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Fruits are essential components of the human diet, valued for their diverse bioactive compounds with potential health-promoting properties. This study focuses on three cold-hardy Actinidia species, namely A. arguta, A. kolomikta, and A. polygama, examining their polyphenolic content, antioxidant/antiradical activities, scavenging capacity and effects on intestinal cell viability (Caco-2 and HT29-MTX). A comprehensive profile of their phenolic compounds was identified, in descending order of total polyphenol content: A. kolomikta > A. arguta > A. polygama. Across species, 16 phenolic acids, 2 flavanols, 2 flavanones, 11 flavonols, and 3 flavones were quantified, with caffeine as a prominent compound. A. kolomikta achieved the highest antioxidant activity, with 'Vitakola' cultivar showing almost double the antioxidant activity compared to 'Tallinn' and 'Pozdni'. By contrast, A. arguta 'Geneva' and A. polygama 'Pomarancheva' exhibited significantly lower activity in both FRAP and DPPH assays. Notably, A. kolomikta cultivars showed distinct radical-scavenging capacities, particularly for superoxide, wherein 'Tallinn' and 'Pozdni' achieved the highest values. Cell viability tests on Caco-2 and HT29-MTX cells revealed a dose-dependent reduction in viability, notably stronger in Caco-2 cells. Overall, this study underscores the therapeutic potential of Actinidia species. [ABSTRACT FROM AUTHOR]

Background/Objectives: The proton-coupled amino acid transporter (PAT1) is an intestinal absorptive solute carrier responsible for the oral bioavailability of some GABA-mimetic drug substances such as vigabatrin and gaboxadol. In the present work, we investigate if non-steroidal anti-inflammatory drug substances (NSAIDs) interact with substrate transport via human (h)PAT1. Methods: The transport of substrates via hPAT1 was investigated in Caco-2 cells using radiolabeled substrate uptake and in X. laevis oocytes injected with hPAT1 cRNA, measuring induced currents using the two-electrode voltage clamp technique. The molecular interaction between NSAIDs and hPAT1 was investigated using an AlphaFold2 model and molecular docking. Results: NSAIDs such as ibuprofen, diclofenac, and flurbiprofen inhibited proline uptake via hPAT1, with IC50 values of 954 (logIC50 2.98 ± 0.1) µM, 272 (logIC50 2.43 ± 0.1) µM, and 280 (logIC50 2.45 ± 0.1) µM, respectively. Ibuprofen acted as a non-competitive inhibitor of hPAT1-mediated proline transport. In hPAT1-expressing oocytes, ibuprofen and diclofenac did not induce inward currents, and inhibited inward currents caused by proline. Molecular modeling pointed to a binding mode involving an allosteric site. Conclusions: NSAIDs interact with hPAT1 as non-translocated non-competitive inhibitors, and molecular modeling points to a binding mode involving an allosteric site distinct from the substrate binding site. The present findings could be used as a starting point for developing specific hPAT1 inhibitors. [ABSTRACT FROM AUTHOR]

Iron uptake in the intestinal epithelium is associated with transport of ferrous iron via the DMT1 transporter (SLC11a2; NRAMP2). In later years, uptake of iron from complex sources, such as nanoparticles, has been found to be mediated through endocytosis. Here we propose that iron from the simple salt ferrous fumarate, a common iron supplement, can be absorbed by clathrin-mediated endocytosis. We used siRNA to silence DMT1 transporter expression, pharmacological inhibition of endocytosis, and Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) to show that iron uptake from ferrous fumarate can be mediated by both transport via DMT1 and by clathrin-dependent endocytosis in Hutu-80 cells. Iron uptake (ferritin L) from ferrous fumarate (0.5 mM, 24 h) in DMT1 silenced cells was significantly decreased (60% ± 11%) in comparison to iron controls while a 1-h dose of ferrous fumarate (0.5 mM) significantly decreased ferritin L formation in the presence of the clathrin inhibitor chlorpromazine (61% ± 10%, in post-confluent cells and 37% ± 9% in non-confluent cells). A pilot showed a similar trend for Ferritin (H) levels (confluent cells) and for total cellular iron load (non-confluent cells). ToF-SIMS analysis revealed diminished membrane-associated iron load in endocytosis-inhibited ferrous fumarate treated cells. The reported results support a clathrin-mediated endocytosis mechanism for uptake of iron from ferrous fumarate in addition to iron uptake by DMT1. More studies are needed to understand what determines which uptake mechanism are employed and to which extent.

