Transepithelial Electrical Resistance Values Research Articles

Delivery of Vitamins through the Intraoral Cavity with Toothpaste Using Penetration Enhancers

Dental health is rarely deemed a significant factor for a healthy life despite its considerable impact on the human body. Many presume that regular dental check-ups and daily toothbrushing are sufficient to prevent oral health diseases. However, consuming certain nutrients is also required to attain a healthy oral cavity. Nutrients necessary for preserving dental health, namely calcium, phosphorus, fluorine, and various vitamins, contribute to the development, maintenance, and repair of oral tissues. Nevertheless, it can be challenging to be conscious of taking such nutrients on a daily basis. In this study, aiming to abolish such inconveniences, vitamins essential for dental health were combined in a toothpaste formulation with penetration enhancers (PEs) to achieve intraoral delivery of the vitamins while toothbrushing. Multiple tests were performed to evaluate the characteristics of the intraoral vitamin-delivery toothpaste (IOVT), such as abrasiveness and antibacterial strength tests. The same tests were also conducted on three different commercial toothpaste to draw comparisons with the IOVT. Additionally, to verify the IOVT’s penetration capability, a mass transport study, and a transepithelial electric resistance (TEER) value test were conducted using nightcrawlers’ skin. The data collected demonstrated congruency between multiple characteristics of the IOVT and those of the commercial toothpaste. Our key finding was that the greater TEER values demonstrated the high plausibility of effective intraoral delivery of vitamins, assisted by PEs, as components of the IOVT.

Differential Effects of Benzalkonium Chloride on Human Trabecular Meshwork Cells Not Treated or Treated with Transforming Growth Factor-β2 or Dexamethasone.

Purpose: The objective of the present study was to evaluate the effects of low concentrations of benzalkonium chloride (BAC) (10-7%, 10-6%, or 10-5%) on healthy and glaucomatous human trabecular meshwork (HTM) cells. For this purpose, we used in vitro models replicating a healthy HTM and HTM with primary open-angle glaucoma (POAG) or steroid-induced glaucoma (SG) using two-dimensional (2D) cultures of HTM cells not treated or treated with a 5 ng/mL solution of transforming growth factor-β2 or 250 nM dexamethasone (DEX). Methods: Analyses were carried out for (1) the intercellular affinity function of 2D HTM monolayers, as determined by transepithelial electrical resistance (TEER) measurements; (2) cell viability; (3) cellular metabolism by using a Seahorse bioanalyzer; and (4) expression of extracellular matrix (ECM) molecules, an ECM modulator, and cell junction-related molecules. Results: In the absence and presence of BAC (10-7% or 10-5%), intercellular affinity function determined by TEER and cellular metabolic activities were significantly and dose dependently affected in both healthy and glaucomatous HTM cells despite the fact that there was no significant decrease in cell viabilities. However, the effects based on TEER values were significantly greater in the healthy HTM. The mRNA expression of several molecules that were tested was not substantially modulated by these concentrations of BAC. Conclusions: The findings reported herein suggest that low concentrations of BAC may have unfavorable adverse effects on cellular metabolic capacity by inducing increases in the intercellular affinity properties of the HTM, but those effects of BAC were different in healthy and glaucomatous HTM cells.

Graphene oxide nanosheets enhancing the photothermal response of a cellular barrier exposed to near infrared light

AbstractRecent developments in targeted photothermal therapy focus on integrating near‐infrared (NIR) light and biocompatible nanostructures. It is yet unclear if graphene oxide nanosheets (GO) can assist in the photothermal response of cellular barriers under NIR irradiation. Herein, this study investigates the photothermal response of a cellular barrier treated with different concentrations of GO under the irradiation of NIR light. The synthesized GO nanosheets show good stability in aqueous media. No toxic effects are imposed onto human umbilical vein endothelial cells (HUVECs) when the cells are treated with GO up to 20 μg/mL for 1 day. In addition, the in vitro cellular barrier model with intercellular tight junctions is formed by using HUVECs, which have a high transepithelial electrical resistance (TEER) value of 89.3 Ω · cm2. Under the irradiation of NIR light (980 nm), the photothermal response on the cellular barrier is enhanced by introducing GO, which has been verified by the decrease of TEER value and the increase in the transport of dextran. The results indicate that the thermal response of a cellular barrier exposed to NIR light is non‐linearly enhanced with increasing concentration of GO.

