Caco-2 Monolayers Research Articles

Gastrointestinal metabolism of Astragalus membranaceus polysaccharides and its related hypoglycemic mechanism based on gut microbial transformation

Astragalus membranaceus polysaccharides (AMP) was reported to exhibit hypoglycemic potential in diabetic host. However, the metabolic fate of AMP in gastrointestinal tract and its underlying hypoglycemic mechanisms remained unclear. Our current study aimed to reveal the structure alteration of AMP in gastrointestinal tract and its hypoglycemic mechanism from the perspective of microbial transformation. Caco-2 monolayer cell model revealed that AMP exhibited poor intestinal absorption. The in-vitro digestion and fermentation study revealed that AMP remained intact after gastrointestinal digestion while it could be degraded and utilized by gut microbiota with increased SCFA formation and decreased levels of all the monosaccharides in AMP except for mannose. Additionally, Diversity of gut microbiota was improved with the increased abundance of Dubosiella and Monoglobus and decreased abundance of Escherichia-Shigella and Acinetobacter after fermentation of AMP. Further hypoglycemic mechanism study for the first time revealed that both AMP and its potential microbial metabolites, SCFA salt mixture, could enhance intestinal integrity significantly on LPS induced Caco-2 cell model, while only SCFA salt mixture rather than AMP could significantly stimulate GLP-1 secretion in NCI-H716 cell model possibly via promoting GPCR43 expression. Such findings provided insights into the hypoglycemic mechanism of AMP from the perspective of microbial transformation.

GYY4137, as a slow-releasing H2S donor, ameliorates sodium deoxycholate-induced chronic intestinal barrier injury and gut microbiota dysbiosis.

Numerous studies have revealed that a long-term high-fat diet can raise intestinal deoxycholate acid concentration, which can harm intestinal mucosal barrier function in several ways. This study aims to verify the protective effect of GYY4137, as a slow-releasing H2S donor, on microbiome disturbance and the chronic injury of the intestinal mucosal barrier function caused by sodium deoxycholate. Caco-2 monolayer and mouse models were treated with a relatively high concentration of sodium deoxycholate (1.0mM and 0.2%, respectively) for longer periods (32h and 12 weeks, respectively) to understand the effects of GYY4137 on sodium deoxycholate-induced chronic intestinal barrier dysfunction and its fundamental mechanisms. A relatively long period of sodium deoxycholate treatment can remarkably increase the intestinal barrier permeability, alter the distribution and expression of tight junction proteins and generate the production of pro-inflammatory cytokines (TNF-α and IL-1β) in the Caco-2 monolayers and mouse models. Moreover, it can activate the MLCK-P-MLC2 pathway in the Caco-2 monolayers, which was further confirmed using RNA sequencing. The body weight, intestinal barrier histological score, and TUNEL index of sodium deoxycholate-treated mice worsened. In addition, an induced microbiome imbalance was observed in these mice. The above variations can be reversed with the administration of GYY4137. This study demonstrates that GYY4137 ameliorates sodium deoxycholate-induced chronic intestinal barrier injury by restricting the MLCK-P-MLC2 pathway while elevating the expression level of tight junction proteins, anti-apoptosis and maintaining the microbiome's homeostasis.

Design of self-emulsifying oral delivery systems for semaglutide: reverse micelles versus hydrophobic ion pairs.

It was the aim of this study to evaluate the potential of reverse micelles (RM) and hydrophobic ion pairs (HIP) for incorporation of semaglutide into self-emulsifying oral drug delivery systems. Reverse micelles loaded with semaglutide were formed with a cationic (ethyl lauroyl arginate, ELA) and an anionic surfactant (docusate, DOC), whereas HIP were formed between semaglutide and ELA. Maximum solubility of the peptide and the rate of dissolution was evaluated in various lipophilic phases (glycerol monocaprylocaprate:caprylic acid 1:4 (m/m), glycerol monolinoleate:caprylic acid 1:4 (m/m) and glycerol monocaprylocaprate:glycerol monolinoleate 1:4 (m/m)). Self-emulsifying drug delivery systems (SEDDS) loaded with RM and HIP were characterized regarding size distribution, zeta potential, cytocompatibility and Caco-2 permeability. Droplet sizes between 50 and 300nm with polydispersity index (PDI) around 0.3 and zeta potentials between - 45 mV (RMDOC) and 36 mV (RMELA) were obtained. RM provided an almost 2-fold higher lipophilicity of semaglutide than HIP resulting in a 4.2-fold higher payload of SEDDS compared to HIP. SEDDS containing RM or HIP showed high cytocompatibilities with a cell survival above 75% for concentrations up to 0.1% on Caco-2 cells and acceptable hemolytic activity. Permeation studies across Caco-2 monolayer revealed an at least 2-fold increase in permeability of semaglutide for the developed formulations.

