Intestinal Alkaline Phosphatase Research Articles

Changes in Digestive Enzyme Activities during Larval Development of Spotted Seatrout (Cynoscion nebulosus)

The spotted seatrout (Cynoscion nebulosus)—an important commercial species—has a high potential for aquaculture in the Gulf of Mexico. To optimize its feeding during larval rearing, this study aims to evaluate the primary gastric (pepsin), intestinal (leucine aminopeptidase and alkaline phosphatase), and pancreatic (alkaline protease, trypsin, chymotrypsin, amylase, and lipase) enzyme activities from hatching to day 30. A multivariate analysis identified three digestive enzyme development stages during the spotted seatrout larval transformation. The first stage occurred between 1 (mean ± standard error (SE) = 1.73 ± 0.14 millimeter (mm) standard length (SL)) and 3 (2.14 ± 0.07 mm SL) days after hatching (DAH); a period of digestive stability showed the highest activity in amylase and bile salt-dependent lipase. The second stage (from 4 (2.53 ± 0.09 mm SL) to 20 (10.92 ± 0.51 mm SL) DAH) was a period of digestive transition, during which leucine aminopeptidase, chymotrypsin, and alkaline proteases were identified as the predominant enzymes from 4 to 5 DAH. In the third stage—a period of digestive stability—pepsin was the major enzyme that occurred between 25 (16.51 ± 0.81 mm SL) and 30 (25.91 ± 0.82 mm SL) DAH. These results indicate that the spotted seatrout larvae have a digestive system adapted to lipids and carbohydrates at the onset of feeding, with an immediate transition to protein digestion when exogenous feeding begins. Additionally, the digestive system of the spotted seatrout may be considered mature at 25 DAH. Further research is needed to elucidate the mechanisms of digestive tract development in the spotted seatrout larvae.

Identifying alkaline phosphatase inhibitory potential of cyclooxygenase-2 inhibitors: Insights from molecular docking, MD simulations, molecular expression analysis in MCF-7 breast cancer cell line and in vitro investigations

Alkaline phosphatases (APs, EC 3.1.3.1) belong to a superfamily of biological macromolecules that dephosphorylate many phosphometabolites and phosphoproteins and their overexpression is intricated in the spread of cancer to liver and bones, neuronal disorders including Alzheimer's disease (AD), inflammation and others. It was hypothesized that cyclooxygenase-2 (COX-2) selective inhibitors may possess anti-APs potential and may be involved in anticancer proceedings. Three COX-2 inhibitors including nimesulide, piroxicam and lornoxicam were evaluated for the inhibition of APs using in silico and in vitro methods. Molecular docking studies against tissue nonspecific alkaline phosphatase (TNAP) offered the best binding affinities for nimesulide (−11.14 kcal/mol) supported with conventional hydrogen bonding and hydrophobic interactions. MD simulations against TNAP for 200 ns and principal component analysis (PCA) reiterated the stability of ligand-receptor complexes. Molecular expression analysis of TNAP enzyme in the breast cancer cell line MCF-7 exhibited 0.24-fold downregulation with 5 μM nimesulide as compared with 0.26-fold standard 10 μM levamisole. In vitro assays against human placental AP (hPAP) displayed potent inhibitions of these drugs with IC50 values of 0.52 ± 0.02 μM to 3.46 ± 0.13 μM and similar results were obtained for bovine intestinal AP (bIAP). The data when generalized collectively emphasizes that the inhibition of APs by COX-2 inhibitors provides another target to work on the development of anticancer drugs.

Necrotizing enterocolitis: a potential protective role for intestinal alkaline phosphatase as lipopolysaccharide detoxifying enzyme.