Novel applications of Yinhua Miyanling tablets in ulcerative colitis treatment based on metabolomics and network pharmacology

BackgroundYinhua Miyanling tablets (YMT), comprising 10 Chinese medicinal compounds, is a proprietary Chinese medicine used in the clinical treatment of urinary tract infections. Medicinal compounds, extracts, or certain monomeric components in YMT all show good effect on ulcerative colitis (UC). However, no evidence supporting YMT as a whole prescription for UC treatment is available. PurposeTo evaluate the anti-UC activity of YMT and elucidate the underlying mechanisms. The objective of the study was to provide evidence for the add-on development of YMT to treat UC. MethodsFirst, YMT's protective effect on the intestinal barrier was evaluated using a lipopolysaccharide (LPS)-induced Caco-2 intestinal injury model. Second, the UC mouse model was established using dextran sodium sulfate (DSS) to determine YMT's influence on symptoms, inflammatory factors, intestinal barrier, and histopathological changes in the colon. Third, an integrated method combining metabolomics and network pharmacology was employed to screen core targets and key metabolic pathways with crucial roles in YMT's therapeutic effect on UC. Molecular docking was employed to identify the key targets with high affinity. Finally, western blotting was performed to validate the mechanism of YMT action against UC. ResultsYMT enhanced the transepithelial electrical resistance value and improved the expression of proteins of the tight junctions dose-dependently in LPS-induced Caco-2 cells. UC mice treated with YMT exhibited alleviated pathological lesions of the colon tissue in the in vivo pharmacodynamic experiments. The colonic lengths tended to be normal, and the levels of inflammatory factors (TNF-α, IL-6, and iNOS) along with those of the core enzymes (MPO, MDA, and SOD) improved. YMT effectively ameliorated DSS-induced colonic mucosal injury; pathological changes along with ultrastructure damage were significantly alleviated (evidenced by a relatively intact colon tissue, recovery of epithelial damage, repaired gland, reduced infiltration of inflammatory cells and epithelial cells arranged closely with dense microvilli). Seven key targets (IL-6, TNF-α, MPO, COX-2, HK2, TPH, and CYP1A2) and four key metabolic pathways (arachidonic acid metabolism, linoleate metabolism, glycolysis, and gluconeogenesis and tyrosine biosynthesis) were identified to play vital roles in the treatment on UC using YMT. ConclusionsYMT exerts beneficial therapeutic effects on UC by regulating multiple endogenous metabolites, targets, and metabolic pathways, suggestive of its potential novel application in UC treatment.

Protective effects of galacto-oligosaccharide and polydextrose on Caco-2 intestinal cell barrier injury model

To explore the protective effect of different ratios of galactose oligosaccharide(GOS) and polydextrose(PDX) on intestinal cell barrier damage model of Caco-2. The same batch of Caco-2 cells were cultured to form a cell barrier model and randomly divided into damaged model group without calcium, calcium-containing blank control group(1.8 mmol/L Ca~(2+)), low-ratio/low-dose group(1.8 mmol/L Ca~(2+)+2 mg/mL GOS+2 mg/mL PDX) and low-ratio/medium-dose group(1.8 mmol/L Ca~(2+)+4 mg/mL GOS+4 mg/mL PDX), low-ratio/high-dose group(1.8 mmol/L Ca~(2+)+8 mg/mL GOS+8 mg/mL PDX) and high-ratio/low-dose group(1.8 mmol/L Ca~(2+)+0.8 mg/mL GOS+3.2mg/mL PDX), high-ratio/medium-dose group(1.8 mmol/L Ca~(2+)+1.6 mg/mL GOS+6.4 mg/mL PDX), high-ratio/high-dose group(1.8 mmol/L Ca~(2+)+3.2mg/mL GOS+12.8 mg/mL PDX), a total of 8 groups, three parallel groups were performed in each group. The Trans Epithelial Electrical Resistance value and apparent permeability coefficient value of each group were determined after 4 d culture, and the morphology of tight junction proteins ZO-1, Occludin and Claudin-1 were observed by immunofluorescence method, and the expression levels of inflammatory related factors in each group were determined by protein microarray method. Compared with damaged model group, TEER ratio in calcium-containing blank control group was significantly increased(P<0.05), while Papp value was significantly decreased(P<0.05);Compared with calcium-containing blank control group, TEER ratio in low-ratio/medium-dose group and high-ratio/high-dose group was significantly increased(P<0.05) while Papp value was significantly decreased(P<0.05), and they could significantly down-regulate some inflammatory response related cytokines. The cell barrier was intact in all groups except for the compact junction protein structure in the model group. Compared with Ca~(2+) alone, the combination of two prebiotics can enhance the density of Caco-2 cell barrier and reduced the permeability of cell bypass. And it can significantly reduce the expression level of some inflammatory cytokines and effectively protect the intestinal cell barrier.

