Search Results (318)

Background/Objectives: Research on postbiotics derived from probiotic fermented milk bases require further expansion, and the mechanisms through which they exert their effects have yet to be fully elucidated. This study utilized in vitro cell co-culture, digestion, and fermentation experiments, combined with targeted T500 technology, to elucidate the mechanism by which postbiotic Pa JY062 safeguards intestinal health. Compared to the LPS group, Pa JY062 boosted phagocytic ability in RAW264.7 macrophages, decreased NO levels, and alleviated LPS-induced excessive inflammation. Pa JY062 suppressed pro-inflammatory cytokines (IL-6, IL-17α, and TNF-α) while elevating anti-inflammatory IL-10. It prevented LPS-induced TEER reduction in Caco-2 monolayers, decreased FITC-dextran permeability, restored intestinal microvilli integrity, and upregulated tight junction genes (ZO-1, occludin, claudin-1, and E-cadherin). The hydrolysis rate of Pa JY062 progressively rose in gastrointestinal fluids in 0–120 min. At 5 mg/mL, it enriched gut microbiota diversity and elevated proportions of Limosilactobacillus, Lactobacillus, Pediococcus, and Lacticaseibacillus while augmenting the microbial production of acetic acid (120.2 ± 8.08 μg/mL), propionic acid (9.9 ± 0.35 μg/mL), and butyric acid (10.55 ± 0.13 μg/mL). Pa JY062 incorporated α_s-casein/β-lactoglobulin hydrolysate (L-glutamic acid, alanine, lysine, tyrosine, phenylalanine, histidine, and arginine) to mitigate protein allergenic potential while harboring bioactive components, including tryptophan metabolites, vitamin B6 (VB6), and γ-aminobutyric acid (GABA). Pa JY062 represented a novel postbiotic with demonstrated intestinal health-promoting properties. These findings advance the current knowledge on postbiotic-mediated gut homeostasis regulation and expedite the translational development of dairy-derived postbiotic formulations. Full article

Irritable Bowel Syndrome (IBS) is a disorder of gut- brain interaction characterized by recurrent abdominal pain associated with altered bowel habits. The therapeutic options for IBS patients include the use of probiotics. The aim of this study was to assess the effect of a multi-strain probiotic made up by Lactobacillus rhamnosus LR 32, Bifidobacterium lactis BL 04, and Bifidobacterium longum BB 536 (Serobioma, Bromatech s.r.l., Milano, Italy) on an in vitro model of the intestinal epithelial barrier in the presence of mucosal mediators that are released by IBS patients. IBS (n = 28; IBS with predominant diarrhea, IBS-D = 10; IBS with predominant constipation, IBS-C = 9; and IBS with mixed bowel habits, IBS-M = 9) patients, diagnosed according to the Rome IV criteria, and asymptomatic controls (ACs, n = 7) were enrolled. Mucosal mediators that were spontaneously released by colonic biopsies were collected (supernatants). Two doses of Serobioma were tested with/without IBS/AC mediators. RNA was extracted from Caco-2 cells to evaluate the tight junction (TJ) expression. Serobioma (10⁶ CFU/mL) significantly reinforced the Caco-2 monolayer compared to growth medium alone (p < 0.05). IBS supernatants significantly increased Caco-2 paracellular permeability compared to the AC supernatants. The co-incubation of Caco-2 cells with IBS supernatants and Serobioma (10⁶ CFU/mL) avoided the paracellular permeability alterations that were induced by IBS supernatants alone (p < 0.001), and, in particular, IBS-D and IBS-M ones. The co-incubation of Serobioma (10⁶ CFU/mL) and IBS-D supernatants significantly increased ZO-1 expression compared to Caco-2 cells incubated with supernatants alone (p < 0.05), as confirmed via qPCR analyses. Serobioma (10⁶ CFU/mL) counteracts the paracellular permeability changes that are induced by IBS supernatants, in particular IBS-D and IBS-M supernatants, likely modulating ZO-1 expression. Full article

