Intestinal Caco-2 Cells Research Articles

Selective transportation and energy homeostasis regulation of dietary advanced glycation end-products in human intestinal Caco-2 cells

Advanced glycation end-products (AGEs) are a chemically heterogeneous set of modifications widely found in processed foods. Due to uncertain bioavailability, dietary AGEs regulate energy homeostasis through mechanisms that largely remain unclear. In this study, selective transmembrane transport of AGEs with different modification types from glycated β-casein digest were identified and compared. The results showed that only a few types of free and peptide-bound AGEs can easily cross the Caco-2 monolayers and thus exert their effects. A combination of biochemical assays, mitochondrial analyses, and comparative experiments identified that the effect of AGEs on cellular energy homeostasis comes mainly from their free fractions. Mechanistically, free AGEs arrest the mitochondrial differentiation and mtDNA repair by intervening in the function of thymidine phosphorylase, and interfering with mitochondrial energy production by inhibiting the activation of AMPK-SIRT6 signaling pathway. These results demonstrate mechanisms by which processed foods cause mitochondrial dysfunction and lead to dysfunctional energy homeostasis.

Control of CDH1/E-Cadherin Gene Expression and Release of a Soluble Form of E-Cadherin in SARS-CoV-2 Infected Caco-2 Intestinal Cells: Physiopathological Consequences for the Intestinal Forms of COVID-19.

COVID-19 is the biggest pandemic the world has seen this century. Alongside the respiratory damage observed in patients with severe forms of the disease, gastrointestinal symptoms have been frequently reported. These symptoms (e.g., diarrhoea), sometimes precede the development of respiratory tract illnesses, as if the digestive tract was a major target during early SARS-CoV-2 dissemination. We hypothesize that in patients carrying intestinal SARS-CoV-2, the virus may trigger epithelial barrier damage through the disruption of E-cadherin (E-cad) adherens junctions, thereby contributing to the overall gastrointestinal symptoms of COVID-19. Here, we use an intestinal Caco-2 cell line of human origin which expresses the viral receptor/co-receptor as well as the membrane anchored cell surface adhesion protein E-cad to investigate the expression of E-cad after exposure to SARS-CoV-2. We found that the expression of CDH1/E-cad mRNA was significantly lower in cells infected with SARS-CoV-2 at 24 hours post-infection, compared to virus-free Caco-2 cells. The viral receptor ACE2 mRNA expression was specifically down-regulated in SARS-CoV-2-infected Caco-2 cells, while it remained stable in HCoV-OC43-infected Caco-2 cells, a virus which uses HLA class I instead of ACE2 to enter cells. It is worth noting that SARS-CoV-2 induces lower transcription of TMPRSS2 (involved in viral entry) and higher expression of B0AT1 mRNA (that encodes a protein known to co-express with ACE2 on intestinal cells). At 48 hours post-exposure to the virus, we also detected a small but significant increase of soluble E-cad protein (sE-cad) in the culture supernatant of SARS-CoV-2-infected Caco-2 cells. The increase of sE-cad release was also found in the intestinal HT29 cell line when infected by SARS-CoV-2. Beside the dysregulation of E-cad, SARS-CoV-2 infection of Caco-2 cells also leads to the dysregulation of other cell adhesion proteins (occludin, JAMA-A, zonulin, connexin-43 and PECAM-1). Taken together, these results shed light on the fact that infection of Caco-2 cells with SARS-CoV-2 affects tight-, adherens-, and gap-junctions. Moreover, intestinal tissues damage was associated to the intranasal SARS-CoV-2 infection in human ACE2 transgenic mice.

The Effect of Glycosylation Modulators on the Trafficking and Interaction of Spike Protein S1 Subunit and Angiotensin‐Converting Enzyme 2

