which Meishan (MS) sows are superior to white crossbred sows in ovarian follicle development remains unclear. Given gut microbiota could regulate female ovarian function and reproductive capacity, this study aimed to determine the role of gut microbiota-ovary axis on follicular development in sows. METHODS: We compared the ovarian follicular development, gut microbiota, plasma metabolome, and follicular fluid metabolome between MS and Landrace × Yorkshire (L × Y) sows. A H2O2-induced cell apoptosis model was used to evaluate the effects of multi-omics identified metabolites on the apoptosis of porcine ovarian granulosa cells in vitro. RESULTS: Compared with L × Y sows, MS sows have greater ovary weight and improved follicular development, including the greater counts of large follicles of diameter ≥ 5 mm, secondary follicles, and antral follicles, but lesser atretic follicles. The ovarian granulosa cells in MS sows had alleviated apoptosis, which was indicated by the increased BCL-2, decreased caspases-3, and decreased cleaved caspases-3 than in L × Y sows. The ovarian follicular fluid of MS sows had higher concentrations of estradiol, progesterone, follicle-stimulating hormone, luteinizing hormone, and insulin like growth factor 1 than L × Y sows. Gut microbiota of MS sows formed a distinct cluster and had improved alpha diversity, including increased Shannon and decreased Simpson than those of L × Y sows. Corresponding to the enhanced function of carbohydrate metabolism and elevated short-chain fatty acids (SCFAs) in feces, the differential metabolites in plasma between MS and L × Y sows are also mainly enriched in pathways of fatty acid metabolism. There were significant correlations among SCFAs with follicular development, ovarian granulosa cells apoptosis, and follicular fluid hormones, respectively. Noteworthily, compared with L × Y sows, MS sows had higher follicular fluid SCFAs concentrations which could ameliorate H2O2-induced porcine granulosa cells apoptosis in vitro. CONCLUSION: MS sows have more secondary and antral follicles, but fewer atretic follicles and apoptotic ovarian granulosa cells, as well as harbored a distinctive gut microbiota than L × Y sows. Gut microbiota may participate in regulating ovarian follicular development via SCFAs affecting granulosa cells apoptosis in sows.

Dietary ellagic acid supplementation attenuates intestinal damage and oxidative stress by regulating gut microbiota in weanling piglets.

Intestinal oxidative stress triggers gut microbiota dysbiosis, which is involved in the etiology of post-weaning diarrhea and enteric infections. Ellagic acid (EA) can potentially serve as an antioxidant supplement to facilitate weaning transition by improving intestinal oxidative stress and gut microbiota dysbiosis. Therefore, we aimed to investigate the effects of dietary EA supplementation on the attenuation of intestinal damage, oxidative stress, and dysbiosis of gut microbiota in weanling piglets. A total of 126 piglets were randomly assigned into 3 groups and treated with a basal diet and 2 mL saline orally (Ctrl group), or the basal diet supplemented with 0.1% EA and 2 mL saline orally (EA group), or the basal diet and 2 mL fecal microbiota suspension from the EA group orally (FEA group), respectively, for 14 d. Compared with the Ctrl group, EA group improved growth performance by increasing average daily feed intake and average daily weight gain (P < 0.05) and decreasing fecal scores (P < 0.05). EA group also alleviated intestinal damage by increasing the tight junction protein occludin (P < 0.05), villus height, and villus height-to-crypt depth ratio (P < 0.05), while decreasing intestinal epithelial apoptosis (P < 0.05). Additionally, EA group enhanced the jejunum antioxidant capacity by increasing the total antioxidant capacity (P < 0.01), catalase (P < 0.05), and glutathione/oxidized glutathione (P < 0.05), but decreased the oxidative metabolite malondialdehyde (P < 0.05) compared to the Ctrl group. Compared with the Ctrl group, EA and FEA groups increased alpha diversity (P < 0.05), enriched beneficial bacteria (Ruminococcaceae and Clostridium ramosum), and increased metabolites short-chain fatty acids (P < 0.05). Correspondingly, FEA group gained effects comparable to those of EA group on growth performance, intestinal damage, and intestinal antioxidant capacity. In addition, the relative abundance of bacteria shifted in EA and FEA groups was significantly related to the examined indices (P < 0.05). Overall, dietary EA supplementation could improve growth performance and attenuate intestinal damage and oxidative stress by regulating the gut microbiota in weanling piglets.

Multi-omics analysis reveals gut microbiota-induced intramuscular fat deposition via regulating expression of lipogenesis-associated genes.