Characteristics and Bioactivities of Protein Hydrolysate from Cricket (Acheta domesticus) Powder Defatted Using Ethanol with Aid of Vacuum Impregnation.

Cricket is a potential proteinaceous source used for protein hydrolysate (PH) preparation, having several biological activities. Nevertheless, cricket has high lipid contents, which are susceptible to oxidation during PH preparation. Thus, ethanol was used together with vacuum impregnation (VI) to enhance defatting efficacy before PH preparation. Also, bioavailability of the digest of PH after gastrointestinal tract (GIT) digestion via the Caco-2 monolayer was assessed. Cricket powder was defatted using ethanol for 1-4 h. Lipid contents were decreased with enhancing time until 2 h. Additionally, the defatting efficacy was augmented when ethanol combined with VI at 4 cycles for 2 h (VI-E-2) was implemented. Lowered mono- and polyunsaturated fatty acid contents were also observed in the VI-E-2 sample. The VI-E-2 sample was used to prepare PH using Alcalase and Flavourzyme (0.2-0.4 units/g dry sample). PH prepared by Alcalase hydrolysis at 0.2 units/g dry sample (A-0.2) showed the higher ABTS radical-scavenging activity and FRAP, compared to that prepared by Flavourzyme hydrolysis (p < 0.05). Thus, the A-0.2 sample was selected for digestion via the GIT system. The obtained digest (500-1000 μg/mL) had bioavailability of peptides, depending on the levels used. Therefore, PH from defatted cricket powder could be a promising ingredient for food applications.

Assessing nanotoxicity of food-relevant particles: A comparative analysis of cellular responses in cell monolayers versus 3D gut epithelial cultures

Engineered nanoparticles (NPs) are extensively used in the food industry, yet safety concerns remain. The lack of validated methodologies is a bottleneck towards resolving this uncertainty. Hence, the current study aims to compare two cell models by examining the toxicological impacts of two food-relevant NPs (SiO2 and Ag) on intestinal epithelia using monolayer Caco-2 cells and full-thickness 3D tissue models of human small intestines (EpiIntestinal™). Comprehensive characterization and dosimetric analysis of the NPs were performed to determine effective doses and model realistic exposures. Neither genotoxicity nor cytotoxicity were detected in the 3D tissues after NP treatment, while the 2D cultures exhibited cytotoxic response from Ag NP treatment for 24 h at 1 μg/ml. Hyperspectral imaging and transmission electron microscopy confirmed uptake of both NPs by cells in both 2D and 3D culture models. Ag NPs caused an increase in autophagy, whereas SiO2 NPs induced increased cytoplasmic vacuolization. Based on realistic exposure levels studied, the 3D small intestinal tissue model was found to be more resilient to NP treatment compared to 2D cell monolayers. This comparative approach towards toxicological assessment of food relevant NPs could be used as a framework for future analysis of NP behavior and nanotoxicity in the gut.

Targeted and non-targeted metabolic characteristics of 6,7-dihydroxy-2,4- dimethoxyphenanthrene during simulated gastrointestinal digestion

6,7-dihydroxy-2,4-dimethoxyphenanthrene (PC4), isolated and identified from Chinese yam, was one of the important characteristic active ingredients. The present study established simulated gastrointestinal digestion and caco-2 intestinal epithelial models to investigate the digestive and metabolic characteristics of PC4. The results indicated that PC4 was mainly digested and metabolized in the intestine, with a digestion rate of 94 %, producing active characteristic metabolites including 2,6-dimethoxy-4-phenanthrenol (MC1) and 1-methoxyphenanthrene (MC2). Molecular docking indicated that the COX-2 enzyme inhibitory activity of MC1 might be superior to that of the prototype PC4. During 24 h co-incubation of PC4 with caco-2 monolayer epithelial, the signal pathway related to lipid decomposition was up-regulated within 0–12 h, while the diabetes complications AGE-RAGE was inhibited just in 0–6 h period significantly. The research database provided scientific basis for the digestion and metabolism of PC4, and laid theoretical foundation for the scientific development of Chinese yam functional foods.

