Ileal Tissue Research Articles

Undernutrition disrupts jejunal and ileal microbiota and epithelial tissue homeostasis in a pregnant sheep model

Nutrition consistently affects microbe-host interactions in the gastrointestinal tract. This study aimed to unravel how undernutrition reshapes the microbial composition and the homeostasis of epithelium in the jejunum and ileum. Sixteen late-gestation Hu-sheep were randomly assigned to the control group (n = 8, 100% ad libitum feeding levels) or the undernutrition group (n = 8, which received 30% ad libitum feeding levels). After 15-d treatment, all ewes were slaughtered, and jejunal and ileal digesta and epithelium samples were collected for 16S rRNA gene sequencing and transcriptome sequencing, respectively. Results indicated that undernutrition decreased the jejunal and ileal tissue weights (P = 0.005 and P = 0.022) and the levels of volatile fatty acids (P = 0.019 and P = 0.007) and microbial protein levels (P = 0.019 and P = 0.031) in jejunal and ileal digesta. The relative abundance of acetate producing microbiota, including Clostridia UCG-014 norank, Ruminococcus, [Ruminococcus] gauvreauii, and Lachnospiraceae_Blautia, were significantly reduced (P < 0.05) in the jejunum and ileum. Undernutrition up-regulated (P < 0.05) the expression of genes involved in amino acid synthesis and fatty acid oxidation, but down-regulated (P < 0.05) the expression of genes associated with amino acid degradation, fatty acid synthesis, and extracellular structures in jejunal and ileal epithelium. In the jejunal epithelium, genes associated with extracellular matrix-receptor interactions, cell growth, and immune response were down-regulated (P < 0.05) upon undernutrition. Taken together, undernutrition changed the microbial community in the jejunum and ileum, which altered the fermentation mode and the production of volatile fatty acids and microbial protein. These affected the energy and protein system in the epithelium and reprogrammed substance metabolism and extracellular structures, which probably further influenced cell growth and immune response. These insights provide a foundation for completely clarifying the crosstalk between small intestinal microbiota and the host.

Mechanism of action of exercise regulating intestinal microflora to improve spontaneous hypertension in rats.

Hypertension is a prevalent cardiovascular disease. Exercise is widely recognized as an effective treatment for hypertension, and it may also influence the composition of the intestinal microflora. However, it remains unclear whether exercise can specifically regulate the intestinal microflora in the context of hypertension treatment. In this study, tail blood pressure in spontaneously hypertensive rats (SHR) was measured using a blood pressure meter after exercise intervention and fecal bacteria transplantation following exercise. Blood lipid levels were assessed using an automatic biochemical analyzer, and 16S rRNA sequencing was employed to analyze the intestinal microflora. Histological examinations of ileal tissue were conducted using HE and Masson staining. Intestinal permeability, inflammatory status, and sympathetic activity were evaluated by measuring the levels of diamine oxidase, D-lactic acid, C-reactive protein, interleukin-6, tumor necrosis factor-α, lipopolysaccharide, norepinephrine, angiotensin II, cyclic adenosine monophosphate, and cyclic guanosine monophosphate. Exercise was found to reduce blood pressure and blood lipid levels in SHR. It also improved the composition of the intestinal microflora, as evidenced by a reduced Firmicutes/Bacteroidetes ratio, an increase in bacteria that produce acetic and butyric acid, and higher Chao 1 and Shannon diversity indices. Furthermore, exercise reduced the thickness of the fibrotic and muscular layers in the ileum, increased the goblet cell/villus ratio and villus length, and decreased intestinal permeability, inflammatory markers, and sympathetic nerve activity. The intestinal microbial flora regulated by exercise demonstrated similar effects on hypertension. In conclusion, exercise appears to regulate the intestinal microflora, and this exercise-induced change in flora may contribute to improvements in hypertension in rats.

Environmental and inflammatory factors influencing concurrent gut and lung inflammation.

Crohn's disease and chronic obstructive pulmonary disease (COPD) are chronic inflammatory diseases that affect the gut and lung respectively and can occur comorbidly. Using the SHIP-1-/- model of Crohn's-like ileitis and chronic lung inflammation, the two diseases were co-investigated. Contrary to prior literature, Crohn's-like ileitis was not fully penetrant in SHIP-1-/- mice, and housing in a specific pathogen-free facility was completely protective. Indeed, ileal tissue from SHIP-1-/- mice without overt ileitis was similar to control ilea. However, SHIP-1-/- mice with ileitis exhibited increased granulocytes in ileal tissue together with T cell lymphopenia and they lacked low abundance Bifidobacteria, suggesting this bacterium protects against ileitis. Lung disease, as defined by inflammation in lung washes, emphysema, and lung consolidation, was present in SHIP-1-/- mice regardless of ileitis phenotype; however, there was a shift in the nature of lung inflammation in animals with ileitis, with increased G-CSF and neutrophils, in addition to type 2 cytokines and eosinophils. Deficiency of G-CSF, which protects against lung disease, protected against the development of ileitis in SHIP-1-/- mice. These studies have defined environmental, immune, and inflammatory factors that predispose to ileitis, and have identified that comorbid lung disease correlates with a granulocyte signature.

Jianpi-Huatan-Huoxue-Anshen formula ameliorates gastrointestinal inflammation and microecological imbalance in chemotherapy-treated mice transplanted with H22 hepatocellular carcinoma.

