Structure Of Villi Research Articles

Establishment and Characterization of Patient-Derived Intestinal Organoids from Pediatric Crohn's Disease Patients.

This study aimed to establish and characterize patient-derived intestinal organoids (PDOs) from children with Crohn's disease (CD). To generate PDOs, endoscopic biopsy specimens were obtained from non-inflamed duodenal bulbs of normal controls and CD patients. To verify the presence of PDOs, histological staining and quantitative reverse transcription polymerase chain reaction (RT-qPCR) analyses were performed. PDOs were successfully established in normal controls (n=2) and CD patients (n=2). Hematoxylin and eosin staining of formalin-fixed, paraffin-embedded PDO sections revealed crypt and villus structures, whereas immunofluorescence staining with EpCAM and DAPI confirmed the epithelial-specific architecture of the PDOs. RT-qPCR results revealed a significant increase in Lgr5, Si, and Chga gene expression and a decrease in Olfm4 and Muc2 expression in CD patients compared to normal controls, suggesting altered stem cell activity and mucosal barrier function (p<0.05). We successfully established and characterized PDOs in children with CD, providing a valuable tool for understanding the pathophysiology of the disease and evaluating potential therapeutic approaches. The differential gene expression of PDOs in CD patients might be caused by the complex interplay between epithelial adaptation and inflammation in the intestinal epithelium.

Limosilactobacillus fermentum SLAM 216-Derived Extracellular Vesicles Promote Intestinal Maturation in Mouse Organoid Models.

Probiotics, when consumed in adequate amounts, can promote the health of the host and beneficially modulate the host's immunity. Particularly during the host's early life, the gut intestine undergoes a period of epithelial maturation in which epithelial cells organize into specific crypt and villus structures. This process can be mediated by the gut microbiota. Recent studies have reported that the administration of probiotics can further promote intestinal maturation in the neonatal intestine. Therefore, in this study, we investigated the effects of extracellular vesicles derived from the Limosilactobacillus fermentum SLAM 216 strain, which is an established probiotic with known immune and anti-aging effects on intestinal epithelial maturation and homeostasis, using mouse small intestinal organoids. As per our findings, treatment with L. fermentum SLAM 216-derived LF216EV (LF216EV) has significantly increased the bud number and size of organoid buds. Furthermore, extracellular vesicle (EV) treatment upregulated the expression of maturation-related genes, including Ascl2, Ephb2, Lgr5, and Sox9. Tight junctions are known to have an important role in the intestinal immune barrier, and EV treatment has significantly increased the expression of genes associated with tight junctions, such as Claudin, Muc2, Occludin, and Zo-1, indicating that it can promote intestinal development. This was supported by RNA sequencing, which revealed the upregulation of genes associated with cAMP-mediated signaling, which is known to regulate cellular processes including cell differentiation. Additionally, organoids exposed to LF216EV exhibited upregulation of genes associated with maintaining brain memory and neurotransmission, suggesting possible future functional implications.

Bovine colostrum prevents formula-induced gut microbiota dysbiosis in preterm pigs.

Preterm birth and formula feeding increase the risk of necrotizing enterocolitis (NEC), a gut inflammatory disease known to be associated with gut microbiota (GM) changes in infants. Supplemental bovine colostrum may protect against formula-induced NEC via GM changes. We hypothesised that feeding colostrum before, after, or during formula feeding affects NEC sensitivity via changes to GM. Colonic GM (profiled by 16S ribosomal RNA gene amplicon sequencing) was compared in preterm pigs fed colostrum for 4 days, either before, after, or together with formula feeding for 4 days. Correlations between GM and gut parameters were assessed on day 5 or 9. Both exclusive and partial colostrum feeding induced higher GM diversity, lower Enterococcus abundance, and improved intestinal maturation parameters (villus structure, digestive enzymeactivities, permeability), relative to exclusive formula feeding (all p < 0.05). Across feeding regimens, Enterococcus abundance was inversely correlated with intestinal maturation parameters. Conversely, there was no correlation between GM changes and early NEC lesions. Bovine colostrum inhibits formula-induced Enterococcus overgrowth and gut dysfunctions just after preterm birth but these effects are not causally linked. Optimising diet-related host responses, not GM, may be critical to prevent NEC in preterm newborn pigs and infants. Supplement of bovine colostrum to formula feeding modified the gut microbiota by increasing species diversity and reducing Enterococcus abundance, while concurrently improving intestinal functions in preterm pigs. Diet-related changes to the gut microbiota were not clearly associated with development of necrotizing enterocolitis (NEC) in preterm pigs, suggesting that diet-related gut microbiota effects are not critical for diet-related NEC protection. The study highlights the potential to use bovine colostrum as a supplement to formula feeding for preterm infants lacking human milk.