Evaluation of Quinoline-Related Carboxylic Acid Derivatives as Prospective Differentially Antiproliferative, Antioxidative, and Anti-Inflammatory Agents.

The higher prevalence of cancer and the unmet need for antioxidant/anti-inflammatory chemotherapeutic compounds with little side effect are of utmost importance. In addition, the increased likelihood of failure in clinical trials along with increasing development costs may have diminished the range of choices among newer drugs for clinical use. This has dictated the necessity to seek out novel medications by repurposing as it needs less time, effort, and resources to explore new uses of a current or unsuccessful medication. In this study, we examined the biological activity of 10 potential quinoline derivatives. Given the half-maximal inhibitory concentration (IC50 value) in lipopolysaccharide (LPS) induced inflammation of RAW264.7 mouse macrophages, all commercial FQs and selected quinolines (quinoline-4-carboxlic and quinoline-3-carboxylic acids) exerted impressively appreciable anti-inflammation affinities versus classical NSAID indomethacin without related cytotoxicities in inflamed macrophages. Conversely, all 14 tested compounds lacked antioxidative DPPH radical scavenging capacities as compared to ascorbic acid. Gemifloxacin, considerably unlike markets FQs, indomethacin and quinoline derivatives, exerted exceptional and differential antiproliferation propensities in colorectum SW480, HCT116, and CACO2, pancreatic PANC1, prostate PC3, mammary T47D, lung A375, and melanoma A549 adherent monolayers using the sulforhodamine B colorimetric method versus antineoplastic cisplatin. All quinoline derivatives and gemifloxacin alike, but not levofloxacin, ciprofloxacin, or indomethacin, displayed substantially selective viability reduction affinities in prolonged tumor incubations of cervical HELA and mammary MCF7 cells. Specifically kynurenic acid (hydrate), quinoline-2-carboxylic acid, quinoline-4-carboxylic acid, quinoline-3-carboxylic acid, and 1,2-dihydro-2-oxo-4-quinoline carboxylic acids possessed the most remarkable growth inhibition capacities against mammary MCF7 cell line, while quinoline-2-carboxylic acid was the only quinoline derivative with significant cytotoxicity on cervical HELA cancer cells. It is highly speculated that chelation with divalent metals via co-planarity with close proximity of the COOH and the N atom could have the potential molecular mechanism for optimally promising repurposed pharmacologies. Conclusively, this study revealed the considerably profound repurposed duality of cytotoxicity and anti-inflammation pharmacologies of quinoline derivatives. Activity-guided structural modifications of the present nuclear scaffolds can be inherently linked to the betterment and enhancement of their repurposed pharmacologies.

Various steaming durations alter digestion, absorption, and fermentation by human gut microbiota outcomes of Polygonatum cyrtonema Hua polysaccharides.