Current in vitro methods for intestinal barrier assessment predominantly utilize two-dimensional (2D) membrane inserts in standard culture plates, which are widely recognized for their inability to replicate the microenvironment critical to intestinal barrier functionality. Our study focuses on creating an alternative method for intestinal barrier function by integrating a 3D-printed transwell device with a paper-based membrane. Caco-2 cells were grown on a Matrigel-modified paper membrane, in which the tight junction formation was evaluated using TEER measurements. Neutrophil-like dHL-60 cells were employed for neutrophil extracellular trap (NET) formation experiments. Furthermore, intestinal barrier dysfunction was demonstrated using NET-isolated and Staurosporine interventions. Intestinal barrier characteristics were investigated through immunofluorescence staining of specific proteins and scanning electron microscopy (SEM). Our paper-based intestinal barrier exhibited an increased resistance in a time-dependent manner, consistent with immunofluorescence images of Zonulin Occludens-1 (ZO-1) expression. Interestingly, immunofluorescence analysis revealed changes in the morphology of the intestinal barrier and the formation of surface villi. These disruptions were found to alter the localization of tight junctions, impacting epithelial polarization and surface functionality. Moreover, we successfully demonstrated the permeability of a paper-based intestinal barrier using FITC-dextran assay. Hence, the 3D-printed transwell device integrated with a paper membrane insert presents a straightforward, cost-effective, and sustainable platform for an in vitro cell model to evaluate intestinal barrier function. Full article

Epithelial linings are crucial for the maintenance of physiological barriers. The intestinal epithelial barrier (IEB) consists of enterocytes through tight junctions and mucus-secreting cells and can undergo physiological modifications throughout life. To reproduce as closely as possible the IEB main features over time, in vitro co-cultures of Caco2/HT-29 70/30 formed by parental Caco2 and HT-29 cells sub-cultivated for more than 40 passages were set up. The measurements of the transepithelial electrical resistance (TEER) identified two populations: physiological TEER co-cultures (PC) with values > 50 Ωcm² formed by parental cells with fewer than 40 passages, and leaky TEER co-cultures (LC) with values < 50 Ωcm² formed by parental cells with more than 40 passages. In LC, paracellular permeability increased in parallel. By immunofluorescence and Western blot analysis, an increase in claudin 2 was observed in LC vs. PC, with no differences in occludin expression. MUC-2 immunoreactivity was stronger in PC than in LC. LC also showed an enhanced vulnerability to TNFα+IFN-γ. These results reproduce the main morpho-functional modifications reported in the human leaky/aged gut and support the usefulness of our in vitro cell model for studying the molecular processes underlying these modifications and testing drug/nutraceutical treatments to ameliorate leaky gut aging. Full article

Background: This study explores the potential of the Pleurotus eryngii mushroom fermentation supernatant (FS-PEWS) as an intervention for mitigating sodium deoxycholate (SDC)-induced intestinal barrier dysfunction and inflammation. Methods: FS-PEWS was assessed for its protective effects against SDC-induced barrier dysfunction and inflammation using an in vitro Caco-2 cell model and ex vivo colonic biopsies from healthy adult donors, where barrier integrity, permeability, immunomodulation and receptor-mediated pathways were evaluated. Results: In Caco-2 cells, SDC exposure downregulated ZO-1, occludin, and claudin-1 expression, with FS-PEWS restoring ZO-1 and claudin-1 levels while maintaining cell viability. In colonic biopsies from healthy adults, FS-PEWS maintained tissue integrity and selectively mitigated transcellular permeability without affecting paracellular permeability when combined with the stressor. Additionally, FS-PEWS exhibited potent anti-inflammatory effects, reducing pro-inflammatory cytokines, e.g., TNF-α, IL-6, and IL-1β and modulating receptor-mediated pathways, i.e., TLR-4, dectin-1. Conclusions: These results demonstrate the potential of FS-PEWS to sustain intestinal barrier function and modulate immune responses under stress, highlighting its therapeutic potential for managing gut barrier dysfunction and inflammation associated with microbial metabolite-induced disruptions. Full article