The betacoronavirus severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is the cause of the ongoing worldwide pandemic. The spike (S) glycoprotein of SARS‐CoV‐2 is a heavily glycosylated trimer that binds to host cell receptor angiotensin‐converting enzyme 2 (ACE2), initiating viral entry. Evidence also shows that the S glycoprotein interacts with other receptors including dipeptidyl peptidase IV (DPPIV). Glycosylation is a post‐translational modification important for proper folding and trafficking of membrane and secretory proteins. It plays a critical role not only for host cell proteins, but also in viral virulence, immunity evasion and receptor binding capabilities. This study focused on the modulation of glycosylation and its effects on the trafficking of the S1 subunit, ACE2 and DPPIV, in addition to its effect on their interactions. For this purpose, the S1 subunit was expressed in COS‐1 cells in the presence or absence of N‐butyldeoxynojirimycin (NB‐DNJ), an inhibitor of the ER‐located a‐glucosidases I and II, 1‐deoxymannojirimycin (dMM), an inhibitor of Golgi‐mannosidase I, or benzyl‐N‐acetyl‐α‐galactosaminide (benzyl‐GalNac), an inhibitor of O‐glycosylation. The data demonstrate that NB‐DNJ affects substantially the trafficking and secretion of S1 protein, while its overall synthetic levels remained unchanged. A similar effect was observed upon treatment of the cells with the benzyl‐GalNac. On the contrary, the presence of dMM in the culture medium did not affect the secretion of S1. Together, the data suggest that folding events implicating calnexin in the ER, the site of action of NB‐DNJ, as well as O‐glycans that are modulated by benzyl‐GalNac are crucial for the secretion of the S1 protein. We further investigated the trafficking of ACE2 and DPPIV in lung Calu‐3 and intestinal Caco‐2 cells in the presence or absence of the glycosylation modulators and their interaction with S1. The data demonstrate a reduced binding capacity of S1 to ACE2 in the presence of NB‐DNJ and dMM, while this interaction was enhanced in Calu‐3 cells upon inhibition of O‐glycosylation by benzyl‐GalNac. We conclude that glycosylation modulators differentially act on the secretory pathway of S1 and the receptors. By virtue of its negative impact on both the secretion of S1 as well as interaction with its receptor ACE2, NB‐DNJ may be considered as a potential therapeutic drug that may act at the level of viral entry and exit.

Mechanistic bottom-up estimation of passive drug absorption from the gastrointestinal tract: Comparison among primary cultured human intestinal cells, Caco-2 cells, artificial membrane, and animal scale-up.

The fraction of drug absorbed (Fa) from the intestine is an important parameter to characterize the pharmacokinetics of a drug. We aimed to search for an experimental system that provides the best parameters for estimating the effective permeability (Peff) used for the bottom-up prediction of Fa. The absorption kinetics of 12 passively absorbed drugs were simulated by a compartment absorption transit (CAT) model using absorption parameters from four different experimental systems: human intestinal epithelial cell (HIEC) monolayer, Caco-2 monolayer, parallel artificial membrane permeability assay (PAMPA), and in situ rat intestinal perfusion. All absorption parameters were obtained from the literature. The in vitro apparent permeability coefficient (Papp) and rat in situ Peff were converted to human Peff using a bottom-up approach for each region, based on the morphological features of the human intestine. The simulated Fa values were compared to the respective observed values. Furthermore, plasma concentration profiles of the drugs were simulated by convolution using the time-course of the absorption rate simulated using the Peff values calculated from the HIEC Papp. The Fa values were best predicted by using the Peff values calculated from HEIC, within a 1.3-fold range of observed Fa in 11 out of 12 drugs. The simulated Cmax values of pharmacokinetic simulation using HIEC Papp fell within a 1.5-fold range of observed values for all the drugs examined. The HIEC monolayer was identified as the most suitable permeation parameter for estimating Fa and Cmax using a morphological feature-based bottom-up approach.

Slc2a5 (GLUT5) upregulation in hyperuricemia drives risk for fructose induced NAFLD