Different steaming durations dramatically alter the structure of Polygonatum cyrtonema polysaccharides (PCPs). This study aimed to compare characteristics of digestion, absorption, and fermentation by gut microbiota across four representative PCPs from different steaming durations (0, 4, 8, and 12 h), each with unique molecular weights and monosaccharide profiles. Chemical composition of the four PCPs was analyzed. Digestibility was evaluated using an in vitro saliva-gastrointestinal digestion model. Absorption characteristics were assessed with a Caco-2 monolayer model, and impacts on gut microbiota composition and short chain fatty acid (SCFA) levels were analyzed using in vitro fermentation with human gut microbiota. Longer steaming durations altered the chemical profiles of PCPs, reducing carbohydrate content (84.87-49.58%) and increasing levels of uronic acid (13.99-19.61%), protein (1.07-5.43%), and polyphenols (0.05-2.75%). Four PCPs were unaffected by saliva digestion but showed enhanced gastrointestinal digestibility, with reducing sugar content rising from 4.06% (P0) to 38.5% (P12). The four PCPs showed varying absorption characteristics, with P0 having the highest permeability coefficient value of 9.59 × 10-8 cm/s. However, all PCPs exhibited poor permeability, favoring gut microbiota fermentation. The four PCPs altered gut microbiota composition and elevated SCFA production, but levels declined progressively with longer steaming durations. All PCPs significantly increased the abundance of Bacteroidota, Firmicutes, and Actinobacteriota, making them the dominant bacterial phyla. Additionally, all PCPs significantly increased the abundance of Bifidobacterium, Prevotella, and Faecalibacterium compared to the control group, which, along with Bacteroides, became the dominant microbiota. Increasing the steaming duration led to a reduction in Prevotella levels, with PCPs from raw rhizomes showing the highest relative abundance at 24.90%. PCPs from moderately steamed rhizomes (4 h) led to a significant rise in Faecalibacterium (7.73%) among four PCPs. P8 and P12, derived from extensively steamed rhizomes (≥8 h), exhibited similar gut microbiota compositions, with significantly higher relative abundances of Bacteroides (20.23-20.30%) and Bifidobacterium (21.05-21.51%) compared to P0 and P4. This research highlights the importance of adjusting steaming durations to maximize the probiotic potential of P. cyrtonema polysaccharides, enhancing their effectiveness in modulating gut microbiota and SCFA levels.

Glucoraphanin and sulforaphane mitigate TNFα-induced Caco-2 monolayers permeabilization and inflammation

Intestinal permeabilization is central to the pathophysiology of chronic gut inflammation. This study investigated the efficacy of glucoraphanin (GR), prevalent in cruciferous vegetables, particularly broccoli, and its derivative sulforaphane (SF), in inhibiting tumor necrosis factor alpha (TNFα)-induced Caco-2 cell monolayers inflammation and permeabilization through the regulation of redox-sensitive events. TNFα binding to its receptor led to a rapid increase in oxidant production and subsequent elevation in the mRNA levels of NOX1, NOX4, and Duox2. GR and SF dose-dependently mitigated both these short- and long-term alterations in redox homeostasis. Downstream, GR and SF inhibited the activation of the redox-sensitive signaling cascades NF-κB (p65 and IKK) and MAPK ERK1/2, which contribute to inflammation and barrier permeabilization. GR (1 μM) and SF (0.5–1 μM) prevented TNFα-induced monolayer permeabilization and the associated reduction in the levels of the tight junction (TJ) proteins occludin and ZO-1. Both GR and SF also mitigated TNFα-induced increased mRNA levels of the myosin light chain kinase, which promotes TJ opening. Molecular docking suggests that although GR is mostly not absorbed, it could interact with extracellular and membrane sites in NOX1. Inhibition of NOX1 activity by GR would mitigate TNFα receptor downstream signaling and associated events. These findings support the concept that not only SF, but also GR, could exert systemic health benefits by protecting the intestinal barrier against inflammation-induced permeabilization, in part by regulating redox-sensitive pathways. GR has heretofore not been viewed as a biologically active molecule, but rather, the benign precursor of highly active SF. The consumption of GR and/or SF-rich vegetables or supplements in the diet may offer a means to mitigate the detrimental consequences of intestinal permeabilization, not only in disease states but also in conditions characterized by chronic inflammation of dietary and lifestyle origin.

Comparison of in vitro screening methods for evaluating the effects of pharmaceutical excipients on membrane permeability