The gut microbiome has great effects on the digestion, absorption, and metabolism of lipids. However, the microbiota composition that can alter the fat deposition and the meat quality of pigs remains unclear. Here, we used Laiwu (LW) pigs (a native Chinese breed with higher intramuscular fat) compared with commercial crossbreed Duroc × (Landrace × Yorkshire) (DLY) pigs to investigate the effects of microbiota on meat quality, especially in intramuscular fat content. A total of 32 DLY piglets were randomly allotted to 4 groups and transplanted with fecal microbiota from healthy LW pigs. The results indicated that the high dose of fecal microbiota transplantation (HFMT) selectively enhanced fat deposition in longissimus dorsi (P < 0.05) but decreased backfat thickness (P < 0.05) compared with control group. HFMT significantly altered meat color and increased feed conversation ratio (P < 0.05). Furthermore, the multi-omics analysis revealed that Bacteroides uniformis, Sphaerochaeta globosa, Hydrogenoanaerobacterium saccharovorans, and Pyramidobacter piscolens are the core species which can regulate lipid deposition. A total of 140 male SPF C57BL/6j mice were randomly allotted into 7 groups and administrated with these 4 microbes alone or consortium to validate the relationships between microbiota and lipid deposition. Inoculating the bacterial consortium into mice increased intramuscular fat content (P < 0.05) compared with control mice. Increased expressions of lipogenesis-associated genes including cluster of differentiation 36 (Cd36), diacylglycerol O-acyltransferase 2 (Dgat2), and fatty acid synthase (FASN) were observed in skeletal muscle in the mice with mixed bacteria compared with control mice. Together, our results suggest that the gut microbiota may play an important role in regulating the lipid deposition in the muscle of pigs and mice.

Integrated analysis of multi-tissues lipidome and gut microbiome reveals microbiota-induced shifts on lipid metabolism in pigs.

Lipid metabolism is very important for meat quality in pigs. Accumulating evidence shows that gut microbiota can contribute to this physiological process. However, the gut microbiota that function in lipid metabolism and adipogenesis remains unclear. Here, we compared the characteristics of fat deposition and gut microbial community between Laiwu pigs and Duroc × (Landrace × Yorkshire) (DLY) pigs. Fecal microbiota transplantation (FMT) was performed to determine the possible impact of gut microbiota on lipid metabolism in pigs. An integrated analysis of the gut microbiome and lipidome of the small intestine, plasma, and liver was conducted to investigate the effects of FMT on host lipid metabolism. The comparative analysis of the gut microbiome showed higher abundance of Bacteroidetes (P = 0.0018) while lower abundance of Firmicutes (P = 0.012) in Laiwu pigs, and the microbial composition can be transferred from Laiwu pigs into DLY pigs. Transmission electron microscope and Oil red-O staining were performed to analyze the effects of FMT on lipid deposition in liver, the main target organ for lipid metabolism. The results showed that FMT significantly increased the number of lipid droplets (P = 0.0035) and lipid accumulation (P = 0.0026) in liver. Furthermore, integrated multi-tissues lipidome analysis demonstrated that the fatty acyls and glycerophospholipids were significantly increased (P < 0.01) in intestine and liver, while glycerolipids and fatty acyls were reduced (P < 0.01) in plasma. In the small intestine, FMT increased (P < 0.01) the relative abundance of polyketides and prenol lipids but reduced (P < 0.01) the saccharolipids. Correlation analysis revealed the potential interactions between microbiota and lipid metabolites. Together, our results indicated that the gut microbiota may regulate the lipid metabolism and enhance the accumulation of lipid droplets in the liver of pigs.

Dietary ε-Polylysine Affects on Gut Microbiota and Plasma Metabolites Profiling in Mice.