Monobutyrin Can Regulate the Gut Microbiota, Which Is Beneficial for the Development of Intestinal Barrier Function and Intestinal Health in Weaned Mice.

In this study, we investigated the effect of monobutyrin (MB) on the gut microbiota and intestinal health of weaned mice. MB was administered via gavage to 21-day-old weaned mice. Samples of small intestinal and ileal contents were collected on day 1, day 7, and day 21 post-administration. Seven days of MB administration enhanced the mucin layer and morphological structure of the intestine and the integrity of the intestinal brush border. Both MB and sodium butyrate (SB) accelerated tight junction development. Compared to SB, MB modulated intestinal T cells in a distinct manner. MB increased the ratio of Treg cells in the small intestine upon the cessation of weaning. After 21 days of MB administration, enhancement of the villus structure of the ileum was observed. MB increased the proportion of Th17 cells in the ileum. MB facilitated the transition of the small intestinal microbiota toward an adult microbial community structure and enhanced the complexity of the microbial community structure. An increase in Th17 cells enhanced intestinal barrier function. The regulatory effect of MB on Th17 cells may occur through the intestinal microbiota. Therefore, MB can potentially be used to promote intestinal barrier function, especially for weaning animals, with promising application prospects.

Cottonseed meal protein hydrolysate influences growth performance, carcass characteristics, serum biochemical indices, and intestinal morphology in yellow-feather broilers.

This study investigated the effects of cottonseed meal protein hydrolysate (CPH) on the growth performance, carcass characteristics, serum biochemical indices, intestinal morphology, and enzyme activities of yellow-feather broilers. We randomly divided 240 chicks into four groups, each with six replicates: a basal diet with 0% (CON), 1% (LCPH), 3% (MCPH), or 5% (HCPH) CPH. The trail spanned 63 days and included three phases: Days 1-21, 22-42, and 43-63. Increased average daily gain (ADG) and decreased ratio of feed to gain (F/G) with LCPH were observed in 21-day-old broilers (P < 0.05). MCPH led to higher ADG and average daily feed intake (ADFI) in 42-day-old broilers (P < 0.05). Additionally, CPH supplementation resulted in increased dressing percentage, percentage of half-eviscerated yield, percentage of eviscerated yield, breast muscle rate, and leg muscle rate were observed (P < 0.05) with diet. The serum levels of total protein (TP), high-density lipoprotein cholesterol (HDL-C), calcium (Ca), and phosphorus (P) were enhanced, and blood urea nitrogen (BUN) and triglyceride (TG) levels decreased with diet and CPH (P < 0.05). CPH increased the length of the jejunum and ileum and the weight of the duodenum, jejunum, and ileum in 21-day-old broilers (P < 0.05). Alterations in the duodenal villus structure in broilers occurred on Days 21 and 42, and the CPH groups performed better; however, a similar change occurred in the jejunum on Days 42 and 63 (P < 0.05). MCPH and HCPH enhanced trypsin activity in the duodenum of 21-day-old and 63-day-old broilers (p < 0.05). Chymotrypsin activity increased (P > 0.05) in the duodenum of 63-day-old broilers fed MCPH. Lipase activity increased (P < 0.05) in the jejuna of 21-day-old broilers treated with HCPH. CPH increased trypsin activity in the ilea of 21-day-old broilers (P < 0.05). These results showed that CPH influenced the growth performance, carcass characteristics, serum biochemical indices, and intestinal morphology of yellow-feather broilers, which are related to growth stage. The recommended CPH level in broilers is 1% before 21 days of age and 3% after 21 days of age.

