Fluorescein Isothiocyanate-dextran Research Articles

Red raspberry (Rubus idaeus) preserves intestinal barrier integrity and reduces oxidative stress in Caco-2 cells exposed to a proinflammatory stimulus.

Growing evidence showed the capacity of (poly)phenols to exert a protective role on intestinal health. Nevertheless, the existing findings are still heterogeneous and the underlying mechanisms remain unclear. This study investigated the potential benefits of a red raspberry (Rubus idaeus) powder on the integrity of the intestinal barrier, focusing on its ability to mitigate the effects of tumor necrosis factor-α (TNF-α)-induced intestinal permeability. Human colorectal adenocarcinoma cells (i.e., Caco-2 cells) were used as a model to assess the impact of red raspberry on intestinal permeability, tight junction expression, and oxidative stress. The Caco-2 cells were differentiated into polarized monolayers and treated with interferon-γ (IFN-γ) (10 ng mL-1) for 24 hours, followed by exposure to TNF-α (10 ng mL-1) in the presence or absence of red raspberry extract (1-5 mg mL-1). The integrity of the intestinal monolayer was evaluated using transepithelial electrical resistance (TEER) and fluorescein isothiocyanate-dextran (FITC-D) efflux assay. Markers of intestinal permeability (claudin-1, occludin, and zonula occludens-1 (ZO-1)) and oxidative stress (8-hydroxy-2-deoxyguanosine (8-OHdG) and protein carbonyl) were assessed using ELISA kits. Treatment with red raspberry resulted in a significant counteraction of TEER value loss (41%; p < 0.01) and a notable reduction in the efflux of FITC-D (-2.5 times; p < 0.01). Additionally, red raspberry attenuated the levels of 8-OHdG (-48.8%; p < 0.01), mitigating the detrimental effects induced by TNF-α. Moreover, red raspberry positively influenced the expression of the integral membrane protein claudin-1 (+18%; p < 0.01), an essential component of tight junctions. These findings contribute to the growing understanding of the beneficial effects of red raspberry in the context of the intestinal barrier. The effect of red raspberry against TNF-α-induced intestinal permeability observed in our in vitro model suggests, for the first time, its potential as a dietary strategy to promote gastrointestinal health.

Particle Engineering via Supramolecular Assembly of Macroscopic Hydrophobic Building Blocks.

Tailoring the hydrophobicity of supramolecular assembly building blocks enables the fabrication of well-defined functional materials. However, the selection of building blocks used in the assembly of metal-phenolic networks (MPNs), an emerging supramolecular assembly platform for particle engineering, has been essentially limited to hydrophilic molecules. Herein, we synthesized and applied biscatechol-functionalized hydrophobic polymers (poly(methyl acrylate) (PMA) and poly(butyl acrylate) (PBA)) as building blocks to engineer MPN particle systems (particles and capsules). Our method allowed control over the shell thickness (e.g., between 10 and 21 nm), stiffness (e.g., from 10 to 126 mN m-1 ), and permeability (e.g., 28-72 % capsules were permeable to 500 kDa fluorescein isothiocyanate-dextran) of the MPN capsules by selection of the hydrophobic polymer building blocks (PMA or PBA) and by controlling the polymer concentration in the MPN assembly solution (0.25-2.0 mM) without additional/engineered assembly processes. Molecular dynamics simulations provided insights into the structural states of the hydrophobic building blocks during assembly and mechanism of film formation. Furthermore, the hydrophobic MPNs facilitated the preparation of fluorescent-labeled and bioactive capsules through postfunctionalization and also particle-cell association engineering by controlling the hydrophobicity of the building blocks. Engineering MPN particle systems via building block hydrophobicity is expected to expand their use.

Magneto-Mechanical Actuation Induces Endothelial Permeability.

