Intestinal Caco-2 Cell Monolayers Research Articles

Lipid-lowering effect and oral transport characteristics study of curculigoside.

The incidence of metabolic disorders during pregnancy is increasing year by year, with diseases including hypertension and hyperlipidemia. Statins are the primary drugs for treating hyperlipidemia or atherosclerosis, yet some patients remain unresponsive to them, and pregnant women are prohibited from taking statins. Curculigoside is the major biologically active natural product present in Curculigo orchioides. In this study, A high-fat mice model was developed to study the lipid-lowering effect of curculigoside. Using intestinal Caco-2 cell monolayer, the curculigoside transport properties at two temperatures and possible transporters were systemically studied. Curculigoside at concentrations used during the experiments have no toxic effect to Caco-2 cells. The curculigoside transfer from the apical to the basolateral side was strongly influenced by temperature. P-glycoprotein, breast cancer resistance protein, and efflux transporters are crucial components of the human intestinal cell line Caco-2. The curculigoside can significantly affect the contents of total cholesterol, triglycerides, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol in mice. The transport properties and potential mechanism of curculigoside offer valuable insights for the design of development of hypolipidemic drugs like anti-atherosclerotic drugs and also be helpful to the further study of the pharmacological activity of curculigoside.

PSIII-28 A Novel Direct-Fed Microbial Supports in Vitro Intestinal Integrity Upon Clostridium Perfringens Challenge

Abstract Gastrointestinal pathogens cause productive, health, welfare, and financial losses to the beef cattle herd. Among these pathogens, Clostridium perfringens type A is of interest given its prevalence, negative effects on intestinal health, and subsequent losses caused in beef animals. Based on this rationale, we hypothesized that a novel direct-fed microbial (DFM) formulation would support the gut barrier integrity upon a challenge with C. perfringens type A. Therefore, our objective was to evaluate how a novel DFM can support the in vitro integrity of intestinal epithelial cell monolayers exposed to C. perfringens type A. Human cancer-derived epithelial intestinal Caco-2 cell monolayers were seeded in transwells for approximately 20 days. A DFM mixture containing Lactobacillus animalis, Propionibacterium freudenreichii, Bacillus licheniformis, and B. subtilis (BOVAMINE DEFEND Plus; Chr. Hansen A/S, Hørsholm, Denmark) was then administered to the apical side of the cell monolayers at 1.0 × 108 CFU/transwell. Then, C. perfringens type A (ATCC #13124) was added at 2.8 × 107 CFU/transwell. Transepithelial electrical resistance (TEER) was measured over a 16-h period, after which area under the curve (AUC) was calculated. By itself, C. perfringens type A led to a TEER decrease that reached its lowest levels at 6.5 h post-pathogen administration. Overall mean decrease in TEER was in the order of 19.3% when compared with control cells. The DFM mixture supported the integrity of the intestinal cell monolayers both with and without the C. perfringens type A challenge, improving AUC by 24.5 and 19.1%, respectively, when compared with non-challenged control cells. In comparison with the negative control cells (C. perfringens-treated), improvements on AUC ranged from 45.6 to 39.4% vs. DFM and DFM + pathogen. In conclusion, these data support our hypothesis that a DFM mixture containing L. animalis, P. freudenreichii, B. licheniformis, and B. subtilis (BOVAMINE DEFEND Plus) supports the gut barrier integrity with or without the pathogenic challenge.

PSIII-29 A Novel Direct-Fed Microbial Supports in Vitro Intestinal Integrity Under Inflamed Conditions

