Intestinal Caco-2 Cells Research Articles

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.

Cannabidiol Isolated From Cannabis sativa L. Protects Intestinal Barrier From In Vitro Inflammation and Oxidative Stress

The relevance and incidence of intestinal bowel diseases (IBD) have been increasing over the last 50 years and the current therapies are characterized by severe side effects, making essential the development of new strategies that combine efficacy and safety in the management of human IBD. Herbal products are highly considered in research aimed at discovering new approaches for IBD therapy and, among others, Cannabis sativa L. has been traditionally used for centuries as an analgesic and anti-inflammatory remedy also in different gastrointestinal disorders. This study aims to investigate the effects of different C. sativa isolated compounds in an in vitro model of intestinal epithelium. The ability of treatments to modulate markers of intestinal dysfunctions was tested on Caco-2 intestinal cell monolayers. Our results, obtained by evaluation of ROS production, TEER and paracellular permeability measurements and tight junctions evaluation show Cannabidiol as the most promising compound against intestinal inflammatory condition. Cannabidiol is able to inhibit ROS production and restore epithelial permeability during inflammatory and oxidative stress conditions, suggesting its possible application as adjuvant in IBD management.

Transport of Dietary Anti-Inflammatory Peptide, γ-Glutamyl Valine (γ-EV), across the Intestinal Caco-2 Monolayer.

The present study analyzed the transepithelial transport of the dietary anti-inflammatory peptide, γ-glutamyl valine (γ-EV). γ-EV is naturally found in dry edible beans. Our previous study demonstrated the anti-inflammatory potency of γ-EV against vascular inflammation at a concentration of 1mM, and that it can transport with the apparent permeability coefficient (Papp) of 1.56 × 10−6 ± 0.7 × 10−6 cm/s across the intestinal Caco-2 cells. The purpose of the current study was to explore whether the permeability of the peptide could be enhanced and to elucidate the mechanism of transport of γ-EV across Caco-2 cells. The initial results indicated that γ-EV was nontoxic to the Caco-2 cells up to 5 mM concentration and could be transported across the intestinal cells intact. During apical-to-basolateral transport, a higher peptide dose (5 mM) significantly (p < 0.01) enhanced the transport rate to 2.5 × 10−6 ± 0.6 × 10−6 cm/s. Cytochalasin-D disintegrated the tight-junction proteins of the Caco-2 monolayer and increased the Papp of γ-EV to 4.36 × 10−6 ± 0.16 × 10−6 cm/s (p < 0.001), while theaflavin 3′-gallate and Gly-Sar significantly decreased the Papp (p < 0.05), with wortmannin having no effects on the peptide transport, indicating that the transport route of γ-EV could be via both PepT1-mediated and paracellular.

Dendrobine Suppresses Lipopolysaccharide-induced Gut Inflammation in a Co-culture of Intestinal Epithelial Caco-2 Cells and RAW264.7 Macrophages

We evaluated dendrobine for its potential effect on Inflammatory Bowel Disease (IBD) progression using the intestinal epithelial Caco-2/RAW264.7 macrophage co-culture cell model. The results showed that dendrobine maintained the tight junction (TJ) proteins in co-cultured Caco-2 cells, suggesting its protective effect on intestinal integrity. Moreover, the findings indicated that the dendrobine group downregulated levels of tumor necrosis factor-α (TNF-α), proinflammatory Interleukin (IL)-1ß and IL-6, and inflammatory markers such as cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in co-cultured RAW264.7 cells. Additionally, dendrobine-mediated anti-inflammatory effects were observed based on the inhibition of LPS-induced proinflammatory mediators of nuclear factor kappa B (NF-κB)-related signaling. These results demonstrated the protective effect of dendrobine at effectively ameliorating intestinal barrier disruption and inhibiting activation of the NF-κB pathway in macrophages through the intestinal barrier.

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.

The effect of hypergravity in intestinal permeability of nanoformulations and molecules

The oral administration of drugs remains a challenge due to rapid enzymatic degradation and minimal absorption in the gastrointestinal tract. Mechanical forces, namely hypergravity, can interfere with cellular integrity and drug absorption, and there is no study describing its influence in the intestinal permeability. In this work, it was studied the effect of hypergravity on intestinal Caco-2 cells and its influence in the intestinal permeability of different nanoformulations and molecules. It was shown that the cellular metabolic activity and integrity were maintained after exposure to different gravity-levels (g-levels). Expression of important drug transporters and tight junctions’ proteins was evaluated and, most proteins demonstrated a switch of behavior in their expression. Furthermore, paracellular transport of FITC-Dextran showed to significantly increase with hypergravity, which agrees with the decrease of transepithelial electrical resistance and the increase of claudin-2 at higher g-levels. The diffusion of camptothecin released from polymeric micelles revealed a significant decrease, which agrees with the increased expression of the P-gp observed with the increase in g-levels, responsible for pumping this drug out. The neonatal Fc receptor-mediated transport of albumin-functionalized nanoparticles loaded with insulin showed no significant changes when increasing the g-levels. Thus, this study supports the effect of hypergravity on intestinal permeability is dependent on the molecule studied and the mechanism by which it is absorbed in the intestine.