Epidemiological and observational studies have found a strong correlation between hyperuricemia (clinically elevated serum urate levels) and the onset of metabolic and systemic diseases. To investigate a potential causal role for urate in metabolic disease, we created a humanized mouse model utilizing a loss of function mutation in a critical intestinal urate transport protein, ABCG2. Male animals from this model were hyperuricemic and displayed fat deposition in the liver and significantly elevated serum glucose and insulin levels, hallmarks of metabolic dysfunction, and similar to what is often observed in high fructose diet models of NAFLD (Nonalcoholic fatty liver disease). However, the relationship between hyperuricemia, liver pathogenesis, and altered intestinal fructose uptake is largely unexplored.Using the Q140K+/+ hyperuricemia model we observed higher gene expression of Slc2a5 as well as increased abundance of its protein product, the apical fructose transporter GLUT5, in enterocytes. In addition, ex‐vivo intestinal fructose transport studies revealed a corresponding significant increase of fructose transport across the intestinal tissue of Q140K+/+ mice (16.497 ±5.955 vs. 34.684 ±5.31 µM/cm2). We hypothesized that HNF4a, the critical transcription factor that regulates the expression of many gut transport proteins, and has potential binding sites in the human and mouse Slc2a5 promoter region, might be sensitive to urate levels. Treatment of the human intestinal Caco‐2 cell monolayers with urate increased the protein abundance of both GLUT5 and HNF4a in a dose‐dependent manner, potentially explaining the observed increased mRNA levels of Slc2a5 in our model. We next explored the possibility that the GLUT5 abundance was further altered due to secondary urate effects on the critical transporter scaffold NEDD4 like E3 ubiquitin‐protein ligase (NEDD4L). Nedd4L gene expression was decreased, and its negative regulator, Sgk2, was significantly increased in intestinal tissue from the hyperuricemic model. Follow‐up experiments demonstrated SGK2 protein abundance is increased in Caco‐2 cells by extracellular urate. Further experiments are needed to confirm if hyperuricemia induced up‐regulation of SGK2, and subsequent down‐regulation of NEDD4L would prevent GLUT5 ubiquitination and increase its residence and abundance at the apical membrane.In conclusion, we find that hyperuricemia induced via common variants in the key intestinal urate transporter, ABCG2, can cause NAFLD and metabolic phenotypes indistinguishable from high fructose diet models of NAFLD. Here we demonstrate that urate may significantly upregulate the capacity for fructose reabsorption through the upregulation of Slc2a5 transcription and GLUT5 abundance, a finding that suggests human carriers of ABCG2 variants are not only at higher risk for hyperuricemia and gout but also the development of metabolic syndrome.

Gut Oxidative Modulation of Polyphenol, Prebiotic, Probiotic, and Postbiotic in Vitro

The study evaluated the individual and combined influence of polyphenol (quercetin), prebiotic (galactooligosaccharide), probiotic ( Lactobacillus acidophilus), and/ or postbiotic (inanimate Lactobacillus acidophilus) on the cellular oxidative status of CACO-2 intestinal epithelial cells. The CACO-2 cells were treated with quercetin (1 µmol L-1), galactooligosaccharide (4 mg mL-1), Lactobacillus acidophilus (2 x 106 CFU mL-1), and/or inanimate Lactobacillus acidophilus (2 x 106 CFU mL-1) individually and in all combinations over twenty four hours. Cellular antioxidant capacities (DPPH radical scavenging activity, Oxygen Radical Absorbance Capacity, Total Peroxyl Trapping Potential, and Trolox Equivalent Antioxidant Capacity), antioxidant enzyme activities (superoxide dismutase and peroxidase), and oxidative damages (F2-isoprostanes and lipid hydroperoxides) were measured. Intracellular quercetin and total short-chain fatty acids (acetic, propionic, and butyric acids) were determined. Treatments with quercetin or inanimate Lactobacillus acidophilus exhibited significant greater cellular antioxidant effects compared to those without quercetin or inanimate Lactobacillus acidophilus. Antioxidant capacities of treatments with quercetin and inanimate Lactobacillus acidophilus were significantly stronger than those with either one. Quercetin and short-chain fatty acids accumulated into the CACO-2 cells incubated with quercetin and inanimate Lactobacillus acidophilus, respectively. Polyphenol, probiotic, and postbiotic, individually or interdependently, influenced the oxidative status of intestinal epithelial CACO-2 cells and protected them from oxidative damage.

Cephalexin loading and controlled release studies on mesoporous silica functionalized with amino groups

KIT-6 mesoporous silica has been synthesized using the sol-gel method and functionalized with 3-aminopropyl triethoxysilane by grafting route to obtain KIT-6/NH2. These samples were used as carriers in the loading and controlled release of cephalexin (CFX). The effect of temperature and gastric and intestinal pH on the stability of pure CFX and loaded in KIT-6 and KIT-6/NH2 were investigated. The properties of the synthesized materials and their CFX loading capacity were studied through FTIR, ads-des N2 at 77 K, TEM, SEM, XPS, and TGA. The controlled release tests were carried out in a simulated physiological medium at gastric (1.2) and intestinal pH (6.8). Furthermore, the biocompatibility of both materials was studied through cell viability tests in Caco-2 intestinal cells. The results revealed that KIT-6 and KIT-6/NH2 had similar CFX loading capacities. It was found that CFX degrades at pH 6.8, however, KIT-6 and KIT-6/NH2 were able to protect it from the aforementioned degradation. Moreover, KIT-6 materials presented a good performance as CFX carriers since both materials provided diffusion-controlled release profiles during 24 h, satisfying the Korsmeyer-Peppas kinetic model. Mesoporous silicas presented in this work are promising candidates to be used in CFX controlled release systems due to their chemical interactions, textural properties, and high cell viability.