IntroductionThe potential for drug-induced gastrointestinal (GI) toxicity is significant, since the GI tract is one of the first barriers which come in to contact with oral drugs. In pharmaceutical research, the complex behavior of human intestinal cells is traditionally investigated using 2D cultures, in which one cell type grows under static conditions. With the development of advanced microphysiological systems (MPS) more in vivo like conditions can be generated which increase the physiological nature and also the predictive validity of these models. Caco-2 cells are known for their capability to build tight junctions. These connections are responsible for the maintenance of intestinal homeostasis and can be used as a specific safety endpoint, by measuring the Trans Epithelial Electrical Resistance (TEER), for the investigation of drug-induced GI toxicity. Compared to a widely used Caco-2 cell 2D Transwell model, the advanced MPS model (Mimetas OrganoPlate®) allows for the recapitulation of the enterocyte cell layer of the intestinal barrier as the Caco-2 cells grow in a tubular structure through which the medium continuously flows.MethodsThe OrganoPlate® intestinal model was qualified to be implemented as a routine test system for the early prediction of drug-induced GI toxicity based on the measurement of the tightness of the cell layer by measuring changes in the TEER. For this qualification 23 well known compounds as well as a positive, negative and solvent control were selected. The compounds were selected based on their known effect on the GI system (chemotherapeutics, tight junction disruptor, liver toxins, controls, NSAIDs and a mixed group of drugs).ResultsThe TEER values were measured 4 h and 24 h after treatment. In parallel the cell viability was determined after 24 h to be able to distinguish between an unspecific cytotoxic effect or direct tight junction damage. Overall, from the 23 tested compounds, 15 showed the expected outcome, i.e., the compound led to a decrease of the TEER for the positive control compounds, or the TEER value remained stable after treatment with non-GI-toxic compounds.ConclusionIn summary, this MPS model allowed the recapitulation of the human intestinal GI barrier and will enable a faster and more robust assessment of drug-induced damage in the GI tract.

This study explores the binding properties of two important constituents from Crocus sativus L (crocin and crocetin) with myosin, examining their influence on antioxidant capacity in myosin and a grilled meat model. And their impact on cytoprotective potential of myosin digestion products was also assessed in Caco-2 cells. Crocin and crocetin exhibited discernible binding affinity to myosin via static quenching, which induced conformational alterations that bolstered the antioxidant capacity of myosin, preventing peroxidation, which also corroborated in a grilled meat model. Crocin resulted in greater enhancement of antioxidant capacity and binding affinity, as confirmed by quantum chemical calculations. Molecular dynamics simulations revealed the stable binding of crocin to GLU:272, GLU:606, GLN:628, and PHE:417 residues of myosin. In addition, crocin substantially enhanced the protective efficacy of myosin digestion products against H 2 O 2 -induced damage in Caco-2 cells by upregulating superoxide dismutase and GSH-Px and simultaneously downregulating reactive oxygen species and malondialdehyde levels. [Display omitted] • Crocin and crocetin can bind to myosin, boosted myosin's antioxidant capacity. • Crocin show stronger antioxidant and binding ability compared to crocetin. • Crocin can stably bind to residue GLU:272, GLU:606, GLN:628, and PHE:417. • Crocin enhanced the protective ability of myosin digestion products. [ABSTRACT FROM AUTHOR]

Permeability is an important molecular property in drug discovery, as it co-determines pharmacokinetics whenever a drug crosses the phospholipid bilayer, e.g., into the cell, in the gastrointestinal tract, or across the blood-brain barrier. Many methods for the determination of permeability have been developed, including cell line assays (CACO-2 and MDCK), cell-free model systems like parallel artificial membrane permeability assay (PAMPA) mimicking, e.g., gastrointestinal epithelia or the skin, as well as the black lipid membrane (BLM) and submicrometer liposomes. Furthermore, many in silico approaches have been developed for permeability prediction: meta-analysis of publicly available databases for permeability data (MolMeDB and ChEMBL) was performed to establish their usability. Four experimental and two computational methods were evaluated. It was shown that repeatability of the reported permeability measurement is not great even for the same method. For the PAMPA method, two different permeabilities are reported: intrinsic and apparent. They can vary in degrees of magnitude; thus, we suggest being extra cautious using literature data on permeability. When we compared data for the same molecules using different methods, the best agreement was between cell-based methods and between BLM and computational methods. Existence of unstirred water layer (UWL) permeability limits the data agreement between cell-based methods (and apparent PAMPA) with data that are not limited by UWL permeability (computational methods, BLM, intrinsic PAMPA). Therefore, different methods have different limitations. Cell-based methods provide results only in a small range of permeabilities (-8 to -4 in cm/s), and computational methods can predict a wider range of permeabilities beyond physical limitations, but their precision is therefore limited. BLM with liposomes can be used for both fast and slow permeating molecules, but its usage is more complicated than standard transwell techniques. To sum up, when working with in-house measured or published permeability data, we recommend caution in interpreting and combining them.