The effects of pharmaceutical excipients on intestinal drug absorption have been highlighted and careful excipient selection is required to develop biologically equivalent formulations. This study aimed to evaluate the effects of excipients on drug permeability and compare the characteristics of in vitro screening methods. Three in vitro models, the commercial precoated parallel artificial membrane permeability assay (PAMPA), PermeaPadTM, and Caco-2 monolayer, were used to evaluate the effects of 14 excipients on the permeability of several drugs with different biopharmaceutical classification system classes. Concentration-dependent effects were analyzed to distinguish non-specific effects. The permeability of low-permeability drugs was increased by excipients such as hydroxypropyl cellulose and povidone K30 in the precoated PAMPA model, whereas PermeaPadTM maintained membrane integrity at higher concentrations. Conversely, croscarmellose sodium and sodium lauryl sulfate (SLS) decreased the permeability of highly permeable drugs in both precoated PAMPA and PermeaPadTM assays in a concentration-dependent manner. In Caco-2 monolayer assays, most excipients showed minimal effects on drug permeability. However, SLS significantly reduces the permeability of highly permeable drugs at concentrations above the critical micelle concentration, thereby compromising the integrity of the cell monolayer. Our results suggested that most of excipients, except SLS, did not affect the membrane permeation of drugs at clinically used concentrations. The pre-coated PAMPA model demonstrated high sensitivity to excipient effects, making it suitable for conservative evaluation. The PermeaPadTM and Caco-2 models allowed assessment at higher excipient concentrations, with PermeaPadTM being particularly useful for excipients that cause toxicity in Caco-2 cells.

Dexmedetomidine ameliorates diabetic intestinal injury by promoting the polarization of M2 macrophages through the MMP23B pathway.

Diabetes is often associated with gastrointestinal dysfunctions, which can lead to hypoglycemia. Dexmedetomidine (DEX) is a commonly used sedative in perioperative diabetic patients and may affect gastrointestinal function. To investigate whether sedative doses of DEX alleviate diabetes-caused intestinal dysfunction. Sedation/anesthesia scores and vital signs of streptozotocin (STZ)-induced diabetic mice under DEX sedation were observed. Diabetic mice were divided into saline and DEX groups. After injecting sedatives intraperitoneally, tight junctions (TJs) and apoptotic levels were evaluated 24 hours later to assess the intestinal barrier function. The role of DEX was validated using Villin-MMP23B flox/flox mice with intestinal epithelial deletion. In vitro, high glucose and hyperosmolarity were used to culture Caco-2 monolayer cells with STZ inter-vention. Immunofluorescence techniques were used to monitor the barrier and mitochondrial functions. MMP23B protein levels in the intestinal tissue of STZ-induced diabetic mice were significantly higher than those in the intestinal tissue of control mice, with the DEX group displaying decreased MMP23B levels. Diabetes-mediated TJ dis-ruption, increased intestinal mucosal permeability, and systemic inflammation in wild-type mice might be reversed by DEX. In Caco-2 cells, MMP23B was associated with increased reactive oxygen species accumulation, mitochondrial membrane potential depolarization, and TJ disruption. DEX reduces MMP23B, which may potentially contribute to STZ-induced intestinal barrier dysfunction, affecting TJ modification through mitochondrial dysfunction.

In vitro ADME, mouse pharmacokinetics of LD14b, and bioanalysis of a novel aβ 17β-HSD10 modulator for the treatment of Alzheimer’s disease

LD14b is an amyloid-β (Aβ) 17β-hydroxysteroid dehydrogenase type 10 (Aβ-17β-HSD10) protein-protein interaction modulator that shows promising in vitro and ex vivo activity to rescue Aβ-induced mitochondrial dysfunction, Aβ-induced toxicity, and Aβ-mediated inhibition of estradiol synthesis. The current study investigated in vitro human S9 fractions metabolic stability, apparent permeability, human and mouse plasma protein binding, in vivo pharmacokinetics, and tissue distribution in Balb/cJ mice. A fast (8-min), sensitive, reliable, and reproducible LC-MS/MS method was developed and validated over the dynamic range of 1-1000 ng/mL for the quantification of LD14b in different biological matrices (plasma, liver, kidney, brain, lungs, heart). LD14b was metabolically stable in human liver S9 fractions with 70% remaining after 90 minutes of incubation, showed intermediate apparent permeability of 3.55 × 10−06 cm/s and 6.16 × 10−06 cm/s for apical-to-basolateral (A-to-B) and basolateral-to-apical (B-to-A), respectively across the Caco-2 monolayer, and was medium/highly bound to human plasma proteins (84.1%), mouse plasma proteins (85.7%), and mouse brain homogenate (95.4%). LD14b showed an in vivo predicted % absorption of 52% in Balb/cJ mice and was well-distributed to the peripheral tissues (liver, kidney, lungs, and heart) including the brain.