Screening of Lactobacillus strains that enhance SCFA uptake in intestinal epithelial cells

The objective was to screen Lactobacillus strains with strong survivability in oro-gastrointestinal tract and adhesion abilities of intestinal mucins and Caco-2 cells, and their effect on absorption of short-chain fatty acids (SCFAs) by epithelial cells was studied. The survival rate of Lactobacillus strains was studied after exposure to oral stress, gastric stress and intestinal stress successively, and then their adhesion ability was also researched. The model of intestinal epithelial cells absorbing SCFAs was established, which was used to evaluate the effect of Lactobacillus strains on Caco-2 cells absorption of SCFAs. The survival rate of Lactobacillus plantarum L58, L67, L97, L123, and L198 and Lactobacillus fermentum L146 was significantly higher than others in oro-gastrointestinal tract (P < 0.05), which also showed high adhesion to mucins and Caco-2 cells. The model was successfully established with the Caco-2 cell line, which formed a polarized cell monolayer and developed tight junctions with an appropriate permeability coefficient for phenol red lower than 1 × 10–6 cm/s after culturing for 15 days, and the viability of Caco-2 cells was significantly higher than other concentrations when the content of propionic acid or butyric acid was 1 mmol/L in the model. The propionic acid content in Caco-2 cells inoculated with L. plantarum L58, L67, L97, L123, and L198 was significantly higher than that of cells without L. plantarum inoculation (P < 0.05), and the butyric acid content in cells inoculated with L. fermentum L146 was significantly higher than that of cells without inoculation (P < 0.05). Our results highlight that L. plantarum L58, L67, L97, L123, L198 and L. fermentum L146 are more resistant to oro-gastrointestinal conditions and their high adhesion to the intestine can enhance SCFAs uptake in intestinal epithelial cells.

Identification of Synbiotics Conducive to Probiotics Adherence to Intestinal Mucosa Using an In Vitro Caco-2 and HT29-MTX Cell Model

The ability of bacteria to adhere to the intestinal mucosa is a critical property necessary for the long-term colonization of the intestinal tract. This ability can be highly sensitive to the presence of prebiotics. However, limited data are available in this respect for beneficial bacteria such as probiotics or resident gut microbiota. We previously demonstrated that the presence of prebiotics may decrease adherence in several pre- and probiotic combinations. Thus, characterizing the interactions between numerous combinations involving different classes of pre- and probiotics can be crucial in identifying new synbiotics. Accordingly, here, we extend our prior analyses to evaluate the adhesion of five lactobacilli, six bifidobacteria, and one probiotic Escherichia coli strains, as commercial probiotics or promising probiotic candidates, together with the cariogenic Bifidobacterium dentium strain. As an in vitro intestinal mucosa model, Caco-2 and mucin-secreting HT29-MTX cells were co-cultured at 9:1 in the presence or absence of prebiotics. Commercial inulin-type fructooligosaccharide prebiotics Orafti® GR, Orafti® P95, and galactooligosaccharide-based prebiotic formula Vivinal®, including purified human milk oligosaccharides (HMOs) were added into the cultivation media as the sole sugar source (2.5% each). Adherence was tested using microtiter plates and was evaluated as the percentage of fluorescently labeled bacteria present in the wells after three washes. Consistent prebiotics-mediated enhanced adherence was observed only for the commercial probiotic strain E. coli O83. For the remaining strains, the presence of HMO or prebiotics Orafti® P95 or Orafti® GR decreased adherence, reaching statistical significance (p &lt; 0.05) for three of out of eight (HMO) or five of out of 11 strains tested, respectively. Conversely, Vivinal® enhanced adhesion in six out of the 12 strains tested, and notably, it significantly attenuated the adherence of the cariogenic Bifidobacterium dentium Culture Collection of Dairy Microorganisms (CCDM) 318. To our knowledge, this represents the first report on the influence of commercial prebiotics and HMOs on the adhesion of the cariogenic Bifidobacterium sp. Vivinal® seems to be a promising prebiotic to be used in the formulation of synbiotics, supporting the adhesion of a wide range of probiotics, especially the strains B. bifidum BBV and BBM and the probiotic Escherichia coli O83.