Necrotizing enterocolitis (NEC) is a life-threatening inflammatory disease. Its onset might be triggered by Toll-Like Receptor 4 (TLR4) activation via bacterial lipopolysaccharide (LPS). We hypothesize that a deficiency of intestinal alkaline phosphatase (IAP), an enzyme secreted by enterocytes that dephosphorylates LPS, may contribute to NEC development. In this prospective pilot study, we analyzed intestinal resection specimens from surgical NEC patients, and from patients undergoing Roux-Y reconstruction for hepatobiliary disease as controls. We assessed IAP activity via enzymatic stainings and assays and explored IAP and TLR4 co-localization through immunofluorescence. The study population consisted of five NEC patients (two Bell's stage IIb and three-stage IIIb, median (IQR) gestational age 25 (24-28) weeks, postmenstrual age at diagnosis 28 (26-31) weeks) and 11 controls (unknown age). There was significantly lower IAP staining in NEC resection specimens [49 (41-50) U/g of protein] compared to controls [115 (76-144), P = 0.03]. LPS-dephosphorylating activity was also lower in NEC patients [0.06 (0-0.1)] than in controls [0.3 (0.2-0.5), P = 0.003]. Furthermore, we observed colocalization of IAP and TLR4 in NEC resection specimens. This study suggests a significantly lower IAP level in resection specimens of NEC patients compared to controls. This lower IAP activity suggests a potential role of IAP as a protective agent in the gut, which needs further confirmation in larger cohorts.

Self-emulsifying drug delivery systems containing sulfate-based surfactants: Are they responsive to alkaline phosphatase?

This study aimed to develop self-emulsifying drug delivery systems (SEDDS) containing sulfate-based surfactants, namely, sodium dodecyl sulfate (SDS), sodium octyl sulfate (SOS) and sodium cetearyl sulfate (SCS), and to evaluate intestinal alkaline phosphatase (IAP)-triggered sulfate release from these SEDDS.Results showed a time-dependent release of para-nitrophenol from para-nitrophenyl phosphate (p-NPP) and para-nitrophenyl sulfate (p-NPS) when incubated with isolated IAP indicating that the enzyme can also cleave sulfate groups. Moreover, sulfate release from p-NPS and sulfate-based surfactants was observed. SEDDS containing sulfate-based surfactants exhibited a narrow droplet size distribution and polydispersity index (PDI). A droplet size of 35.31 ± 0.41 nm to 41.15 ± 0.98 nm, PDI of 0.17 ± 0.02 to 0.26 ± 0.02 and zeta potential of 9.59 ± 0.85 mV to −18.53 ± 2.75 mV were recorded. Droplet size and PDI showed a minor increase upon contact with isolated IAP. Incubation with isolated IAP caused a rapid sulfate release from sulfate-based surfactant-containing SEDDS (SDS-SEDDS, SOS-SEDDS and SCS-SEDDS). Furthermore, resulting SEDDS exhibited a non-toxic profile. According to these results, SEDDS containing sulfate-based surfactants can be considered as valid alternative to IAP-responsive drug delivery systems containing phosphorylated auxiliary agents.

Investigation of the relationship between atopic dermatitis of dogs and intestinal epithelial damage.

A significant association between atopic dermatitis and leaky gut syndrome has been demonstrated in humans. No studies have been conducted to determine whether there is an association between atopic dermatitis and intestinal damage in dogs. This study aimed to determine whether there is an association between canine atopic dermatitis and intestinal damage using selected intestinal-related biomarkers. Twenty-six dogs with atopic dermatitis and 10 healthy dogs were included. Moderate-to-severe pruritus, erythema, erosion and alopecia on different parts of the body were sought in dogs to suspect atopic dermatitis. The presence of atopic dermatitis was confirmed by an allergic skin test.Serum biomarkers including intestinal fatty acid binding protein (I-FABP), intestinal alkaline phosphatase (IAP), trefoil factor-3 (TFF-3), immunoglobulin E (IgE), interleukin-4 (IL-4) and interleukin-13 (IL-13) concentrations were measured from venous blood samples. Of the 26 dogs tested for allergens, 16 were found to be sensitive to mould mites, 10 to vernal grass, eight to house dust mites, five to wheat dust and five to grass pollen mix allergens. Significant increases in serum IAP, TFF-3, IgE, IL-4 and IL-13 concentrations were determined. It was thought that the increase in TFF-3 and IAP concentrations may be due to the presence of intestinal epithelial damage and the repair of this damage. In addition, the development of atopic dermatitis may be predisposed to the entry of allergens into the body through sites of intestinal damage.