Method for Two-Dimensional Epithelial Monolayer Formation Derived from Mouse Three-Dimensional Small Intestinal Organoids.

The intestinal epithelium is composed of two distinct structures, namely, the villi and crypts. The base of the crypts contains intestinal stem cells (ISCs), which support the high regenerative capacity of the intestinal epithelium. With the establishment of the three-dimensional (3D) organoid culture method, the cellular and molecular mechanisms of differentiation, proliferation, and maintenance of ISCs have been widely analyzed. However, the sphere-like morphology of the 3D organoids prevents access to the apical side of the epithelium. To overcome this limitation, two-dimensional (2D) monolayer cultures derived from 3D organoids have been attempted; however, 2D culture methods for the mouse small intestine have not been well established. In this study, we developed a simple method that uses only commercially available materials, for the formation of 2D epithelial monolayers from mouse 3D small intestinal organoids. Using this method, confluent 2D epithelial monolayers were established within 4days. This monolayer showed stable tight junction and included ISCs and differentiated intestinal cells. It also showed physiologically relevant transepithelial electrical resistance values. On the basis of these findings, this method opens a novel platform for analyzing the physiology of the intestinal epithelium, its interaction with microbes, and mechanisms of villus formation.

Beneficial effects of GABA-producing potential probiotic Limosilactobacillus fermentum L18 of human origin on intestinal permeability and human gut microbiota

BackgroundGamma-aminobutyric acid (GABA) is a non-protein amino acid with neuroinhibitory, antidiabetic, and antihypertensive properties and is used as a drug for treating anxiety and depression. Some strains of lactobacilli are known to produce GABA and strengthen the gut barrier function which play an important role in ameliorating the effects caused by the pathogen on the gut barrier. The probiotic bacteria are also known to modulate the human fecal microbiota, however, the role of GABA-producing strains on the gut epithelium permeability and gut microbiota is not known.ResultsIn this study, we report the production of high levels of GABA by potential probiotic bacterium Limosilactobacillus fermentum L18 for the first time. The kinetics of the production of GABA by L18 showed that the maximum production of GABA in the culture supernatant (CS) occurred at 24 h, whereas in fermented milk it took 48 h of fermentation. The effect of L18 on the restoration of lipopolysaccharide (LPS)-disrupted intestinal cell membrane permeability in Caco-2 monolayers showed that it significantly restored the transepithelial electrical resistance (TEER) values, by significantly increasing the levels of junction proteins, occludin and E-cadherin in L18 and LPS-treated Caco-2 cells as compared to only LPS-treated cells. The effect of GABA-secreting L18 on the metataxonome of human stool samples from healthy individuals was investigated by a batch fermentor that mimics the conditions of the human colon. Although, no differences were observed in the α and β diversities of the L18-treated and untreated samples at 24 h, the relative abundances of bacterial families Lactobacillaceae and Bifidobacteriaceae increased in the L18-treated group, but both decreased in the untreated groups. On the other hand, the relative abundance of Enterobacteriaceae decreased in the L18 samples but it increased in the untreated samples.ConclusionThese results indicate that Li. fermentum L18 is a promising GABA-secreting strain that strengthens the gut epithelial barrier by increasing junction protein concentrations and positively modulating the gut microbiota. It has the potential to be used as a psychobiotic or for the production of functional foods for the management of anxiety-related illnesses.

The Effects of ROCK Inhibitor on Prevention of Dexamethasone-Induced Glaucoma Phenotype in Human Trabecular Meshwork Cells.