Current Status of Organic Matters in Bottled Drinking Water in Korea

Microplastics may be ubiquitous across ecosystems, yet the current state of exposure to microplastics in daily life remains poorly understood. We focused on drinking water in plastic bottles purchased from a local market in South Korea. The composition, amount, and size of organic matter in solid residues obtained by freeze-drying drinking water in a plastic bottle were determined using analytical methods including dry ashing, centrifugation, Raman analysis, and electron microscopy. A significant amount of organic matter was found in the plastic bottles (0.25–2.0 mg/L), and once the organic matter was concentrated and dried as a solid, it was not soluble in water. In addition, a series of stress to the plastic bottle, such as light, heat, and mechanical stress, did not significantly increase the organic matter within a month. The potential adverse effects of organic matter on humans were evaluated using the human intestinal Caco-2 cell line. The results showed no significant toxicity in cell viability even at relatively high concentrations of organic matter (1.0 mg/mL). However, the monitoring of the accumulation of organic matter in the human body over a longer period of time will be needed because of the higher probability of nanoscale material uptake in the long term.

Hyperthermia-Induced In Situ Drug Amorphization by Superparamagnetic Nanoparticles in Oral Dosage Forms.

Superparamagnetic iron oxide nanoparticles (SPIONs) generate heat upon exposure to an alternating magnetic field (AMF), which has been studied for hyperthermia treatment and triggered drug release. This study introduces a novel application of magnetic hyperthermia to induce amorphization of a poorly aqueous soluble drug, celecoxib, in situ in tablets for oral administration. Poor aqueous solubility of many drug candidates is a major hurdle in oral drug development. A novel approach to overcome this challenge is in situ amorphization of crystalline drugs. This method facilitates amorphization by molecular dispersion of the drug in a polymeric network inside a tablet, circumventing the physical instability encountered during the manufacturing and storage of conventional amorphous solid dispersions. However, the current shortcomings of this approach include low drug loading, toxicity of excipients, and drug degradation. Here, doped SPIONs produced by flame spray pyrolysis are compacted with polyvinylpyrrolidone and celecoxib and exposed to an AMF in solid state. A design of experiments approach was used to investigate the effects of SPION composition (Zn0.5Fe2.5O4 and Mn0.5Fe2.5O4), doped SPION content (10–20 wt %), drug load (30–50 wt %), and duration of AMF (3–15 min) on the degree of drug amorphization. The degree of amorphization is strongly linked to the maximum tablet temperature achieved during the AMF exposure (r = 0.96), which depends on the SPION composition and content in the tablets. Complete amorphization is achieved with 20 wt % Mn0.5Fe2.5O4 and 30 wt % celecoxib in the tablets that reached the maximum temperature of 165.2 °C after 15 min of AMF exposure. Furthermore, manganese ferrite exhibits no toxicity in human intestinal Caco-2 cell lines. The resulting maximum solubility of in situ amorphized celecoxib is 5 times higher than that of crystalline celecoxib in biorelevant intestinal fluid. This demonstrates the promising capability of SPIONs as enabling excipients to magnetically induce amorphization in situ in oral dosage forms.

Impact of simulated in vitro gastrointestinal digestion on bioactive compounds, bioactivity and cytotoxicity of melon (Cucumis melo L. inodorus) peel juice powder

The objectives of this research work were to evaluate the effect of in vitro gastrointestinal digestion (GIT) on melon peel juice (MPJ) powder from fruit processing industry by-products, considering (i) the recovery and accessibility indexes, (ii) the changes on antioxidant activity, and (iii) the prebiotic effect. Throughout exposition to GIT conditions a decrease on the total phenolic content (TPC = 65.31%) and antioxidant activity by ABTS = 39.77% and DPPH = 45.91% were observed. However, these both parameters exhibited stable accessibility, accounting with 81.89%, 76.55%, and 54.07% for TPC, ABTS and DPPH, respectively. After gastrointestinal digestion, the non-absorbed fraction exhibited a positive impact on the growth of Bifidobacterium and Lactobacillus strains, possibly associated with the high content of simple sugar (glucose and fructose). This fraction also showed to be safe on Caco-2 intestinal cells. These findings suggest that MPJ might be used as a potential food functional ingredient.