Given the antibacterial effects of ε-polylysine acting on cell membranes, and that glycerol phospholipids are important components of the cell membrane, we hypothesized that ε-polylysine may regulate glycerophospholipid metabolism by modifying the gut microbiota. To test this hypothesis, we treated post-weaning C57 mice with different levels of ε-polylysine (0, 300, 600, and 1,200 ppm) in their basic diet. The growth performance and morphology of intestine were then determined. Modification of the gut microbiota and their function were analyzed using 16S rDNA sequencing. Metabolite identification was performed using the LC-MS method. The results showed that body weight decreased with an increasing supplemental level of ε-polylysine from 5 to 7 weeks (P < 0.05), but no significant difference was observed after 8 weeks (P > 0.05). Supplementation with 1,200 ppm ε-polylysine changed the morphology of the jejunum and ileum, increased the villus length, decreased the crypt depth of the jejunum, and decreased the villus length and crypt depth of the ileum (P < 0.05). ε-Polylysine shifted the intestine microbiota by changing alpha diversity (Chao 1, observed species, Shannon, and Simpson indices) and varied at different times. ε-polylysine decreased Firmicutes and increased Bacteroidetes at 4 week, but increased Firmicutes and decreased Bacteroidetes at 10 week. ε-Polylysine regulated genera associated with lipid metabolism such as Parabacteroides, Odoribacter, Akkermansia, Alistipes, Lachnospiraceae UCG-001, Collinsella, Ruminococcaceae, and Intestinimonas. During the adult period, the genera Alistipes, Lachnospiraceae UCG-001, and Streptomyces were positively associated with PC, PE, LysoPC, LysoPE, 1-Arachidonoylglycerophosphoinositol and OHOHA-PS (R > 0.6, P < 0.001), but changes in Blautia, Christensenellaceae R-7 group, Odoribacter, Allobaculum, Ruminococcaceae UCG-004, Ruminococcaceae UCG-005, and Lachnospiraceae UCG-010 were negatively correlated with glycerophospholipid metabolites (R < -0.6, P < 0.001). The abundance of glycerophospholipid metabolites, including PC, PE, lysoPC, and lysoPE, were decreased by ε-polylysine. Furthermore, ε-polylysine reduced the incidence of the genera including Ruminococcus, Prevotella, Prevotellaceae, Butyricimonas, and Escherichia-Shigella and reduced the abundance of Faecalibaculum, Christensenellaceae R-7 group, Coriobacteriaceae UCG-002. In conclusion, ε-polylysine modified gut microbiota composition and function while also restraining pathogenic bacteria. The glycerophospholipid metabolism pathway and associated metabolites may be regulated by intestinal bacteria.

Increased Ingestion of Hydroxy-Methionine by Both Sows and Piglets Improves the Ability of the Progeny to Counteract LPS-Induced Hepatic and Splenic Injury with Potential Regulation of TLR4 and NOD Signaling.

Methionine, as an essential amino acid, play roles in antioxidant defense and the regulation of immune responses. This study was designed to determine the effects and mechanisms of increased consumption of methionine by sows and piglets on the capacity of the progeny to counteract lipopolysaccharide (LPS) challenge-induced injury in the liver and spleen of piglets. Primiparous sows (n = 10/diet) and their progeny were fed a diet that was adequate in sulfur amino acids (CON) or CON + 25% total sulfur amino acids as methionine from gestation day 85 to postnatal day 35. A total of ten male piglets were selected from each treatment and divided into 2 groups (n = 5/treatment) for a 2 × 2 factorial design [diets (CON, Methionine) and challenge (saline or LPS)] at 35 d old. After 24 h challenge, the piglets were euthanized to collect the liver and spleen for the histopathology, redox status, and gene expression analysis. The histopathological results showed that LPS challenge induced liver and spleen injury, while dietary methionine supplementation alleviated these damages that were induced by the LPS challenge. Furthermore, the LPS challenge also decreased the activities of GPX, SOD, and CAT and upregulated the mRNA and(or) protein expression of TLR4, MyD88, TRAF6, NOD1, NOD2, NF-kB, TNF-α, IL-8, p53, BCL2, and COX2 in the liver and (or) spleen. The alterations of GPX and SOD activities and the former nine genes were prevented or alleviated by the methionine supplementation. In conclusion, the maternal and neonatal dietary supplementation of methionine improved the ability of piglets to resist LPS challenge-induced liver and spleen injury, potentially through the increased antioxidant capacity and inhibition of TLR4 and NOD signaling pathway.

Dietary Quercetin Supplementation Attenuates Diarrhea and Intestinal Damage by Regulating Gut Microbiota in Weanling Piglets.