Exploration of newly synthesized azo-thiohydantoins as the potential alkaline phosphatase inhibitors via advanced biochemical characterization and molecular modeling approaches

In the current study, Azo-Thiohydantoins derivatives were synthesized and characterized by using various spectroscopic techniques including FTIR, 1H-NMR, 13C-NMR, elemental and HRMS analysis. The compounds were evaluated for alkaline phosphatase activity and it was observed that among all the synthesized compounds, derivative 7e exhibited substantial inhibitory activity (IC50 = 0.308 ± 0.065 µM), surpassing the standard inhibitor (L–Phenyl alanine, IC50 = 80.2 ± 1.1 µM). Along with this, these derivatives were comprehensively examined regarding the electronic properties and reactivity of the synthesized compounds using Density Functional Theory (DFT) calculations, where the results were found very promising and the synthesized compound were found stable. After that, SwissADME evaluations highlighted compounds for their favorable physicochemical properties, including solubility and drug-likeness. Molecular docking exhibited the strong binding affinities of 7f and 7e derivatives with intestinal alkaline phosphatase (IAP), further supported by Molecular Dynamics (MD) simulations. This comprehensive integration of experimental and computational approaches sheds the light on the potential therapeutic applications of the synthesized compounds. By providing a detailed investigation of these aspects, this research opens the avenues for the development of novel pharmacologically active compounds with diverse applications.

Adapting recombinant bacterial alkaline phosphatase for nucleotide exchange of small GTPases

The small GTPase Rat sarcoma virus proteins (RAS) are key regulators of cell growth and involved in 20–30% of cancers. RAS switches between its active state and inactive state via exchange of GTP (active) and GDP (inactive). Therefore, to study active protein, it needs to undergo nucleotide exchange to a non-hydrolysable GTP analog. Calf intestine alkaline phosphatase bound to agarose beads (CIP-agarose) is regularly used in a nucleotide exchange protocol to replace GDP with a non-hydrolysable analog. Due to pandemic supply problems and product shortages, we found the need for an alternative to this commercially available product. Here we describe how we generated a bacterial alkaline phosphatase (BAP) with an affinity tag bound to an agarose bead. This BAP completely exchanges the nucleotide in our samples, thereby demonstrating an alternative to the commercially available product using generally available laboratory equipment.

Self‐Activating Phosphorus‐Rich Substrate Import Enzyme Catalytic Domains for Bone Regeneration

AbstractEnzymes play a key role in natural mineralization, but their function is disabling in bone metabolism when patients suffer severe degenerations like hypophosphatasia. To regulate bone homeostasis, a self‐activating phosphorus‐rich system is proposed on calcium glycerophosphate (CaGP) modified polyurethane (PU) fibrous substrate, wherein ample enzyme catalytic domains are imported with intestinal alkaline phosphatase (IALP) chelating and activating by calcium in situ that is testified by chemical potential evaluation. Therefore, Ca‐bonding IALP catalyzes organophosphate to phosphate locally to achieve a calcified symbiotic state, regulating mineralization and accelerating osteoinduction in vitro and in vivo. The self‐activating enzyme system promotes early osteogenic differentiation and angiogenesis, resulting in rapid healing of cranial defect with a “long‐tail” effect compared to an enzyme‐assisted system. Transcriptomic and metabolomic analyses demonstrate positive associations with neovascularization and calcium/phosphorus metabolism, specifically upregulated glycerophospholipids and calcium ions in favor of osteogenic differentiation under cytokine‐free conditions. These results provide a potential enzyme‐activating therapy in bone homeostasis and regeneration.

Hydroethanolic extract of Piliostigma thonningii leaves extenuates the severity of diarrhoea in female Wistar rats.