Jianpi-Huatan-Huoxue-Anshen formula [Tzu-Chi cancer-antagonizing & life-protecting II decoction (TCCL)] is a Chinese medical formula that has been clinically shown to reduce the gastrointestinal side effects of chemotherapy in cancer patients and improve their quality of life. However, its effect and mechanism on the intestinal microecology after chemotherapy are not yet clear. To discover the potential mechanisms of TCCL on gastrointestinal inflammation and microecological imbalance in chemotherapy-treated mice transplanted with hepatocellular carcinoma (HCC). Ninety-six mice were inoculated subcutaneously with HCC cells. One week later, the mice received a large dose of 5-fluorouracil by intraperitoneal injection to establish a HCC chemotherapy model. Thirty-six mice were randomly selected before administration, and feces, ileal tissue, and ileal contents were collected from each mouse. The remaining mice were randomized into normal saline, continuous chemotherapy, Yangzheng Xiaoji capsules-treated, and three TCCL-treated groups. After treatment, feces, tumors, liver, spleen, thymus, stomach, jejunum, ileum, and colon tissues, and ileal contents were collected. Morphological changes, serum levels of IL-1β, IL-6, IL-8, IL-10, IL-22, TNF-α, and TGF-β, intestinal SIgA, and protein and mRNA expression of ZO-1, NF-κB, Occludin, MUC-2, Claudin-1, and IκB-α in colon tissues were documented. The effect of TCCL on the abundance and diversity of intestinal flora was analyzed using 16S rDNA sequencing. TCCL treatment improved thymus and spleen weight, thymus and spleen indexes, and body weight, decreased tumor volumes and tumor tissue cell density, and alleviated injury to gastric, ileal, and colonic mucosal tissues. Among proteins and genes associated with inflammation, IL-10, TGF-β, SIgA, ZO-1, MUC-2, and Occludin were upregulated, whereas NF-κB, IL-1β, IL-6, TNF-α, IL-22, IL-8, and IκB-α were downregulated. Additionally, TCCL increased the proportions of fecal Actinobacteria, AF12, Adlercreutzia, Clostridium, Coriobacteriaceae, and Paraprevotella in the intermediate stage of treatment, decreased the proportions of Mucipirillum, Odoribacter, RF32, YS2, and Rikenellaceae but increased the proportions of p_Deferribacteres and Lactobacillus at the end of treatment. Studies on ileal mucosal microbiota showed similar findings. Moreover, TCCL improved community richness, evenness, and the diversity of fecal and ileal mucosal flora. TCCL relieves pathological changes in tumor tissue and chemotherapy-induced gastrointestinal injury, potentially by reducing the release of pro-inflammatory factors to repair the gastrointestinal mucosa, enhancing intestinal barrier function, and maintaining gastrointestinal microecological balance. Hence, TCCL is a very effective adjuvant to chemotherapy.

The unseen spread: a case of disseminated tuberculosis with renal manifestation in a healthy adult

Disseminated tuberculosis (TB), resulting from the hematogenous spread of tubercle bacilli, typically affects immunocompromised individuals, such as those infected with the human immunodeficiency virus. However, risk factors in immunocompetent populations are not well understood. Here, we report a rare case of disseminated TB with CD4+ T-cell depletion in a previously healthy 35-year-old man. The patient presented with a 2-month history of intermittent gross hematuria, dysuria, loose stools, and weight loss. His medical history was unremarkable except for a herpes zoster infection 4 years prior to presentation. Laboratory tests revealed microscopic hematuria and pyuria; however, the urine culture was negative. Urine specimens tested positive for TB-polymerase chain reaction. Abdominal computed tomography revealed a focal filling defect in the left kidney, segmental wall thickening of the terminal ileum, and multiple enlarged lymph nodes with central necrosis. Chest computed tomography revealed active pulmonary TB. Colonoscopy confirmed intestinal TB in the terminal ileum and ileocecal valve, with positive TB-polymerase chain reaction results from sputum and ileal ulcer tissue. The patient was diagnosed with disseminated TB and was treated with standard anti-TB drugs. Although the human immunodeficiency virus test results were negative, the patient’s CD4+ T-cell count was significantly low (278/μL). Follow-up tests after 1 month showed negative TB cultures; however, the patient’s CD4+ T-cell depletion persisted, with counts remaining low after 1 year. This case highlights the rare occurrence of disseminated TB in immunocompetent individuals with CD4+ T-cell depletion and emphasizes the importance of CD4+ T-cell assessment in healthy patients presenting with disseminated TB.

Intestinal microbiota composition in broilers fed protein-free or casein-based diets