This study investigated the effects of dexamethasone (Dex) on human trabecular meshwork (TM) cells, a model of glucocorticoid-induced glaucoma, and evaluated the impact of ripasudil (Rip) as a co-delivery or sequential dosing strategy. In vitro experiments were conducted to assess the effects of Dex and Rip on TM cells. Confocal microscopy was used to evaluate the impact of Dex and Rip on F-actin staining signals. Contractility of the TM cells upon Dex and Rip treatment mimicking co-delivery and sequential delivery was quantified using collagen gel contraction assay. Transepithelial electrical resistance (TEER) values and fluorescein isothiocyanate (FITC)-dextran permeability were also measured to assess the impact of Dex and Rip on TM cells. Dex and Rip did not exhibit cytotoxicity at the maximum tested concentration (20 µM). Dex-treated TM cells exhibited higher F-actin staining signals compared to controls, which were reduced when co-treated with Rip. Rip inhibited Dex-induced collagen gel contraction activity in both co-delivery and sequential treatments. Dex resulted in increased TEER values as the dose increased, whereas TEER values were maintained when co-treated with Rip. Co-delivery of Rip has the potential to prevent glaucoma symptoms when patients are treated with Dex. This study highlights the importance of identifying strategies to reduce the side effects of prolonged use of glucocorticoids, such as Dex, in the treatment of various diseases. This study demonstrates the potential of co-delivering ripasudil with dexamethasone to mitigate glucocorticoid-induced ocular hypertension and a secondary glaucoma that resembles primary open-angle glaucoma, providing insights for the development of novel preventive strategies in clinical care.

Evaluation of arsenic metabolism and tight junction injury after exposure to arsenite and monomethylarsonous acid using a rat in vitro blood-Brain barrier model.

Experimental verification of impairment to cognitive abilities and cognitive dysfunction resulting from inorganic arsenic (iAs) exposure in children and adults is challenging. This study aimed to elucidate the effects of arsenite (iAsIII; 1, 10 and 20 μM) or monomethylarsonous acid (MMAIII; 0.1, 1 and 2 μM) exposure on arsenic metabolism and tight junction (TJ) function in the blood-brain barrier (BBB) using a rat in vitro-BBB model. The results showed that a small percentage (~15%) of iAsIII was oxidized or methylated within the BBB, suggesting the persistence of toxicity as iAsIII. Approximately 65% of MMAIII was converted to low-toxicity monomethylarsonic acid and dimethylarsenic acid via oxidation and methylation. Therefore, it is estimated that MMAIII causes TJ injury to the BBB at approximately 35% of the unconverted level. TJ injury of BBB after iAsIII or MMAIII exposure could be significantly assessed from decreased expression of claudin-5 and decreased transepithelial electrical resistance values. TJ injury in BBB was found to be significantly affected by MMAIII than iAsIII. Relatedly, the penetration rate in the BBB by 24 h of exposure was higher for MMAIII (53.1% ± 2.72%) than for iAsIII (43.3% ± 0.71%) (p < 0.01). Exposure to iAsIII or MMAIII induced an antioxidant stress response, with concentration-dependent increases in the expression of nuclear factor-erythroid 2-related factor 2 in astrocytes and heme oxygenase-1 in a group of vascular endothelial cells and pericytes, respectively. This study found that TJ injury at the BBB is closely related to the chemical form and species of arsenic; we believe that elucidation of methylation in the brain is essential to verify the impairment of cognitive abilities and cognitive dysfunction caused by iAs exposure.

Microfluidic-assisted preparation of PLGA nanoparticles loaded with insulin: a comparison with double emulsion solvent evaporation method

Poly lactic-co-glycolic acid (PLGA) is an ideal polymer for the delivery of small and macromolecule drugs. Conventional preparation methods of PLGA nanoparticles (NPs) result in poor control over NPs properties. In this research, a microfluidic mixer was designed to produce insulin-loaded PLGA NPs with tuned properties. Importantly; aggregation of the NPs through the mixer was diminished due to the coaxial mixing of the precursors. The micromixer allowed for the production of NPs with small size and narrow size distribution compared to the double emulsion solvent evaporation (DESE) method. Furthermore, encapsulation efficiency and loading capacity indicated a significant increase in optimized NPs produced through the microfluidic method in comparison to DESE method. NPs prepared by the microfluidic method were able to achieve a more reduction of trans-epithelial electrical resistance values in the Caco-2 cells compared to those developed by the DESE technique that leads to greater paracellular permeation. Compatibility and interaction between components were evaluated by differential scanning calorimetry and fourier transform infrared analysis. Also, the effect of NPs on cell toxicity was investigated using MTT test. Numerical simulations were conducted to analyze the effect of mixing patterns on the properties of the NPs. It was revealed that by decreasing flow rate ratio, i.e. flow rate of the organic phase to the flow rate of the aqueous phase, mixing of the two streams increases. As an alternative to the DESE method, high flexibility in modulating hydrodynamic conditions of the microfluidic mixer allowed for nanoassembly of NPs with superior insulin encapsulation at smaller particle sizes.

Characterization of critical parameters using an air-liquid interface model with RPMI 2650 cells for permeability studies of small molecules.