Abstract Ruminants are often exposed to pathogenic and non-pathogenic challenges during daily handling procedures and management activities. These responses, in turn, may trigger the release of pro-inflammatory compounds that can have negative effects on the host. Based on this rationale, we hypothesized that 1) pro-inflammatory cytokines (TNF-α and IFN-γ) would compromise the in vitro gut barrier integrity, and 2) a novel direct-fed microbial (DFM) formulation would support the gut barrier integrity under inflammatory conditions. Therefore, our objective was to evaluate the ability of a novel DFM mixture to support barrier integrity of intestinal epithelial cell monolayers in the absence or presence of TNF-α and IFN-γ by measuring transepithelial electrical resistance (TEER). Human cancer-derived epithelial intestinal Caco-2 cell monolayers were seeded on transwells for approximately 20 days. A DFM mixture containing Lactobacillus animalis, Propionibacterium freudenreichii, Bacillus licheniformis, and B. subtilis (BOVAMINE DEFEND Plus; Chr. Hansen A/S, Hørsholm, Denmark) at 1.0 × 108 CFU/transwell was then administered to the apical side of the cell monolayers. Then, two hours later a cocktail containing TNF-α / IFN-γ at a 10:1 ratio (100:10 ng/mL, respectively) was added to the basolateral side after which TEER was measured over a 16-h period and used to calculate the area under the curve (AUC). Following the administration of the pro-inflammatory challenge, TEER was decreased, reaching its least concentrations at 5.5 h post-challenge, whereas overall AUC decreased by 21.4% compared with non-challenged control cells. The DFM mixture by itself was able to support the integrity of the intestinal cells by stimulating TEER increase over time and increasing overall AUC vs. non-treated cells (P < 0.01). Moreover, when cells receiving the DFM were challenged with the pro-inflammatory cocktail, TEER concentrations were still above 100% and no decrease was observed over the evaluation period (16 h). Moreover, when compared with non-DFM-treated but challenged cells, AUC was greater for DFM vs. challenged control cells (P < 0.05). In conclusion, these data support our hypothesis that a DFM mixture containing L. animalis, P. freudenreichii, B. licheniformis, and B. subtilis (BOVAMINE DEFEND Plus) supports the gut barrier integrity with or without the pro-inflammatory challenge.

Caco-2 Cell Sheet Partially Laminated with HT29-MTX Cells as a Novel In Vitro Model of Gut Epithelium Drug Permeability.

The intestinal epithelial Caco-2 cell monolayer is a well-established in vitro model useful for predicting intestinal drug absorption in humans. Coculture models of Caco-2 and goblet-cell-like HT29-MTX cells have been developed to overcome the lack of a mucus layer; however, those models are much leakier compared to the intestinal epithelium. Here, we developed a partially laminated culture model where HT29-MTX cells were superimposed onto a Caco-2 monolayer to overcome this issue. A morphological study showed that the piled HT29-MTX cells were voluntarily incorporated into the Caco-2 monolayer, and mucus production was confirmed via periodic acid-Schiff and mucin protein 2 staining. Permeability was evaluated in terms of transepithelial electrical resistance (TEER) and the apparent permeability of paracellular markers with different molecular sizes. The partially laminated model maintained the high barrier function of the Caco-2 monolayer, whose permeability appeared adjustable according to the HT29-MTX/Caco-2 cell ratio. In contrast, the coculture models showed abnormally high permeability of those markers, correlated with low TEER. Thus, the partially laminated model enabled in vitro recapitulation of effective mucosal barrier function. Consequently, this novel model may be useful as an in vitro high-throughput evaluation system for enteral mucosal permeability and mucus-penetrating efficiency of drugs and nanocarriers.

Purification, Identification, Chelation Mechanism, and Calcium Absorption Activity of a Novel Calcium-Binding Peptide from Peanut (Arachis hypogaea) Protein Hydrolysate.

A novel calcium-binding peptide was purified from peanut protein hydrolysate using gel filtration chromatography and identified using HPLC-MS/MS. Its amino acid sequence was determined as Phe-Pro-Pro-Asp-Val-Ala (FPPDVA, named as FA6) with the calcium-binding capacity of 15.67 ± 0.39 mg/g. Then, the calcium chelating characteristics of FPPDVA were investigated using ultraviolet-visible absorption spectroscopy, fluorescence spectroscopy, Fourier transform infrared spectroscopy, particle size, and zeta potential. The results showed that FPPDVA interacted with calcium ions, the chelation of calcium ions induced FPPDVA to fold and form a denser structure, the calcium-binding sites may mainly involve oxygen atoms from the carboxyl residues of Asp and Ala, and Phe possessed contact energy and carbonyl residues of Val. Microstructure analysis showed that FPPDVA-calcium chelate exhibited a regularly ordered and tightly aggregated sheets or block structures. Additionally, FPPDVA-calcium chelate had good gastrointestinal digestive stability and thermal stability. The results of everted rat intestinal sac and Caco-2 cell monolayer experiments showed that FPPDVA-calcium chelate could promote calcium absorption and transport through the Cav1.3 and TRPV6 calcium channels. These data suggest that FPPDVA-calcium chelate possesses the potential to be developed and applied as calcium supplement.