Growing broiler chickens of the Cobb500 strain were used to determine the effects on intestinal microbiota composition of a protein-free (PF) diet as compared to a diet based in casein (CAS) as the only protein source. CAS was formulated to contain the same amount of protein (190 g kg-1) as a commercial Maize-soy diet which was used as a practical reference. The ileal AA flow (g kg-1 dry matter intake) was significantly higher (P < 0.001) than PF in birds fed protein containing diets (CAS or Maize-soy). Taken as a whole (discriminant and ANOSIM analysis), the intestinal (ileal and caecal contents and ileal tissue) microbiota composition of PF and CAS were significantly (P < 0.001) different from Maize-soy and not different from each other in some cases. RT-qPCR and sequencing analysis of the ileal and caecal microbiota revealed significant (P < 0.05) differences in a number of bacterial groups between broilers fed PF, CAS or Maize-soy diets. The main result was that the lack of protein in the intestinal medium of PF birds resulted in a drop of Lactobacillus spp. counts (on average, 43 in PF vs 1,734 in the Maize-soy diet) and increased Enterobacteriaceae (on average, 419 in PF vs 172 in the Maize-soy diet) and other potentially pathogenic bacterial groups (in both intestinal contents and tissue). Thus, the lack of protein in the intestinal medium of PF birds resulted in a microbiota composition compatible with a pro-inflammatory state, and this effect was somewhat less marked in birds fed CAS. The results reported here suggest that the adverse effects on microbiota composition in broilers fed CAS were less marked than in those fed PF, which would be in line with a preferential use of a highly digestible protein containing diet to determine endogenous AA excretion instead of a PF diet.

Activation of GABABR Attenuates Intestinal Inflammation by Reducing Oxidative Stress through Modulating the TLR4/MyD88/NLRP3 Pathway and Gut Microbiota Abundance.

Oxidative stress emerges as a prominent factor in the onset and progression of intestinal inflammation, primarily due to its critical role in damaging cells and tissues. GABAergic signaling is important in the occurrence and development of various intestinal disorders, yet its effect on oxidative stress remains unclear. We attempted to assess whether GABAergic signaling participated in the regulation of oxidative stress during enteritis. The results showed that lipopolysaccharide (LPS) significantly decreased γ-aminobutyric acid (GABA) levels in the ileal tissues of mice. Interestingly, the application of GABA significantly repressed the shedding of intestinal mucosal epithelial cells and inflammatory cell infiltration, inhibited the expressions of proinflammatory factors, including granulocyte colony-stimulating factor and granulocyte-macrophage colony stimulating factor, and enhanced the levels of anti-inflammatory cytokines interleukin (IL)-4 and IL-10, indicating that GABA could alleviate enteritis in mice. This observation was further supported by transcriptome sequencing, revealing a total of 271 differentially expressed genes, which exhibited a marked enrichment of inflammatory and immune-related pathways, alongside a prominent enhancement of GABA B receptor (GABABR) signaling following GABA administration. Effectively, Baclofen pretreatment alleviated intestinal mucosal damage in LPS-induced mice, suppressed proinflammatory cytokines IL-1β, IL-6, and tumor necrosis factor alpha expressions, and boosted total antioxidant capacity, superoxide dismutase (SOD), and glutathione (GSH) levels. Moreover, Baclofen notably enhanced the viability of LPS-stimulated IPEC-J2 cells, contracted the proinflammatory secretion factors, and reinforced SOD, GSH, and catalase levels, emphasizing the anti-inflammatory and antioxidant effects associated with GABABR activation. Mechanistically, Baclofen restrained the mRNA and protein levels of toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing 3 (NLRP3), and inducible nitric oxide synthase, while elevating nuclear factor erythroid 2-related factor 2 and heme oxygenase-1 in both mice and IPEC-J2 cells, indicating that activating GABABR strengthened antioxidant abilities by interrupting the TLR4/MyD88/NLRP3 pathway. Furthermore, 16S rDNA analysis demonstrated that Baclofen increased the relative abundance of probiotic, particularly Lactobacillus, renowned for its antioxidant properties, while reducing the relative richness of harmful bacteria, predominantly Enterobacteriaceae, suggesting that GABABR signaling may have contributed to reversing intestinal flora imbalances to relieve oxidative stress in LPS-induced mice. Our study identified previously unappreciated roles for GABABR signaling in constricting oxidative stress to attenuate enteritis, thus offering novel insights for the treatment of intestinal inflammation.

Comparison of human breast milk vs commercial formula-induced early trophic enteral nutrition during postoperative prolonged starvation in an animal model

The present study aimed to characterize the changes in macromolecular composition and structure in ileal tissue induced by postoperative prolonged starvation (PS), human breast milk feeding (HM) and commercial formula feeding (CF) for 48 and 72 h (h). Forty-two Wistar albino rats underwent an ileal transection and primary anastomosis and were then divided into six subgroups. Two groups of seven rats were food-deprived for 48 and 72 h with free access to water only in metabolic cages (48 h PS, 72 h PS). Then, two groups of seven rats received early enteral trophic nutrition (EEN) either using HM, and CF at 48 h post-operation (48 h HM, 48 h CF). The other two groups of seven rats received the same trophic enteral nutrition at 72 h post-operation (72 h HM, 72 h CF). An additional seven rats were fed normal rat chow (control), after which the ileal tissues were harvested and freeze-dried overnight. Then sample spectra were recorded by Fourier transform infrared (FTIR) spectroscopy. PS at 48 and 72 h resulted in an increase in the concentration of lipids and a decrease in the concentration of proteins. CF and HM trophic feeding induced a decrease in membrane fluidity and an increase in lipid order. Ileal tissues showed similar compositional and structural changes in lipids and proteins in the PS and CF groups after 48 and 72 h. A marked decrease in nucleic acid concentration was seen in CF at 48 h compared to HM. The human milk feeding groups did not induce any significant alterations and showed compositional and structural data similar to the controls. In conclusion, EEN application seems to be safer when introduced at 48 h rather than 72 h and time of this nutrition is crucial to maintain ileum structure and therefore immunity and well-being. HM-induced trophic nutrition is seen to protect the ileal tissue from significant alterations within lipid and protein compositions, whereas CF caused notable changes. HM is absolutely the best nutritional source for gut health in this animal model.