Nigeria ranks second in the global prevalence of diarrhoea with most cases concentrated in the Northern-region of the country. This research explored the antidiarrhoeal efficacy of the hydroethanolic extract of Piliostigma thonningii leaves (HEPTL), locally used to manage diarrhoeal conditions in Kebbi State, Nigeria. P.thonningii leaves were screened for their secondary metabolites and mineral constituents. Using 3 standard-diarrhoea models, female Wistar rats completely-randomised into six-groups of six animals each were utilised for probing the antidiarrhoeal activity of HEPTL. Animals in groups I and II served as the negative and positive controls, whereas the rats in groups III, IV and V respectively received 50, 100, and 200 mg/kg body weight-(bw) of HEPTL. Six secondary metabolites and eight minerals were found in the extract, with flavonoids and calcium being the most abundant while steroids and zinc were the least prevalent, respectively. High performance liquid chromatographic analysis revealed the presence of 19 bioactive substances. Furthermore, there was a significant (p<0.05) and dose-related reduction in diarrhoea onset, water content, and wet faeces count. Similarly, the amount of intestinal fluid and average distance traversed by the charcoal-meal were decreased dose-dependently by the HEPTL with a commensurate rise in the suppression of intestinal fluid accrual andperistalsis. Acetylcholinesterase, Na+/K+-ATPase, reduced glutathione, intestinal-alkaline phosphatase and protein levels increased significantly (p<0.05) whereas superoxide-dismutase, catalase, intestinal-nitric oxide and malondialdehyde levels all fell significantly (p<0.05). However, the level of intestinal glucose was not significantly altered. Overall, the HEPTL exhibited a profound effect in the alleviation of the severity of diarrhoea, notably at 200 mg/kg bw.

Alkaline Phosphatase Relieves Colitis in Obese Mice Subjected to Forced Exercise via Its Anti-Inflammatory and Intestinal Microbiota-Shaping Properties.

Intestinal alkaline phosphatase (IAP) is an enzyme that plays a protective role in the gut. This study investigated the effect of IAP treatment on experimental colitis in mice subjected to forced exercise on a high-fat diet. C57BL/6 mice with TNBS colitis were fed a high-fat diet and subjected to forced treadmill exercise with or without IAP treatment. Disease activity, oxidative stress, inflammatory cytokines, and gut microbiota were assessed. Forced exercise exacerbated colitis in obese mice, as evidenced by increased disease activity index (DAI), oxidative stress markers, and proinflammatory adipokines and cytokines. IAP treatment significantly reduced these effects and promoted the expression of barrier proteins in the colonic mucosa. Additionally, IAP treatment altered the gut microbiota composition, favoring beneficial Verrucomicrobiota and reducing pathogenic Clostridia and Odoribacter. IAP treatment ameliorates the worsening effect of forced exercise on murine colitis by attenuating oxidative stress, downregulating proinflammatory biomarkers, and modulating the gut microbiota. IAP warrants further investigation as a potential therapeutic strategy for ulcerative colitis.

Effects of Different Protein Sources on Growth Performance, Muscle Flavor Substances and Quality Structure in Triploid Crucian Carp

A 56-day feeding trial was conducted to evaluate the effect of different dietary protein sources on the growth performance, muscle flavor substances, and quality structure of Triploid Crucian Carp. Three isonitrogenous (32.00%), isolipidic (8.00%), and isoenergetic (18.00 MJ kg−1) practical diets were formulated. These diets consisted of fishmeal as the animal-derived protein source (AP), a combination of soybean meal and rapeseed meal as the plant-derived protein source (PP), and a mixture of fishmeal, soybean meal, and rapeseed meal as the mixed protein source (MP). Each diet was randomly assigned to triplicate tanks of fish and each tank was stocked with 25 fish (11.5 ± 0.4 g). The fish were fed until apparent satiation twice a day. The results showed a significant enhancement in the growth performance of Triploid Crucian Carp in the AP group compared with both the MP and PP groups (p &lt; 0.05). Dietary plant derived protein can remarkably reduce the crude lipid content and increase the moisture content of the whole body and the dorsal muscle (p &lt; 0.05). The antioxidant ability of fish in the PP group and MP group was better than that in the AP group (p &lt; 0.05). Regarding free amino acids composition of muscle, the contents of glycine, methionine, and lysine were significantly enhanced in the AP group, while the histidine content was significantly increased in the PP group (p &lt; 0.05). In terms of texture, dietary plant protein significantly improved the muscular hardness, gumminess, and chewiness. The activities of intestinal trypsin and alkaline phosphatase (ALP) of fish in the PP group were significantly higher than that in the AP group (p &lt; 0.05). The expression levels of hepatopancreas TOR and IGF1 genes in the PP group were significantly higher than that in the MP group (p &lt; 0.05). The present results indicate dietary fishmeal significantly improved the growth performance and muscular flavor glycine content, while dietary plant-based protein increased crude protein content, antioxidant ability, and muscular texture performance of Triploid Crucian Carp.