Antioxidant polyphenols from plants are potential dietary supplementation to alleviate early weaning-induced intestinal disorders in piglets. Recent evidences showed polyphenol quercetin could reshape gut microbiota when it functioned as anti-inflammation or antioxidation agents in rodent models. However, the effect of dietary quercetin supplementation on intestinal disorders and gut microbiota of weanling piglets, along with the role of gut microbiota in this effect, both remain unclear. Here, we determined the quercetin's effect on attenuating diarrhea, intestinal damage, and redox imbalance, as well as the role of gut microbiota by transferring the quercetin-treated fecal microbiota to the recipient piglets. The results showed that dietary quercetin supplementation decreased piglets' fecal scores improved intestinal damage by increasing tight junction protein occludin, villus height, and villus height/crypt depth ratio but decreased crypt depth and intestinal epithelial apoptosis (TUNEL staining). Quercetin also increased antioxidant capacity indices, including total antioxidant capacity, catalase, and glutathione/oxidized glutathione disulfide but decreased oxidative metabolite malondialdehyde in the jejunum tissue. Fecal microbiota transplantation (FMT) from quercetin-treated piglets had comparable effects on improving intestinal damage and antioxidative capacity than dietary quercetin supplementation. Further analysis of gut microbiota using 16S rDNA sequencing showed that dietary quercetin supplementation or FMT shifted the structure and increased the diversity of gut microbiota. Especially, anaerobic trait and carbohydrate metabolism functions of gut microbiota were enriched after dietary quercetin supplementation and FMT, which may owe to the increased antioxidative capacity of intestine. Quercetin increased the relative abundances of Fibrobacteres, Akkermansia muciniphila, Clostridium butyricum, Clostridium celatum, and Prevotella copri but decreased the relative abundances of Proteobacteria, Lactobacillus coleohominis, and Ruminococcus bromii. Besides, quercetin-shifted bacteria and carbohydrate metabolites short chain fatty acids were significantly related to the indices of antioxidant capacity and intestinal integrity. Overall, dietary quercetin supplementation attenuated diarrhea and intestinal damage by enhancing the antioxidant capacity and regulating gut microbial structure and metabolism in piglets.

Gut microbiota contributes to the development of endometrial glands in gilts during the ovary-dependent period.

BACKGROUND: The hyper-prolificacy Meishan gilts achieved a superior endometrial gland development (EGD) than white crossbred gilts during the ovary-independent period (before 60 d of age). Then, the EGD continues under the management of ovary-derived steroid hormones that regulated by gut microbiota (after 60 d of age). However, whether Meishan gilts' superiority in EGD lasting to the ovary-dependent period (after 60 d of age) and the role of gut microbiota in this period both remain unclear. METHODS: Meishan gilts and Landrace x Yorkshire (LxY) gilts were raised under the same housing and feeding conditions until sexual maturity and then we compared their EGD and gut microbiota. Meanwhile, we transplanted fecal microbiota from Meishan gilts to L×Y gilts to explore the role of gut microbiota in EGD. We sampled plasma every 3 weeks and collected the uterus, ovary, liver, and rectal feces after the sacrifice. We then determined the hormone concentrations and expressions of the EGD-related genes. We also profiled the gut microbiota using 16S rDNA sequencing and metabolites of plasma and liver tissue using untargeted metabolomics. Finally, the correlation analysis and significant test was conducted between FMT-shifted gut microbes and EGD-related indices. RESULTS: Meishan gilts have larger endometrial gland area (P < 0.001), longer uterine horn length (P < 0.01) but lighter uterine horn weight (P < 0.05), a distinctive gut microbiota compared with L×Y gilts. Fecal microbiota transplantation (FMT) increased endometrial gland area (P < 0.01). FMT markedly shifted the metabolite profiles of both liver and plasma, and these differential metabolites enriched in steroid hormone biosynthesis pathway. FMT increased estradiol and insulin-like growth factor 1 but decreased progesterone dynamically. FMT also increased the expression of the EGD-related genes estrogen receptor 1 gene, epithelial cadherin, and forkhead box protein A2. There is a significant correlation between FMT-shifted gut microbes and EGD-related indices. CONCLUSION: Sexually matured Meishan gilts achieved a superior EGD than LxY gilts. Meanwhile, gut microbiota contribute to the EGD potentially via regulating of steroid hormones during the ovary-dependent period.

Dietary glycyl-glutamine supplementation ameliorates intestinal integrity, inflammatory response, and oxidative status in association with the gut microbiota in LPS-challenged piglets.

