Search Results (215)

Impedance-based cellular assays allow determination of biological functions of cell populations in real-time by measuring electrical impedance. As compared to end-point assays, such as trans-epithelial electrical resistance assays, for example, they enable fast, non-invasive, and easy detection of cell kinetics—their growth, attachment, and interaction can be monitored over time. In our experiment, Caco-2 cells were cultured on E-plates 16. Next, fully differentiated cells were treated with either TNF-α or 3,4-dihydroxy-L-phenylalanine (L-DOPA). We aimed to verify the possibility of real-time testing of the viability, monolayer formation, and integrity (i.e., the presence of a functional and polarized monolayer) of Caco-2 cells by the xCELLigence real-time cell analyzer (RTCA) S16 system (Agilent Technologies). Full article

Sarcopenia, the age-related loss of skeletal muscle mass and function, is associated with inflammation, mitochondrial dysfunction, and gut barrier impairment. This study investigates the postbiotic effects of heat-killed Lactiplantibacillus plantarum HY7715 (HY7715) and its extracellular vesicles (EVs) on muscle health and intestinal integrity. In C2C12 myotubes, both treatments enhanced myogenic differentiation by upregulating Myf5 and MYOG, and improved mitochondrial activity and biogenesis via increased PGC1α and mTOR expression. Under TNFα-induced muscle atrophy, they suppressed expression of atrophy-related markers (Fbox32, MuRF1, and myostatin). EVs showed stronger anti-inflammatory effects by reducing IL6 expression in muscle cells. In intestinal Caco-2 cells, HY7715-derived EVs improved barrier function by upregulating tight junction proteins (ZO-1, occludin, and claudins), and effectively reduced LPS-induced inflammation. These findings suggest that heat-killed HY7715 and its EVs may alleviate sarcopenia by enhancing muscle regeneration and maintaining intestinal homeostasis, highlighting their potential as safe, gut–muscle axis-targeting postbiotic interventions for healthy aging. Full article

MicroRNA (miR)-200c enhances osteogenesis, modulates inflammation, and participates in dentin development. This study was to investigate the beneficial potential of miR-200c in vital pulp therapy (VPT) by mitigating pulpitis and promoting dentin regeneration. We explored the miR-200c variations in inflamed pulp tissues from patients with symptomatic irreversible pulpitis and primary human dental pulp-derived cells (DPCs) challenged with P.g. lipopolysaccharide (Pg-LPS). We further assessed the functions of overexpression of miR-200c on odontogenic differentiation, pulpal inflammation, and dentin regeneration in vitro and in vivo. Our findings revealed a noteworthy downregulation of miR-200c expression in inflamed pulp tissues and primary human DPCs. Through the overexpression of miR-200c via transfecting plasmid DNA (pDNA), we observed a substantial downregulation of proinflammatory cytokines interleukin (IL)-6 and IL-8 in human DPCs. Furthermore, this overexpression significantly enhanced the transcript and protein levels of odontogenic differentiation markers, including Runt-related transcription factor (Runx)2, osteocalcin (OCN), dentin matrix protein (DMP)1, and dentin sialophosphoprotein (DSPP). In a rat model of pulpitis induced by Pg-LPS, we demonstrated notable benefits by local application of pDNA encoding miR-200c delivered by CaCO₃-based nanoparticles to reduce pulpal inflammation and promote dentin formation. These results underscore the significant impact of locally applied miR-200c in modulating pulpal inflammation and facilitating dentin repair, showcasing its ability to improve VPT outcomes. Full article