The field of nasal drug delivery gained enormously on interest over the past decade. Performing nasal in vivo studies is expensive and time-consuming, but also unfeasible for an initial high-throughput compound and formulation screening. Therefore, the development of fast and high-throughput in vitro models to screen compounds for their permeability through the nasal epithelium and mucosa is constantly expanding. Yet, the protocols used for nasal in vitro permeability studies are varying, which limits the comparability and reproducibility of generated data. This project aimed to elucidate the influence of different culture and assay parameters of RPMI 2650 cells grown under air-liquid interface (ALI) conditions on the transepithelial electrical resistance (TEER) and apparent permeability (Papp) values of five selected reference compounds, covering the range of low to moderate to high permeability. The influence of the passage number, seeding density, and timepoint of airlift was minimal in our approach, while the substrate pore density had a significant influence on the Papp values of carbamazepine, propranolol, and metoprolol, classified as highly permeable compounds, but not on atenolol and aciclovir. Elevation of the experimental concentration of carbamazepine, propranolol, and metoprolol in the donor compartment had an increasing effect on the Papp values, while prolonging the assay time did not have a significant influence. Based on the results reported here, RPMI 2650 cells cultured under ALI conditions offer the possibility of a standardized high-throughput screening model for small molecules and their formulations for in vitro drug permeation studies to predict and select optimal conditions for their nasal delivery.

Sex-Dependent Differences in Blood-Urine Barrier Are Subtle but Significant in Healthy and Chronically Inflamed Mouse Bladders.

The urothelium is a vital permeability barrier that prevents the uncontrolled flow of urinary components into and out of the bladder interstitium. Our study addressed the question of possible sex-specific variations in the urothelium of healthy mice and their impact on chronic bladder inflammation. We found that healthy female bladders have a less robust barrier function than male bladders, as indicated by significant differences in transepithelial electrical resistance (TEER) values. These differences could be attributed to detected higher claudin 2 mRNA expression and a less pronounced glycocalyx in females than in males. In addition, TEER measurements showed delayed barrier recovery in chronically inflamed female bladders. We found subtle differences in the expressions of genes involved in the regulation of the actin cytoskeleton between the sexes, as well as pronounced urothelial hyperplasia in females compensating for attenuated barrier function. The identified genetic variations in glycosylation pathways may also contribute to this divergence. Our findings add to the growing body of literature on the intricate sex-specific nuances of urothelial permeability function and their implications for chronic bladder inflammation. Understanding these differences could lead to tailored diagnostic and therapeutic approaches in the treatment of bladder disorders in the future.

Evaluation of Nephrotoxic Properties of Favipiravir Using the RPTEC cell model

Scientific relevance. Favipiravir is an antiviral RNA polymerase inhibitor used to treat COVID-19. An adverse drug reaction associated with the use of favipiravir is renal disorder.Aim. This study aimed to investigate favipiravir nephrotoxicity by assessing its effects on the integrity of a monolayer formed by renal proximal tubular epithelial cells (RPTECs).Materials and methods. This study focused on an RPTEC monolayer culture that was seeded at a density of 6×104 cells/cm2 on plates with membrane inserts with 0.4 μm pores. Favipiravir was added to the plate wells at a concentration of 5, 10, or 15 μg/mL. The nephrotoxicity evaluation relied on measuring the transepithelial electrical resistance (TEER) of the RPTEC monolayer. A TEER value of 120–140 Ω×cm2 was considered an indication of nephrotoxicity.Results. RPTEC incubation with favipiravir led to a dose-dependent decrease in the TEER values. However, the TEER values after 6 days of incubation ranged within 250–280 Ω×cm2 and were above the critical thresholdof 120–140 Ω×cm2.Conclusions. The results of this study indicate that favipiravir has no pronounced effect on the TEER of the RPTEC monolayer.

PSVI-9 In Vitro Effects of Monoglycerides and Zinc Glycinate: Anti-Inflammatory and Epithelial Barrier Function