Bioavailability of Citrulline in Watermelon Flesh, Rind, and Skin Using a Human Intestinal Epithelial Caco-2 Cell Model

Watermelon produces many byproducts (watermelon rind and skin) even though those components contain various bioactive compounds, including citrulline. This study evaluated the citrulline concentration, total phenolic content, and antioxidant activity (DPPH and FRAP assays) of different parts of watermelon and investigated the bioavailability of citrulline from different parts of watermelon using an in vitro human intestinal epithelial Caco-2 cell monolayer model. Solid-phase extracted watermelon flesh, rind, and skin samples were treated on a Caco-2 cell monolayer for 1, 2, and 4 h. The collected basolateral solution at each time point was analyzed for the percentage of citrulline transport. Watermelon flesh had the highest citrulline content, but the watermelon skin had the highest total phenolic content and antioxidant activity compared to other watermelon parts. The citrulline bioavailability showed greater % transport in watermelon skin than in watermelon flesh, rind, and L-citrulline standard. It may be due to the different food matrices of watermelon parts. This suggests that the utilization of watermelon by-products such as skin would help develop value-added products with better bioavailability of citrulline. However, since this study was conducted with an in vitro cell model, more extensive research with in vivo studies will be needed.

TEER and Ion Selective Transwell-Integrated Sensors System for Caco-2 Cell Model.

Monitoring of ions in real-time directly in cell culture systems and in organ-on-a-chip platforms represents a significant investigation tool to understand ion regulation and distribution in the body and ions' involvement in biological mechanisms and specific pathologies. Innovative flexible sensors coupling electrochemical stripping analysis (square wave anodic stripping voltammetry, SWASV) with an ion selective membrane (ISM) were developed and integrated in Transwell™ cell culture systems to investigate the transport of zinc and copper ions across a human intestinal Caco-2 cell monolayer. The fabricated ion-selective sensors demonstrated good sensitivity (1 × 10-11 M ion concentration) and low detection limits, consistent with pathophysiological cellular concentration ranges. A non-invasive electrochemical impedance spectroscopy (EIS) analysis, in situ, across a selected spectrum of frequencies (10-105 Hz), and an equivalent circuit fitting were employed to obtain useful electrical parameters for cellular barrier integrity monitoring. Transepithelial electrical resistance (TEER) data and immunofluorescent images were used to validate the intestinal epithelial integrity and the permeability enhancer effect of ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) treatment. The proposed devices represent a real prospective tool for monitoring cellular and molecular events and for studies on gut metabolism/permeability. They will enable a rapid integration of these sensors into gut-on-chip systems.

Hesperidin enhances intestinal barrier function in Caco-2 cell monolayers via AMPK-mediated tight junction-related proteins.

The intestinal epithelium is a single-cell layer on the mucosal surface that absorbs food-derived nutrients and functions as a barrier that protects mucosal integrity. Hesperidin (hesperetin-7-rhamnoglucoside) is a flavanone glycoside composed of the flavanone hesperetin and the disaccharide rutinose, which has various physiological benefits, including antioxidative, anti-inflammatory, and antiallergic effects. Here, we used human intestinal Caco-2 cell monolayers to examine the effect of hesperidin on intestinal barrier function. Hesperidin-treated Caco-2 cell monolayers displayed enhanced intestinal barrier integrity, as indicated by an increase in transepithelial electrical resistance (TEER) and a decreased apparent permeability (Papp ) for fluorescein. Hesperidin elevated the mRNA and protein levels of occludin, MarvelD3, JAM-1, claudin-1, and claudin-4, which are encoded by tight junction (TJ)-related genes. Moreover, hesperidin significantly increased the phosphorylation of AMP-activated protein kinase (AMPK), indicating improved intestinal barrier function. Thus, our results suggest that hesperidin enhances intestinal barrier function by increasing the expression of TJ-related occludin, MarvelD3, JAM-1, and claudin-1 via AMPK activation in human intestinal Caco-2 cells.