Enhancement of the in vitro bioavailable carotenoid content of a citrus juice combining crossflow microfiltration and high-pressure treatments

High-pressure treatments combined with crossflow microfiltration were used to obtain citrus concentrates enriched in carotenoids. The aim of this study was to investigate the effect of this process combination on carotenoid bioaccessibility and uptake by intestinal Caco-2 cells. Two high-pressure processes, high hydrostatic pressure treatment (HHP) and ultra-high-pressure homogenization (UHPH) were compared to conventional pasteurization. Processing effects on carotenoid content and bioaccessibility, on physicochemical and structural characteristics of the product, on methylation degree of pectins and micelle size after in vitro digestion were assessed. UHPH at 400 MPa drastically enhanced carotenoid bioaccessibility compared to HHP and pasteurization. Moreover, carotenoid uptake by Caco-2 cells was significantly improved by UHPH underlining the importance of the micelle size after in vitro digestion and the degree of methylation of pectins in this uptake. Finally, the in vitro bioavailable carotenoid content of different concentrates was evaluated, taking into account carotenoid content, bioaccessibility and uptake. Combining crossflow microfiltration with UHPH increased by 4–6 fold the bioavailable carotenoid content in the final product. The process led to a concentrate of high nutritional quality compared to the original juice, raw or pasteurized concentrates.

Grape Pomace Extract Attenuates Inflammatory Response in Intestinal Epithelial and Endothelial Cells: Potential Health-Promoting Properties in Bowel Inflammation.

Inflammatory bowel disease (IBD) implies the chronic inflammation of the gastrointestinal tract, combined with systemic vascular manifestations. In IBD, the incidence of cardiovascular disease appears to be related to an increase of oxidative stress and endothelial dysfunction. Grape pomace contains high levels of anti-oxidant polyphenols that are able to counteract chronic inflammatory symptoms. The aim of this study was to determine whether grape pomace polyphenolic extract (GPE) was able to mitigate the overwhelming inflammatory response in enterocyte-like cells and to improve vascular function. Intestinal epithelial Caco-2 cells, grown in monolayers or in co-culture with endothelial cells (Caco-2/HMEC-1), were treated with different concentrations of GPE (1, 5, 10 µg/mL gallic acid equivalents) for 2 h and then stimulated with lipopolysaccharide (LPS) and tumor necrosis factor (TNF)-α for 16 h. Through multiple assays, the expression of intestinal and endothelial inflammatory mediators, intracellular reactive oxygen species (ROS) levels and NF-κB activation, as well as endothelial-leukocyte adhesion, were evaluated. The results showed that GPE supplementation prevented, in a concentration-dependent manner, the intestinal expression and release of interleukin (IL)-6, monocyte chemoattractant protein (MCP)-1, and matrix metalloproteinases (MMP)-9 and MMP-2. In Caco-2 cells, GPE also suppressed the gene expression of several pro-inflammatory markers, such as IL-1β, TNF-α, macrophage colony-stimulating factor (M-CSF), C-X-C motif ligand (CXCL)-10, intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, and cyclooxygenase (COX)-2. The GPE anti-inflammatory effect was mediated by the inhibition of NF-κB activity and reduced intracellular ROS levels. Furthermore, transepithelial GPE suppressed the endothelial expression of IL-6, MCP-1, VCAM-1, and ICAM-1 and the subsequent adhesion of leukocytes to the endothelial cells under pro-inflammatory conditions. In conclusion, our findings suggest grape pomace as a natural source of polyphenols with multiple health-promoting properties that could contribute to the mitigation of gut chronic inflammatory diseases and improve vascular endothelial function.

Sweet Biotechnology: Enzymatic Production and Digestibility Screening of Novel Kojibiose and Nigerose Analogues.