Abstract The objectives of this study were to investigate the in vitro anti-inflammatory effects of monoglycerides and zinc glycinate with porcine alveolar macrophages (PAMs) and their impacts on epithelial barrier function using the IPEC-J2 cell line. MTT assays were performed for each compound to determine cell viability and appropriate treatment doses. PAMs were isolated from 6 piglets at 7 weeks of age, and then seeded into 24-well plates with 106 cells/mL. After 24 h incubation, cells were treated with each compound or lipopolysaccharide (LPS). The experimental design was 2 × 6 factorial arrangement with 2 doses of LPS (0 or 1 μg/mL) and 6 doses of each compound (monoglycerides: 0, 50, 100, 250, 500, and 1,000 µg/mL; zinc glycinate: 0, 25, 50, 100, 250, and 500 µg/mL). The supernatants were collected after another 24 h incubation to analyze concentration of tumor necrosis factor α (TNF-α) and interleukin-1β (IL-1β) by ELISA. IPEC-J2 cells (5 × 105 cells/mL) were seeded into 12-well plates on transwell cell culture inserts and cultured until confluence, and then were treated with each compound (monoglycerides: 0, 25, 100, 250, 500, and 1,000 µg/mL; zinc glycinate: 0, 2, 5, 12.5, 25, and 50 µg/mL). Transepithelial electrical resistance (TEER) was measured in Ωcm2 using a Millicell ERS-2 voltohmmeter at 0 h (before treatment) and at 24, 48, and 72 h post-treatment. Data were analyzed by ANOVA using PROC MIXED of SAS with a randomized complete block design. LPS challenge increased (P &amp;lt; 0.05) the production of TNF-α and IL-1β from PAMs. In the non-challenge group, supplementing 50 or 100 μg/mL of monoglycerides stimulated (P &amp;lt; 0.05) TNF-α and IL-1β production from PAMs. Treatment with 25 or 100 μg/mL of zinc glycinate also enhanced (P &amp;lt; 0.05) TNF-α production from PAMs. In LPS-treated PAMs, 1,000 μg/mL of monoglycerides increased (P &amp;lt; 0.05) IL-1β production, while zinc glycinate suppressed (P &amp;lt; 0.0001) the secretion of TNF-α and IL-1β at the doses of 100, 250, and 500 μg/mL. IPEC-J2 cells treated with 250 or 1,000 μg/mL of monoglycerides, or 5 μg/mL of zinc glycinate had increased (P &amp;lt; 0.0001) TEER values at 48 and 72 h post-treatment, compared with control. However, cells treated with 50 μg/mL of zinc glycinate reduced (P &amp;lt; 0.0001) TEER values at 24, 48, and 72 h post-treatment compared with other treatments. In conclusion, results of this in vitro study indicate that monoglycerides have a positive effect on the barrier function of epithelium, while zinc glycinate may have strong immune regulatory benefits. Future in vivo animal studies will be needed to verify their impacts on animal gut health, immune responses, and growth performance.

PSXI-23 Effects of Dietary Threonine, Tryptophan, and Glycine Supplementation on Growth Performance and Intestinal Barrier Function in Broiler Chickens Raised Under Multiple Stress Conditions

Abstract The objective of the current study was to compare the effects of dietary threonine, tryptophan, and glycine supplementation on growth performance and intestinal barrier function in broiler chickens raised under multiple stress conditions. All birds were allotted to 1 of 5 treatments with 8 replicates in a completely randomized design. Positive control (PC) treatment was normal condition and other treatments are multiple stress conditions with heat stress and high stocking density. Multiple stress conditions were exposed to a cyclic heat stress condition (30 ± 0.3°C for 10 h/d and 23 ± 0.2°C for 14 h/d). In addition, 10 birds per cage (16.9 birds/m2) were allotted in multiple stress conditions, but 15 birds per cage (25.3 birds/m2) were allotted in normal conditions. Basal diet was fed to broiler chickens in PC (normal condition) and negative control (NC; multiple stress conditions) treatments. Three additional diets were prepared by adding 0.68% threonine (99%), 0.19% tryptophan (99%), or 1.60% glycine (99%) to the basal diet. Four treatment diets such as NC, threonine, tryptophan, and glycine treatments were provided to birds in multiple stress conditions. All experimental diets and water were provided ad libitum for 14 d. The body weight gain (BWG) and feed intake (FI) were recorded at the end of the experiment. The feed efficiency (FE, g/kg) was calculated by dividing BWG with FI after mortality correction for FI. The intestinal barrier function was analyzed by trans-epithelial electrical resistance (TEER) value in a two-channel Ussing chamber system. Statistical analysis was conducted as a completely randomized design by using the PROC MIXED procedure of SAS (SAS Institute Inc., Cary, NC). The replicate was considered an experimental unit. Outlier data were confirmed by the UNIVARIATE procedure of SAS. The LSMEANS procedure was conducted to calculate treatment means. In addition, if the difference was significant, the PDIFF option of SAS was conducted to separate the means. The significance for statistical tests was considered at P &amp;lt; 0.05. Results indicated that all treatments under multiple stress condition (i.e., NC, Thr, Trp, and Gly treatments) caused a significant reduction (P &amp;lt; 0.05) in BW, BWG, FI, and FE as compared with PC treatment (Table 1). Dietary Thr supplementation under multiple stress conditions was similar FE compared with PC treatment (Table 2). Dietary threonine, tryptophan, and glycine supplementation under multiple stress condition induced intermediated values for TEER with dietary threonine supplementation having similar values to PC treatment. In conclusion, dietary supplementation of threonine seems the most effective functional amino acid. In addition, it can be a novel dietary treatment to improve growth performance and gut health in broiler chickens raised under multiple stress condition.