PSIV-19 The use of microencapsulated zinc oxide improves the growth performance and intestinal health of newly weaned piglets

Abstract The application of microencapsulation technology to protect zinc may support inclusion of reduced levels with similar benefits to elevated doses of free zinc oxide. This study aimed to compare the effects of different doses of a microencapsulated ZnO (Zinco-Plus S) and free ZnO in nursery diets on growth performance and intestinal health of pigs. A total of 480 newly weaned piglets [26 d of age, initial body weight (BW) = 7.39 kg] were randomly assigned to one of four dietary treatments for 35d. Treatments were as follow: free ZnO (3,000, 1,500, and 0 ppm at phase 1, 2, and 3, respectively) or one of three doses of the microencapsulated ZnO (400, 600, and 800 ppm) maintained consistently across all 3 nursery phases (total amount of zinc in diets were within limits allowed by Canadian regulation). Each dietary treatment had 10 replicates, with 12 pigs/pen, gender balanced. All treatments had protease (Jefo Protease; 125 g/t), xylanase (Belfeed Xylanase; 100 g/t) and protected organic acids and essential oils (Porcinat+; 2 kg/t in phase 1 and 2, 1 kg/t in phase 3). Body weight, BW gain and feed intake were measured to determine feed conversion. At the end of phase 1, fecal sample was collected (pooled of 4 samples/pen) to determine zinc excretion. At the end of phase 2, blood serum was collected (1 pig/pen) to measure calprotectin and diamine oxidase concentrations and, ileal tissue was collected (1 pig/pen) to measure expression of zonular occludens (ZO) -1. Data were submitted to ANOVA and Fisher test with significance set at P &amp;lt; 0.05. There was no difference (P &amp;gt; 0.05) in final BW of pigs across all treatments. Nonetheless, piglets receiving 800 ppm of the microencapsulated ZnO had improved (P &amp;lt; 0.05) feed conversion ratio compared with pigs fed diet containing free ZnO. There was no difference (P &amp;gt; 0.05) in serum concentrations of calprotectin and diamine oxidase across all treatments, indicating improved intestinal health regardless of the form of ZnO. Piglets supplemented with microencapsulated ZnO at 800 ppm had an increased (P &amp;lt; 0.05) intestinal expression of ZO-1 in comparison with piglets receiving free ZnO, indicating better intestinal integrity. Fecal zinc concentrations were reduced (P &amp;lt; 0.05) by 75 % with the inclusion of microencapsulated ZnO compared with free ZnO. Piglets supplemented with microencapsulated ZnO at 800 ppm improved (P &amp;lt; 0.05) feed cost per kg BW gain by 10.7 % when compared with free ZnO. These findings show that supplementing lower levels of zinc in nursery diets via microencapsulation technology improves growth performance and supports intestinal health at levels comparable with increased free ZnO supplementation. Moreover, the utilization of microencapsulated zinc oxide has the additional advantage of minimizing environmental impact through the reduction of zinc wastage.

476 Induced hindgut acidosis in sheep affected gut permeability and ruminal fermentation

Abstract Leaky gut may be caused by acidotic conditions in the hindgut and lead to systemic inflammation and negative effects on the gastrointestinal tract. The objective was to determine the effects of induced hindgut acidosis in sheep on cecal pH, ruminal fermentation, and gut permeability. Ruminally and cecally cannulated ewes [n = 11; body weight (BW) = 49 ± 4 kg] were assigned to one of two treatments: control (CON; n = 5) or induced hindgut acidosis (HGA; n = 6). To induce hindgut acidosis, 3 g wheat starch/kg BW per 24 h was continuously infused via the cecal cannula for 4 d. Control ewes received a constant infusion of deionized water. The diet contained 40% grass hay pellets, 35% whole shelled corn, 15% dried distillers grains, and 10% alfalfa cubes. Chromium EDTA was dosed once daily via the cecal cannula as a marker of gut permeability. Rumen, cecal, and fecal samples were collected to determine pH and volatile fatty acids (VFA). Rumen fluid was collected on d 4 for an ex vivo fermentation. Flasks were incubated for 24 h to determine pH, VFA, ammonia and in vitro dry matter digestibility. Tissue samples from the ileum, cecum, and colon were mounted in Ussing chambers to measure transepithelial electrical resistance (TEER). There was a treatment × time effect (P = 0.05) for cecal pH with HGA ewes having lesser cecal pH after d 1. By d 4, cecal pH had declined to 5.07 for HGA ewes compared with CON ewes which remained above 6.40 throughout the experiment. A treatment × time interaction was also observed (P &amp;lt; 0.01) for fecal pH and followed the same trend as cecal pH. Rumen pH was not affected (P = 0.87) by the interaction of treatment and time but was affected (P &amp;lt; 0.01) by treatment as ewes on the HGA treatment had a lesser rumen pH than CON ewes. Control ewes had lesser ruminal VFA and ammonia concentrations than HGA ewes (P &amp;lt; 0.01). Urinary Cr recovery was not affected by the interaction of treatment and time, or treatment (P ≥ 0.13). In vitro dry matter disappearance was not affected by cecal infusion treatment (P = 0.60). There was no effect (P = 0.78) of treatment on ex vivo pH. There were no effects (P ≥ 0.11) of treatment, time, or their interaction on ex vivo ammonia or total VFA concentration. In cecal tissue, TEER tended (P = 0.09) to be greater in CON ewes than HGA ewes. In contrast, TEER was not different (P ≥ 0.83) in ileal or colonic tissues between treatments. Induced hindgut acidosis in sheep altered rumen fermentation and tended to increase ex vivo measures of cecal permeability, but did not affect ex vivo rumen fermentation.