Cancer treatment is one of the major health problems that burden our society. According to the American Cancer Society, over 1.9 million new cancer cases and ∼0.6 million deaths from cancer are expected in the US in 2023. Therapeutic targeting is considered to be the gold standard in cancer treatment. However, when a tumor grows beyond a critical size, its vascular system differentiates abnormally and erratically, creating a heterogeneous endothelial barrier that further restricts drug delivery into tumors. While several methods exist, these prompt tumor migration and the appearance of new metastatic sites. Herein, we propose an innovative method based on magneto-mechanical actuation (MMA) to induce endothelial permeability. This method employs FDA-approved PEGylated superparamagnetic iron oxide nanoparticles (PEG-SPIONs) and alternating nonheating magnetic fields. MMA lies in the translation of magnetic forces into mechanical agitation. As a proof of concept, we developed a 2D cell culture model based on human umbilical vein endothelial cells (HUVEC), which were incubated with PEG-SPIONs and then exposed to different magnetic doses. After adjusting the particle concentration, incubation times, and parameters (amplitude, frequency, and exposure time) of the magnetic field generator, we induced actin filament remodeling and subsequent vascular endothelial-cadherin junction disruption. This led to transient gaps in cell monolayers, through which fluorescein isothiocyanate-dextran was translocated. We observed no cell viability reduction for 3 h of particle incubation up to a concentration of 100 μg/mL in the presence and absence of magnetic fields. For optimal permeability studies, the magnetic field parameters were adjusted to 100 mT, 65 Hz, and 30 min in a pulse mode with 5 min OFF intervals. We found that the endothelial permeability reached the highest value (33%) when 2 h postmagnetic field treatment was used. To explain these findings, a magneto-mechanical transduced stress mechanism mediated by intracellular forces was proposed. This method can open new avenues for targeted drug delivery into anatomic regions within the body for a broad range of disease interventions.

CircMIRLET7BHG, upregulated in an m6A-dependent manner, induces the nasal epithelial barrier dysfunction in allergic rhinitis pathogenesis

ObjectiveAllergic rhinitis (AR) remains a frequent aspiratory allergic inflammatory disorder with a high incidence. Circular RNAs (circRNAs) have been revealed to participate in the pathogenesis of AR. This study investigated the biological function of circMIRLET7BHG (hsa_circ_0008668) in AR progression. MethodsOvalbumin (OVA)-exposed human nasal epithelial cell line (HNEpC) and mice were adopted as the in vitro and in vivo models of AR. Immunofluorescence staining was used to determine epithelial tight junction protein expression. Target molecule levels were assessed by RT-qPCR and Western blotting. Localization of circMIRLET7BHG and IGF2BP1 was observed by RNA-FISH and immunofluorescence. Epithelial barrier damage was determined by transepithelial electrical resistance and fluorescein isothiocyanate-dextran (FD4) permeability. Serum concentrations of IgE, sIgE, IFN-γ, IL-4, and IL-5 were detected by ELISA. Apoptosis, pathological changes, and eosinophil infiltration in nasal mucosa tissues were evaluated by TUNEL, H&E, and Sirius red staining, respectively. Molecular mechanism was analyzed by RNA pull-down, RIP, and MeRIP assays. ResultsAn increased expression of circMIRLET7BHG was found in AR patients and experimental models. Down-regulation of circMIRLET7BHG attenuated OVA-induced allergic symptoms via relieving epithelial thicknesses, eosinophil infiltration, apoptosis, and inflammatory response in mice. Subsequently, circMIRLET7BHG deficiency prevented OVA-induced epithelial barrier dysfunction by reducing epithelial permeability, and inhibiting tight junction proteins. Mechanistically, methyltransferase-like 3 (METTL3) enhanced circMIRLET7BHG expression via m6A methylation, which enhanced ADAM10 mRNA stability via interaction with IGF2BP1. ConclusionMETTL3-mediated m6A modification increased circMIRLET7BHG expression that consequently raised ADAM10 mRNA stability via interplay with IGF2BP1, thereby promoting AR by inducing epithelial barrier dysfunction.

Lactic acid enhances vaginal epithelial barrier integrity and ameliorates inflammatory effects of dysbiotic short chain fatty acids and HIV-1

The vaginal microenvironment is key in mediating susceptibility to sexually transmitted infections. A polymicrobial environment with reduced Lactobacilllus spp. is characteristic of vaginal dysbiosis, associated with increased production of several short chain fatty acids (SCFAs), vaginal inflammation and an increased risk of HIV-1 acquisition. In contrast, a eubiotic vaginal microbiome (VMB), dominated by Lactobacillus spp. correlates with increased production of lactic acid (LA), an acidic milieu and protection against HIV-1. Vaginal metabolites, specifically LA and SCFAs including butyric, succinic and acetic acids are associated with modulation of HIV-1 risk. We assessed the impact of combined and individual SCFAs and LA on vaginal epithelial cells (VK2) grown in air–liquid interface cultures. Treatment of VK2 cells with eubiotic SCFA + LA mixture showed increased epithelial barrier integrity, reduced FITC dextran leakage and enhanced expression of cell–cell adhesion proteins. Treatment with dysbiotic SCFA + LA mixture diminished epithelial barrier integrity, increased NFκB activation and inflammatory mediators: TNF-α, IL-6, IL-8 and RANTES. LA was found to be the primary contributor of the beneficial effects. Eubiotic SCFA + LA mixture ameliorated HIV-1 mediated barrier disruption and HIV-1 leakage, whereas dysbiotic SCFA + LA treatment exacerbated HIV-1 effects. These findings indicate a key role for LA in future prophylactic strategies.