Evaluation of the Bioavailability of Iodine and Arsenic in Raw and Cooked Saccharina japonica Based on Simulated Digestion/Caco-2 Cell Model.

Kelp is a traditional healthy food due to its high nutritional content; however, its relatively high contents of iodine and arsenic have raised concerns about its edible safety. This study explored the effects of different cooking treatments on the contents of iodine and arsenic in kelp, evaluated the bioaccessibility and bioavailability of iodine and arsenic in kelp using in vitro digestion, and compared the differences in the transport characteristics of iodine in kelp and KIO3 using a Caco-2 monolayer cell transport model. The results show that the content of target elements that reached systemic circulation could be reduced by cooking and gastrointestinal digestion. The highest reductions in iodine and arsenic were 94.4% and 74.7%, respectively, which were achieved by boiling for 10 min. The bioaccessibility and bioavailability of iodine and arsenic were significantly improved by a cooking treatment. However, the contents of iodine and arsenic decreased significantly, with the bioaccessibility of iodine reducing from 3188.2 μg/L to 317.0 μg/L and that of arsenic reducing from 32.5 μg/L to 18.1 μg/L in the gastric phase after boiling. The findings also show that the efficiency of iodine transport in kelp and KIO3 was positively correlated with the transport time and negatively correlated with the concentration of iodine. With the increase in the iodine concentration, the rate of iodine transport in kelp decreased from 63.93% to 3.14%, but that of KIO3 was stable at around 35%, which indicates that the absorption efficiency of iodine from kelp was limited, even when too much kelp was ingested. In conclusion, the edible safety of kelp is significantly improved after cooking. The risk of excessive iodine and arsenic intake caused by consuming kelp is extremely low, and as an effective iodine supplement source, kelp has higher edible safety compared with KIO3. This study clarifies the safety of algae based on iodine and arsenic contents and also provides a basis for the formulation of food safety standards.

Absorption and transport mechanism of colloidal nanoparticles (CNPs) in lamb soup based on Caco-2 cell

Soup is an important presence in diet, but its absorption and transport mechanism by the human body remains unclear. In this study, Caco-2 intact cell and monolayer cell models were constructed to simulate small intestine absorption on colloidal nanoparticles (CNPs) isolated from lamb soup. The intracellular localization of CNPs was viewed by laser confocal microscopy (LSCM). CNPs uptake and release pathways were explored by differences in CNPs concentrations in 5 endocytosis inhibitor models and 4 exocytosis inhibitor models. Results indicated that CNPs endocytosis by Caco-2 cells was restrained by Nystatin and Cytochalasin D, with exocytosis being inhibited by Nocodazole and Monensin. Therefore, the major absorption pathways for CNPs were caveolin-dependent endocytosis, macropinocytosis and phagocytosis. The major transport pathways were microtubule-vesicle-mediated protein transport to the membrane and transportation between the Golgi apparatus and membrane. This study may provide theoretical support for the transport mechanism of soup products in the small intestine.

Studies on absorption mechanism and pharmacokinetic properties of albendazole-bile acid conjugate: In vivo and in vitro

PurposeTo improve the oral bioavailability of albendazole (ABZ), a series of albendazole-bile acid conjugates (ABCs) were synthesized. ABC's transmembrane transport mechanism and in vivo pharmacokinetic properties were preliminarily studied. MethodsThe transmembrane transport mechanism of ABCs was studied using the Caco-2 monolayer cell model and intestinal perfusion model. The concentration of ABCs and ABZ were evaluated using High-Performance Liquid Chromatography (HPLC) and HPLC-Mass Spectrometry (HPLC-MS/MS). ResultsCompared to ABZ, better permeability was observed for different types and concentrations of ABCs using the Caco-2 monolayer cell model, with ABC-C8 showing the highest permeability. The transmembrane transport of ABCs was affected by ASBT inhibitors, indicating an ASBT-mediated active transport mechanism. Additionally, introducing cholic acid resulted in ABZ no longer being a substrate for P-gp, MRP2, and BCRP, effectively reversing ABZ efflux. In vivo unidirectional intestinal perfusion results in rats showed that ABCs altered the absorption site of ABZ from the jejunum to the ileum. The absorption efficiency of ABCs in each intestinal segment was higher than that of ABZ, and the transmembrane transport efficiency decreased with increasing concentrations of ASBT inhibitors. This further confirmed the presence of both passive diffusion and ASBT-mediated active transport mechanisms in the transport of ABCs. The solubility of ABCs in gastric juice and pharmacokinetics in rats showed that ABZ-C4 exhibited enhanced solubility. Moreover, ABCs significantly increased oral bioavailability compared to ABZ, with ABC-C4 showing an approximately 31-fold increase in bioavailability. ConclusionThe transmembrane transport mechanism of ABCs involves a combination of ASBT-mediated active transport and passive diffusion. Moreover, the incorporation of BAs successfully reverses the efflux of ABZ by efflux proteins. Among the synthesized conjugates, ABC-C4 demonstrated superior dissolution behavior both in vitro and in vivo.