Gut mucosae are composed of stratified layers of microbes, a selectively permeable mucus, an epithelial lining, and connective tissue homing immune cells. Studying cellular and chemical interactions between the gut mucosal components has been limited without a good model system. We have engineered a three-dimensional (3D) multi-cellular co-culture system we coined "3D Flipwell system" using cell culture inserts stacked against each other. This system allows an assessment of the impact of a gut mucosal environmental change on interactions between gut bacteria, epithelia, and immune cells. As such, this system can be utilized in examining the effects of exogenous stimuli, such as dietary nutrients, bacterial infection, and drugs, on the gut mucosa that could predetermine how these stimuli might influence the rest of body. Here, we describe the methods of construction and application of the new 3D Flipwell system we utilized previously in assessing the crosstalk between the gut mucosa and macrophage polarization. We demonstrate the physiological responses of different components of the co-cultures to Sepiapterin (SEP), the precursor of the nitric oxide synthase cofactor tetrahydrobiopterin (BH 4 ). We reported previously that SEP induces a pro-immunogenic shift of macrophages having acquired an immune suppressive phenotype. We also showed that SEP induces a defense mechanism of commensal gut bacteria. The protocol describing the assembly and use of the 3D Flipwell co-culture system herein would grant its utility in evaluating the concurrent effects of pharmacologic and microbiologic stimuli on gut mucosal components. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: 3D Flipwell construction, assembly, and collagen coating Basic Protocol 2: Flipwell cell seeding and cell culture Basic Protocol 3: Addition of bacterial culture to the Flipwell system Basic Protocol 4: Flipwell disassembly for scanning electron microscopy (SEM) studies Basic Protocol 5: Immunofluorescence antibody staining for confocal microscopy., (© 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.)

Colorectal cancer (CRC) continues to be a significant health challenge, necessitating the development of efficient therapeutic strategies. Drug repurposing, which involves the use of existing medications for new purposes, presents a promising opportunity. Metformin, a widely used antidiabetic drug, has demonstrated potential anticancer effects. To enhance its efficacy, we formulated nano-metformin, metformin encapsulated within pectin nanoparticles. Our study aimed to evaluate the superiority of nano-metformin over free metformin in treating CRC. The cytotoxicity of both metformin and nano-metformin on Caco-2 CRC cells was assessed using the MTT assay, revealing a significant dose-dependent inhibition of cell growth using nano-metformin. The anti-inflammatory potential was evaluated by measuring the levels of nitric oxide and the pro-inflammatory cytokines IL-2 and IL-6 following lipopolysaccharide (LPS) induction, and the results revealed that treating LPS-induced cells with nano-metformin significantly reduced the production of these inflammatory mediators. To elucidate the mechanism of cell death, we employed an acridine orange/ethidium bromide staining assay, which revealed the enhancement of apoptotic cell death following treatment with nano-metformin. Additionally, we examined the expression of key apoptotic regulators using real-time qPCR. Nano-metformin, in particular, significantly downregulated the expression of the antiapoptotic markers Bcl-2 and Survivin while upregulating the proapoptotic caspases 3, 7, and 9. The comet assay revealed significant DNA damage induced by treatment with the nano-metformin compared with that in the free form. Moreover, nano-metformin significantly reduced the migration ability of cells. In conclusion, our work revealed the superior efficacy of our formulated nanoform over free metformin, highlighting its potential as a promising therapeutic agent for CRC treatment., (© 2024 Deutsche Pharmazeutische Gesellschaft.)