Durvillaea incurvata, a Chilean brown seaweed, exhibits high antioxidant activity and polyphenol content, positioning it as a promising candidate for developing bioactive food ingredients. This study evaluated the anti-inflammatory activity of an ethanolic extract of Durvillaea incurvata, produced via ultrasound-assisted extraction, and its subsequent microencapsulation to obtain a functional food-grade ingredient. The extract’s anti-inflammatory capacity was assessed in vitro through hyaluronidase inhibition, and its cytotoxicity was evaluated using gastrointestinal cell models (HT-29 and Caco-2). Microencapsulation was performed by spray-drying with maltodextrin, and encapsulation efficiency (EE) was optimized using response surface methodology. Characterization included scanning electron microscopy, differential scanning calorimetry, and X-ray diffraction. The extract exhibited low cytotoxicity (cell viability > 75%). Optimal encapsulation conditions (inlet temperature: 198.28 °C, maltodextrin: 23.11 g/100 g) yielded an EE of 72.7% ± 1.2% and extract recovery (R) of 45.9% ± 2.4%. The microparticles (mean diameter, 2.75 µm) exhibited a uniform morphology, shell formation, glassy microstructure, and suitable physicochemical properties (moisture, 3.4 ± 0.1%; water activity, 0.193 ± 0.004; hygroscopicity, 30.3 ± 0.4 g/100 g) for food applications. These findings support the potential of microencapsulated Durvillaea incurvata extract as an anti-inflammatory ingredient for functional food development. Full article

Aberrant DNA methylation is a hallmark of colorectal cancer (CRC), contributing to tumor progression through the silencing of tumor suppressor genes and activation of oncogenes. Indicaxanthin (IND), a dietary betalain pigment from Opuntia ficus indica, has shown antiproliferative effects in CRC models, yet its epigenetic impact remains unexplored. In this study, we investigated the effects of IND on the methylome of Caco-2 cells using Reduced Representation Bisulfite Sequencing (RRBS). IND induced a global hypermethylation profile, particularly at gene promoters and CpG islands. Among the differentially methylated genes, 60% were protein-coding, and 10% encoded transcription factors, including PAX5 and TFAP4, both hypermethylated at active enhancers. Functional enrichment analysis revealed pathways beyond canonical intestinal functions, suggesting altered cell identity and plasticity. Transcription factor targets (SOX10, NFKB1, AHR, ARNT) were significantly enriched among the affected genes, several of which are involved in transdifferentiation processes. Methylation changes also indicated potential reprogramming toward epithelial cell types from pulmonary or neuroectodermal origin. Moreover, IND induced selective hypomethylation of Alu elements on chromosome 21 and hypermethylation of rDNA loci, hinting at suppressed ribosomal biogenesis. Overall, these findings highlight the epigenetic remodeling potential of IND and its possible role in modulating cell fate and metabolism in CRC cells. Full article

Probiotics have been widely adopted due to their beneficial health properties. Here, we investigated the interactions of a probiotic Limosilactobacillus (Lactobacillus) reuteri M4-100, with a translocating Escherichia coli strain HMLN-1, in a co-culture of cells, representing the intestinal epithelium, and identified molecular mechanisms associated with the host response. A co-culture of Caco-2:HT29-MTX cells was exposed to the HMLN-1 strain and the route of translocation was studied. Scanning and transmission electron microscopy revealed the adhesion of the strain to the microvilli, the establishment of close contact with the co-culture prior to being taken up by membrane-bound vesicles, and translocation via the intracellular pathway. When the HMLN-1 strain was challenged with L. reuteri M4-100 in co- and pre-inoculation experiments, its adhesion to the co-culture of cells was significantly reduced (p < 0.0001). A significant reduction in the invasion of the HMLN-1 strain was also observed upon the inoculation of L. reuteri M4-100 with the co-culture 60 min prior to HMLN-1 exposure (p < 0.0001). The L. reuteri M4-100 strain also significantly (p < 0.0001) reduced the translocation of the HMLN-1 strain in both co- and pre-inoculation experiments. Differential gene expression studies identified key cellular responses to the interaction with these bacteria, both alone. These data demonstrate the efficacy of L. reuteri M4-100 to reduce or inhibit the interaction of E. coli HMLN-1 with the intestinal epithelium. A prophylactic role of this probiotic strain is postulated as these effects were more pronounced in pre-inoculation experiments. Full article