A hybridized mechano-electroporation technique for efficient immune cell engineering

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White-Light-Responsive Prussian Blue Nanophotonic Particles for Effective Eradication of Bacteria and Improved Healing of Infected Cutaneous Wounds.

The increasing burden of cutaneous wound infections with drug-resistant bacteria underlines the dire need for novel treatment approaches. Here, we report the preparation steps, characterization, and antibacterial efficacy of novel chitosan-coated Prussian blue nanoparticles loaded with the photosensitizer fluorescein isothiocyanate-dextran (CHPB-FD). With excellent photothermal and photodynamic properties, CHPB-FD nanoparticles can effectively eradicate both Gram-positive methicillin-resistant Staphylococcus aureus and Gram-negative Pseudomonas aeruginosa in vitro and in vivo. The antibacterial efficacy of CHPB-FD nanophotonic particles further increases in the presence of white light. Using a bacteria-infected cutaneous wound rat model, we demonstrate that CHPB-FD particles upregulate genes involved in tissue remodeling, promote collagen deposition, reduce unwanted inflammation, and enhance healing. The light-responsive CHPB-FD nanophotonic particles can, therefore, be potentially used as an economical and safe alternative to antibiotics for effectively decontaminating skin wounds and for disinfecting biomedical equipment and surfaces in hospitals and other places.

25 Measuring Intestinal Permeability When Urine is not an Option

Abstract The need to understand intestinal health is of increasing importance as the livestock industry seeks to maintain performance efficiency while eliminating prophylactic antibiotic usage. Intestinal permeability is a biomarker of intestinal health as it measures the movement of an indigestible marker across the intestinal barrier with excretion in the urine as the primary biological specimen collected for measurement. The adoption of these methods into poultry has been slow due to the lack of urine excretion. Therefore, the objective of this study is to compare differences in intestinal permeability assays in serum of poultry to determine if these assays could be a reliable method to detect changes in intestinal permeability in poultry under a known stressor. To compare the two commonly used methods [fluorescein isothiocyanate-dextran (FITC-D) method and lactulose/mannitol (LMS) dual sugar method], 2 independent trials were conducted with 40 pens split across 2 fed status (fed or fasted for 12 hours) and 4 sugar treatments (no sugar, FITC-D, LMS, FITC-D+LMS). All pens were sampled at 3 timepoints (14, 28, and 42 days of age) with 5 birds sampled per timepoint. Serum FITC-D was determined through fluorescent spectrophotometry, while mannitol and lactulose concentrations were determined using ion chromatography. Data were analyzed using PROC Glimmix. Fed status, sugar treatment, age, and all 2-and 3-way interactions were included as fixed effects. Trial was included in the model as a random effect with pen included as a repeated effect. Serum lactulose and FITC-D increased in fasted compared with fed birds (P &amp;lt; 0.006). Surprisingly, mannitol, which was expected to be similar across fed and fasted, was increased in fed compared with fasted birds (P &amp;lt; 0.001). The increased concentrations of mannitol in fed birds and measurable concentrations of mannitol in birds not given mannitol suggest another molecule eluted at the same rate as mannitol. Therefore, we propose under these conditions that mannitol, and its ratio with lactulose, are not appropriate methods for measuring intestinal permeability in poultry. Lactulose and FITC-D were increased in serum when the birds were provided those sugars and serum concentration of both sugars decreased at 28 days compared with 14 and 42 days (P &amp;lt; 0.003). FITC-D had a significant 2-way interaction for the sugar by fed status (P &amp;lt; 0.05) whereas other 2-and 3-way interactions were similar across treatments (P &amp;gt; 0.202). Serum lactulose concentrations were affected by all 2-and 3-way interactions with elevated serum lactulose concentrations in broilers provided lactulose and fasted (P &amp;lt; 0.001). Birds that did not receive lactulose had very low to undetectable concentrations of lactulose. This reduced background noise for the detection of serum lactulose and the ability statistically to detect small changes in intestinal permeability make lactulose a potential serum biomarker for intestinal permeability in poultry.