In view of the global pandemic of obesity and related metabolic diseases, there is an increased interest in alternative carbohydrates with promising physiochemical and health-related properties as a potential replacement for traditional sugars. However, our current knowledge is limited to only a small selection of carbohydrates, whereas the majority of alternative rare carbohydrates and especially their properties remain to be investigated. Unraveling their potential properties, like digestibility and glycemic content, could unlock their use in industrial applications. Here, we describe the enzymatic production and in vitro digestibility of three novel glycosides, namely, two kojibiose analogues (i.e., d-Glcp-α-1,2-d-Gal and d-Glcp-α-1,2-d-Rib) and one nigerose analogue (i.e., d-Glcp-α-1,3-l-Ara). These novel sugars were discovered after an intensive acceptor screening with a sucrose phosphorylase originating from Bifidobacterium adolescentis (BaSP). Optimization and upscaling of this process led to roughly 100 g of these disaccharides. Digestibility, absorption, and caloric potential were assessed using brush border enzymes of rat origin and human intestinal Caco-2 cells. The rare disaccharides showed a reduced digestibility and a limited impact on energy metabolism, which was structure-dependent and even more pronounced for the three novel disaccharides in comparison to their respective glucobioses, translating to a low-caloric potential for these novel rare disaccharides.

Characterization of the MicroRNA Profile of Ginger Exosome-like Nanoparticles and Their Anti-Inflammatory Effects in Intestinal Caco-2 Cells

Plant-derived exosome-like nanoparticles (PELNs) have been shown to enter mammalian cells for disease treatment. Although abundant miRNAs are contained in ginger exosome-like nanoparticles (GELNs), little is known about their type and function. Herein, we extracted GELNs with desirable particle sizes (156 ± 36 nm) and a negative surface charge (-26.6 ± 5 mV). The miRNA profiles in ginger and GELNs were analyzed using high-throughput sequencing, and the results of the sequencing were validated by real-time quantitative polymerase chain reaction (RT-qPCR). There were 27 miRNAs with higher expression levels in the GELNs, and they were mainly involved in the regulation of inflammatory and cancer-related pathways. Furthermore, GELNs could be specifically internalized by intestine cells via caveolin-mediated endocytosis and micropinocytosis, as well as counteract lipopolysaccharide (LPS)-induced inflammation by downregulating NF-κβ, IL-6, IL-8, and TNF-α expression. Importantly, the positive effects were further proved to be possibly related to the miRNAs enriched in the GELNs. Overall, these results indicated that PELNs could target human digestive organs and play a cross-kingdom physiological regulation role through miRNAs.

Exposure to the mycotoxin deoxynivalenol reduces the transport of conjugated bile acids by intestinal Caco-2 cells

Conjugated bile acids are synthesized in liver and subsequently secreted into the intestinal lumen from which they are actively reabsorbed and transported back to liver. The efficient enterohepatic circulation of conjugated bile acids is important to maintain homeostasis. The mycotoxin deoxynivalenol (DON) is a fungal secondary metabolite that contaminates cereal food. Upon human exposure, it can cause intestinal dysfunction. We explored the effects of DON exposure on the intestinal absorption of conjugated bile acids and the expression of bile acid transporters using an in vitro model based on Caco-2 cell layers grown in transwells. Our study shows that the transport rate of taurocholic acid (TCA) is decreased after 48-h pre-exposure of the Caco-2 cells to 2 µM DON, which is a realistic intestinal DON concentration. Exposure to DON downregulates expression of the genes coding for the apical sodium-dependent bile acid transporter (ASBT), the ileal bile acid-binding protein (IBABP) and the organic solute transporter α (OSTα), and it counteracts the agonist activity of Farnesoid X receptor (FXR) agonist GW4064 on these genes. In addition, the transport of ten taurine or glycine-conjugated bile acids in a physiological relevant mixture by the intestinal Caco-2 cell layers was decreased after pre-exposure of the cells to DON, pointing at a potential for DON-mediated accumulation of the conjugated bile acids at the intestinal luminal side. Together the results reveal that DON inhibits intestinal bile acid reabsorption by reducing the expression of bile acid transporters thereby affecting bile acid intestinal kinetics, leading to bile acid malabsorption in the intestine. Our study provides new insights into the hazards of DON exposure.

Lactobacillus crispatus Strain KT-11 S-Layer Protein Inhibits Rotavirus Infection.