Dietary supplementation of valine, isoleucine, and tryptophan may overcome the negative effects of excess leucine in diets for weanling pigs containing corn fermented protein

BackgroundDiets with high inclusion of corn co-products such as corn fermented protein (CFP) may contain excess Leu, which has a negative impact on feed intake and growth performance of pigs due to increased catabolism of Val and Ile and reduced availability of Trp in the brain for serotonin synthesis. However, we hypothesized that the negative effect of using CFP in diets for weanling pigs may be overcome if diets are fortified with crystalline sources of Val, Trp, and (or) Ile.MethodsThree hundred and twenty weanling pigs were randomly allotted to one of 10 dietary treatments in a completely randomized design, with 4 pigs per pen and 8 replicate pens per treatment. A corn-soybean meal diet and 2 basal diets based on corn and 10% CFP or corn and 20% CFP were formulated. Seven additional diets were formulated by fortifying the basal diet with 20% CFP with Ile, Trp, Val, Ile and Val, Ile and Trp, Trp and Val, or Ile, Trp and Val. A two-phase feeding program was used, with d 1 to 14 being phase 1 and d 15 to 28 being phase 2. Fecal scores were recorded every other day. Blood samples were collected on d 14 and 28 from one pig per pen. On d 14, fecal samples were collected from one pig per pen in 3 of the 10 treatments to determine volatile fatty acids, ammonium concentration, and microbial protein. These pigs were also euthanized and ileal tissue was collected.ResultsThere were no effects of dietary treatments on any of the parameters evaluated in phase 1. Inclusion of 10% or 20% CFP in diets reduced (P < 0.05) final body weight on d 28, and average daily gain (ADG) and average daily feed intake (ADFI) in phase 2 and for the entire experimental period. However, pigs fed the CFP diet supplemented with Val, Ile, and Trp had final body weight, ADFI, ADG and gain to feed ratio in phase 2 and for the entire experiment that was not different from pigs fed the control diet. Fecal scores in phase 2 were reduced (P < 0.05) if CFP was used.ConclusionsCorn fermented protein may be included by up to 20% in diets for weanling pigs without affecting growth performance, gut health, or hindgut fermentation, if diets are fortified with extra Val, Trp, and Ile. Inclusion of CFP also improved fecal consistency of pigs.

Hawthorn-leaf flavonoid alleviate intestinal health and microbial dysbiosis problems induced by glyphosate

Glyphosate is the active ingredient in the herbicide (i.e., Roundup, Touchdown and Erasure), the safety of which has become a social concern. Hawthorn-leaf flavonoid (HF) possesses various biological functions, including antioxidant, regulating lipid metabolism and intestinal microbiota. Whether HF could reduce the health risk of pure glyphosate to birds remain unknown. The experiment aimed to evaluate the effects of pure glyphosate (25 mg/kg added to water) on the intestinal health and microbiota of chicks and the protective roles of HF (60 mg/kg added to the diet). Exposure to glyphosate decreased growth performance, ileal morphology structure, and antioxidant capacity, and increased the serum level of lipid and pro-inflammatory factors. 16S rRNA sequencing indicated that glyphosate decreased bacterial richness and the abundance of Lactobacillus, and increased proportions of pathogens in the ileum. Metabolomic results revealed that glyphosate increased the level of the cholic acid and fatty acids in the ileac digesta. Meanwhile, glyphosate down-regulated the protein expression associated with lipid transport, antioxidant and tight junction in the ileal mucosal tissue, and up-regulated the pro-inflammatory, oxidative stress proteins. However, dietary HF supplementation effectively mitigated the adverse effects of glyphosate and improved intestinal health of chicks. Therefore, dietary HF can ameliorate the harmful effects of glyphosate on birds, which highlights the potential application of HF in reducing the health risks.

An IL-17-DUOX2 axis controls gastrointestinal colonization by Candida albicans.

Candida albicans is a ubiquitous fungus in the human gut microbiome as well as a prevalent cause of opportunistic mucosal and systemic disease. There is currently little understanding, however, as to how crosstalk between C. albicans and the host regulates colonization of this key niche. Here, we performed expression profiling on ileal and colonic tissues in germ-free mice colonized with C. albicans to define the global response to this fungus. We reveal that Duox2 and Duoxa2 , encoding dual NADPH oxidase activity, are upregulated in both the ileum and colon, and that induction requires the C. albicans yeast-hyphal transition and the hyphal-specific toxin candidalysin. Hosts lacking the IL-17 receptor failed to upregulate Duox2/Duoxa2 in response to C. albicans , while addition of IL-17A to colonoids induced these genes together with the concomitant production of hydrogen peroxide. To directly define the role of Duox2/Duoxa2 in fungal colonization, antibiotic-treated mice lacking intestinal DUOX2 activity were evaluated for C. albicans colonization and host responses. Surprisingly, loss of DUOX2 function reduced fungal colonization at extended time points (>17 days colonization) and increased the proportion of hyphal cells in the gut. IL-17A levels were also elevated in C. albicans -colonized mice lacking functional DUOX2 highlighting cross-regulation between this cytokine and DUOX2. Together, these experiments reveal novel links between fungal cells, candidalysin toxin and the host IL-17-DUOX2 axis, and that a complex interplay between these factors regulates C. albicans filamentation and colonization in the gut.