Abstract 2125: The use of intestinal organoids as a preclinical screen to assess gastrointestinal (GI) toxicity and barrier integrity

Abstract Gastrointestinal (GI) toxicity is a common and often severe limiting factor in the development of antineoplastic drugs. Symptoms include diarrhoea, dehydration and ulceration which increase susceptibility to infection, partly due to damage of crypt and/or villus structures in the small intestine, impairing the barrier function. As novel targeted therapies are emerging, assessment of their potential GI toxicity remains crucial. Small intestinal (SI) organoids are 3D in vitro models of the epithelium which recapitulate the structure and function of the small intestine. We have further developed and validated the organoid model as a screening tool to predict GI toxicity and subsequent mucosal regeneration in four species: mouse, rat, dog and human. The culture conditions have been designed to mimic the stem cell niche and allow cell differentiation and proliferation to occur. All intestinal lineages were present in the organoids derived from each species and the epithelial hierarchy closely resembled that observed in vivo. The toxic effects of antineoplastic drugs on the organoids were assessed by quantification of the total cell viability upon treatment, coupled to morphometric analysis of the organoids (size, area, perimeter, and branching efficiency). Immunofluorescence and RNAseq can be used to identify the targeted cells and the toxicity mechanisms of the drugs. To further assess the effect of drugs on barrier integrity, we have developed transwell-grown cell monolayers derived from normal human small intestinal organoids. This model allows direct access to both apical and basal surfaces, which is not possible when using organoids where their geometry prevents access to the apical surface. The monolayer formation was followed by live imaging and Trans-Epithelial Electrical Resistance (TEER) measurements and treatments with toxic drugs were applied after TEER reached a plateau. The direct effect on barrier integrity was evaluated by TEER reduction, increased FITC-Dextran permeability and analysis of TJ proteins by immunofluorescence. Furthermore, effects on cell death were assessed by measuring the levels of Lactate Dehydrogenase (LDH) released into the medium by dying cells. In conclusion, our models provide an innovative in vitro solution to further study the toxic effects of drugs on the normal small intestine and to analyse direct effects on barrier integrity. The monolayer assay may also be useful for developing models of transient TJ impairment to improve the bioavailability of orally administered antineoplastic drugs. The model can be considered more representative of the normal intestine than transformed cell lines such as CaCo2. Citation Format: Valentina Ubertini, Sarah Hall, Frida Ponthan, James Wilson. The use of intestinal organoids as a preclinical screen to assess gastrointestinal (GI) toxicity and barrier integrity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2125.

P019 Development of hydrogel systems for targeted rectal delivery of intestinal cells to injured mucosa in inflammatory bowel disease

Abstract Background Crohn’s Disease (CD) and Ulcerative Colitis (UC) are Inflammatory Bowel Diseases (IBD) affecting over 6.8 million people worldwide. CD and UC are chronic and progressive diseases, characterised by destructive inflammation of the intestinal tract. IBD management consists of drugs such as steroids, aminosalicylates, immunosuppressants, and biologics that aim to induce disease maintenance and remission. However, frequent, and systemic drug administration leads to limited local effects, severe side effects, and low patient compliance. Ultimately, about 20% of UC and up to 80% of CD patients require surgical colon removal. Thus, new approaches to locally restore the inflamed mucosa and initiate colonic wound repair are required. Human intestinal organoids (HIOs) are 3D cell aggregates derived from primary tissue or stem cells grown in vitro. They demonstrate a similar composition architecture to the primary intestinal tissue. Studies showed that HIOs can engraft into injured colonic mucosa resulting in wound repair. Thus, delivery of HIOs to damaged intestinal epithelium represents a promising therapeutic option in IBD treatment. The project aims to develop hydrogel systems with embedded HIOs and obtain human-relevant information on their efficacy in the restitution of injured colonic mucosa using an improved and human-relevant organ-on-a-chip technology. Methods A series of thermoresponsive hydrogels were fabricated (based on methylcellulose/hyaluronic acid polymers) and characterised. Following the embedment of HIOs into hydrogels, the morphology and viability of HIOs were determined using microscopy and MTT assays. Inflammation was induced through the administration of LPS to the basolateral side of the gut chip. Hydrogel/HIO systems were applied to the apical chip compartment via flow for 24 hours. Subsequently, hydrogel/HIO systems were removed from the chip, and HIO engraftment into injured mucosa was determined utilising permeability (4 kDa dextran) and cytokine release assays (ELISA IL-8). Results Our findings show that the gels were liquid at 25 °C and solidified at 37 °C and demonstrated low cytotoxicity. Additionally, our results suggest that the hydrogel systems had a restorative and possibly protective effect on the villi structures in the gut chip. Microscopy results clearly demonstrated the presence of fully differentiated villi structures in the chips that were treated with the hydrogel/cell systems (Figure 1B). These villi structures were absent in the not-treated chips (Figure 1A). Conclusion This study suggests that development of organoid-based hydrogels that can serve as a safe, effective, and inexpensive therapy (compared to surgery) and significantly improve the treatment of IBD.