NPC1L1-dependent transport of 27-alkyne cholesterol in intestinal epithelial cells.

Niemann-Pick C1 Like-1 (NPC1L1) mediates the uptake of micellar cholesterol by intestinal epithelial cells and is the molecular target of the cholesterol-lowering drug ezetimibe (EZE). The detailed mechanisms responsible for intracellular shuttling of micellar cholesterol are not fully understood due to the lack of a suitable NPC1L1 substrate that can be traced by fluorescence imaging and biochemical methods. 27-Alkyne cholesterol has been previously shown to serve as a substrate for different cellular processes similar to native cholesterol. However, it is not known whether alkyne cholesterol is absorbed via an NPC1L1-dependent pathway. We aimed to determine whether alkyne cholesterol is a substrate for NPC1L1 in intestinal cells. Human intestinal epithelial Caco2 cells were incubated with micelles containing alkyne cholesterol in the presence or absence of EZE. Small intestinal closed loops in C57BL/6J mice were injected with micelles containing alkyne cholesterol with or without EZE. Alkyne cholesterol esterification in Caco2 cells was significantly inhibited by EZE and by inhibitor of clathrin-mediated endocytosis Pitstop 2. The esterification was similarly reduced by inhibitors of the acyl-CoA cholesterol acyltransferase (ACAT). Alkyne cholesterol efficiently labeled the apical membrane of Caco2 cells and the amount retained on the membrane was significantly increased by EZE as judged by accessibility to exogenous cholesterol oxidase. In mouse small intestine, the presence of EZE reduced total alkyne cholesterol uptake by ∼75%. These data show that alkyne cholesterol acts as a substrate for NPC1L1 and may serve as a nonradioactive tracer to measure cholesterol absorption in both in vitro and in vivo models.

Effect of skimmed milk on intestinal tract: Prevention of increased reactive oxygen species and nitric oxide formation

The capacity of skimmed milk to neutralise increased reactive oxygen species (ROS) and to attenuate nitric oxide (NO) production, as well as to present cytoprotective effect at the intestinal level was assessed after in vitro gastro-intestinal digestion. The impact on ROS modulation was evaluated at a non-cytotoxic concentration of hydrogen peroxide (H2O2) in a co-culture of Caco-2 and HT-29 intestinal cells. In parallel, a cytotoxic concentration of H2O2 was used to study the effect of digested milk against induced cell apoptosis. Concerning induced NO production, it was evaluated using the model lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells. Results showed that digested milk prevented the increase of basal ROS level in the intestinal epithelium and attenuated NO production by LPS-stimulated macrophage cells. In the H2O2-induced cytotoxicity assay, digested milk had no protection against apoptosis, confirmed by the failure in attenuating activated caspase-3/7.

Trans-Epithelial Transport, Metabolism, and Biological Activity Assessment of the Multi-Target Lupin Peptide LILPKHSDAD (P5) and Its Metabolite LPKHSDAD (P5-Met).

P5 (LILPKHSDAD) is a hypocholesterolemic peptide from lupin protein with a multi-target activity, since it inhibits both 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCoAR) and proprotein convertase subtilisin/kexin type-9 (PCSK9). This work shows that, during epithelial transport experiments, the metabolic transformation mediated by intestinal peptidases produces two main detected peptides, ILPKHSDAD (P5-frag) and LPKHSDAD (P5-met), and that both P5 and P5-met are linearly absorbed by differentiated human intestinal Caco-2 cells. Extensive comparative structural, biochemical, and cellular characterizations of P5-met and the parent peptide P5 demonstrate that both peptides have unique characteristics and share the same mechanisms of action. In fact, they exert an intrinsically multi-target behavior being able to regulate cholesterol metabolism by modulating different pathways. The results of this study also highlight the dynamic nature of bioactive peptides that may be modulated by the biological systems they get in contact with.

Elucidating the mechanism of cellular uptake of fullerene nanoparticles

Understanding the molecular interactions between biological cells and engineered nanoparticles is a key to evaluating potential toxicities to humans and the environment. This study developed a method to determine the mechanisms by which fullerene aggregates are distributed into a representative cell line, human intestinal Caco-2 cells. First, we determined that the presence of fetal bovine serum (FBS) in the cell culture media changes the particle characteristics and inhibits particle adsorptions onto cell surfaces. Second, significantly lower amounts of fullerene were internalized at 4°C, a temperature at which active transport mechanisms are effectively impeded, than at 37°C. Third, metabolic inhibitors of active transport and a microtubule transport inhibitor decreased fullerene uptake at 37°C. Fourth, cellular uptake of fullerene increased with increasing fullerene concentration, suggesting that passive diffusion into lipid membranes contributed to uptake over the broad concentration range used in this study. Together, these results indicate fullerene transport into cells occurs via two mechanisms: passive diffusion across the lipid bilayer and active transport including microtubule involved endocytosis. The results also suggest that simple physical-chemical partitioning models do not fully describe fullerene uptake, and instead, active transport models are also required to estimate the cellular uptake and toxicity of fullerene.