Enhancement of prostaglandin D2-D prostanoid 1 signaling reduces intestinal permeability by stimulating mucus secretion

IntroductionThe intestinal barrier plays a crucial role in distinguishing foods from toxins. Prostaglandin D2 (PGD2) is one of the lipid-derived autacoids synthesized from cell membrane-derived arachidonic acid. We previously reported that pharmacological stimulation of PGD2 receptor, D prostanoid 1 (DP1) attenuated the symptoms of azoxymethane/dextran sodium sulfate-induced colitis and ovalbumin-induced food allergy in mouse models. These observations suggested that DP1 stimulation protects the intestinal barrier. The present study aimed to uncover the effects of DP1 stimulation on intestinal barrier function and elucidate the underlying mechanisms.Materials and methodsIntestinal permeability was assessed in mice by measuring the transfer of orally administered fluorescein isothiocyanate-dextran (40 kDa) into the blood. The DP1 agonist BW245C (1 mg/kg) was administered 10 min prior to dextran administration. The intestinal permeability was confirmed using the ex vivo everted sac method. Tight junction integrity was evaluated in vitro by measuring the transepithelial electrical resistance (TER) in the human intestinal epithelial cell line Caco-2. Mucus secretion was assessed by observing Alcian Blue-stained intestinal sections.ResultsPharmacological DP1 stimulation reduced intestinal permeability both in vivo and ex vivo. Immunohistochemical staining showed that DP1 was strongly expressed on the apical side of the epithelial cells. DP1 stimulation did not affect TER in vitro but induced mucus secretion from goblet cells. Mucus removal by a mucolytic agent N-acetyl-l-cysteine canceled the inhibition of intestinal permeability by DP1 stimulation.ConclusionThese observations suggest that pharmacological DP1 stimulation decreases intestinal permeability by stimulating mucus secretion.

Quantitative analysis of gastrointestinal fluid absorption and secretion to estimate luminal fluid dynamics in rats

The drug absorption profile is dependent on the luminal drug concentration, which in turn is influenced by the gastrointestinal (GI) fluid dynamics. In the present study, therefore, we aimed to examine the luminal fluid dynamics by kinetically analyzing fluid absorption and secretion along the GI tract in rats using the in situ closed-loop technique with non-absorbable fluorescein isothiocyanate-dextran 4000 (FD-4) and tritium water labeling ([3H]water) under different osmotic conditions. We found that the luminal fluid volume in the jejunum and ileum, but not the colon, gradually decreased and reached a steady state. In contrast, [3H]water almost completely disappeared in all intestinal regions. Kinetic analysis revealed the following rank order for the rate constant of fluid secretion: jejunum > ileum > colon, whereas a negligible regional difference was observed in the rate constant of fluid absorption. Fluid secretion under an isosmotic condition (300 mOsm/kg) was higher than that at 0 mOsm/kg in all intestinal regions, though no significant changes in fluid absorption were observed. Thus, the fluid secretion process appears to be the major determinant of the regional differences in GI fluid dynamics. Our findings indicate that the luminal fluid volume is altered as a result of water ingestion, absorption, and secretion, and finally reaches an apparent steady state, which is regulated mainly by the process of fluid secretion.

NEDD4L Promotes IκBα Ubiquitination and Degradation in the Pathogenesis of Diabetic Retinopathy