Candida albicans is an opportunistic pathogenic yeast commonly found in the gastrointestinal tract of healthy humans. Under certain conditions, it can become invasive and cause life-threatening systemic infections. One mechanism used by C.albicans to breach the epithelial barrier is the secretion of candidalysin, a cytolytic peptide toxin. Candidalysin damages epithelial membranes and activates the innate immune response, making it key to C.albicans’ pathogenicity and a promising therapeutic target. Although candidalysin mediates C. albicans translocation through intestinal layers, its impact on epithelial responses is not fully understood. This study aims to characterize this response and develop scalable, quantitative methodologies to assess candidalysin’s toxicological effects using gut-on-chip models. We used the OrganoPlate^® platform to expose Caco-2 tubules to candidalysin and evaluated their response with trans-epithelial electrical resistance (TEER), protein detection, and immunostaining. We then validated our findings in a proof-of-concept experiment using human intestinal organoid tubules. Candidalysin impaired barrier integrity, induced actin remodeling, and increased cell permeability. It also induced the release of LDH, cytokines, and the antimicrobial peptide LL37, suggesting cellular damage, inflammation, and antimicrobial activity. This study strengthens our understanding of candidalysin’s role in C. albicans pathogenesis and suggests new therapeutic strategies targeting this toxin. Moreover, patient-derived organoids show promise for capturing patient heterogeneity and developing personalized treatments. Full article

The pinnatoxins (PnTXs) and portimines, produced by Vulcanodinium rugosum, have been detected in several countries, raising concerns for human health. Although no human poisoning from these toxins has been reported so far, they have been shown to distribute throughout the rodent body after oral administration. Therefore, we investigated the impact of PnTX analogs (PnTX-A, -E, -F, -G, and -H) and portimine (8, 16, and 32 ng/mL) on intestinal barrier integrity and their oral bioavailability using human Caco-2 cell monolayers treated for 2, 6, and 24 h. Our results demonstrated that all of the toxins could impair barrier integrity after 24 h, with differences observed for PnTX-A, -E, and -F, as well as portimine, the most potent of all. While PnTX-A and -E exhibited poor permeability, the other PnTXs were more penetrative, with a Papp > 1.5 × 10⁻⁶ cm·s⁻¹. Portimine was the only toxin displaying both a time- and concentration-dependent passage, likely involving a passive diffusion process. The experimental results were compared to predictions obtained by QSAR tools. Although only qualitative, our results suggest that some of these compounds may be more likely to be distributed throughout the body. Further in vivo studies are required to estimate oral bioavailability and potential public health concerns. Full article

The present study aimed to investigate the ability of an aqueous extract derived from mustard seed meal to counteract the effects of E. coli endotoxin lipopolysaccharide (LPS) on the intestinal epithelium. Caco-2 cells were cultured together with HT29-MTX and used as a cellular model to analyze critical intestinal parameters, such as renewal, integrity, innate immunity, and signaling pathway. Byproducts of mustard seed oil extraction are rich in soluble polysaccharides, proteins, allyl isothiocyanates, and phenolic acids, which are known as powerful antioxidants with antimicrobial and antifungal properties. Cells were seeded at a ratio of nine (Caco-2) to one (HT29-MXT) and treated for 2 h with mustard meal extract (ME, dilution 1/50) and zinc oxide (ZnO, 50 μM) after reaching 80–100% confluence. Then, they were challenged with 5 μg/mL E. coli-LPS and incubated for another 4 h. The results show that LPS did not alter the cell viability but decreased proliferation compared to the control, ME and ZnO treatments. LPS altered the cell membrane integrity and monolayer permeability by decreasing the transepithelial electrical resistance and tight-junction protein expression. In addition, LPS increased the activity of LDH and the expression of Toll-like receptors. The mechanisms by which LPS induces these disturbances involves the overexpression of PKC, p38 MAPK, and NF-κB signaling molecules. The pretreatment with mustard meal and ZnO succeeded in counteracting the impairment of epithelial renewal, the damage of the membrane integrity and permeability as well as in restoring the gene expression of tight-junction proteins. Full article