During weaning transition, mammalian newborns suffer severe enteric infections and thus induced gut microbiota dysbiosis, which in turn aggravates enteric disorder. The synthetic dipeptide glycyl-glutamine (GlyGln) has been used as a diet supplement to improve the weaning transition of newborns. However, the effect of dietary GlyGln supplementation on the gut microbiota of piglets with enteric infection remains unclear. Here, weaned piglets received a basal diet or a basal diet supplemented with 0.25% GlyGln for 3 weeks. Five piglets in each group received an intraperitoneal injection of lipopolysaccharide (LPS) (100 µg per kg BW) (LPS and GlyGln + LPS groups) and meanwhile five piglets in a control group received an intraperitoneal injection of saline (Ctrl group). The results showed that dietary GlyGln supplementation improved the LPS induced inflammation response and damage to the ileum morphology by increasing interleukin 10, tight junction proteins, villus height, and the ratio villus height/crypt depth, but decreasing the crypt depth. For the oxidative status, dietary GlyGln supplementation increased the ileal superoxide dismutase and meanwhile reduced the malondialdehyde and nitric oxide synthase activity (NOS) (total NOS and inducible NOS), compared with that in the LPS group. LPS challenge reduced the diversity of gut microbiota and enriched the facultative anaerobic Escherichia coli. The GlyGln restored alpha diversity and the structure of the gut microbiota by enriching obligate anaerobes and short-chain fatty acid (SCFA)-producing bacteria, including Clostridium, Lachnospira, Phascolarctobacterium, Roseburia, Lachnospiraceae, and Synergistetes. GlyGln enriched the gut microbiota function of carbohydrate metabolism and elevated the ileal SCFA concentrations of propionic acid and butyric acid that had been decreased by the LPS challenge. The beneficial effects of dietary GlyGln supplementation are closely associated with its enriched bacteria and SCFAs. Taken together, dietary GlyGln supplementation improved the gut microbiota dysbiosis induced by LPS challenge and enriched obligate anaerobes and SCFA-producing bacteria, which contributed to the amelioration of intestinal integrity, inflammatory responses, and oxidative status.

Dietary Supplementation of ε-Polylysine Beneficially Affects Ileal Microbiota Structure and Function in Ningxiang Pigs.

Intestinal microbiota plays an important role in the health of animals. However, little is known about the gut microbiota in Ningxiang pigs. Thus, we investigated how dietary supplementation with different ε-polylysine concentrations (0, 20, 40, 80, and 160 ppm) affected the ileal microbiota in Ningxiang pigs using a replicated 5 × 5 Latin square method. Each experimental period included 10 days for diet adaptation, 3 days for feces collection and 2 days for digesta collection. The ileal contents were collected and used for sequencing of the V3-V4 hypervariable region of the 16S rRNA gene. The results revealed that ε-polylysine significantly decreased the digestibility of crude protein and crude fiber, as well as the utilization of metabolizable energy (P < 0.05). The relative abundances of 19 bacterial genera significantly increased, while those of 26 genera significantly decreased (P < 0.05). In addition, ε-polylysine increased the abundance of some bacteria (e.g., Faecalibacterium, Bifidobacterium, and lactic acid bacteria) and inhibited some other bacteria (e.g., Micrococcaceae, Acinetobacter, Anaerococcus, Peptoniphilus, Dehalobacterium, Finegoldia, Treponema, and Brevundimonas). Furthermore, based on the 16S rRNA gene data and data from the precalculated GreenGenes database, bacterial communities in the ileal contents exhibited enhanced functional maturation, including changes in the metabolism of carbohydrates, amino acids (e.g., alanine, lysine, tryptophan, cysteine, and methionine), cofactors, and vitamins (e.g., biotin, thiamine, and folate), as well as in the activity of the insulin signaling pathway. This study suggests that ε-polylysine may influence the utilization of feed nutrients by Ningxiang pigs, including proteins, lipids, metabolizable energy, and fiber, by regulating the gut microbiota.

Anti­inflammatory mechanism of berberine on lipopolysaccharide­induced IEC­18 models based on comparative transcriptomics.

Intestinal surface epithelial cells (IECs) have long been considered as an effective barrier for maintaining water and electrolyte balance, and are involved in the mechanism of nutrient absorption. When intestinal inflammation occurs, it is often accompanied by IEC malfunction. Berberine (BBR) is an isoquinoline alkaloid found in numerous types of medicinal plants, which has been clinically used in China to treat symptoms of gastrointestinal pathogenic bacterial infection, especially bacteria­induced diarrhea and inflammation. In the present study, IEC­18 rat intestinal epithelial cells were treated with lipopolysaccharide (LPS) to establish an in vitro model of epithelial cell inflammation, and the cells were subsequently treated with BBR in order to elucidate the anti­inflammatory mechanism. Transcriptome data were then searched to find the differentially expressed genes (DEGs) compared between two of the treatment groups (namely, the LPS and LPS+BBR groups), and DEGs were analyzed using Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, Weighted Gene Correlation Network Analysis and Interactive Pathways Explorer to identify the functions and pathways enriched with DEGs. Finally, reverse transcription­quantitative PCR was used to verify the transcriptome data. These experiments revealed that, comparing between the LPS and LPS+BBR groups, the functions and pathways enriched in DEGs were 'DNA replication', 'cell cycle', 'apoptosis', 'leukocyte migration' and the 'NF­κB and AP­1 pathways'. The results revealed that BBR is able to restrict DNA replication, inhibit the cell cycle and promote apoptosis. It can also inhibit the classic inflammatory pathways, such as those mediated by NF­κB and AP­1, and the expression of various chemokines to prevent the migration of leukocytes. According to transcriptomic data, BBR can exert its anti­inflammatory effects by regulating a variety of cellular physiological activities, including cell cycle, apoptosis, inflammatory pathways and leukocyte migration.