Translocation of probiotics via gut–lung axis enhanced pulmonary immunity of weaned piglets exposed to low concentrations of ammonia

ObjectiveLactobacillus salivarius is a probiotic bacteria strain in human and animal diets. The administration of probiotics to weaned piglets may improve their growth by optimizing the gastrointestinal bacterial composition. To further investigate the effect of bacterial communication between the gastrointestinal tract and lungs on bodily immunity, we reared weaned piglets in a low-ammonia gas environment. L. salivarius was supplemented to explore its effects on pulmonary immunity and its potential for bacterial translocation. ResultsOne hundred sixty weaned piglets were allocated to four groups: L. salivarius-supplemented, L. reuteri-supplemented, control, and antibiotic drug (aureomycin)-supplemented. The feeding duration was 28 d. The body weights of piglets administered a strain of Lactobacillus were better than those of the control (P < 0.01). The transcription level of immune factors interleukin 2 (IL-2), IL-4, interferon α (IFN-α), and tumor necrosis factor α (TNF-α) in cells of the ileum and lung was significantly higher (P < 0.01). Lung and ileal mucus tissues were isolated to sequence the bacterial composition, which suggested a higher richness in the lungs at the phylum level, which was not significant in the ileum. Functional bacteria were more abundant in the ileum and lungs. The proportion of the genera of Lactobacillus, Prevotella, Actinobacillus, and Prevotellaceae_ NK3B31_group increased in two tissues, and a lower ratio of Streptococcus, Escherichia-Shigella, and mycoplasma was detected. The correlation between the microbial genus composition and the levels of immune factors suggests that the abundance of Lactobacillus plays the same positive role in the lungs and ileum. Mycoplasmas play a negative role in ileal and pulmonary immunity. More Lactobacillus reuteri and anaerobic probiotic bacteria were detected in the lungs. ConclusionThe colonization of Lactobacillus salivarius and Lactobacillus reuteri in the membrane of the ileum optimized the ileal microbial composition, enrolled other probiotic bacteria translating to the lung, improved the abundance of pulmonary microbiota, and enhanced immunity after exposure to low concentrations of ammonia.

Identifying the importance of PCK1 in maintaining ileal epithelial barrier integrity in Crohn’s disease

BackgroundCrohn’s disease (CD) is marked by disruption of intestinal epithelial barrier, with unclear underlying molecular mechanisms. This study aimed to investigate key genes regulating the intestinal barrier in CD patients. MethodsDifferential gene expression analysis and gene set enrichment analysis were conducted to identify potential key genes involved in CD within the GEO database. Single-cell RNA sequencing from ileum samples in GSE134809 of 59,831 inflamed and uninflamed cells from 11 CD patients and microarray data from ileal tissues in GSE69762 (3 controls and 4 CD patients) and GSE75214 (11 controls and 51 CD patients) with GSE179285 (49 uninflamed and 33 inflamed from CD patients) as the validation set. Protein-protein interaction and logistic regression analyses identified key downregulated genes in CD. A key gene was then investigated through immunohistochemistry of ileal tissues from 5 CD patients and in the Caco-2 cell line with RNA interference and treatment with IFN-γ and TNF-α to stimulate inflammation. ResultsSingle-cell RNA-seq identified 33 genes and microarray identified 167 genes with significant downregulation in inflamed CD samples. PCK1 was identified and validated as one of the most promising candidate genes. Reduced PCK1 expression was evident in inflamed ileal tissues. In vitro, knockdown of PCK1 resulted in decreased cell viability, increased apoptosis, and reduced nectin-2 production, while combination of IFN-γ and TNF-α significantly reduced PCK1. ConclusionsPCK1 is downregulated in inflamed ileal tissues of CD patients and may be a key factor in maintaining epithelial integrity during inflammation in Crohn’s disease.

Oxymatrine alleviates NSAID-associated small bowel mucosal injury by regulating MIP-1/CCR1 signalling and gut microbiota