S-layer proteins (SLPs), which are present in the external layer of certain strains of lactic acid bacteria isolated from the intestinal tract, are known to recognize and bind to specific proteins and glycan structures and contribute to adsorption to the host intestinal mucosa. The binding properties of certain SLPs are considered to exert a competitive inhibitory effect on infection because similar properties are involved in the infection mechanisms of several viruses. However, little is known regarding whether SLPs directly inhibit viral infection. In the present study, we investigated the effect of an SLP of the Lactobacillus crispatus KT-11 strain, a probiotic strain isolated from a healthy human infant, on human rotavirus infection. The impact of KT-11 lithium chloride extract (KT-11 LE), which contains SLP, on the infection of the P[4] genotype human rotavirus strain DS-1 was evaluated by monitoring the amplification of viral protein 6 (VP6) expression in human intestinal epithelial Caco-2 cells by quantitative reverse transcription-polymerase chain reaction assay after infection. KT-11 LE showed a significant suppressive effect on DS-1 infection in a dose-dependent manner with pre-infection treatment, whereas post-infection treatment was not effective. A 45 KDa protein isolated from KT-11 LE was investigated for homology using the BLAST database and was found to be a novel SLP. KT-11 SLP concentrate (KT-11 SLP) significantly inhibited the proliferative process of the DS-1 strain but not that of the P[8] genotype human rotavirus strain Wa. KT-11 SLP exerted significant inhibitory effect on DS-1 infection by pre-infection treatment even after digestion with gastric juice up to 2 h. Our results provided crucial evidence that SLPs from certain Lactobacillus strains can inhibit human rotavirus infection of intestinal epithelial cells.

Somatostatin attenuates intestinal epithelial barrier injury during acute intestinal ischemia–reperfusion through Tollip/Myeloiddifferentiationfactor 88/Nuclear factor kappa-B signaling

ABSTRACT In the process of ischemia–reperfusion injury, intestinal ischemia and inflammation interweave, leading to tissue damage or necrosis. However, oxygen radicals and inflammatory mediators produced after reperfusion cause tissue damage again, resulting in severe intestinal epithelial barrier dysfunction. The aim of this study was to determine the protective effect of somatostatin on intestinal epithelial barrier function during intestinal ischemia–reperfusion injury and explore its mechanism. By establishing a rat intestinal ischemia–reperfusion model, pretreating the rats with somatostatin, and then detecting the histopathological changes, intestinal permeability and expression of tight junction proteins in intestinal tissues, the protective effect of somatostatin on the intestinal epithelial barrier was measured in vivo. The mechanism was determined in interferon γ (IFN-γ)-treated Caco-2 cells in vitro. The results showed that somatostatin could ameliorate ischemia–reperfusion-induced intestinal epithelial barrier dysfunction and protect Caco-2 cells against IFN-γ-induced decreases in tight junction protein expression and increases in monolayer cell permeability. The expression of Tollip was upregulated by somatostatin both in ischemia–reperfusion rats and IFN-γ-treated Caco-2 cells, while the activation of TLR2/MyD88/NF-κB signaling was inhibited by somatostatin. Tollip inhibition reversed the protective effect of somatostatin on the intestinal epithelial barrier. In conclusion, somatostatin could attenuate ischemia–reperfusion-induced intestinal epithelial barrier injury by inhibiting the activation of TLR2/MyD88/NF-κB signaling through upregulation of Tollip.

Identification and characterization of metal-chelating bioenhancer peptide derived from fermented Citrullus lanatus seed milk.

In the present investigation, a metal-chelating bioactive peptide was derived from Citrullus lanatus seed milk fermented with Lactococcus lactis. The cationic fermented milk peptide (FMP) thus obtained was purified using the HiTrap-chelating column followed by rpHPLC. The FMP possessed the ability to chelate multiple divalent cations like Cu2+ , Ca2+ , and Fe2+ with 86.81%, 61.04%, and 24.32% of chelation respectively and further it exhibited 78.03% of DPPH free radical scavenging activity. Interaction of FMP with metal ions was assessed by change in the absorption spectra and was analyzed by ultraviolet-visible and fluorescence spectroscopy. The FMP-metal complexes were found stable at simulated gastric conditions. In vitro analysis using intestinal Caco-2 cell lines revealed that there was an increase in metal bioavailability in the presence of the FMP and was least influenced by the addition of a dietary inhibitor, phytic acid. By LC-MS analysis the molecular mass of FMP was found to be 11.6kD and it contains oxygen-rich and nitrogen-rich amino acids that favor the metal chelation. In our study, we have found that the fermented C. lanatus seed milk can serve as a potential functional food with bioenhancer peptides that increase metal bioavailability and enhance human health. PRACTICAL APPLICATIONS: Chelated metals are preferred over non-chelated ones by most nutritionists for their better absorption rate. Chelation protects the minerals from the digestive process and increases their bioavailability. Fermentation with lactic acid bacteria produces bioactive peptides with metal-chelating and antioxidant ability which provides additional health benefits beyond supplying basic nutrients. Lactococcus lactis fermented milk acts as a probiotic product with bioenhancer peptide that increases mineral bioavailability. Consumption of metals in chelated form can reduce excess intake of metal. Fermented watermelon seed milk can be a promising probiotic drink rich in bioenhancer peptides and can enhance the bioavailability of divalent cations of a high therapeutic index.