The inflammatory injury of porcine small intestinal epithelial cells induced by deoxynivalenol is related to the decrease in glucose transport.

The present study aimed to investigate the effects of deoxynivalenol (DON) stimulation on inflammatory injury and the expression of the glucose transporters sodium-dependent glucose transporter 1 (SGLT1) and glucose transporter protein 2 (GLU2) in porcine small intestinal epithelial cells (IPEC-J2). Additionally, the study aimed to provide initial insights into the connection between the expression of glucose transporters and the inflammatory injury of IPEC-J2 cells. DON concentration and DON treatment time were determined using the CCK‑8 assay. Accordingly, 1.0 µg/mL DON and treatment for 24h were chosen for subsequent experiments. Then IPEC-J2 cells were treated without DON (CON, N = 6) or with 1 μg/mL DON (DON, N = 6). Lactate dehydrogenase (LDH) content, apoptosis rate, and proinflammatory cytokines including interleukin (IL)-1β, Il-6, and tumor necrosis factor α (TNF-α) were measured. Additionally, the expression of AMP-activated protein kinase α1 (AMPK-α1), the content of glucose, intestinal alkaline phosphatase (AKP), and sodium/potassium-transporting adenosine triphosphatase (Na+/K+-ATPase) activity, and the expression of SGLT1 and GLU2 of IPEC-J2 cells were also analyzed. The results showed that DON exposure significantly increased LDH release and apoptosis rate of IPEC-J2 cells. Stimulation with DON resulted in significant cellular inflammatory damage, as evidenced by a significant increase in proinflammatory cytokines (IL-1β, IL-6, and TNF-α). Additionally, DON caused damage to the glucose absorption capacity of IPEC-J2 cells, indicated by decreased levels of glucose content, AKP activity, Na+/K+-ATPase activity, AMPK-α1 protein expression, and SGLT1 expression. Correlation analysis revealed that glucose absorption capacity was negatively correlated with cell inflammatory cytokines. Based on the findings of this study, it can be preliminarily concluded that the cell inflammatory damage caused by DON may be associated with decreased glucose absorption.

Bound polyphenols in insoluble dietary fiber of navel orange peel modulate LPS-induced intestinal-like co-culture inflammation through CSF2-mediated NF-κB/JAK-STAT pathway.

Our laboratory previously extracted bound polyphenols (BPP) in insoluble dietary fiber from navel orange peel (NOP-IDF), and the aim of this study was to investigate the anti-inflammatory activity and potential molecular mechanisms of BPP by establishing an LPS-induced intestinal-like Caco-2/RAW264.7 co-culture inflammation model. The results demonstrated that BPP reduced the expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), as well as the production of pro-inflammatory cytokines, nitric oxide (NO), and reactive oxidative species (ROS) during the inflammatory damage process. Furthermore, BPP alleviated the lipopolysaccharides (LPS)-induced intestinal barrier damage by attenuating the decrease in trans-epithelial electrical resistance (TEER), diamine oxidase (DAO) activity, and intestinal alkaline phosphatase (IAP) activity, as well as the downregulation of ZO-1, Occludin, and Claudin-1 protein expression levels. RNA-seq results on RAW264.7 cells in the co-culture model showed that the NF-κB and JAK-STAT pathways belonged to the most significantly affected signaling pathways in the KEGG analysis, and western blot confirmed that they are essential for the role of BPP in intestinal inflammation. Additionally, overexpression of the granulocyte-macrophage colony-stimulating factor (CSF2) gene triggered abnormal activation of the NF-κB and JAK-STAT pathways and high-level expression of inflammatory factors, while BPP effectively improved this phenomenon. The above results suggested that BPP could inhibit intestinal inflammatory injury and protect intestinal barrier integrity through CSF2-mediated NF-κB and JAK-STAT pathways.