Organic anion transporting polypeptide 3a1 is a novel influx pump for Perfluorooctane sulfonate in Sertoli cells and contributes to its reproductive toxicity

Persistent organic pollutant perfluorooctane sulfonate (PFOS) is strongly associated with male reproductive disorders, but the related mechanisms are still not fully understood. In this study, we used in vivo and in vitro models to explore the role of organic anion transporting polypeptide 3a1 (Oatp3a1) on PFOS-induced male reproductive injury. Thirty male C57BL/6 (B6) mice were orally given PFOS (0–10 mg/kg/bw) for 28 days. Body weight, organ index, sperm count, histology, and blood-testis barrier (BTB) integrity were evaluated. Primary Sertoli cells were used to describe the related molecular mechanisms of male reproductive injury caused by PFOS. Our results showed that PFOS induced a decrease in sperm count, morphological damage to testicular Sertoli cells, and disruption of BTB. In the in vitro model, exposure to PFOS significantly increased Oatp3a1 mRNA and protein levels and decreased miR-23a-3p expression in Sertoli cells, accompanied by reduced trans-epithelial electrical resistance (TEER) value. By performing the 14C-PFOS uptake experiment, we showed that 14C-PFOS uptake in HEK293-Oatp3a1 cells was apparently higher than in HEK293-MOCK cells. Meanwhile, treating Sertoli cells with Oatp3a1 siRNA significantly decreased Oatp3a1 expression and rescued PFOS-induced decreases in TEER value. As such, the present study highlights that Oatp3a1 may play an important role in the toxic effect of PFOS on Sertoli cells, advancing our understanding of molecular mechanisms for PFOS-induced male reproductive disorders.

Passiflora edulis leaf extract inhibits inflammatory response and preserves intestinal barrier function in Caco-2 and RAW264.7 co-culture model

Inflammatory bowel disease (IBD) is a chronic and incurable illness that affects people all over the world. Conventional therapies usually cause adverse side effects that may affect patients’ quality of life. This study assessed the effect of the aqueous extract of Passiflora edulis leaves (PELE) in co-culture of Caco-2 and RAW264.7 cells, simulating an in vitro model of IBD. After being stimulated with bacterial-derived lipopolysaccharides (LPS), PELE’s treatment inhibited the release of the pro-inflammatory cytokines interleukin (IL) 6 and 8, although was not able to reduce nitric oxide (NO) production. In the co-culture system, PELE was also able to preserve the intestinal barrier function by decreasing paracellular permeability and increasing transepithelial electrical resistance (TEER) values, compromised after the LPS stimulus. The beneficial activity seen in this in vitro study may suggest that PELE has a possible anti-inflammatory role in IBD and can alleviate inflammatory events.

Cannabidiol attenuates inflammatory impairment of intestinal cells expanding biomaterial-based therapeutic approaches

Cannabis-based biomaterials have the potential to deliver anti-inflammatory therapeutics specifically to desired cells, tissues, and organs, enhancing drug delivery and the effectiveness of anti-inflammatory treatment while minimizing toxicity. As a major component of Cannabis, Cannabidiol (CBD) has gained major attention in recent years because of its potential therapeutic properties, e.g., for restoring a disturbed barrier resulting from inflammatory conditions. The aim of this study was to test the hypothesis that CBD has beneficial effects under normal and inflammatory conditions in the established non-transformed intestinal epithelial cell model IPEC-J2. CBD induced a significant increase in transepithelial electrical resistance (TER) values and a decrease in the paracellular permeability of [³H]-D-Mannitol, indicating a strengthening effect on the barrier. Under inflammatory conditions induced by tumor necrosis factor alpha (TNFα), CBD stabilized the TER and mitigated the increase in paracellular permeability. Additionally, CBD prevented the barrier-disrupting effects of TNFα on the distribution and localization of sealing TJ proteins. CBD also affected the expression of TNF receptors. These findings demonstrate the potential of CBD as a component of Cannabis-based biomaterials used in the development of novel therapeutic approaches against inflammatory pathogenesis.