Effects of Cortex Phellodendri extract on post-weaning piglets diarrhoea.

The diarrhoea incidence rate is often high among weaning piglets. In light of the fact that Cortex phellodendri has long been used to treat diarrhoea in China, this study aimed to evaluate the effects of Cortex Phellodendri Extract (CPE) on diarrhoea in weaning piglets and the mechanism behind such effects. In the first trial, 36 diarrhoeal weaning piglets were randomly divided into three groups. The control group was injected with 20 mg oxytetracycline/kg BW, while the two treatment groups were orally administered with 10 mg and 20 mg CPE/kg BW respectively. In the second trial, 96 weaning piglets were randomly divided into two groups. The control group was fed basal diet, while 300 mg CPE/kg BW was added to the diet of the treatment group. The pathogenic bacteria were then isolated and identified from the diarrhoeal faecal samples. Cell adhesion and RT-PCR tests were used to investigate the effect of CPE on the adhesion of pathogenic bacteria to IPEC-J2 cells. 16S rDNA-based high-throughput sequencing was used to analyse faecal microflora. The results showed that CPE reduced the diarrhoea incidence rate (p < 0.05) and diarrhoea index (p < 0.05) compared to control group, and increased the richness and evenness of weaning piglets' gut microbiota. Escherichia coli (E. coil) was identified as the causative organism. Cell adhesion and RT-PCR tests suggested that CPE reduced the adhesion of E. coli to IPEC-J2 cells (p < 0.05) and the expression of fae and faeG gene (p < 0.05) responsible for encoding E. coli fimbriae protein.

Cortex Phellodendri extract's anti-diarrhea effect in mice related to its modification of gut microbiota.

Cortex Phellodendri extract (CPE) has been used in China to treat diarrhea whereas the underlying mechanisms remain poorly understood. Given that dysbiosis of gut microbiota is a potential reason for diarrhea, and that oral CPE has a low absorption rate in intestine, we hypothesized that modification of gut microbiota is an important factor in CPE's anti-diarrhea effect. To test this hypothesis, we established a diarrhea model by challenging post-weaning mice with oral Enterotoxigenic-Escherichia coli (ETEC), and then the mice were treated with two doses of CPE (80 mg/kg bodyweight and 160 mg/kg bodyweight) or the vehicle control (phosphate buffered saline). Diarrhea indices, inflammatory factors, morphology of jejunum, short-chain fatty acids (SCFAs), and serum endocrine were determined. Modification of gut microbiota was analyzed using 16S rDNA high-throughput sequencing. The changes in functional profiles of gut microbiota were predicted using software PICRUSt. We then explored the association between CPE-responding bacteria and the symptoms indices with the spearman's rank correlation coefficient and significance test. Compared with diarrheal mice, CPE decreased Gut/Carcass ratio and water content of stool, increased goblet cell density and villus height/crypt depth of jejunum, as well as decreased inflammatory indices (Tumour Necrosis Factor-α, Myeloperoxidase and Interleukin-1α). CPE shifted the gut microbiota significantly by increasing alpha diversity (observed species, ace, Shannon, and Simpson) and restoring the gut microbiota. CPE increased Firmicutes and decreased Bacteroidetes. The reduced genus Prevotella, Acinetobacter, and Morganella were positively associated with the diarrhea indices, whereas increased genus Odoribacter, Rikenella, and Roseburia were negatively associated with the diarrhea indices. The abundance of carbohydrate metabolism-related gene and SCFAs-producing bacteria were increased, which was evidenced by increased butyric acid and total SCFAs concentration in the caecum. Consequently, endocrine peptides glucagon-like peptide-1, epidermal growth factor, and peptide tyrosine tyrosine in serum were elevated. CONCLUSIONS: CPE shows a shift function on the gut microbiota in alleviating the diarrhea of mice in a dose-dependent manner. In addition, the microbial metabolites SCFAs may mediate CPE's anti-diarrhea effect by enhancing endocrine secretion in mice.