Developing an adult stem cell derived microphysiological intestinal system for predicting oral prodrug bioconversion and permeability in humans.

Microphysiological systems (MPS) incorporating human intestinal organoids have shown the potential to faithfully model intestinal biology with the promise to accelerate development of oral prodrugs. We hypothesized that an MPS model incorporating flow, shear stress, and vasculature could provide more reliable measures of prodrug bioconversion and permeability. Following construction of jejunal and duodenal organoid MPS derived from 3 donors, we determined the area under the concentration-time (AUC) curve for the active drug in the vascular channel and characterized the enzymology of prodrug bioconversion. Fosamprenavir underwent phosphatase mediated hydrolysis to amprenavir while dabigatran etexilate (DABE) exhibited proper CES2- and, as anticipated, not CES1-mediated de-esterification, followed by permeation of amprenavir to the vascular channel. When experiments were conducted in the presence of bio-converting enzyme inhibitors (orthovanadate for alkaline phosphatase; bis(p-nitrophenyl)phosphate for carboxylesterase), the AUC of the active drug decreased accordingly in the vascular channel. In addition to functional analysis, the MPS was characterized through imaging and proteomic analysis. Imaging revealed proper expression and localization of epithelial, endothelial, tight junction and catalytic enzyme markers. Global proteomic analysis was used to analyze the MPS model and 3 comparator sources: an organoid-based transwell model (which was also evaluated for function), Matrigel embedded organoids and finally jejunal and duodenal cadaver tissues collected from 3 donors. Hierarchical clustering analysis (HCA) and principal component analysis (PCA) of global proteomic data demonstrated that all organoid-based models exhibited strong similarity and were distinct from tissues. Intestinal organoids in the MPS model exhibited strong similarity to human tissue for key epithelial markers via HCA. Quantitative proteomic analysis showed higher expression of key prodrug converting and drug metabolizing enzymes in MPS-derived organoids compared to tissues, organoids in Matrigel, and organoids on transwells. When comparing organoids from MPS and transwells, expression of intestinal alkaline phosphatase (ALPI), carboxylesterase (CES)2, cytochrome P450 3A4 (CYP3A4) and sucrase isomaltase (SI) was 2.97-, 1.2-, 11.3-, and 27.7-fold higher for duodenum and 7.7-, 4.6-, 18.1-, and 112.2-fold higher for jejunum organoids in MPS, respectively. The MPS approach can provide a more physiological system than enzymes, organoids, and organoids on transwells for pharmacokinetic analysis of prodrugs that account for 10% of all commercial medicines.

Роль щелочной фосфатазы кишечника в развитии ожирения. Модуляция активности фермента высокожировой диетой и пищевыми волокнами

Роль щелочной фосфатазы кишечника в развитии ожирения. Модуляция активности фермента высокожировой диетой и пищевыми волокнами

Anti-enzymatic and DNA docking studies of montelukast: A multifaceted molecular scaffold with in vitro investigations, molecular expression analysis and molecular dynamics simulations