Nonsteroidal anti-inflammatory drugs (NSAIDs) can induce serious adverse effects in gastrointestinal (GI) mucosa, increasing intestinal permeability and leading to mitochondrial dysfunction, oxidative stress, apoptosis and inflammation. As proton pump inhibitors are effective in protecting against NSAID-induced gastropathy but not NSAID-induced enteropathy, current research is focused on natural products as protective substances for therapy and prevention of intestinal injury. Herein, through the use of an in vitro model based on intestinal epithelial cell (Caco-2) damage caused by indomethacin (INDO), we examined the protective activity of a commercially available standardized extract (OFI+OE) from Opuntia ficus-indica (L.) Mill. cladodes and Olea europaea L. leaves. Pre-treatment with OFI+OE prevented INDO-induced intestinal epithelial barrier damage, as demonstrated by TEER measurement, fluorescein permeability, and tight junction protein expression. The extract showed positive effects against INDO-induced oxidative stress and correlated activation of apoptosis, decreasing pro-apoptotic markers BAX and Caspase-3 and increasing anti-apoptotic factor Bcl-2. Moreover, the extract inhibited the NF-κB pathway and pro-inflammatory cascade. In conclusion, these data support the use of OFI+OE extract as a natural strategy for therapy and prevention of intestinal mucosal damage, demonstrating its beneficial effects against INDO-induced intestinal damage, through modulation of oxidative, apoptotic, and inflammatory pathways. Full article

The aryl hydrocarbon receptor (AhR) and the peroxisome proliferator-activated receptor γ (PPARγ) are ligand-activated transcription factors that have in recent years been investigated for their anti-inflammatory properties for treatment of inflammatory bowel diseases (IBDs). These are globally prevalent chronic maladies of the gut that lack cost-efficient therapeutical options capable of inducing long-term remission. In the present study, we used an in vitro Transwell^® co-culture model composed of Caco-2 epithelial cells in the apical compartment and lipopolysaccharide-treated (LPS) THP-1 macrophages in the basolateral compartment. Secretion of cytokines, disruption of epithelial integrity, and expression of surface markers and junctional proteins were assessed in order to investigate interactions between AhR and PPARγ on the ligand-elicited effects on the control of inflammation. The results revealed that the potent AhR ligand 6-formylindolo[3,2-b]carbazole (FICZ) attenuated LPS-induced IL-6 release by macrophages, which then stabilized Caco-2 monolayer permeability by decreasing claudin-2 expression. These effects were disrupted by GW9662 and to some extent by CH223191, inhibitors of PPARγ and AhR, respectively. Our main findings evidence PPARγ might be a downstream regulator of AhR activation essential for its ligand-based anti-inflammatory effects, suggesting it might be employed as either an auxiliary target or as a biomarker of therapeutical efficacy on AhR-based IBD pharmacotherapy. Full article

Background/Objectives: In addition to oncological applications, poly(ADP-ribose) polymerase (PARP) inhibitors have potential as anti-inflammatory agents. Colon-targeted delivery of PARP inhibitors has been evaluated as a pharmaceutical strategy to enhance their safety and therapeutic efficacy against gut inflammation. Methods: Colon-targeted PARP inhibitors 5-aminoisoquinoline (5-AIQ) and 3-aminobenzamide (3-AB) were designed and synthesized by azo coupling with salicylic acid (SA), yielding 5-AIQ azo-linked with SA (AQSA) and 3-AB azo-linked with SA (ABSA). Additional conjugation of AQSA with acidic amino acids yielded glutamic acid-conjugated AQSA (AQSA-Glu) and aspartic acid-conjugated AQSA, which further increased the hydrophilicity of AQSA. Results: The distribution coefficients of PARP inhibitors were lowered by chemical modifications, which correlated well with drug permeability via the Caco-2 cell monolayer. All derivatives were effectively converted to their corresponding PARP inhibitors in the cecal contents. Compared with observations in the oral administration of PARP inhibitors, AQSA-Glu and ABSA resulted in the accumulation of much greater amounts of each PARP inhibitor in the cecum. ABSA accumulated mesalazine (5-ASA) in the cecum to a similar extent as sulfasalazine (SSZ), a colon-targeted 5-ASA prodrug. In the DNBS-induced rat colitis model, AQSA-Glu enhanced the anticolitic potency of 5-AIQ. Furthermore, ABSA was more effective against rat colitis than SSZ or AQSA-Glu, and the anticolitic effects of AQSA-Glu were augmented by combined treatment with a colon-targeted 5-ASA prodrug. In addition, the colon-targeted delivery of PARP inhibitors substantially reduced their systemic absorption. Conclusions: Colon-targeted PARP inhibitors may improve the therapeutic and toxicological properties of inhibitors and synergize the anticolitic effects of 5-ASA. Full article