Purpose The dysregulation of NF-κB signaling activity plays an important role in the pathogenesis of diabetic retinopathy (DR). This study explored the association between NEDD4L and IκBα in DR. Methods The rat model of diabetes was established and altered retinal vascular permeability in these rats was examined through an Evans blue dye assay. A range of glucose concentrations were used to treat retinal vascular endothelial cells (RVECs). The cells viability and apoptosis were assessed through MTT and flow cytometry, while shifts in cell permeability were examined by transendothelial resistance (TEER) and FITC dextran assay. The interaction of NEDD4L and IκBα was tested by Co-IP, while mRNA and protein levels were assessed via qPCR and Western blotting, respectively. Results High glucose suppressed proliferative activity of RVECs, and promoted apoptosis and the protein level of NEDD4L and NF-κB p65, but decreased IκBα. NEDD4L knockdown reversed the changes in inflammation, oxidative stress, and permeability in RVECs exposed to high glucose. Similarly, NEDD4L silencing reverted observed TEER decreases, increased monolayer permeability to FITC dextran, and ZO-1 and Claudin-5 downregulation in response to high glucose. Conversely, the impact of NEDD4L overexpression was reversed by the NF-κB inhibitor PDTC treatment. NEDD4L induced the ubiquitination of IκBα in an IKK-2-dependent manner. Moreover, siNEDD4L treatment alleviated the symptoms of DR through the inactivation of NF-κB signaling in vivo. Conclusions NEDD4L could enhance inflammation, oxidative stress, and permeability in the retinal vascular endothelium by facilitating the ubiquitination of IκBα in an IKK-2-dependent manner. Our results support a role for NEDD4L in the pathogenesis of DR.

Engineered Knockout of TRPA1 Inhibits Laser-Induced Choroidal Neovascularization Along With Associated TGFβ1 Expression and Neutrophil Infiltration

We examined the effects of gene ablation and chemical inhibition of transient receptor potential ankyrin 1 (TRPA1) on the growth of experimental argon laser–induced choroidal neovascularization (CNV) in mice. CNV was induced in the eyes of 6- to 8-week-old TRPA1-null (knockout [KO]) and wild-type (WT) mice by argon laser irradiation. Gene expression analysis was performed in laser-injured tissues at days 1 and 3. CNV growth was evaluated at day 14. Reciprocal bone marrow transplantation was performed between each genotype to identify the components responsible for either recipient tissue or bone marrow–derived inflammatory cells. Our results show that laser irradiation successfully induced CNV growth at the site of laser injury. The size of induced CNV was significantly smaller in KO mice than in WT mice at day 14, as determined by angiography with fluorescein isothiocyanate-dextran. Invasion of neutrophils, but not macrophages, was suppressed in association with suppression of the expression of transforming growth factor β1 and interleukin 6 in laser-irradiated KO tissue. Bone marrow transplantation indicated that the genotype of the recipient mouse, but not of inflammatory cells, is attributable to the KO phenotype. Systemic administration of a TRPA1 antagonist also reduced the CNV in a WT mouse. In conclusion, TRPA1 signaling in local cells is involved in growth of laser-induced CNV. The phenotype was not attributable to vascular endothelial cells and inflammatory cells. Blocking TRPA1 signal may therefore be a potential treatment strategy for CNV-related ocular diseases.

Evaluation of Aspergillus meal prebiotic in productive parameters, bone mineralization and intestinal integrity in broiler chickens

This study investigates the effects of Aspergillus meal prebiotic on a spectrum of parameters, including productive performance, bone mineralization, and intestinal integrity, in broiler chickens. Day-of-hatch Cobb 500 by-product male chicks (320 total) were randomly assigned to two experimental groups (G1 and G2) in floor pens (150×300 cm), each pen containing separate feeders and watering systems (8 replicates per treatment, 20 birds/pen). Chickens kept in G1 (control) were fed a basal diet, while those of G2 (treated) were fed a basal diet supplemented with 0.2% PRI-A-FERM. Performance parameters, including body weight (BW), body weight gain (BWG), feed intake (FI), and feed conversion ratio (FCR), were evaluated weekly from the 7th day to the end of the trial at 42 d. Fluorescein isothiocyanate-dextran (FITC-d) was administered via oral gavage to 3 chickens/replicate (n = 24), and blood was collected on 21d and on 42d to determine serum concentration of FITC-d as a biomarker to evaluate intestinal permeability and peptide YY as an indirect biomarker to evaluate short chain fatty acids. Chickens that received the \textit{Aspergillus} meal prebiotic showed a significant increase in BW, BWG, and FI on days 14, 21, 28, 35, and 42 (P&lt;0.05) and a significant reduction in FCR on days 28, 35, and 42 of evaluation compared to control. Moreover, chickens that were supplemented with the Aspergillus meal prebiotic showed a significant reduction in serum concentrations of FITC-d on both days of evaluation, as well as a significant increase in serum concentration of peptide YY. Additionally, treated animals significantly increased bone strength, total bone ash, and calcium and phosphorous content at both evaluation times compared to the control. In summary, the results of this study suggest that Aspergillus meal prebiotic supplementation can positively influence productive parameters, bone mineralization, and intestinal integrity in broiler chickens. The observed improvements underscore the importance of gut health in overall poultry performance. The findings contribute to the growing body of knowledge on the benefits of prebiotics (0.2%, PRI-A-FERM) in animal nutrition and highlight the potential for their practical application in enhancing poultry production systems. Further research is warranted to elucidate the underlying mechanisms and optimize prebiotic utilization for improved broiler health and productivity

Lidocaine ameliorates intestinal barrier dysfunction in irritable bowel syndrome by modulating corticotropin-releasing hormone receptor 2.