Montelukast, an approved leukotriene receptor 1 (Cys-LT 1) antagonist with anti-inflammatory properties is used for the treatment of asthma and allergic rhinitis. In the present studies, montelukast was subjected to in vitro inhibitory assays followed by kinetic and in silico investigations. Montelukast demonstrated inhibitory activity against yeast α-glucosidase (IC50 44.31 ± 1.21 μM), jack bean urease (JB urease, IC50 8.72 ± 0.23 μM), human placental alkaline phosphatase (hPAP, IC50 17.53 ± 0.19 μM), bovine intestinal alkaline phosphatase (bIAP, IC50 15.18 ± 0.23 μM) and soybean 15-lipoxygenase (15-LOX, IC50 2.41 ± 0.13 μM). Kinetic studies against α-glucosidase and urease enzymes revealed its competitive mode of inhibition. Molecular expression analysis of montelukast in breast cancer cell line MCF-7 down-regulated AP by a factor of 0.27 (5 μM) compared with the 0.26 value for standard inhibitor levamisole (10 μM). Molecular docking estimated a binding affinity ranging −8.82 to −15.65 kcal/mol for the enzymes. Docking against the DNA dodecamer (ID: 1BNA) observed −9.13 kcal/mol via minor groove binding. MD simulations suggested stable binding between montelukast and the target proteins predicting strong inhibitory potential of the ligand. Montelukast features a chloroquinoline, phenyl ring, a cyclopropane group, a carboxylic group and a sulfur atom all of which collectively enhance its inhibitory potential against the said enzymes. These in vitro and computational investigations demonstrate that it is possible and suggested that the interactions of montelukast with more than one targets presented herein may be linked with the side effects presented by this drug and necessitate additional work. The results altogether suggest montelukast as an important structural scaffold possessing multitargeted features and warrant further investigations in repurposing beyond its traditional pharmacological use.

Anti-Salmonella Defence and Intestinal Homeostatic Maintenance In Vitro of a Consortium Containing Limosilactobacillus fermentum 3872 and Ligilactobacillus salivarius 7247 Strains in Human, Porcine, and Chicken Enterocytes.

Limosilactobacillus fermentum strain 3872 (LF3872) was originally isolated from the breast milk of a healthy woman during lactation and the breastfeeding of a child. Ligilactobacillus salivarius strain 7247 (LS7247) was isolated at the same time from the intestines and reproductive system of a healthy woman. The genomes of these strains contain genes responsible for the production of peptidoglycan-degrading enzymes and factors that increase the permeability of the outer membrane of Gram-negative pathogens. In this work, the anti-Salmonella and intestinal homeostatic features of the LF3872 and LS7247 consortium were studied. A multi-drug resistant (MDR) strain of Salmonella enteritidis (SE) was used in the experiments. The consortium effectively inhibited the adhesion of SE to intact and activated human, porcine, and chicken enterocytes and reduced invasion. The consortium had a bactericidal effect on SE in 6 h of co-culturing. A gene expression analysis of SE showed that the cell-free supernatant (CFS) of the consortium inhibited the expression of virulence genes critical for the colonization of human and animal enterocytes. The CFS stimulated the production of an intestinal homeostatic factor-intestinal alkaline phosphatase (IAP)-in Caco-2 and HT-29 enterocytes. The consortium decreased the production of pro-inflammatory cytokines IL-8, TNF-α, and IL-1β, and TLR4 mRNA expression in human and animal enterocytes. It stimulated the expression of TLR9 in human and porcine enterocytes and stimulated the expression of TLR21 in chicken enterocytes. The consortium also protected the intestinal barrier functions through the increase of transepithelial electrical resistance (TEER) and the inhibition of paracellular permeability in the monolayers of human and animal enterocytes. The results obtained suggest that a LF3872 and LS7247 consortium can be used as an innovative feed additive to reduce the spread of MDR SE among the population and farm animals.

Ketogenic Diet High in Saturated Fat Promotes Colonic Claudin Expression without Changes in Intestinal Permeability to Iohexol in Healthy Mice.