Increased Consumption of Sulfur Amino Acids by Both Sows and Piglets Enhances the Ability of the Progeny to Adverse Effects Induced by Lipopolysaccharide.

This study determined the effects of increased consumption of sulfur amino acids (SAA), as either DL-Met or Hydroxy-Met (OH-Met), by sows and piglets on their performance and the ability of the progeny to resist a lipopolysaccharide (LPS) challenge. Thirty primiparous sows were fed a diet adequate in SAA (CON) or CON + 25% SAA, either as DL-Met or OH-Met from gestation day 85 to postnatal day 21. At 35 d old, 20 male piglets from each treatment were selected and divided into 2 groups (n = 10/treatment) for a 3 × 2 factorial design [diets (CON, DL-Met or OH-Met) and challenge (saline or LPS)]. OH-Met and/or DL-Met supplementation increased (p ≤ 0.05) piglets' body weight gain during day 0-7 and day 7-14. Sow's milk quality was improved in the supplemented treatments compared to the CON. The LPS challenge decreased (p ≤ 0.05) piglets' performance from 35 to 63 d and increased (p ≤ 0.05) the levels of aspartate aminotransferase, total bilirubin, IL-1ß, IL-6, TNF-a, and malondialdehyde. Plasma albumin, total protein, total antioxidant capacity and glutathione peroxidase decreased post-challenge. The results were better with OH-Met than DL-Met. The increase of Met consumption, particularly as OH-Met increased piglets' growth performance during the lactation phase and the challenging period.

Modulation of Gut Microbial Community and Metabolism by Dietary Glycyl-Glutamine Supplementation May Favor Weaning Transition in Piglets.

Gut microbiota plays a crucial role in diet nutrient metabolism and maintaining host health. The synthetic dipeptides glycyl-glutamine (Gly-Gln) used as diet supplementation to improve the weaning transition of newborns could be metabolized by certain bacteria in vitro. However, the effect of diet Gly-Gln supplementation on gut microbiota in vivo remains largely unknown. 240 piglets at the age of 28 days (day 28) were randomly assigned to two groups that received a basal diet (Ctrl group) or a basal diet supplemented with 0.25% Gly-Gln (Gly-Gln group) for 3 weeks. Five piglets from each group were euthanized for sampling after overnight fasting on day 38 and day 49, respectively. We determined their structure shifts of the gut microbiota using 16S rDNA-based high-throughput sequencing analysis. Microbial metabolites short-chain fatty acids (SCFAs) in the ileum and the colon were determined with high-performance gas chromatography. The concentrations of endocrine peptides including epidermal growth factor, glucagon-like peptide-1, and glucagon-like peptide-2 in ileal mucosa, as well as the serum concentration of interleukin 1 beta, interleukin 6, interleukin 10, and tumor necrosis factor alpha were determined using Enzyme-Linked Immunosorbent Assay. In addition, we also checked the diarrhea ratio, growth performance, and intestinal morphology to assess the favorable effect of dietary Gly-Gln supplementation during the weaning transition. Dietary Gly-Gln supplementation beneficially altered the gut microbiota by increasing bacterial loading, elevating alpha diversity, and increasing the relative abundance of anaerobes and fiber-degrading bacteria (Phylum Fibrobacteres). Accordingly, the microbial metabolites SCFAs in both colon and ileum, as well as the downstream endocrine peptides in the ileum increased. Meanwhile, dietary Gly-Gln's favorable weaning transition was reflected in the increase of growth performance indices and the reduced inflammatory response in a time dependent manner. There were significant correlations among the bacteria which responded to dietary Gly-Gln supplementation and these checked indices. Taken together, dietary Gly-Gln supplementation selectively modulated the gut microbiota, which may favor piglets' weaning-transition. These findings suggest that gut microbiota targeted approaches can be potentially used to improve weaning transition of piglets by dietary functional amino acid.

Standardized Preparation for Fecal Microbiota Transplantation in Pigs.