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.

Transmucosal Solid Lipid Nanoparticles to Improve Genistein Absorption via Intestinal Lymphatic Transport.

Genistein (GEN) is a soy-derived isoflavone that exhibits several biological effects, such as neuroprotective activity and the prevention of several types of cancer and cardiovascular disease. However, due to its poor water solubility and the extensive first-pass metabolism, the oral bioavailability of GEN is limited. In this work, solid lipid nanoparticles (SLN) were developed to preferentially reach the intestinal lymphatic vessels, avoiding the first-pass metabolism of GEN. GEN-loaded SLN were obtained by a hot homogenization process, and the formulation parameters were chosen based on already formulated studies. The nanoparticles were characterized, and the preliminary in vitro chylomicron formation was evaluated. The cell uptake of selected nanocarriers was studied on the Caco-2 cell line and intestinal mucosa. The SLN, characterized by a spherical shape, showed an average diameter (about 280 nm) suitable for an intestinal lymphatic uptake, good stability during the testing time, and high drug loading capacity. Furthermore, the intestinal mucosa and Caco-2 cells were found to uptake SLN. The approximately two-fold increase in particle size suggested a possible interaction between SLN and the lipid components of chylomicrons like phospholipid; therefore, the results may support the potential for these SLN to improve oral GEN bioavailability via intestinal lymphatic absorption.

Intestinal and hepatic effects of iron oxide nanoparticles

Iron oxide nanoparticles gain increasing attention due to their broad industrial use. However, safety concerns exist since their effects on human cells are still under investigation. The presence of iron oxide nanoparticles in the food pigment E172 has been shown recently. Here, we studied four iron oxide nanoparticles, one food pigment E172 and the ionic control FeSO4 regarding dissolution in biological media, uptake and transport, and cellular effects in vitro in human intestinal Caco-2 and HepaRG hepatocarcinoma cells. The iron oxide nanoparticles passed the gastrointestinal passage without dissolution and reached the intestine in the form of particles. Minor uptake was seen into Caco-2 cells but almost no transport to the basolateral site was detected for any of the tested particles. HepaRG cells showed higher particle uptake. Caco-2 cells showed no alterations in reactive oxygen species production, apoptosis, or mitochondrial membrane potential, whereas two particles induced apoptosis in HepaRG cells, and one altered mitochondrial membrane potential at non-cytotoxic concentrations. No correlation between physicochemical particle characteristics and cellular effects was observed, thus emphasizing the need for case-by-case assessment of iron oxide nanoparticles.

MTORC1 silencing during intestinal epithelial Caco-2 cell differentiation is mediated by the activation of the AMPK/TSC2 pathway

The mechanistic target of rapamycin complex 1 (mTORC1) signaling is the prototypical pathway regulating protein synthesis and cell proliferation. The level of mTORC1 activity is high in intestinal stem cells located at the base of the crypts and thought to gradually decrease as transit-amplifying cells migrate out of the crypts and differentiate into enterocytes, goblet cells or enteroendocrine cells along the epithelium. The unknown mechanism responsible for the silencing of intestinal epithelium mTORC1 during cell differentiation was investigated in Caco-2 cells, which spontaneously differentiate into enterocytes in standard growth medium. The results show that TSC2, an upstream negative regulator of mTORC1 was central to mTORC1 silencing in differentiated Caco-2 cells. AMPK-mediated activation of TSC2 (Ser1387) and repression of Raptor (Ser792), an essential component of mTORC1, were stimulated in differentiated Caco-2 cells. ERK1/2-mediated repression of TSC2 (Ser664) seen in undifferentiated Caco-2 cells was lifted in differentiated cells. IRS-1-mediated activation of AKT (Thr308) phosphorylation was stimulated in differentiated Caco-2 cells and may be involved in cross-pathway repression of ERK1/2. Additionally, PRAS40 (Thr246) phosphorylation was decreased in differentiated Caco-2 cells compared to undifferentiated cells allowing dephosphorylated PRAS40 to displace Raptor thereby repressing mTORC1 kinase activity.

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.