Oxymatrine (OMT) as a quinazine alkaloid extracted from matrine has been shown to exhibit anti-inflammatory and anti-tumour effects. However, the protective mechanism of OMT on NSAID-associated small bowel mucosal injury remains unreported. We found that OMT could improve the clinical symptoms and pathological inflammation scoring, reduce the secretion of proinflammatory cytokines IL-1β, IL-6 and TNF-α and cell apoptosis, promote cell proliferation and protect intestinal mucosal barrier as compared with the Diclofenac Sodium (DS) group. Further RNA-seq and KEGG analysis uncovered that the differentially expressed genes between DS and control groups were mainly enriched in immune regulation, of which MIP-1γ and its receptor CCR1 expression were validated to be repressed by OMTH. MAPK/NF-κB as the MIP-1 upstream signalling was also inactivated by OMT treatment. In this study, OMT regulated gut microbiota. Venn diagrams visualized and identified 1163 shared OTUs between DS group and OMTH group. The results showed that the α diversity index in the DS group was lower than that in the OMTH group, indicating that the complexity of the flora was reduced in the intestinal inflammatory state. β diversity mainly includes Principal Component Analysis (PCA) and Principal Co-ordinates Analysis (PCoA). The differences between groups can be observed through PCA. The more similar the composition of the flora, the closer the samples are. We found that the difference was smaller in the DS group than in the OMTH group. The results of PcoA showed that the sample similarity between OMTH groups was the highest. Moreover, gut microbiota analysis unveiled that the abundances of Ruminococcus 1, Oscillibacter and Prevotellaceae at the genus level as well as Lactobacillus SP-L-Yj at the species level were increased in OMTH group as compared with the DS group but the abundance of Allobaculum, Ruminococceos-UCG-005, Ruminococceos-NK4A214 and Clostridium associated with DS-induced small bowel mucosal injury could be decreased by OMTH. MIP-1α and CCR1 were upregulated in human small bowel injury samples as compared with the normal ileal mucosa tissues. In conclusion, our findings demonstrated that OMT could alleviate NSAID-associated small bowel mucosal injury by inhibiting MIP-1γ/CCR1 signalling and regulating gut microbiota.

Quantifying Forms and Functions of Enterohepatic Bile Acid Pools in Mice

Backgrounds & AimsBile acids (BAs) are core gastrointestinal metabolites with dual functions in lipid absorption and cell signaling. BAs circulate between the liver and distal small intestine (i.e., ileum), yet the dynamics through which complex BA pools are absorbed in the ileum and interact with host intestinal cells in vivo remain poorly understood. As ileal absorption is rate-limiting in determining which BAs in the intestinal lumen gain access to host intestinal cells and receptors, and at what concentrations, we hypothesized that defining the rates and routes of ileal BA absorption in vivo would yield novel insights into the physiological forms and functions of mouse enterohepatic BA pools. MethodsUsing ex vivo mass spectrometry, we quantified 88 BA species and metabolites in the intestinal lumen and superior mesenteric vein of individual wild type mice, as well as cage-mates lacking the ileal BA transporter, Asbt/Slc10a2. ResultsUsing these data, we calculated that the pool of BAs circulating through ileal tissue (i.e., the ‘ileal BA pool’) in fasting C57BL/6J female mice is ∼0.3 μmoles/g. Asbt-mediated transport accounted for ∼80% of this pool and amplified size. Passive permeability explained the remaining ∼20% and generated diversity. Compared with wild type mice, the ileal BA pool in Asbt-deficient mice was ∼5-fold smaller, enriched in secondary BA species and metabolites normally found in the colon, and elicited unique transcriptional responses upon addition to ex vivo-cultured ileal explants. ConclusionsThis study defines quantitative traits of the mouse enterohepatic BA pool and reveals how aberrant BA metabolism can impinge directly on host intestinal physiology.

Mechanistic study of fructus aurantii (Quzhou origin) in regulating ileal reg3g in the treatment for NASH

BackgroundNon-alcoholic steatohepatitis (NASH) is a critical stage in the progression of non-alcoholic fatty liver disease (NAFLD), characterized by obvious inflammation and fibrosis. Because of its high incidence rate and serious consequences, NASH is becoming a global health problem. The influence of endotoxin translocation on NASH is receiving attention. As a traditional Chinese herb that effectively improves hepatic inflammation, Fructus Aurantii (Quzhou origin, FAQ) is widely used in the clinical treatment of NASH. However, the intervention mechanism of FAQ on reg3g and related endotoxin translocation remains unclear. AimTo study the mechanism of the impact by which ileal regenerating family member 3 gamma (reg3g) deficiency and subsequent endotoxin translocation impact the progression of NASH; To elucidate the efficacy and mechanism of FAQ in the treatment of NASH. MethodsClinical serum, ileal tissue, and dynamic NASH model-related analyses collectively confirmed that reg3g is a pivotal gene associated with NASH. Reg3g−/− mice were used to assess the impact of reg3g on liver injury, inflammation, and fibrosis, as well as the underlying mechanism involved. In vitro studies elucidated the regulatory effects of FAQ on reg3g, intestinal barrier function, and intestinal permeability. Subsequently, the efficacy of FAQ was investigated in NASH mouse models. Pathological examinations combined with Western blotting (WB), immunohistochemistry (IHC), and multiplex immunohistochemical (mIHC) analyses were used to evaluate the effects of FAQ on mucosal repair and barrier function. Transepithelial electrical resistance (TEER), fluorescein isothiocyanate-dextran 4 (FD-4) experiments, coupled with enzyme linked immunosorbent assay (ELISA) and chromogenic LAL endotoxin assay were used to confirm intestinal permeability and endotoxin translocation. The results of WB and mIHC reflected the levels of endotoxin recruitment and M1 macrophage polarization in the liver. Parameters such as body weight, transaminases, and cholesterol were utilized to assess the metabolic effects of FAQ. ResultsDecreased expression of reg3g was associated with the progression of NASH. Ileal deficiency in reg3g resulted in damage to the intestinal barrier and permeability, leading to the recruitment of endotoxins via the 'gut-liver' axis to the liver, causing the polarization of M1 macrophages, release of inflammatory factors, excessive inflammation, and activation of hepatic stellate cells (HSCs), leading to fibrosis. FAQ significantly upregulated ileal reg3g expression and the expression of intestinal barrier-related proteins tight junction protein 1 (ZO-1) and occludin (OLCN) in mice (p < 0.05), thereby improving intestinal barrier function and permeability. Reduced intestinal permeability led to decreases in endotoxins entering the bloodstream and accumulating in the liver (p < 0.05). The expression of CD68 suggested reduced polarization of M1 macrophages. Expression levels of actin alpha 2, smooth muscle actin (α-SMA) and extracellular matrix (ECM)-related proteins also decreased, indicating improved liver fibrosis. ConclusionFAQ ameliorates NASH by upregulating the expression of reg3g. The upregulation of reg3g contributes to the repair of the intestinal barrier and permeability, reducing the recruitment of endotoxins and subsequent polarization of M1 macrophages, excessive inflammation, and fibrosis.