Excessive glucose absorption is a major contributing factor of metabolic disorders that necessitates effective therapeutic strategies. This study investigates the potential of fermented Aralia cordata extract (FACE) in regulating glucose transport and immune responses under high-glucose stress conditions. Caco-2 intestinal cells and L cells were treated with FACE to determine effects on key glucose-regulating proteins and cytokines. FACE treatment inhibited the expression of glucose transporters SGLT1 and GLUT2 while promoting GLP-1 secretion. This effect was associated with HDAC and somatostatin suppression, along with AMPK-γ upregulation. Notably, FACE inhibited DPP-4 expression, further enhancing GLP-1 stability and function. Immunomodulatory effects also occurred, specifically FACE promotion of T lymphocyte differentiation, with a stronger influence on Th2 cell development. Additionally, FACE increased the secretion of essential molecules for immune balance and inflammation control, including antimicrobial peptides LL-37 and defensin, along with cytokines IL-4 and IL-13. These findings suggest that FACE exerts dual effects of improving glucose regulation and modulating immune responses, highlighting its potential as a novel bioactive material for managing metabolic disorders and enhancing intestinal immunity. Further research is warranted to explore its clinical applicability in therapeutic formulations. Full article

Background/Objectives: COVID-19 infection continues globally, with frequent emergence of unfamiliar SARS-CoV-2 variants acting to impair immunity. The competitive binding of SARS-CoV-2 spike proteins and angiotensin-converting enzyme 2 (ACE-2) can decrease the binding of the virus on native ACE-2 receptors on healthy human cells. It remains a practical approach to lessen viral spread. In this study, a method to encapsulate ACE-2 in the form of chitosan/tripolyphosphate cross-linked nanoparticles (NPs) was developed with emphasis placed on the best dehydration method to secure functional ACE-2 nanoparticles. Methods: Methods: Preparation conditions were assessed by varying pH (4.0–6.5) and the ratio between chitosan and ACE-2 mixing ratios (1:1, 1.5:1, 2:1, 2.5:1, and 3:1). The formulated NPs were then dehydrated using different approaches that included spray-drying (SD), freeze-drying (FD), and spray-freeze drying (SFD) and used varying mannitol concentrations (0, 1:1, and 5:1 of total weight). The mannitol was served as a cryoprotectant in this study. Results: The best formulation achieved used a pH 5.5 with a mixing chitosan–ACE-2 ratio of 2:1, where ACE-2-loaded NPs had an average particle size of 303.7 nm, polydispersity index (PDI) of 0.21, encapsulation efficiency (EE) of 98.4%, and ACE-2 loading content (LC) of 28.4%. After reconstitution, all SD samples had a relatively low yield rate, but the ACE-2 NPs dehydrated specifically using SFD required a lower amount of added mannitol (1:1 of its total weight) and produced a higher yield rate (p < 0.05) and similar PDI and EE values, along with relatively good particle size and LC. This formulation also produced a high ACE-2 release and uptake in differentiated Caco-2 cells, thus representing an effective ACE-2 encapsulation procedure for use with dry powders. Conclusions: This work showed that spray-freeze drying was the best method to dehydrate ACE-2 NPs, using less cryoprotectant to create a significant advantage in terms of greater loading capacity with lower additive requirements. Full article