Background: To accurately measure permeability of compounds in the intestine, there is a need for preclinical in vitro models that accurately represent the specificity, integrity and complexity of the human small intestinal barrier. Intestine-on-chip systems hold considerable promise as testing platforms, but several characteristics still require optimization and further development. Methods: An established intestine-on-chip model for tissue explants was adopted for intestinal cell monolayer culture. A 3D-printed culture disc was designed to allow cell culture in static conditions and subsequent permeability studies in a dynamic environment. Membrane characteristics and standardized read-outs were investigated and compared to traditional permeability studies under static conditions. Results: By starting cultures outside the chip in conventional wells plates, the new cell disc design could support accurate cell monolayer formation for both Caco-2 and human enteroids. When transferred to the chip with laminar flow, there was accurate detection of barrier integrity (FD4 and Cascade Blue) and permeability (atenolol/antipyrine). Both flow and membrane characteristics had a significant impact on permeability outcomes. Conclusions: This novel intestinal cell-on-chip system offers large flexibility for intestinal permeability studies, although it still requires validation with more compounds to reveal its full potential. Full article

Background/Objectives: Campylobacter jejuni (CJ) is the etiological agent of the world’s most common intestinal infectious food-borne disease, ranging from mild symptoms to fatal outcomes. The development of innovative synbiotics that inhibit the adhesion and reproduction of multidrug-resistant (MDR) CJ in animals and humans, thereby preserving intestinal homeostasis, is relevant. We have created a synbiotic based on the consortium of Lactobacillus crispatus 2029 (LC2029), Ligilactobacillus salivarius 7247 (LS7247), and a mannan-rich prebiotic (Actigen^®). The purpose of this work was to study the in vitro anti-adhesive and antagonistic activities of the created synbiotic against MDR CJ strains, along with its role in preventing intestinal barrier dysfunction, which disrupts intestinal homeostasis. Methods: A complex of microbiological, immunological, and molecular biological methods was used. The ability of the LC2029 and LS7247 consortium to promote intestinal homeostasis in vitro was assessed by the effectiveness of controlling CJ-induced TLR4 activation, secretion of pro-inflammatory cytokines, development of intestinal barrier dysfunction, and production of intestinal alkaline phosphatase (IAP). Results: All MDR CJ strains showed marked adhesion to human Caco-2, pig IPEC-J2, chicken CPCE, and bovine BPCE enterocytes. For the first time, we found that the prebiotic and cell-free culture supernatant (CFS) from the consortium of LC2029 and LS7247 strains exhibit an additive effect in inhibiting the adhesion of MDR strains of CJ to human and animal enterocytes. CFS from the LC2029 and LS7247 consortium increased the permeability of the outer and inner membranes of CJ cells, which led to extracellular leakage of ATP and provided access to the peptidoglycan of the pathogen for the peptidoglycan-degrading bacteriocins nisin and enterolysin A produced by LS7247. The LC2029 and LS7247 consortium showed a bactericidal effect on CJ strains. Co-cultivation of the consortium with CJ strains resulted in a decrease in the viability of the pathogen by 6 log. CFS from the LC2029 and LS7247 consortium prevented the growth of CJ-induced TLR4 mRNA expression in enterocytes. The LC2029 and LS7247 consortium inhibited a CJ-induced increase in IL-8 and TNF-α production in enterocytes, prevented CJ-induced intestinal barrier dysfunction, maintained the transepithelial electrical resistance of the enterocyte monolayers, and prevented an increase in intestinal paracellular permeability and zonulin secretion. CFS from the consortium stimulated IAP mRNA expression in enterocytes. The LC2029 and LS7247 consortium and the prebiotic Actigen represent a new synergistic synbiotic with anti-CJ properties that prevents intestinal barrier dysfunction and preserves intestinal homeostasis. Conclusions: These data highlight the potential of using a synergistic synbiotic as a preventive strategy for creating feed additives and functional nutrition products based on it to combat the prevalence of campylobacteriosis caused by MDR strains in animals and humans. Full article