Biological analysis of constituents in Spatholobi Caulis by UFLC-MS/MS: Enhanced quantification and application to permeability properties study in Caco-2 cell monolayer model

Major chemical constituents in medicinal materials are often used as the marker compounds of traditional Chinese medicine (TCM) for treating various diseases. For spatholobi caulis (SPC), it contains a variety of flavones, phenolic acid esters, and lignans which exert many pharmacological effects. However, the absorption and permeability properties of these constituents of SPC are still unclear and require further investigation. Different types and major compounds of SPC were chosen as representative constituents to study their absorption and transepithelial transport characteristics in the human intestinal epithelium-like Caco-2 cell monolayer model. 35 constituents of SPC were evaluated by using ultra fast liquid chromatography combined with electrospray ionization triple quadrupole tandem mass spectrometry (UFLC-MS/MS) method, acetonitrile and water containing with 0.5 mM ammonium acetate were used as mobile phase, these analytes with good linear relationships (R2 was within 0.9967−0.9998), precision (CV values were less than 10.23 %, LLOQ was less than 13.69 %), accuracy (Mean of inter- and intra-day were within 85.02 %−111.61 % and 85.50−112.97 %, respectively) and stability (The mean was within 85.07 %−113.93 %), among which 16 analytes showed good permeability, 5 analytes were considered to be poorly permeable compounds, and the other 14 analytes were assigned for the moderately absorbed compounds in Caco-2 cell monolayer model. The further results showed that the absorption mechanism of 7 well absorbed compounds, 8-O-methylretusin (1), genistein (7), spasuberol B (16), naringenin (18), isoliquiritigenin (19), 4-hydroxy-3-methoxy cinnamic acid methyl ester (23) and (+)-epipinoresinol (31) in SPC was mainly passive diffusion, their bidirectional transport rate was correlated with the concentration and transport time. The chemical structures of these compounds could affect the permeability properties on the cell monolayer. This study demonstrated the utility of Caco-2 cell monolayer model for evaluating the absorption properties and initial mechanisms of compounds in SPC in vitro, and provided important basis for predicting oral bioavailability of SPC compounds.

Cytoskeletal Responses and Aif-1 Expression in Caco-2 Monolayers Exposed to Phorbol-12-Myristate-13-Acetate and Carnosine.

The dis(re)organization of the cytoskeletal actin in enterocytes mediates epithelial barrier dys(re)function, playing a key role in modulating epithelial monolayer's integrity and remodeling under transition from physiological to pathological states. Here, by fluorescence-based morphological and morphometric analyses, we detected differential responses of cytoskeletal actin in intestinal epithelial Caco-2 cell monolayers at two different stages of their spontaneous differentiation, i.e., undifferentiated cells at 7 days post-seeding (dps) and differentiated enterocyte-like cells at 21 dps, upon challenge in vitro with the inflammation-mimicking stimulus of phorbol-12-myristate-13-acetate (PMA). In addition, specific responses were found in the presence of the natural dipeptide carnosine detecting its potential counteraction against PMA-induced cytoskeletal alterations and remodeling in differentiated Caco-2 monolayers. In such an experimental context, by both immunocytochemistry and Western blot assays in Caco-2 monolayers, we identified the expression of the allograft inflammatory factor 1 (AIF-1) as protein functionally related to both inflammatory and cytoskeletal pathways. In 21 dps monolayers, particularly, we detected variations of its intracellular localization associated with the inflammatory stimulus and its mRNA/protein increase associated with the differentiated 21 dps enterocyte-like monolayer compared to the undifferentiated cells.

Development of Paeonol Liposomes: Design, Optimization, in vitro and in vivo Evaluation.