Bazi Bushen capsule improves the deterioration of the intestinal barrier function by inhibiting NLRP3 inflammasome-mediated pyroptosis through microbiota-gut-brain axis.

The senescence-accelerated prone mouse 8 (SAMP8) is a widely used model for accelerating aging, especially in central aging. Mounting evidence indicates that the microbiota-gut-brain axis may be involved in the pathogenesis and progression of central aging-related diseases. This study aims to investigate whether Bazi Bushen capsule (BZBS) attenuates the deterioration of the intestinal function in the central aging animal model. In our study, the SAMP8 mice were randomly divided into the model group, the BZ-low group (0.5 g/kg/d BZBS), the BZ-high group (1 g/kg/d BZBS) and the RAPA group (2 mg/kg/d rapamycin). Age-matched SAMR1 mice were used as the control group. Next, cognitive function was detected through Nissl staining and two-photon microscopy. The gut microbiota composition of fecal samples was analyzed by 16S rRNA gene sequencing. The Ileum tissue morphology was observed by hematoxylin and eosin staining, and the intestinal barrier function was observed by immunofluorescence. The expression of senescence-associated secretory phenotype (SASP) factors, including P53, TNF-α, NF-κB, IL-4, IL-6, and IL-10 was measured by real-time quantitative PCR. Macrophage infiltration and the proliferation and differentiation of intestinal cells were assessed by immunohistochemistry. We also detected the inflammasome and pyroptosis levels in ileum tissue by western blotting. BZBS improved the cognitive function and neuronal density of SAMP8 mice. BZBS also restored the intestinal villus structure and barrier function, which were damaged in SAMP8 mice. BZBS reduced the expression of SASP factors and the infiltration of macrophages in the ileum tissues, indicating a lower level of inflammation. BZBS enhanced the proliferation and differentiation of intestinal cells, which are essential for maintaining intestinal homeostasis. BZBS modulated the gut microbiota composition, by which BZBS inhibited the activation of inflammasomes and pyroptosis in the intestine. BZBS could restore the dysbiosis of the gut microbiota and prevent the deterioration of intestinal barrier function by inhibiting NLRP3 inflammasome-mediated pyroptosis. These results suggested that BZBS attenuated the cognitive aging of SAMP8 mice, at least partially, by targeting the microbiota-gut-brain axis.

Entero-toxigenic Bacteroides fragilis contributes to intestinal barrier injury and colorectal cancer progression by mediating the BFT/STAT3/ZEB2 pathway

ABSTRACT Our previous findings confirmed the high enrichment of Bacteroides fragilis (BF) in fecal samples from patients with colorectal cancer (CRC). The intestinal mucosal barrier is the first defense of the organism against commensal flora and intestinal pathogens and is closely associated with the occurrence and development of CRC. Therefore, this study aimed to investigate the molecular mechanisms through which BF mediates intestinal barrier injury and CRC progression. SW480 cells and a Caco2 intestinal barrier model were treated with entero-toxigenic BF (ETBF), its enterotoxin (B. fragilis toxin, BFT), and non-toxigenic BF (NTBF). Cell counting kit-8, flow cytometry, wound healing and transwell assays were performed to analyze the proliferation, apoptosis, migration, and invasion of SW480 cells. Transmission electron microscopy, FITC-dextran, and transepithelial electrical resistance (TEER) were used to analyze damage in the Caco2 intestinal barrier model. The Azoxymethane/Dextran Sulfate Sodium (AOM/DSS) animal model was established to evaluate the effect of ETBF on intestinal barrier injury and CRC progression in vivo. ETBF and BFT enhanced the viability, wound healing ratio, invasion, and EMT of SW480 cells. In addition, ETBF and BFT disrupted the tight junctions and villus structure in the intestinal barrier model, resulting in increased permeability and reduced TEER. Similarly, the expression of intestinal barrier-related proteins (MUC2, Occludin and Zo-1) was restricted by ETBF and BFT. Interestingly, the STAT3/ZEB2 axis was activated by ETBF and BFT, and treatment with Brevilin A (a STAT3 inhibitor) or knockdown of ZEB2 limited the promotional effect of ETBF and BFT on the SW480 malignant phenotype. In vivo experiments also confirmed that ETBF colonization accelerated tumor load, carcinogenesis, and intestinal mucosal barrier damage in the colorectum of the AOM/DSS animal model, and that treatment with Brevilin A alleviated these processes. ETBF-secreted BFT accelerated intestinal barrier damage and CRC by activating the STAT3/ZEB2 axis. Our findings provide new insights and perspectives for the application of ETBF in CRC treatment.