Background: Oral administration remains the most common route for drug absorption. Emerging evidence highlights the important role of gut microbiome in the pharmacokinetics of oral medications. Glycyrrhizic acid (GL), a widely used hepatoprotective drug, is orally administrated and subsequently biotransformed by the gut microbiota into its active metabolite, glycyrrhetinic acid (GA), which exerts a therapeutic effect. However, it remains unclear whether the disturbance of the gut microbiome directly impacts the metabolism of GL. Methods: Antibiotic cocktail and probiotic Lacticaseibacillus rhamnosus R0011 were applied as two interventions targeting the gut microbiome. Pharmacokinetic parameters were evaluated by LC-MS, and 16S rRNA sequencing was applied to analyze the gut microbiome composition. The transcriptome analysis of Caco-2 cells was used to elucidate the regulation mechanism of polar metabolites resulting from gut microbiome perturbation. Results: R0011 supplementation could significantly increase the Area Under Curve (AUC) value of GA, which was positively correlated with the change in gut microbiome composition. In contrast, the plasma levels of GA were nearly undetectable following antibiotic intervention. Furthermore, the relative expressions of transporter multidrug resistance gene 1 (MDR1) in the ileum were site specifically downregulated under the probiotic intervention. The polar gut microbial metabolites may play a crucial role in differentiated regulating MDR1 expression, likely through the modulation of transcription factors FoxO1 and TP53. Conclusions: Our research provides new insights into the regulatory mechanism by which the gut microbiome affects the bioabsorption of orally administrated drugs, potentially offering strategies to optimize drug bioavailability and improve clinical efficacy. Full article

Background: Herein, we review the Cotton Top Tamarin (CTT), Saguinus oedipus, a unique spontaneous model for colorectal cancer (CRC). Despite its predisposition to inflammatory bowel disease (IBD) and frequent development of CRC, the CTT is adept at avoiding colorectal metastasis in the liver. In contrast, the common marmoset (CM), Callithrix jacchus, is a natural negative control, in that it also contracts IBD, but usually not CRC. We review our findings in these New World monkeys in terms of the expression of CEACAM adhesion models and their related molecules to contrast them with human disease. Methods: Specimens were collected from aforementioned monkey colorectal and other tissues, colonic washings, serum for analysis of tissue extraction, and colonic washings via ELISA, using a battery of antibodies. Fixed tissues were analyzed using immunohistochemistry and CEACAMs were extracted via Western blotting. Serum CEA levels were analyzed using ELISA, and DNA was extracted via a Bigblast genomics sequencing kit. Results: Serum CEA was significantly elevated in CTTs, and one-third of them die from CRC. Unlike others, we were unable to stain for CEA in tissues. The sialylated carbohydrate antigen recognized by monoclonal antibody (MAb) SPAN-1 does stain in 16.7% of CTT tissues, but the anti-aminoproteoglycan MAb, CaCo.3/61, stained 93.3% (OR70·00[CI6.5–754.5] p < 0.0001). The common CEA kits from Abbott and Roche were non-conclusive for CEA. We later adopted a CEA AIA-PACK from Tosoh Medics, which identified a 50 Kda band via Western blotting in humans and CTTs. The CEA levels were higher using the CEA AIA-PACK than the Pharmatrope kit (932 ± 690 versus 432 ± 407 ng/mL (p < 0.05)) in human patient colonic effluent, not statistically significant (NSS) for CTT extracts or effluent (733 ± 325 and 739 ± 401 ng/mL, respectively). It was suggested that the smaller CTT CEA moiety might lack components that facilitate the spread of liver metastasis. Later, using more specific CEA assays and increased numbers of specimens, we were able to show higher CEA serum expression in CTTs than in CMs (632.1 ± 306.1 vs. 81.6 ± 183.6, p < 0.005), with similar differences in the serum samples. Western blotting with the anti-CEA T84.66 MAb showed bands above 100 KDa in CTTs. The profiles in CTTs were similar to human patients with inflammatory bowel disease. We established that the CEA anchorage to the cell was a GPI-linkage, advantageous for the inhibition of differentiation and anoikis. With further CEA DNA analysis, we were able to determine at least five different mechanisms that may inhibit liver metastasis, mostly related to CEA, but later expanded this to seven, and increased the relationships to CEACAM1 and other related molecules. Recently, we obtained CTT liver mRNA transcriptomes that implicated several pathways of interest. Conclusions: With efforts spanning over three decades, we were able to characterize CEA and other changes that allow us to better understand the CTT phenomenon of liver metastasis inhibition. We are in the process of characterizing the CTT liver mRNA transcriptome to compare it with that of the common marmoset. Currently, liver CTT gene expression patterns suggest that ribosomes, lipoproteins, and antioxidant defense are related to differences between CTTs and CMs. Full article