BackgroundUlcerative colitis (UC) is one of the intractable diseases recognized by the World Health Organization, and paeonol has been proven to have therapeutic effects. However, the low solubility of paeonol limits its clinical application. To prepare and optimize paeonol liposome, study its absorption mechanism and the anti-inflammatory activity in vitro and in vivo, in order to provide experimental basis for the further development of paeonol into an anti-inflammatory drug in the future.MethodsPaeonol loaded liposomes were prepared and optimized by thin film dispersion-ultrasonic method. The absorption mechanism of paeonol-loaded liposomes was studied by pharmacokinetics, in situ single-pass intestinal perfusion and Caco-2 cell monolayer model, the anti-inflammatory activity was studied in a mouse ulcerative model.ResultsBox-Behnken response surface methodology permits to screen the best formulations. The structural and morphological characterization showed that paeonol was entrapped inside the bilayer in liposomes. Pharmacokinetic studies found that the AUC0-t of Pae-Lips was 2.78 times than that of paeonol suspension, indicating that Pae-Lips significantly improved the absorption of paeonol. In situ single intestinal perfusion and Caco-2 monolayer cell model results showed that paeonol was passively transported and absorbed, and was the substrate of P-gp, MRP2 and BCRP, and the Papp value of Pae-Lips was significantly higher than that of paeonol. In vitro and in vivo anti-inflammatory experiments showed that compared with paeonol, Pae-Lips exhibited excellent anti-inflammatory activity.ConclusionIn this study, Pae-Lips were successfully prepared to improve the oral absorption of paeonol. Absorption may involve passive diffusion and efflux transporters. Moreover, Pae-Lips have excellent anti-inflammatory activity in vitro and in vivo, which preliminarily clarifies the feasibility of further development of Pae-Lips into oral anti-inflammatory drugs.

Bioavailability of Peptides Derived from the In Vitro Digestion of Human Milk Assessed by Caco-2 Cell Monolayers.

Human milk-protein-derived peptides exhibit an array of bioactivities. Certain bioactivities cannot be exerted unless the peptides are absorbed across the gastrointestinal lumen into the bloodstream. The purpose of study was to determine which peptides derived from in vitro digestion of human milk could cross human intestinal Caco-2 cell monolayers. Our results showed that the numbers of peptides absorbed by the Caco-2 cell monolayer were different at different concentrations (44 peptides out of 169 peptides detected at 10 μg/mL, 124 peptides out of 204 peptides detected at 100 μg/mL, and 175 peptides out of 236 peptides detected at 1000 μg/mL). Four peptides (NLHLPLP (β-casein [138-144]), PLAPVHNPI (β-casein [216-224]), PLMQQVPQPIPQ (β-casein [148-159]), and FDPQIPK (β-casein [126-132])) crossed to the basolateral chamber of the Caco-2 monolayer incubated with peptides at all three concentrations. Among the peptides identified in the basolateral chambers, three peptides (NLHLPLP (β-casein [138-144]), LENLHLPLP (β-casein [136-144]), and QVVPYPQ (β-casein [182-188])) are known ACE-inhibitors; one peptide (LLNQELLLNPTHQIYPV (β-casein [197-213])) is antimicrobial, and another peptide (QVVPYPQ (β-casein [182-188])) has antioxidant activity. These findings indicate that specific milk peptides may be able to reach the bloodstream and exert bioactivity.

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

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

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

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

Ferulic Acid Metabolites Attenuate LPS-Induced Inflammatory Response in Enterocyte-like Cells.

Ferulic acid (FA) is a polyphenol pertaining to the class of hydroxycinnamic acids present in numerous foods of a plant origin. Its dietary consumption leads to the formation of several phase I and II metabolites in vivo, which represent the largest amount of ferulates in the circulation and in the intestine in comparison with FA itself. In this work, we evaluated their efficacy against the proinflammatory effects induced by lipopolysaccharide (LPS) in intestinal Caco-2 cell monolayers, as well as the mechanisms underlying their protective action. LPS-induced overexpression of proinflammatory enzymes such as inducible nitric oxide synthase (iNOS) and the consequent hyperproduction of nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) were limited by physiological relevant concentrations (1 µM) of FA, its derivatives isoferulic acid (IFA) and dihydroferulic acid (DHFA), and their glucuronidated and sulfated metabolites, which acted upstream by limiting the activation of MAPK p38 and ERK and of Akt kinase, thus decreasing the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-ĸB) translocation into the nucleus. Furthermore, the compounds were found to promote the expression of Nrf2, which may have contributed to the downregulation of NF-ĸB activity. The overall data show that phase I/II metabolites retain the efficacy of their dietary free form in contrasting inflammatory response.