The impact of gut microbiota on vascularization of the small intestine

The commensal microbiota resides in a mutualistic relationship within the mammalian gut. It significantly influences the formation of capillary networks in the small intestine, not only during development but also in adulthood. Mucosal capillaries in small intestinal villus structures play a critical role for the uptake of dietary nutrients and immune regulation. Emerging studies have elucidated how the composition of gut microbiota can influence not only postnatal gut development regarding immune tolerance, nutrient absorption, and morphology but also the development and maintenance of blood and lymphatic capillaries within the small intestine. In particular, the analysis of gnotobiotic mouse models affirmed the importance of the gut microbiota, or even only single gut bacteria, in the remodeling of the small intestinal capillaries. Here, different epithelial-to-endothelial cross talk pathways, e.g. Paneth cell-derived signals, Toll-like receptor signaling, or tissue factor–protease activated receptor-1 signaling, have been reported to affect intestinal villus vascular remodeling in a microbiota-dependent fashion. In this review article, we will provide a comprehensive overview on the relevant microbiota–host interaction pathways, which have been revealed to influence angiogenesis and vascular remodeling in the small intestine.

OC 19.5 Epithelial Neuropilin-1 is Regulated by the Gut Microbiota Promoting Vascularization of Small Intestinal Villus Structures

OC 19.5 Epithelial Neuropilin-1 is Regulated by the Gut Microbiota Promoting Vascularization of Small Intestinal Villus Structures

Gallic acid treatment protects intestinal tissue against ischaemia-reperfusion.

This study aimed to investigate the protective effects of gallic acid (GA) in the rat intestine against ischaemia-reperfusion (IR) injury. Thirty male Wistar albino rats with a mean weight of 200-250 g were used. Animals were categorized into the sham, IR, and IR+GA groups. Ischaemia of the intestine was induced for 3 h by occluding the superior mesenteric artery (SMA) and then left for 3 h of reperfusion. In the IR+GA group, after ischaemia induction, 50 mg/kg GA was orally administered to the animals. Blood samples were collected for biochemical assays. Intestinal tissues were excised for histopathologic and immunohistochemical processing. Malondialdehyde (MDA) levels were increased, and catalase (CAT) and glutathione (GSH) levels were decreased in the IR group compared to the sham group. After GA treatment, MDA levels decreased and CAT and GSH levels increased in the GA-treated group compared to the IR group. In the sham group, normal intestinal histology was observed. In the IR group, the villi structures were completely degenerated. In the IR+GA group, histology was improved after GA treatment. In the sham group, the caspase-3 reaction was generally negative in the epithelium and glands. In the IR group, the caspase-3 reaction increased in apoptotic bodies and inflammatory cells. The caspase-3 reaction was negative in goblet cells and the epithelium. A moderate caspase-3 reaction was observed in the IR+GA group. The beclin-1 reaction was negative in epithelial cells and goblet cells in villi in the sham group. In the IR group, the beclin-1 reaction was positive in the degenerated villi. An intense beclin-1 reaction was also observed in some inflammatory cells. After GA treatment, the beclin-1 reaction was positive in a few cells. In general, moderate beclin-1 positivity was observed. Gallic acid, with its antioxidative effect, inhibited the apoptotic pathway (caspase-3) through beclin-1 regulation.

The fecal biomarker ovotransferrin associates with broiler performance under field conditions