In Vitro Protective Effects of a Standardized Extract From Cynara Cardunculus L. Leaves Against TNF-α-Induced Intestinal Inflammation.

Inflammatory bowel disease (IBD) represents a group of progressive disorders characterized by recurrent chronic inflammation of the gut. New unconventional therapies based on plant derived compounds capable of preventing and/or reducing acute or chronic inflammation could represent a valid alternative for the treatment or prevention of IBDs. Cynara cardunculus L. leaves, considered a food-waste suitable as a rich source of bioactive polyphenols including luteolin and chlorogenic acid, has been reported for its positive effects in digestive tract. The aim of the present work was to evaluate the in vitro molecular mechanisms of beneficial effects of a standardized polyphenol-rich extract obtained from the leaves of Cynara cardunculus L (CCLE) against acute intestinal inflammation induced by TNF-α on intestinal epithelial Caco-2 cells. CCLE prevented TNF-α-induced NF-κB inflammatory pathway and the overexpression of IL-8 and COX-2. In addition, CCLE was able to improve basal intracellular antioxidant power in both TNF-α-unexposed or -exposed Caco-2 cells and this effect was associated to the activation of Nrf2 pathway, a master regulator of redox homeostasis affecting antioxidant and phase II detoxifying genes, stimulating an adaptive cellular response. In conclusion, our data clearly evidenced that, although considered a waste, Cynara cardunculus leaves may be used to obtain extracts rich in bioactive polyphenols potentially useful for prevention and treatment of inflammatory intestinal diseases.

A flavonoid rich standardized extract of Glycyrrhiza glabra protects intestinal epithelial barrier function and regulates the tight-junction proteins expression

BackgroundIntestinal epithelial barrier dysfunction predisposes to many gastrointestinal, metabolic, and psychological disorders. A flavonoid rich extract of Glycyrrhiza glabra (FREG) has previously been reported to possess anti-inflammatory, antioxidant, and antiulcer properties.AimTo investigate the effect of FREG (GutGard®) on restoring intestinal barrier function in tumor necrosis factor-alpha (TNF-α) stimulated human colonic adenocarcinoma cell monolayer (Caco-2) and 2,4,6-Trinitrobenzenesulfonic acid (TNBS) induced ulcerative colitis in rats.MethodsIn in vitro, human intestinal Caco-2 cell monolayers were treated with TNF-α in the presence or absence of FREG and the paracellular permeability to FITC-conjugated 4-kD dextran (FD4) was measured to evaluate protection against the barrier dysfunction. In in vivo, intestinal barrier dysfunction was induced in male albino Wistar rats via intrarectal instillation of TNBS. Subsequently, the rats were treated orally with either FREG at 6.25, 12.5, and 25 mg/kg body weight, or Mesacol (250 mg/kg) for 5 days. On day 5, intestinal epithelial permeability was assessed with FD4 leakage into the serum. Also, colonic inflammation, colon morphology, histology and macroscopic score, weight to length ratio were evaluated. The activity of myeloperoxidase (MPO), TNF- α, secretory IgA levels and tight junction proteins expression were evaluated in rat’s colon.ResultsFREG protected the intestinal epithelial barrier integrity in human intestinal Caco-2 cells in vitro. FREG administration significantly improved the intestinal epithelial barrier function as evident from significant reduction in FD4 leakage. The colon morphology, histology score, macroscopic score, colon weight to length ratio also indicates beneficial effects of FREG on barrier function. In addition, FREG regulated the tight junction proteins, and markedly decreased TNF-α, MPO levels and significantly increased the secretory IgA levels in TNBS induced colitis rats.ConclusionThe study findings support the protective action of FREG on intestinal epithelial barrier integrity indicating its potential in protecting from implications of leaky gut.