Design and Investigation of Alginate Coated Solid Lipid Nanoparticles for Oral Insulin Delivery

Abstract: Introduction: The conventional subcutaneous administration of insulin has been associated with several limitations leading to poor patient compliance. The poor oral bioavailability of insulin due to degradation by gastrointestinal enzymes and secretions can be countered by the use of protective carriers such as solid lipid nanoparticles that are capable of being taken up by the Peyer’s patches. The aim of the investigation was to design and investigate alginate coated solid lipid nanoparticles (SLN) of insulin for oral administration. Materials and Methods: The SLN were prepared from glyceryl behenate and glyceryl monostearate and coated with mucoadhesive polymer, sodium alginate. The SLN were evaluated for size, shape, zeta potential, drug content, in vitro release and ex vivo drug permeation through goat intestinal mucosa and Caco-2 cell monolayer model. Results and Discussion: Transmission electron microscopy revealed spherical particles of uniform size distribution. In vitro drug release using the reverse dialysis method revealed that the alginate coating maintained the potency of insulin in simulated GI fluids and also provided sustained release. Absorption enhancement was demonstrated in ex vivo permeation studies in the goat intestinal mucosal model as well as in the Caco-2 cell monolayer model. The oral administration of alginate-coated insulin SLN in streptozotocin induced diabetic rats resulted in a significant hypoglycemic effect as compared to that of uncoated insulin-loaded SLN. The percentage glycemia at the end of 10 h was statistically significant (p<0.05) to oral insulin and the hypoglycemic levels reached were comparable to that of the conventional subcutaneous insulin. Conclusion: Alginate coated SLN has the potential of improving the absorption of insulin through intestinal mucosa and possibly its bioavailability. Key words: Solid lipid nanoparticle, Insulin, Oral delivery, Sodium alginate, Glycerol monostearate, Hypoglycemia.

A small copper carrier in blood plasma: purification, characterization, and metabolism

In mammals, concentrations of copper in tissues and fluids are kept very constant. The main mechanism underlying this constancy is the ability of the liver to modulate excretion of copper by varying the amounts entering the bile – which end up in the feces. When biliary excretion of copper is hindered, as occurs when one of the proteins involved is defective, copper accumulates in the liver and later in other organs, with fatal consequences if untreated. Major examples are the copper “pump”, ATP7B, defective in Wilson Disease, and COMMD1, which also aids biliary excretion. With copper overload, there is increased copper in the urine, which normally is very low. Studies by Gray et al.[1] with the mouse Atp7b knockout model of Wilson disease determined that the increase in urine is due to a small copper carrier (SCC) of about 2 kDa, which our laboratory then determined was also in the blood plasma of these mice. Moreover, canines with defective ATP7B also had large increases in SCC-Cu in their blood and urine. We have been researching ways of purifying significant amounts of SCC, using commercially available porcine plasma, so it can be further characterized. Based on size exclusion chromatography (SEC) of 3 kDa ultrafiltrates in small pore gels, we know that usually most of the Cu is in a single component (SCC), which by ESI-MS appears to have a mass/charge ratio of 845, one atom of Cu(II) – detected in EPR as bound to N and O ligands, and a pI in the range of 3-4. New ways of purifying SCC beyond the ultrafiltration stage have been developed, involving concentration by lyophilization and special forms of dialysis using very small pore membranes. Hydrophilic interaction chromatography and additional SEC are providing much purer SCC, with more reasonable yields. Meanwhile, we have also been investigating the secretion and uptake of SCC-Cu, using cultured cells. SCC is secreted by hepatic cells as well as those modeling intestinal and kidney epithelium, particularly when preloading with Cu(II)-diHis. Intestinal (Caco2) cell monolayers with tight junctions secreted SCC mainly across the apical membrane (representing the gut lumen), but this was dramatically reversed when albumin was present in the basal medium (which is more physiological). Kidney epithelial cells also secreted SCC-Cu and some additional copper components mainly into the apical fluid. Other studies, with 67Cu-labeled SCC from hepatic secretions, confirmed not only that SCC was secreted, but showed that it was also taken up by all cell types examined. These findings suggest that metabolism of SCC and its copper is very active, and that SCC may play an important role in mammalian copper homeostasis. [1]Gray et al. PLoS ONE 7(6): e38327.

Oligonucleotide Delivery across the Caco-2 Monolayer: The Design and Evaluation of Self-Emulsifying Drug Delivery Systems (SEDDS).