Broilers often suffer from subclinical intestinal health problems of ill-defined etiology, which have a negative impact on performance. Macroscopic and microscopic evaluations can be used to monitor intestinal health, but because these are subjective and time-consuming, respectively, objective and easy-to-measure biomarkers are urgently needed. Fecal biomarkers can potentially be used as noninvasive, objective measures to evaluate gut health in broilers. The aim of the current study was to evaluate ovotransferrin (OVT) as a biomarker in fecal/colonic samples derived from broilers from 27 industrial farms by investigating associations between OVT, broiler performance and gut histology parameters. Eight chickens per farm were randomly selected, weighed and euthanized on d 28 of the production round. A duodenal section was collected to measure the intestinal villus structure (villus length, crypt depth) and the inflammatory status of the gut (CD3+ T-lymphocytes area percentage). The coefficient of variation for the OVT (between farms; 83.45%, within farms; 95.13%) was high compared to the villus length (between farms; 10.91%, within farms; 15.48%), crypt depth (between farms; 15.91%, within farms; 14.10%), villus-to-crypt ratio (between farms; 22.08%, within farms; 20.53%), and CD3+ (between farms; 36.38%, within farms; 26.13%). At farm level, colonic OVT was significantly associated with the average slaughter weight (P=0.005), daily weight gain (P=0.007) and the European production index (EPI) (P=0.009). At broiler level, significant associations were found between colonic OVT and the villus length (P=0.044) and between the colonic OVT and villus-to-crypt ratio (P=0.050). These results thus show that quantifying OVT in colon can have merit for evaluation of intestinal health in broilers under field conditions.

Intestinal Villi-Inspired Mathematically Base-Layer Engineered Microneedles (IMBEMs) for Effective Molecular Exchange during Biomarker Enrichment and Drug Deposition in Diversified Mucosa.

The mucosa-interfacing systems based on bioinspired engineering design for sampling/drug delivery have manifested crucial potential for the monitoring of infectious diseases and the treatment of mucosa-related diseases. However, their efficiency and validity are severely restricted by limited contact area for molecular transfer and dissatisfactory capture/detachment capability. Herein, inspired by the multilayer villus structure of the small intestine that enables high nutrient absorption, a trigonometric function-based periodic pattern was fabricated and integrated on the base layer of the microneedle patch, exhibiting a desirable synergistic effect with needle tips for deep sample enrichment and promising molecular transfer, significantly improving the device-mucosa bidirectional interaction. Moreover, mathematical modeling and finite element analysis were adopted to visualize and quantify the microcosmic molecular transmission process, guiding parameter optimization in actual situation. Encouragingly, these intestinal villi-inspired mathematically base-layer engineered microneedles (IMBEMs) have demonstrated distinguished applicability among mucosa tissue with varying surface curvatures, tissue toughness, and local environments, and simultaneously, have gained favorable support from healthy volunteers receiving preliminary test of IMBEMs patches. Overall, validated by numerous in vitro and in vivo tests, the IMBEMs were confirmed to act as a promising candidate to facilitate mucosa-based sampling and topical drug delivery, indicating highly clinical translation potential.

Consumption of honey ameliorates lipopolysaccharide-induced intestinal barrier dysfunction via upregulation of tight junction proteins.

The leaky gut barrier is an important factor leading to various inflammatory gastrointestinal disorders. The nutritional value of honey and variety of its health benefits have long been recognized. This study was undertaken to assess the role of Indian mustard honey in preventing lipopolysaccharide (LPS)-induced intestinal barrier dysfunction using a combination of in vitro and in vivo experimental model systems. LPS was used to induce intestinal barrier damage in a trans-well model of Caco-2 cells (1µg/ml) and in Swiss albino mice (5mg/kg body weight). Gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) were used to analyse sugar and phenolic components in honey samples. The Caco-2 cell monolayer integrity was evaluated by transepithelial electrical resistance (TEER) and paracellular permeability assays. The histopathology of intestinal tissue was analysed by haematoxylin and eosin dual staining. The quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to quantify the transcription of genes. The protein expression was analysed by immunofluorescence, western blot and ELISA-based techniques. The in vitro data showed that honey prevented LPS-induced intestinal barrier dysfunction dose dependently as was measured by TEER and paracellular flux of FITC-dextran dye. Further, the in vivo data showed a prophylactic effect of orally administered honey as it prevented the loss of intestinal barrier integrity and villus structure. The cellular localization and expression of tight junction (TJ) proteins were upregulated along with downregulation of pro-inflammatory cytokines in response to the administration of honey with LPS. The findings of this study suggest a propitious role of honey in the maintenance of TJ protein integrity, thereby preventing LPS-induced intestinal barrier disintegration.