The intestine of pigs harbors a mass of microorganisms which are essential for intestinal homeostasis and host health. Intestinal microbial disorders induce enteric inflammation and metabolic dysfunction, thereby causing adverse effects on the growth and health of pigs. In the human medicine, fecal microbiota transplantation (FMT), which engrafts the fecal microbiota from a healthy donor into a patient recipient, has shown efficacy in intestinal microbiota restoration. In addition, it has been used widely in therapy for human gastrointestinal diseases, including Clostridium difficile infection, inflammatory bowel diseases, and irritable bowel syndrome. Given that pigs share many similarities with humans, in terms of anatomy, nutritional physiology, and intestinal microbial compositions, FMT may also be used to restore the normal intestinal microbiota of pigs. However, feasible procedures for performing FMT in pigs remains unclear. Here, we summarize a standardized preparation for FMT in pigs by combining the standard methodology for human FMT with pig production. The key issues include the donor selection, fecal material preparation, fecal material transfer, stool bank establishment, and the safety for porcine FMT. Optimal donors should be selected to ensure the efficacy of porcine FMT and reduce the risks of transmitting infectious diseases to recipients during FMT. Preparing for fresh fecal material is highly recommended. Alternatively, frozen fecal suspension can also be prepared as an optimal choice because it is convenient and has similar efficacy. Oral administration of fecal suspension could be an optimal method for porcine fecal material transfer. Furthermore, the dilution ratio of fecal materials and the frequency of fecal material transfer could be adjusted according to practical situations in the pig industry. To meet the potential large-scale requirement in the pig industry, it is important to establish a stool bank to make porcine FMT readily available. Future studies should also focus on providing more robust safety data on FMT to improve the safety and tolerability of the recipient pigs. This standardized preparation for porcine FMT can facilitate the development of microbial targeted therapies and improve the intestinal health of pigs.

Lactobacillus frumenti Facilitates Intestinal Epithelial Barrier Function Maintenance in Early-Weaned Piglets.

Increased intestinal epithelial barrier function damages caused by early weaning stress have adverse effects on swine health and feed utilization efficiency. Probiotics have emerged as the promising antibiotic alternatives used for intestinal barrier function damage prevention. Our previous data showed that Lactobacillus frumenti was identified as a predominant Lactobacillus in the intestinal microbiota of weaned piglets. However, whether the intestinal epithelial barrier function in piglets was regulated by L. frumenti is still unclear. Here, piglets received a PBS vehicle or PBS suspension (2 ml, 108 CFU/ml) containing the L. frumenti by oral gavage once a day during the period of 6-20 days of age prior to early weaning. Our data demonstrated that oral administration of L. frumenti significantly improved the intestinal mucosal integrity and decreased the serum endotoxin and D-lactic acid levels in early-weaned piglets (26 days of age). The intestinal tight junction proteins (including ZO-1, Occludin, and Claudin-1) were significantly up-regulated by L. frumenti administration. The serum immunoglobulin G (IgG) levels, intestinal secretory immunoglobulin A (sIgA) levels, and interferon-γ (IFN-γ) levels were significantly increased by L. frumenti administration. Furthermore, our data revealed that oral administration of L. frumenti significantly increased the relative abundances of health-promoting microbes (including L. frumenti, Lactobacillus gasseri LA39, Parabacteroides distasonis, and Kazachstania telluris) and decreased the relative abundances of opportunistic pathogens (including Desulfovibrio desulfuricans and Candida humilis). Functional alteration of the intestinal bacterial community by L. frumenti administration was characterized by the significantly increased fatty acids and protein metabolism and decreased diseases-associated metabolic pathways. These findings suggest that L. frumenti facilitates intestinal epithelial barrier function maintenance in early-weaned piglets and may be a promising antibiotic alternative used for intestinal epithelial barrier function damage prevention in mammals.

Effects of recombinant Agrocybe aegerita lectin as an immunoadjuvant on immune responses.

CONTEXT: Accumulated evidence has indicated that recombinant Agrocybe aegerita lectin (AAL) possesses immunoadjuvant activity to enhance antigen-specific immune responses. However, the mechanism of how AAL regulates immune response remains poorly defined. AIM: This study is aimed to reveal the mechanism of AAL's immunoadjuvant activity. METHODS: In this study, AAL alone or combined with inactivated avian influenza virus H9N2 was immunized to mice and the transcriptome profile of immunized mice was analyzed. RESULTS: In line with previous studies, our results showed that H9N2-specific IgG level was significantly increased in AAL-treated mice, suggesting the immunoadjuvant activity of AAL. More importantly, transcriptome data revealed that genes participating in the primary adherence, lymphocyte activation, secondary adherence and transmembrane migration of leukocyte migration, were up-regulated by AAL. CONCLUSION: These findings suggest that AAL exerts immunoadjuvant effects by promoting chemotaxis and phagotrophy activity of neutrophil leucocyte and macrophage to improve innate immunity and antigen presentation.