Oligonucleotides (OND) represent a promising therapeutic approach. However, their instability and low intestinal permeability hamper oral bioavailability. Well-established for oral delivery, self-emulsifying drug delivery systems (SEDDS) can overcome the weakness of other delivery systems such as long-term instability of nanoparticles or complicated formulation processes. Therefore, the present study aims to prepare SEDDS for delivery of a nonspecific fluorescently labeled OND across the intestinal Caco-2 monolayer. The hydrophobic ion pairing of an OND and a cationic lipid served as an effective hydrophobization method using either dimethyldioctadecylammonium bromide (DDAB) or 1,2-dioleoyl-3-trimethylammonium propane (DOTAP). This strategy allowed a successful loading of OND-cationic lipid complexes into both negatively charged and neutral SEDDS. Subjecting both complex-loaded SEDDS to a nuclease, the negatively charged SEDDS protected about 16% of the complexed OND in contrast to 58% protected by its neutral counterpart. Furthermore, both SEDDS containing permeation-enhancing excipients facilitated delivery of OND across the intestinal Caco-2 cell monolayer. The negatively charged SEDDS showed a more stable permeability profile over 120 min, with a permeability of about 2 × 10−7 cm/s, unlike neutral SEDDS, which displayed an increasing permeability reaching up to 7 × 10−7 cm/s. In conclusion, these novel SEDDS-based formulations provide a promising tool for OND protection and delivery across the Caco-2 cell monolayer.

Permeability of the Cyanotoxin Microcystin-RR across a Caco-2 Cells Monolayer.

Microcystins (MCs) are toxins produced by several cyanobacterial species found worldwide. While MCs have a common structure, the variation of two amino acids in their structure affects their toxicity. As toxicodynamics are very similar between the MC variants, their differential toxicity could rather be explained by toxicokinetic parameters. Microcystin-RR (MC-RR) is the second most abundant congener and induces toxicity through oral exposure. As intestinal permeability is a key parameter of oral toxicokinetics, the apparent permeability of MC-RR across a differentiated intestinal Caco-2 cell monolayer was investigated. We observed a rapid and large decrease of MC-RR levels in the donor compartment. However, irrespective of the loaded concentration and exposure time, the permeabilities were very low from apical to basolateral compartments (from 4 to 15 × 10−8 cm·s−1) and from basolateral to apical compartments (from 2 to 37 × 10−8 cm·s−1). Our results suggested that MC-RR would be poorly absorbed orally. As similar low permeability was reported for the most abundant congener microcystin-LR, and this variant presented a greater acute oral toxicity than MC-RR, we concluded that the intestinal permeability was probably not involved in the differential toxicity between them, in contrast to the hepatic uptake and metabolism.

Extract of Unifloral Camellia sinensis L. Pollen Collected by Apis mellifera L. Honeybees Exerted Inhibitory Effects on Glucose Uptake and Transport by Interacting with Glucose Transporters in Human Intestinal Cells.

Bee pollen possesses potential hypoglycemic effects but its inhibitory mechanisms on glucose absorption and transportation in intestinal cells still need to be clarified. Here, we determined the inhibitory effects of bee pollen extract originating from Camellia sinensis L. (BP-Cs) as well as its representative phenolic compounds on glucose uptake and transport through a human intestinal Caco-2 cell monolayer model. It showed that three representative phenolic compounds, including gallic acid (GA), 3-O-[6'-O-(trans-p-coumaroyl)-β-d-glucopyranosyl]kaempferol (K1), and 3-O-[2',6'-di-O-(trans-p-coumaroyl)-β-d-glucopyranosyl]kaempferol (K2), with contents of 27.7 ± 0.86, 9.88 ± 0.54, and 7.83 ± 0.46 μg/mg in BP-Cs extract, respectively, exerted mutual antagonistic actions interacting with glucose transporters to inhibit glucose uptake and transport based on their combination index (CI) and molecular docking analysis. K1, K2, and GA might compete with d-glucose to form hydrogen bonds with the same active residues including GLU-412, GLY-416, GLN-314, and TRP-420 in GLUT2. These findings provide us a deep understanding of the mechanisms underlying the anti-hyperglycemia by bee pollen, which provide a new sight on dietary intervention strategies against diabetes.