Intestinal barrier dysfunction is a prevalent pathogenic factor underlying various disorders. Currently there is no effective resolution. Previous studies have reported the potential anti-inflammatory properties of lidocaine and its ability to alleviate visceral hypersensitivity in individuals with irritable bowel syndrome (IBS). Therefore, our study will further verify the effect of lidocaine on intestinal barrier dysfunction in IBS and investigate the underlying mechanisms. In this study, we investigated the role of lidocaine by assessing visceral hypersensitivity, body weight, inflammatory factors, fluorescein isothiocyanate-dextran 4000 (FD4) flux, tight junctions (TJs) and spleen and thymus index in rats subjected to water avoidance stress (WAS) to mimic intestinal barrier dysfunction in IBS with and without lidocaine. In vitro, we investigated the role of corticotropin-releasing hormone receptor 2 (CRHR2) in lidocaine-treated Caco2 cells using small interfering RNA (siRNA) targeting CRHR2. In WAS rats, lidocaine significantly restored weight loss, damaged TJs, spleen index and thymus index and inhibited abdominal hypersensitivity as well as blood levels of markers indicating intestinal permeability, such as diamine oxidase (DAO), D-lactic acid (D-Lac) and lipopolysaccharide (LPS). Consequently, the leakage of FD4 flux from intestine was significantly attenuated in lidocaine group, and levels of intestinal inflammatory factors (IL-1β, IFN-γ, TNF-α) were reduced. Interestingly, lidocaine significantly suppressed corticotropin-releasing hormone (CRH) levels in lamina propria cells, while the CRH receptor CRHR2 was upregulated in intestinal epithelial cells. In vitro, lidocaine enhanced the expression of CRHR2 on Caco-2 intestinal epithelial cells and restored disrupted TJs and the epithelial barrier caused by LPS. Conversely, these effects were diminished by a CRHR2 antagonist and siRNA-CRHR2, suggesting that the protective effect of lidocaine depends on CRHR2. Lidocaine ameliorates intestinal barrier dysfunction in IBS by potentially modulating the expression of CRHR2 on intestinal epithelial cells.

Taraxacum coreanum Nakai extract attenuates lipopolysaccharide-induced inflammatory responses and intestinal barrier dysfunction in Caco-2 cells

Ethnopharmacological relevanceTaraxacum coreanum Nakai (TC) is a dandelion native to Korea that has long been used as a medicinal herb with antioxidant and anti-inflammatory properties. Intestinal inflammation is closely associated with intestinal epithelial barrier disruption, which leads to the progression of various intestinal diseases. Aim of the studyThe aim of this study was to investigate the protective effects of TC extract on inflammatory responses and intestinal barrier dysfunction in lipopolysaccharide (LPS)-stimulated Caco-2 cells. Materials and methodsThe inhibitory effect of TC on nitric oxide (NO) and pro-inflammatory cytokines production were determined by Griess reagent and enzyme-linked immunosorbent assay, respectively. The epithelial permeability was evaluated by transepithelial electrical resistance (TEER) assay, and inflammation- and tight junction (TJ)-related protein expression were analyzed by Western blotting. In addition, the presence of ten active compounds was identified and quantified using UHPLC-ESI-MS and HPLC-DAD analyses. ResultsTreatment with TC significantly reduced NO production and pro-inflammatory cytokines production [interleukin (IL)-6 and tumor necrosis factor (TNF)-α] compared to the group treated with LPS only, particularly at 100 μg/mL. TC significantly decreased monolayer permeability as detected by TEER. In addition, the transmission of fluorescein isothiocyanate-dextran 4 across the barrier was decreased after treatment with TC. Inflammation-related proteins (inducible NO synthase, cyclooxygenase-2, TNF-α, IL-6, and IL-1β) were down-regulated after treatment with TC. In contrast, TC significantly increased the protein levels of the TJ-related protein, claudin-5. Ten phytochemicals (protocatechuic acid, chlorogenic acid, caffeic acid, scopoletin, chicoric acid, hyperoside, nicotiflorin, luteoloside, sophoricoside, and luteolin) were identified by UHPLC-ESI-MS and HPLC-DAD analysis. ConclusionOur findings suggest that ethanolic extract of TC could attenuate the LPS-induced intestinal barrier dysfunction by increasing the TJ protein and suppressing inflammatory responses.