Rosa canina L. Can Restore Endoplasmic Reticulum Alterations, Protein Trafficking and Membrane Integrity in a Dextran Sulfate Sodium-Induced Inflammatory Bowel Disease Phenotype.

Rosa canina L. is a natural polyphenol-rich medicinal plant that exhibits antioxidant and anti-inflammatory activities. Recent in vivo studies have demonstrated that a methanol extract of Rosa canina L. (RCME) has reversed an inflammatory bowel disease (IBD)-like phenotype that has been triggered by dextran sulfate sodium (DSS) in mice. In the current study, we investigated the effects of RCME on perturbations of cellular mechanisms induced by DSS-treatment of intestinal Caco-2 cells, including stress response in the endoplasmic reticulum (ER), protein trafficking and sorting as well as lipid rafts integrity and functional capacities of an intestinal enzyme. 6 days post-confluent cells were treated for 24 h with DSS (3%) or simultaneously with DSS (3%) and RCME (100 µg/mL) or exclusively with RCME (100 µg/mL) or not treated. The results obtained demonstrate the ability of RCME to counteract the substantial increase in the expression levels of several ER stress markers in DSS-treated cells. Concomitantly, the delayed trafficking of intestinal membrane glycoproteins sucrase-isomaltase (SI) and dipeptidyl peptidase 4 (DPP4) induced by DSS between the ER and the Golgi has been compromised by RCME. Furthermore, RCME restored the partially impaired polarized sorting of SI and DPP4 to the brush border membrane. An efficient sorting mechanism of SI and DPP4 is tightly associated with intact lipid rafts structures in the trans-Golgi network (TGN), which have been distorted by DSS and normalized by RCME. Finally, the enzymatic activities of SI are enhanced in the presence of RCME. Altogether, DSS treatment has triggered ER stress, impaired trafficking and function of membrane glycoproteins and distorted lipid rafts, all of which can be compromised by RCME. These findings indicate that the antioxidants in RCME act at two major sites in Caco-2 cells, the ER and the TGN and are thus capable of maintaining the membrane integrity by correcting the sorting of membrane-associated proteins.

A clinical pilot study on the effect of the probiotic Lacticaseibacillus rhamnosus TOM 22.8 strain in women with vaginal dysbiosis

Lactobacilli with probiotic features play an essential role in maintaining a balanced vaginal microbiota and their administration has been suggested for the treatment and prevention of vaginal dysbiosis. The present study was aimed to in vitro and in vivo investigate the probiotic potential of the Lacticaseibacillus rhamnosus TOM 22.8 strain, isolated from the vaginal ecosystem of a healthy woman. For this purpose, safety and functional properties were in depth evaluated. The strain exhibited a broad spectrum of antagonistic activity against vaginal pathogens; adhesion capacity to both the vaginal VK2/E6E7 and the intestinal Caco-2 cells; anti-inflammatory and antioxidant activities, suggesting its promising probiotic features. In addition, an in vivo pilot-study was planned. Based on both clinical and microbiological parameters, the oral or vaginal strain administration, determined a significant pathogens reduction after 10 days of administration and a maintenance of eubiosis up to 30 days after the end of the treatment. Therefore, the L. rhamnosus TOM 22.8 strain can be proposed as valuable oral and/or vaginal treatment for vaginal dysbiosis.

Investigation of the Cellular Effects of Beta- Cyclodextrin Derivatives on Caco-2 Intestinal Epithelial Cells.

Cyclodextrins are widely used excipients for increasing water-solubility, delivery and bioavailability of lipophilic drugs. By using fluorescent cyclodextrin derivatives, we showed previously that cyclodextrins are able to enter Caco-2 intestinal cells by endocytosis, but the influence of different fluorescent labeling on the same cyclodextrin derivative has not been studied. The consequences of the cellular internalization of cyclodextrins have not been revealed yet either. The aims of this study were to compare the cellular internalization of fluorescein- and rhodamine-labeled (2-hydroxypropyl)-, (HPBCD) and randommethyl-β-cyclodextrins (RAMEB) and to investigate the intracellular effects of these derivatives on Caco-2 cells. Stimulation of the NF-kappa B pathway and autophagy and localization of these derivatives in lysosomes were tested. The endocytosis of these derivatives was examined by fluorescence microscopy and flow cytometry. Both fluorescein- and rhodamine-labeled derivatives entered the cells, therefore the type of the fluorescent labeling did not influence their internalization. Cyclodextrin pretreatment did not activate the translocation of the p65 subunit of the NF-kappa B heterodimer into the cell nuclei from the cytoplasm. After HPBCD or RAMEB treatment, formation of the autophagosomes did not increase compared to the control sample and at the same time these derivatives could be detected in lysosomes after internalization.