Glycated walnut meal peptide‑calcium chelates (COS-MMGGED-Ca): Preparation, characterization, and calcium absorption-promoting

This study isolated a novel peptide MMGGED with strong calcium-binding capacity from defatted walnut meal and synthesized a novel peptide‑calcium chelate COS-MMGGED-Ca with high stability via glycation. Structural characterization and computer simulation identified binding sites, while in vitro digestion stability and calcium transport experiments explored the chelate's properties. Results showed that after glycation, COS-MMGGED bound Ca2+ with 88.75 ± 1.75 %, mainly via aspartic and glutamic acids. COS-MMGGED-Ca released Ca2+ steadily (60.27 %), with thermal denaturation temperature increased by 18 °C and 37 °C compared to MMGGED-Ca, indicating good processing performance. Furthermore, COS-MMGGED significantly enhanced Ca2+ transport across Caco-2 monolayers, 1.13-fold and 1.62-fold higher than CaCl2 and MMGGED, respectively, at 240 h. These findings prove glycation enhances structural properties, stability, calcium loading, and transport of peptide‑calcium chelates, providing a scientific basis for developing novel efficient calcium supplements and high-value utilization of walnut meal.

Development of mucoadhesive 3D-printed Carbopol/Eudragit/SNAC tablets for the oral delivery of enoxaparin: In vitro and ex vivo evaluation

3D-printed dosage forms comprised of Carbopol and Eudragit were fabricated through semi-solid extrusion, combining Enoxaparin (Enox) and the permeation enhancer SNAC in a single-step process without subsequent post-processing. Inks were characterized using rheology and Fourier-transform infrared (FTIR) spectroscopy. The stability of Enox in the fabricated dosage forms was assessed by means of Nuclear Magnetic Resonance (NMR) and Circular Dichroism (CD) analysis. In vitro release studies revealed the release of Enox in a sustained manner, whereas ex vivo experiments demonstrated the mucoadhesive properties of the 3D-printed dosage forms and their ability to enhance Enox permeability across intestinal mucosa. Cellular assays (CCK-8 assay) revealed a dose- and time-dependent response following incubation with the 3D-printed dosage forms. The encapsulation of SNAC in the 3D-printed dosage forms demonstrated their capacity to increase the transcellularly transport of macromolecule across Caco-2 monolayer in a reversible manner, as confirmed by Transepithelial Resistance (TEER) measurements.

Poly(2-Deoxy-2-Methacrylamido-D-Glucose)-Based Complex Conjugates of Colistin, Deferoxamine and Vitamin B12: Synthesis and Biological Evaluation.