Commensal bacteria weaken the intestinal barrier by suppressing epithelial neuropilin-1 and Hedgehog signaling

The gut microbiota influences intestinal barrier integrity through mechanisms that are incompletely understood. Here we show that the commensal microbiota weakens the intestinal barrier by suppressing epithelial neuropilin-1 (NRP1) and Hedgehog (Hh) signaling. Microbial colonization of germ-free mice dampens signaling of the intestinal Hh pathway through epithelial Toll-like receptor (TLR)-2, resulting in decreased epithelial NRP1 protein levels. Following activation via TLR2/TLR6, epithelial NRP1, a positive-feedback regulator of Hh signaling, is lysosomally degraded. Conversely, elevated epithelial NRP1 levels in germ-free mice are associated with a strengthened gut barrier. Functionally, intestinal epithelial cell-specific Nrp1 deficiency (Nrp1ΔIEC) results in decreased Hh pathway activity and a weakened gut barrier. In addition, Nrp1ΔIEC mice have a reduced density of capillary networks in their small intestinal villus structures. Collectively, our results reveal a role for the commensal microbiota and epithelial NRP1 signaling in the regulation of intestinal barrier function through postnatal control of Hh signaling.

Effect of Fish Meal Substitution with Black Soldier Fly (Hermetia illucens) on Growth Performance, Feed Stability, Blood Biochemistry, and Liver and Gut Morphology of Siamese Fighting Fish (Betta splendens)

Insects such as black soldier fly larvae (BSFL) are gaining interest among researchers and the aquafeed industry due to the fluctuating price and supply of fish meal (FM). This study evaluated the growth performance, feed stability, blood biochemistry, and liver and gut morphology of Betta splendens using BSFL as an alternative to FM. Five formulated diets were prepared: 0% BSFL, 6.5% BSFL, 13% BSFL, 19.5% BSFL, and 24.5% BSFL. The expansion rate, pellet durability index, floatability, bulk density, and water stability of the prepared feed have been assessed. Except for the diameter of the feed, all the parameters studied differed significantly ( p &lt; 0.05 ) across the experimental diets. After 60 days, the fish fed with 13% BSFL had the highest final length, final weight, net weight gain, specific growth rate, weight gain, and gastrointestinal weight, with mean and standard deviation values of 3.97 ± 0.43 cm, 3.95 ± 0.1 g, 2.78 ± 0.1 g, 4.63 ± 0.17 , 4.65 ± 0.13 , 237.26 ± 7.9 % , and 0.04 ± 0.01 mg, respectively. Similar blood haematology and biochemical properties, including corpuscular volume, lymphocytes, white blood cells, red blood cells, haematocrit, albumin, and alkaline phosphatase, were the highest ( p &lt; 0.05 ) in the 13% BSFL diet group compared to the other treatment groups. In addition, BSFL had a significant impact ( p &lt; 0.05 ) on villus length, width, and crypt depth for the anterior and posterior guts of B. splendens. The 13% BSFL diet group had an intact epithelial barrier in the goblet cell arrangement and a well-organized villus structure and tunica muscularis, compared to the other treatment groups. Furthermore, the liver cell was altered with different BSFL inclusions; the 13% FM group demonstrated better nuclei and cytoplasm structure than the other treatment groups. In conclusion, replacing 13% FM with BSFL could improve the growth performance, blood parameters, and liver and intestine morphology of B. splendens, thus providing a promising alternative diet for ornamental freshwater fish.

Effects of Si-Miao-Yong-An decoction on myocardial I/R rats by regulating gut microbiota to inhibit LPS-induced TLR4/NF-κB signaling pathway

BackgroundCoronary Artery Disease (CAD) is primarily caused by inflammation which is closely linked to the gut microbiota. Si-Miao-Yong-An (SMYA) decoction is a traditional Chinese herbal formula with anti-inflammatory properties that found to be effective against CAD. However, it is still unclear whether SMYA can modulate gut microbiota and whether it contributes to the improvement of CAD by reducing inflammation and regulating the gut microbiota.MethodsThe identification of components in the SMYA extract was conducted using the HPLC method. A total of four groups of SD rats were orally administered with SMYA for 28 days. The levels of inflammatory biomarkers and myocardial damage biomarkers were measured through ELISA, while echocardiography was used to assess heart function. Histological alterations in the myocardial and colonic tissues were examined following H&E staining. Western blotting was performed to evaluate protein expression, whereas alterations in gut microbiota were determined by 16 s rDNA sequencing.ResultsSMYA was found to enhance cardiac function and decrease the expression of serum CK-MB and LDH. SMYA was also observed to inhibit the TLR4/NF-κB signaling pathway by downregulating the protein expression of myocardial TLR4, MyD88, and p-P65, leading to a reduction in serum pro-inflammatory factors. SMYA modified the composition of gut microbiota by decreasing the Firmicutes/Bacteroidetes ratio, modulating Prevotellaceae_Ga6A1 and Prevotellaceae_NK3B3 linked to the LPS/TLR4/NF-κB pathway, and increasing beneficial microbiota such as Bacteroidetes, Alloprevotella, and other bacterial species. Moreover, SMYA was found to safeguard the intestinal mucosal and villi structures, elevate the expression of tight junction protein (ZO-1, occludin), and reduce intestinal permeability and inflammation.ConclusionsThe results indicate that SMYA has the potential to modulate the gut microbiota and protect the intestinal barrier, thereby reducing the translocation of LPS into circulation. SMYA was also found to inhibit the LPS-induced TLR4/NF-κB signaling pathway, leading to a decrease in the release of inflammatory factors, which ultimately mitigated myocardial injury. Hence, SMYA holds promise as a therapeutic agent for the management of CAD.