Quantitative intravitreal pharmacokinetics in mouse as a step towards inter-species translation

Although mouse models are widely used in retinal drug development, pharmacokinetics in mouse eye is poorly understood. In this study, we applied non-invasive in vivo fluorophotometry to study pharmacokinetics of intravitreal fluorescein sodium (molecular weight 0.38 kDa) and fluorescein isothiocyanate–dextran (FD-150; molecular weight 150 kDa) in mice. Intravitreal half-lives of fluorescein and FD-150 in mouse eyes were 0.53 ± 0.06 h and 2.61 ± 0.86 h, respectively. These values are 8–230 times shorter than the elimination half-lives of similar compounds in the human vitreous. The apparent volumes of distribution in the mouse vitreous were close to the anatomical volume of the mouse vitreous (FD-150, 5.1 μl; fluorescein, 9.6 μl). Dose scaling factors were calculated from mouse to rat, rabbit, monkey and human translation. Based on pharmacokinetic modelling and compound concentrations in the vitreous and anterior chamber, fluorescein is mainly eliminated posteriorly across blood-retina barrier, but FD-150 is cleared via aqueous humour outflow. The results of this study improve the knowledge of intravitreal pharmacokinetics in mouse and facilitate inter-species scaling in ocular drug development.

Treatment with S-adenosylmethionine ameliorates irinotecan-induced intestinal barrier dysfunction and intestinal microbial disorder in mice

This study aimed to investigate the protective effects of S-adenosylmethionine (SAM) on irinotecan-induced intestinal barrier dysfunction and microbial ecological dysregulation in both mice and human colon cell line Caco-2, which is widely used for studying intestinal epithelial barrier function. Specifically, this study utilized Caco-2 monolayers incubated with 7-ethyl-10-hydroxycamptothecin (SN-38) as well as an irinotecan-induced diarrhea model in mice.Our study found that SAM pretreatment significantly reduced body weight loss and diarrhea induced by irinotecan in mice. Furthermore, SAM inhibited the increase of intestinal permeability in irinotecan-treated mice and ameliorated the decrease of Zonula occludens-1(ZO-1), Occludin, and Claudin-1 expression. Additionally, irinotecan treatment increased the relative abundance of Proteobacteria compared to the control group, an effect that was reversed by SAM administration. In Caco-2 monolayers, SAM reduced the expression of reactive oxygen species (ROS) and ameliorated the decrease in transepithelial electrical resistance (TER) and increase in fluorescein isothiocyanate–dextran 4000 Da (FD-4) flux caused by SN-38. Moreover, SAM attenuated changes in the localization and distribution of ZO-1and Occludin in Caco-2 monolayers induced by SN-38 and protected barrier function by inhibiting activation of the p38 MAPK/p65 NF-κB/MLCK/MLC signaling pathway. These findings provide preliminary evidence for the potential use of SAM in treating diarrhea caused by irinotecan.

Study Transportation of Drugs within Newly Established Murine Colon Organoid Systems.

The development of 3D organoids of the small intestine is a tremendous breakthrough in drug development and biological research. However, the development of colonic organoids (i.e., colonoids) is particularly challenging due to a lack of simple, cost-effective protocols for colonoid cultivation. Here, intestinal homogenates are described as a supplement to the culture medium for maintaining and replicating colonic stem cells. Colonoids generated by this cultivation protocol demonstrate substantial proliferation and differentiation (3 months). There is a similarity between cultured colonoids and primary colon tissue regarding structure and functionality. To evaluate the functionality of colonoids, permeability testing is performed with suspensions of 4 and 40kDa fluorescein isothiocyanate-dextran (FITC-DEX). It is observed that neither can permeate the healthy epithelial barrier. The P-glycoprotein receptor, a vital drug efflux pump mitigating potential drug toxicity, is functionally manipulated, as evidenced by its inhibition function by verapamil and monitoring uptake of Rhodamin 123. In addition, Forskolin treatment which affects chloride transport results in organoid swelling; this confirms the functional expression of the CFTR transporter in the colonoids. This protocol to generate colonoids is promising for high-throughput drug screening, toxicity testing, and oral drug development.