Yeast mixture supplementation modulates faecal microbiota and ileum morphology of weaning pigs

Different yeast strains benefit postweaning piglets by promoting intestinal health. The objective of this study was to investigate the effect of a yeast mixture containing Kluyveromyces marxianus fragilis, Pichia guilliermondii, and Saccharomyces cerevisiae (Vetoquinol italia s.r.l., Italy) on gut health parameters and growth performance traits of weaned piglets. Forty-eight postweaning castrated male piglets (27 ± 1.7 days, 7.19 ± 0.54 kg) were randomly allocated to two homogeneous experimental groups and involved in a 28-day trial. Both the groups received a basal diet with (yeast mixture, YM) or without (control, CTR) the inclusion of 0.8% yeast mixture during weeks 1 and 2, and 0.6% during weeks 3 and 4. Individual BW and box feed intake were determined on days 0, 14, and 28, and average daily gain and Gain:Feed ratio were subsequently calculated for each administration period (0–14, 14–28). Individual faecal samples were collected for microbiota analysis on days 4, 14, 21, and 28, and faecal score was evaluated on the same days. At the end of the trial, 12 piglets for each group were sacrificed, and ileal tissue was sampled for morphological analysis and the evaluation of mucins profile, using Alcian-Blue/Periodic Acid-Shiff (PAS) staining. On ileum samples, dividing and differentiated epithelial cells were also identified using proliferating cell nuclear antigen and alkaline phosphatase expression, respectively. Differences in the means between the experimental groups were determined by ANOVA, while the metataxonomics analyses were performed by sequencing for V3 and V4 hypervariable regions of the 16S rRNA gene. Growth performance traits were not different among the two experimental groups when considering the whole trial period, while treated animals showed increased faecal consistency on weeks 1 and 4 (P = 0.036 and 0.021, respectively). Yeast mixture administration increased the abundance of Bifidobacterium (P = 0.006) and Coprococcus 2 (P = 0.015), and decreased Clostridium Sensu Stricto 1 (P = 0.019) at all the considered timepoints. Ileum villous height, villous width, and crypt depth were significantly increased by yeast mixture supplementation (P = 0.019; P = 0.013; P = 0.036, respectively), while no differences were observed for the villous:crypt ratio among the groups. The mucin profile showed no differences among experimental groups for acid and neutral glycoconjugates. However, a higher presence of PAS-positive mucins was highlighted in the villi of YM piglets (P < 0.001) compared to CTR. Overall, the administration of a yeast mixture to postweaning piglets showed positive effects on gut health when compared to piglets not receiving the tested product, improving beneficial genera and intestinal morphology.

Impact of dietary fat types on expression levels of dopamine and serotonin transporters in the ileum of broiler chickens

Various types of dietary fats undergo distinct fermentation processes by gut microbes, potentially leading to the production of neurotransmitters that can influence the gut. Serotonin and dopamine are recognized neurotransmitters with positive effects on gut function. A broiler chicken trial was conducted to evaluate the influence of dietary fat types on protein expression of 2 neurotransmitter transporters, dopamine (DAT) and serotonin (5-HTT). A total of 560 day-old (Ross 708) male broiler chicks were randomly assigned to 7 dietary treatments. The experimental treatments included a basal diet of corn-soybean meal (SBM), supplemented with 3% of various fats: poultry fat (CON), olive oil (OLIV), fish oil (FISH), canola oil (CANO), lard (LARD), coconut oil (COCO), or flaxseed oil (FLAX). Bodyweight (BW) and feed conversion ratio (FCR) were recorded. Ileal tissues were aseptically collected to determine the expression levels of DAT and 5-HTT through western blot analysis. In addition, plasma samples were analyzed for reactive oxygen metabolites (d-ROM) tests on d 55. Results showed that dietary fat type inclusion did not have any detrimental effect on growth performance parameters. The expression levels of DAT were higher (P < 0.05) in FLAX treatments compared to CON treatments on d 20 and d 55, respectively. Similarly, with 5-HTT levels, FLAX, CANO, and LARD treatments were higher (P < 0.05) than CON treatments on d 20 and d 55. However, higher levels of oxidative stress (d-ROM values) were recorded in COCO (32.75 Carr U), CANO (29 Carr U), and CON treatments (25.5 Carr U) compared to FLAX (18.5 Carr U; P < 0.05) treatment. These findings suggest that incorporating dietary flaxseed oil at a 3% level in the diet has significant potential to elevate the expression levels of intestinal DAT and 5-HTT without inducing oxidative stress.