Amino Acid Digestibility of Different Formulations of Torula Yeast in an In Vitro Porcine Gastrointestinal Digestion Model and Their Protective Effects on Barrier Function and Inflammation in a Caco-2/THP1Co-Culture Model.

Single-cell protein from torula yeast (Cyberlindnera jadinii) grown on lignocellulosic biomass has been proven to be an excellent alternative protein source for animal feed. This study aimed to evaluate the amino acid (AA) digestibility by estimating intestinal absorption from three yeast-based ingredients, produced by cultivating C. jadinii on hydrolysate, using either mixed woody species (drum- (WDI) or spray-dried (WSI)) or corn dextrose (drum-dried (DDI)) as the carbon source. Further, the protective effect of intestinal digests on activated THP1-Blue™-induced epithelial damage and cytokine profile was evaluated. Total protein content from these three ingredients ranged from 34 to 45%, while the AA dialysis showed an estimated bioaccessibility between 41 and 58%, indicating good digestibility of all test products. A protective effect against epithelial-induced damage was observed for two of the three tested products. Torula yeast cultivated on wood and drum-dried (WDI) and torula yeast cultivated on wood and spray-dried (WSI) significantly increased transepithelial electrical resistance (TEER) values (111-147%, p < 0.05), recovering the epithelial barrier from the inflammation-induced damage in a dose-dependent manner. Further, WSI digests significantly reduced IL8 (250.8 ± 28.1 ng/mL), IL6 (237.9 ± 1.8 pg/mL) and TNF (2797.9 ± 216.3 pg/mL) compared to the blank control (IL8 = 485.7 ± 74.4 ng/mL, IL6 = 478.7 ± 58.9 pg/mL; TNF = 4273.5 ± 20.9 pg/mL) (p < 0.05). These results align with previous in vivo studies, supporting torula yeast-based ingredients as a high-quality protein source for pigs, protecting the intestinal barrier from inflammatory damage, and reducing the pro-inflammatory response. We provided novel insights into the mechanisms behind the health improvement of pigs fed on torula yeast-based ingredients, with potential applications for designing nutritional interventions to recover intestinal homeostasis during critical production periods, such as weaning.

Effects of functional nutrients on chicken intestinal epithelial cells induced with oxidative stress.

The objective of this study was to investigate the protective effects of functional nutrients including various functional amino acids, vitamins, and minerals on chicken intestinal epithelial cells (cIECs) treated with oxidative stress. The cIECs were isolated from specific pathogen free eggs. Cells were exposed to 0 mM supplement (control), 20 mM threonine (Thr), 0.4 mM tryptophan (Trp), 1 mM glycine (Gly), 10 μM vitamin C (VC), 40 μM vitamin E (VE), 5 μM vitamin A (VA), 34 μM chromium (Cr), 0.42 μM selenium (Se), and 50 μM zinc (Zn) for 24 h with 6 replicates for each treatment. After 24 h, cells were further incubated with fresh culture medium (positive control, PC) or 1 mM H2O2 with different supplements (negative control, NC and each treatment). Oxidative stress was measured by cell proliferation, whereas tight junction barrier function was analyzed by fluorescein isothiocyanate (FITC)-dextran permeability and transepithelial electrical resistance (TEER). Results indicated that cell viability and TEER values were less (p < 0.05) in NC treatments with oxidative stress than in PC treatments. In addition, FITC-dextran values were greater (p < 0.05) in NC treatments with oxidative stress than in PC treatments. The supplementations of Thr, Trp, Gly, VC, and VE in cells treated with H2O2 showed greater (p < 0.05) cell viability than the supplementation of VA, Cr, Se, and Zn. The supplementations of Trp, Gly, VC, and Se in cells treated with H2O2 showed the least (p < 0.05) cellular permeability. In addition, the supplementation of Thr, VE, VA, Cr, and Zn in cells treated with H2O2 decreased (p < 0.05) cellular permeability. At 48 h, the supplementations of Thr, Trp, and Gly in cells treated with H2O2 showed the greatest (p < 0.05) TEER values among all treatments, and the supplementations of VC and VE in cells treated with H2O2 showed greater (p < 0.05) TEER values than the supplementations of VA, Cr, Se, and Zn in cells treated with H2O2. In conclusion, Thr, Trp, Gly, and VC supplements were effective in improving cell viability and intestinal barrier function of cIECs exposed to oxidative stress.