Although rare, the ability to produce surface S-layer proteins is beneficially associated with particular Lactobacillus strains being investigated as probiotics. Therefore, this work aimed to study specific probiotic functionalities of selected Levilactobacillus brevis strains MB1, MB2, MB13 and MB20, isolated from human milk microbiota, and to assess the contribution of S-proteins. Firstly, Rapid Annotation using Subsystem Technology revealed that cell wall-related genes were abundant in analysed L. brevis genomes. Furthermore, the results demonstrated that S-proteins mediate aggregation capacity and competitive exclusion of selected pathogens by L. brevis strains. The improvement of Caco-2 epithelial monolayer barrier function was demonstrated by the increase in JAM-A and occludin expressions when L. brevis strains or S-proteins were added, with the effect being most pronounced after treatment with MB2 and S-proteins of MB1. L. brevis strains, especially MB20, exerted the potential to adhere to recombinant human ZG16. Strain MB2 and MB20-S-proteins improved the barrier function of HT29 epithelial monolayer, as evidenced by increased ZG16 expression. Analysed L. brevis strains and S-proteins differentially affected the protein expression of IL-1β, IL-6 and IL-8, and IL-10 cytokines. The most prominent effect was observed by S-proteins of MB20, since IL-1β production was decreased while IL-10 production was significantly increased. Full article

Background/Objectives: Intestinal alkaline phosphatase (IAP) is an enzyme expressed in the intestinal brush border, which may exert anti-inflammatory effects by detoxifying lipopolysaccharides (LPSs), thereby preventing metabolic disorders. Various food components have been reported to influence IAP activity. However, few studies have evaluated the effects of fermented milk on IAP activity. In this study, we aimed to investigate fermented milk with high IAP-activating capacity and investigate its effect. Methods: We screened a skim milk culture (SC), a fermented milk model, using differentiated Caco-2 cells. We investigated the effect of SC on IAP activity and gene expression in the Drosophila midgut. Quantitative PCR and immunoblot assays were conducted to examine gene and protein levels. Results: Among the SC samples from different lactic acid bacteria or bifidobacteria, the SC of Lactobacillus johnsonii SBT0309 (LJ0309 SC) demonstrated a particularly strong capacity to activate IAP in Caco-2 cells, demonstrated by significantly increased IAP gene expression and protein levels in Caco-2 cells. Additionally, LJ0309 SC inhibited increased secretion of IL-8 in LPS-stimulated Caco-2 cells. Finally, in Drosophila melanogaster fed LJ0309 SC, we observed an increase in both IAP activity and gene expression in the midgut. Conclusions: LJ0309 SC increased IAP activity and gene expression in both Caco-2 cells and the Drosophila midgut, and inhibited the inflammatory response in LPS-stimulated Caco-2 cells. Although further in vivo studies are required, LJ0309 SC might help to ameliorate LPS-induced inflammation and disease via IAP activation. Full article