Growing resistance to traditional antibiotics poses a global threat to public health. In this regard, modification of known antibiotics, but with limited applications due to side effects, is one of the extremely promising approaches at present. In this study, we proposed the synthesis of novel complex polymeric conjugates of the peptide antibiotic colistin (CT). A biocompatible and water-soluble synthetic glycopolymer, namely, poly(2-deoxy-2-methacrylamido-D-glucose) (PMAG), was used as a polymer carrier. In addition to monoconjugates containing CT linked to PMAG by hydrolyzable and stable bonds, a set of complex conjugates also containing the siderophore deferoxamine (DFOA) and vitamin B12 was developed. The structures of the conjugates were confirmed by 1H NMR and FTIR-spectroscopy, while the compositions of conjugates were determined by UV-Vis spectrophotometry and HPLC analysis. The buffer media with pH 7.4, corresponding to blood or ileum pH, and 5.2, corresponding to the intestinal pH after ingestion or pH in the focus of inflammation, were used to study the release of CT. The resulting conjugates were examined for cytotoxicity and antimicrobial activity. All conjugates showed less cytotoxicity than free colistin. A Caco-2 cell permeability assay was carried out for complex conjugates to simulate the drug absorption in the intestine. In contrast to free CT, which showed very low permeability through the Caco-2 monolayer, the complex polymeric conjugates of vitamin B12 and CT provided significant transport. The antimicrobial activity of the conjugates depended on the conjugate composition. It was found that conjugates containing CT linked to the polymer by a hydrolyzable bond were found to be more active than conjugates with a non-hydrolyzable bond between CT and PMAG. Conjugates containing DFOA complexed with Fe3+ were characterized by enhanced antimicrobial activity against Pseudomonas aeruginosa compared to other conjugates.

Human enteroid monolayers as a potential alternative for Ussing chamber and Caco-2 monolayers to study passive permeability and drug efflux.

After oral administration, the intestine is the first site of drug absorption, making it a key determinant of the bioavailability of a drug, and hence drug efficacy and safety. Existing non-clinical models of the intestinal barrier in vitro often fail to mimic the barrier and absorption of the human intestine. We explore if human enteroid monolayers are a suitable tool for intestinal absorption studies compared to primary tissue (Ussing chamber) and Caco-2 cells. Bidirectional drug transport was determined in enteroid monolayers, fresh tissue (Ussing chamber methodology) and Caco-2 cells. Apparent permeability (Papp) and efflux ratios for enalaprilat (paracellular), propranolol (transcellular), talinolol (P-glycoprotein (P-gp)) and rosuvastatin (Breast cancer resistance protein (BCRP)) were determined and compared between all three methodologies and across intestinal regions. Bulk RNA sequencing was performed to compare gene expression between enteroid monolayers and primary tissue. All three models showed functional efflux transport by P-gp and BCRP with higher basolateral to apical (B-to-A) transport compared to apical-to-basolateral (A-to-B). B-to-A Papp values were similar for talinolol and rosuvastatin in tissue and enteroids. Paracellular transport of enalaprilat was lower and transcellular transport of propranolol was higher in enteroids compared to tissue. Enteroids appeared show more region- specific gene expression compared to tissue. Fresh tissue and enteroid monolayers both show active efflux by P-gp and BCRP in jejunum and ileum. Hence, the use of enteroid monolayers represents a promising and versatile experimental platform to complement current in vitro models.

Escherichia coli Nissle 1917 Protects against Sepsis-Induced Intestinal Damage by Regulating the SCFA/GPRs Signaling Pathway.

This study explores whether Escherichia coli Nissle 1917 (EcN) can preserve the integrity of the intestinal barrier by modulating the metabolism pathway of short-chain fatty acids (SCFAs) in a C57BL/6J mouse model of lipopolysaccharide (LPS)-induced acute enteritis and a model of a Caco-2 monolayer. The study involved establishing a septic shock model in mice through lipopolysaccharide (LPS) injection. Clinical scores and intestinal permeability were meticulously documented. Immunofluorescence was utilized to localize the tight junction proteins. A quantitative real-time polymerase chain reaction (qRT-PCR) was employed to assess the expression of G protein-coupled receptors (GPRs) signaling. Additionally, the supplement of acetate and butyrate with Caco-2 monolayers to elucidate the potential of EcN in augmenting the intestinal barrier primarily via the modulation of SCFAs and qRT-PCR was performed to detect the expression of tight junction proteins and the activation of the GPRs protein signaling pathway. EcN mitigated the clinical symptoms and reduced intestinal permeability in the colon of LPS-induced mice. It also enhanced the production of SCFAs in the gut and upregulated the expression of SCFA receptor proteins GPR41 and GPR43 in the colon tissue. Our findings reveal that EcN activates the SCFA/GPRs pathway, thereby preserving intestinal barrier function and alleviating inflammation in a mouse sepsis model.