3D structural quantification of equine placental villi through multiscale correlative imaging

As placental processes occur within the bounds of a three-dimensional (3D) anatomical structure, the quantification of placental structures across different scales is necessary for a proper understanding of placental function and disease (Lewis and Pearson-Farr, Placenta. 2020;102:55-60). Three-dimensional imaging techniques have recently identified and quantified novel structures in human placental villi. In equidae, fetal growth relies on feto-maternal exchanges taking place in placental villi facing the entire uterine endometrium. Villi length increases throughout pregnancy, but branching and internal blood flow have been poorly explored so far. Here, we present a workflow for correlative multiscale 3D imaging combining X-ray Computed Microtomography (microCT) and Serial Block Face Scanning Electron Microscopy (SBF-SEM) applied to villi from sections (∼3 × 3 mm) of the chorioallantois taken from term placentas in horses (n=2),zebras (n=2), donkeys (n=3), and mules (n=5). Although this work is still ongoing and biological conclusions are pending, this workflow, resulting in 3D villi reconstructions, enabled the quantification of the 3D villus structure across multiple orders of magnitude for metrics such as surface area, volume, and branching angle. Not only were the reconstructions able to identify novel nanoscale structures in equine villi, such as stromal macrovesicles previously only observed in human placental villi, but they also allowed the computational modelling of physiological processes such as blood flow and diffusion through realistic tissue geometries. Taken together, the structural data generated from this correlative workflow will help us to better resolve equine placental organization, contribute towards a holistic understanding of equine placental function in health and disease, and identify potential differences between different equid species.

Protective effect of mussel polysaccharide on cyclophosphamide-induced intestinal oxidative stress injury via Nrf2-Keap1 signaling pathway.

The hard-shelled mussel (Mytilus coruscus) has been used as a traditional Chinese medicine and health food in China for centuries. Polysaccharides from mussel has been reported to have multiple biological functions, however, it remains unclear whether mussel polysaccharide (MP) exerts protective effects in intestinal functions, and the underlying mechanisms of action remain unclear. The aim of this study was to investigate the protective effects and mechanism of MP on intestinal oxidative injury in mice. In this study, 40 male BALB/C mice were used, with 30 utilized to produce an animal model of intestinal oxidative injury with intraperitoneal injection of cyclophosphamide (Cy) for four consecutive days. The protective effects of two different doses of MP (300 and 600 mg/kg) were assessed by investigating the change in body weight, visceral index, and observing colon histomorphology. Moreover, the underlying molecular mechanisms were investigated by measuring the antioxidant enzymes and related signaling molecules through ELISA, real-time PCR, and western blot methods. The results showed that MP pretreatment effectively protected the intestinal from Cy-induced injury: improved the colon tissue morphology and villus structure, increased superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) activities, and reduced malondialdehyde (MDA) content in serum and colon tissues. Meanwhile, MP also significantly increased the expression levels of SOD, GSH-Px, heme oxygenase-1 (HO-1), and nuclear factor E2-related factor 2 (Nrf2) mRNA in colon tissues. Further, western blot results showed that the expression of Nrf2 protein was significantly upregulated while kelch-like ECH-associated protein 1 (Keap1) was significantly downregulated by MP in the colonic tissues. This study indicates that MP can ameliorate Cy-induced oxidative stress injury in mice, and Nrf2-Keap1 signaling pathway may mediate these protective effects.