Inflammation is associated with the pathophysiology of type 2 diabetes and COVID-19. Phytochemicals have the potential to modulate inflammation, expression of SARS-CoV-2 viral entry receptors (angiotensin-converting enzyme 2 (ACE2) and transmembrane protease, serine 2 (TMPRSS2)) and glucose transport in the gut. This study assessed the impact of phytochemicals on these processes. We screened 12 phytochemicals alongside 10 pharmaceuticals and three plant extracts, selected for known or hypothesised effects on the SARS-CoV-2 receptors and COVID-19 risk, for their effects on the expression of ACE2 or TMPRSS2 in differentiated Caco-2/TC7 human intestinal epithelial cells. Genistein, apigenin, artemisinin and sulforaphane were the most promising ones, as assessed by the downregulation of TMPRSS2, and thus they were used in subsequent experiments. The cells were then co-stimulated with pro-inflammatory cytokines interleukin-1 beta (IL-1β) and tumour necrosis factor-alpha (TNF-α) for ≤168 h to induce inflammation, which are known to induce multiple pathways, including the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway. Target gene expression (ACE2, TMPRSS2, SGLT1 (sodium-dependent glucose transporter 1) and GLUT2 (glucose transporter 2)) was measured by droplet digital PCR, while interleukin-1 (IL-6), interleukin-1 (IL-8) and ACE2 proteins were assessed using ELISA in both normal and inflamed cells. IL-1β and TNF-α treatment upregulated ACE2, TMPRSS2 and SGLT1 gene expression. ACE2 increased with the duration of cytokine exposure, coupled with a significant decrease in IL-8, SGLT1 and TMPRSS2 over time. Pearson correlation analysis revealed that the increase in ACE2 was strongly associated with a decrease in IL-8 (r = −0.77, p < 0.01). The regulation of SGLT1 gene expression followed the same pattern as TMPRSS2, implying a common mechanism. Although none of the phytochemicals decreased inflammation-induced IL-8 secretion, genistein normalised inflammation-induced increases in SGLT1 and TMPRSS2. The association between TMPRSS2 and SGLT1 gene expression, which is particularly evident in inflammatory conditions, suggests a common regulatory pathway. Genistein downregulated the inflammation-induced increase in SGLT1 and TMPRSS2, which may help lower the postprandial glycaemic response and COVID-19 risk or severity in healthy individuals and those with metabolic disorders. Full article

Gut microbial metabolism of dietary flavonoids leads to a diverse array of bioactive products that are closely associated with human health. Combining enzyme promiscuity prediction, metabolomics, and in vitro model systems, we identified a chalcone-synthase-like bacterial polyketide synthase that can initiate the metabolism of naringenin by catalyzing the C-ring cleavage. This was validated using a mutant strain of the model organism Bacillus subtilis (ATCC 23857). Our prediction–validation methodology could be used to systematically characterize the products of gut bacterial flavonoid metabolism and identify the responsible enzymes and species. In vitro experiments with Caco-2 cells revealed that naringenin and its bacterial metabolites differentially engage the aryl hydrocarbon receptor (AhR) and orphan nuclear receptor 4A (NR4A). These results suggest that metabolism by gut bacterial species could directly impact the profile of bioactive flavonoids and influence inflammatory responses in the intestine. These results are significant for understanding gut-microbiota-dependent physiological effects of dietary flavonoids. Full article

With the rise in global plastic production and the presence of plastic waste in the environment, microplastics are considered an emerging environmental contaminant. Human exposure and the impact of microplastics on human health are not well studied. Recent studies have observed the presence of microplastics in human tissues and several studies have noted toxicity in in vitro and in vivo mammalian models. We examined the impact of polystyrene nano- and microplastics in increasingly complex intestinal cell models. Using an undifferentiated Caco-2 mono-culture model, we assessed particle association, cytotoxicity, and particle clearance/retention, whereas in differentiated mono- and tri-culture transwell models, we assessed membrane integrity and particle translocation. Only 50 nm and 500 nm particles were internalized in the undifferentiated cells; however, no signs of cellular toxicity were observed at any concentrations tested. Additionally, polystyrene particles had no impact on barrier integrity, but the 50 nm particles were able to cross to the basolateral side, albeit attenuated in the tri-culture model that had a mucus layer. This study reduced some of the variability common to MNPL testing across various in vitro models, but further testing is needed to fully understand the potential effects of human MNPL exposure. Full article