Background/Objectives: Lactoferrin (Lf) is an iron-binding glycoprotein with multiple bioactivities, including promotion of cell proliferation and differentiation, immunomodulation, and antimicrobial activity. Lf, a basic glycoprotein, can bind to α-lactalbumin (α-Lac), an acidic whey protein. The current study aimed to evaluate whether Lf forms protein complexes with α-Lac and proteins/peptides from whey protein hydrolysate (WPH) and nonfat bovine milk powder (MP) and whether forming protein complexes influences resistance to gastrointestinal digestion and affects the bioactivities of Lf in human intestinal epithelial cells (HIECs and differentiated Caco-2 cells). Methods: Lf was blended with α-Lac, WPH, or MP. Assays were conducted to evaluate the bioactivities of proteins (Lf, α-Lac, WPH, or MP) and Lf–protein blends on HIECs and Caco-2 cells. Results: (1) Lf forms complexes with α-Lac and proteins/peptides from WPH and MP; (2) compared with Lf alone, complexed Lf shows greater resistance to in vitro digestion; (3) forming protein complexes does not affect Lf’s binding to the Lf receptor or its uptake by HIECs; and (4) forming protein complexes does not impact Lf’s bioactivities, including the promotion of cell proliferation and differentiation, reduction of cell permeability by upregulating tight-junction proteins, immune modulation through the regulation of IL-18, inhibition of enteropathogenic Escherichia coli growth, and modulation of immune responses to EPEC infection. Conclusions: Lf forms complexes with α-Lac and other milk proteins/peptides from WPH and MP in protein blends, and forming complexes does not affect the functionalities of Lf. Full article

Damage to intestinal epithelial cells is present in obesity and other diseases because of inflammatory and oxidative processes. This damage compromises the gastrointestinal barrier, killing enterocytes, altering intestinal permeability, and eliciting abnormal immune responses that promote chronic inflammation. Recent evidence shows that spirulina is a potent natural agent with antioxidant and anti-inflammatory properties. Objectives: This study was conducted to evaluate the effect of spirulina aqueous extract (SPAE) on the alterations of the intestinal epithelium induced by lipid micelles (LMs) and/or inflammation induced by lipopolysaccharides (LPSs) in the Caco-2 cell line. Methods: In the current research, we assessed the protective actions of SPAE against cytotoxicity, oxidative stress, inflammation, and epithelial barrier perturbation by using an in vitro model, the intestinal Caco-2 cells, treated with LPSs and/or LMs. We also performed an in silico molecular docking analysis with spirulina’s bioactive compound, phycocyanobilin. Results: Our results showed that SPAE has no cytotoxic effect on Caco-2 cells. On the contrary, it improved cell viability and exhibited anti-inflammatory and antioxidant actions. SPAE also protected against endoplasmic reticulum stress and tight junction proteins, thus improving the epithelial barrier. The in silico study revealed a strong binding affinity of the phycocyanobilin compound with human SOD and NADPH oxidase and a good binding affinity towards COX-2 and iNOS. Conclusions: Taken together, these findings demonstrate the beneficial actions of SPAE on Caco-2 cells, suggesting it may be useful in preserving the epithelial intestinal barrier in human conditions involving oxidative stress and inflammation such as obesity. Full article