A Physiologically Relevant Dose of 50% Egg-Phosphatidylcholine Is Sufficient in Improving Gut Permeability while Attenuating Immune Cell Dysfunction Induced by a High-Fat Diet in Male Wistar Rats

BackgroundObesity is associated with increased intestinal permeability and a diminished immune response. Phosphatidylcholine (PC), a form of choline found in eggs, has been shown to beneficially modulate T-cell response in the context of obesity when provided as the sole form of choline in the diet. ObjectiveThis study aimed to determine the impact of varying doses of PC as part of a high-fat diet (HFD) on immune cell function and intestinal permeability. MethodsMale Wistar rats 4 wk of age were randomly assigned to consume 1 of 6 diets for 12 wk containing the same amount of total choline but differing in the forms of choline: 1—control low-fat (CLF, 20% fat, 100% free choline [FC]); 2—control high-fat (CHF, 50% fat, 100% FC); 3—100% PC (100PC, 50% fat, 100% egg-PC); 4—75% PC (75PC, 50% fat, 75% egg-PC+25% FC); 5—50% PC (50PC, 50% fat, 50% egg-PC+50% FC); and 6—25% PC (25PC; 50% fat, 25% egg-PC+75% FC). Intestinal permeability was measured by fluorescein isothiocyanate–dextran. Immune function was assessed by ex vivo cytokine production of splenocytes and cells isolated from the mesenteric lymph node (MLN) after stimulation with different mitogens. ResultsFeeding the CHF diet increased intestinal permeability compared with the CLF diet, and doses of PC 50% or greater returned permeability to levels similar to that of the CLF diet. Feeding the CHF diet lowered splenocyte production of interleukin (IL)-1β, IL-2, IL-10, and tumor necrosis factor-alpha, and MLN production of IL-2 compared with the CLF group. The 50PC diet most consistently significantly improved cytokine levels (IL-2, IL-10, tumor necrosis factor-alpha) compared with the CHF diet. ConclusionsOur results show that a dose of 50% of total choline derived from egg-PC can ameliorate HFD-induced intestinal permeability and immune cell dysfunction.

A mannitol-modified emodin nano-drug restores the intestinal barrier function and alleviates inflammation in a mouse model of DSS-induced ulcerative colitis

BackgroundUlcerative colitis (UC) is an inflammatory disease of the colon that is characterized by mucosal ulcers. Given its increasing prevalence worldwide, it is imperative to develop safe and effective drugs for treating UC. Emodin, a natural anthraquinone derivative present in various medicinal herbs, has demonstrated therapeutic effects against UC. However, low bioavailability due to poor water solubility limits its clinical applications.MethodsEmodin-borate nanoparticles (EmB) were synthesized to improve drug solubility, and they modified with oligomeric mannitol into microgels (EmB-MO) for targeted delivery to intestinal macrophages that express mannose receptors. UC was induced in a mouse model using dextran sulfate sodium (DSS), and different drug formulations were administered to the mice via drinking water. The levels of inflammation-related factors in the colon tissues and fecal matter were measured using enzyme-linked immunosorbent assay. Intestinal permeability was evaluated using fluorescein isothiocyanate dextran. HE staining, in vivo imaging, real-time PCR, and western blotting were performed to assess intestinal barrier dysfunction.ResultsBoth EmB and EmB-MO markedly alleviated the symptoms of UC, including body weight loss, stool inconsistency, and bloody stools and restored the levels of pro- and anti-inflammatory cytokines. However, the therapeutic effects of EmB-MO on the macroscopic and immunological indices were stronger than those of EmB and similar to those of 5-aminosalicylic acid. Furthermore, EmB-MO selectively accumulated in the inflamed colon epithelium and restored the levels of the gut barrier proteins such as ZO-1 and Occludin.ConclusionsEmB-MO encapsulation significantly improved water solubility, which translated to greater therapeutic effects on the immune balance and gut barrier function in mice with DSS-induced UC. Our findings provide novel insights into developing emodin-derived drugs for the management of UC.