Ketogenic diets (KDs) have been studied in preclinical models of intestinal diseases. However, little is known of how the fat source of these diets influences the intestinal barrier. Herein, we studied the impact of four-week feeding with KD high either in saturated fatty acids (SFA-KD) or polyunsaturated linoleic acid (LA-KD) on paracellular permeability of the intestine to iohexol in healthy male C57BL/6J mice. We investigated jejunal and colonic tight junction protein expression, histological changes, and inflammatory markers (Il1b, Il6, Tnf, and Lcn2), as well as the activity and expression of intestinal alkaline phosphatase (IAP) in feces and jejunal tissue, respectively, and plasma lipopolysaccharide. KDs did not change intestinal permeability to iohexol after two or twenty-six days of feeding regardless of fat quality. SFA-KD, but not LA-KD, upregulated the colonic expression of tight junction proteins claudin-1 and -4, as well as the activity of IAP. Both KDs resulted in increased epithelial vacuolation in jejunum, and this was pronounced in SFA-KD. Jejunal Il1β expression was lower and colonic Il6 expression higher in LA-KD compared to SFA-KD. In colon, Tnf mRNA was increased in LA-KD when compared to controls. Overall, the results suggest that KDs do not influence intestinal permeability to iohexol but elicit changes in colonic tight junction proteins and inflammatory markers in both jejunum and colon. Future research will show whether these changes become of importance upon proinflammatory insults.

Postprandial kinetics of digestive function in rainbow trout (Oncorhynchus mykiss): genes expression, enzymatic activity and blood biochemistry as a practical tool for nutritional studies

Postprandial kinetics of genes expression of gastric (chitinase, pepsinogen) and intestinal (alkaline phosphatase, maltase) digestive enzymes and nutrient transporters (peptide transporter 1, sodium-glucose transporter 1), Brush Border Membrane (BBM) enzymes activity (alkaline phosphatase, leucine aminopeptidase, maltase, saccharase) and blood biochemistry (triglycerides, cholesterol, protein, albumin, glucose, amino acids) through NMR spectroscopy, were investigated in rainbow trout (Oncorhynchus mykiss) fed a commercial aquafeed. For this purpose, fish were starved 72 h and digestive tract and blood were sampled before the meal and at 1.5, 3, 6, 9, 12, and 24 h after feeding (T0, T1.5, T3, T6, T9, T12 and T24). The postprandial kinetic showed that the expression of the genes involved in digestion and nutrient transport, the activity of BBM enzymes, and the presence of metabolites in blood were stimulated in different ways by the presence of feed in the digestive tract. The expression of most genes peaked 3 h after meal except gastric pepsinogen and maltase in distal intestine that peaked at T9 and T12, respectively. The activity of BBM enzymes were stimulated differently based on the intestine tract. The plasma proteins level increased from T1.5 until T9, while the other blood parameters unvariated during the postprandial period. This study supplied useful information about the physiological effects a single meal as a potential tool for planning nutritional studies involving the digestive functions.

Pectin Modulates Calcium Absorption in Polarized Caco-2 Cells via a Pathway Distinct from Vitamin D Stimulation.

Pectin, a type of soluble fiber, promotes morphological changes in the small intestinal villi. Although its physiological significance is unknown, we hypothesized that changes in villus morphology enhance the efficiency of nutrient absorption in the small intestine and investigated the effect of pectin derived from persimmon on calcium absorption using polarized Caco-2 cells. In polarized Caco-2 cells, pectin altered the mRNA expression levels of substances involved in calcium absorption and the regulation of intracellular calcium concentration and significantly reduced calcium absorption. Although this was comparable to the results of absorption and permeability associated with the addition of active vitamin D, the simultaneous action of pectin and active vitamin D did not show any additive effects. Furthermore, as active vitamin D significantly increases the activity of intestinal alkaline phosphatase (ALP), which is known to be involved in the regulation of intestinal absorption of calcium and lipids, we also investigated the effect of pectin on intestinal ALP activity. As a result, it was found that, unlike the effect of active vitamin D, pectin significantly reduced intestinal ALP activity. These results suggest that pectin stimulates polarized Caco-2 cells through a mechanism distinct from the regulation of calcium absorption by vitamin D, modulating total calcium absorption from the elongated villi through morphological changes in the small intestine by suppressing it at the cellular level.