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

Endothelial cells of mammalian blood vessels have multiple levels of heterogeneity along the vascular tree and among different organs. Further heterogeneity results from blood flow turbulence and variations in shear stress. In the aorta, vascular endothelial protein tyrosine phosphatase (VE-PTP), which dephosphorylates tyrosine kinase receptor Tie2 in the plasma membrane, undergoes downstream polarization and endocytosis in endothelial cells exposed to laminar flow and high shear stress. VE-PTP sequestration promotes Tie2 phosphorylation at tyrosine992 and endothelial barrier tightening. The present study characterized the heterogeneity of VE-PTP polarization, Tie2-pY992 and total Tie2, and claudin-5 in anatomically defined regions of endothelial cells in the mouse descending thoracic aorta, where laminar flow is variable and IgG extravasation is patchy. We discovered that VE-PTP and Tie2-pY992 had mosaic patterns, unlike the uniform distribution of total Tie2. Claudin-5 at tight junctions also had a mosaic pattern, whereas VE-cadherin at adherens junctions bordered all endothelial cells. Importantly, the amounts of Tie2-pY992 and claudin-5 in aortic endothelial cells correlated with downstream polarization of VE-PTP. VE-PTP and Tie2-pY992 also had mosaic patterns in the vena cava, but claudin-5 was nearly absent and extravasated IgG was ubiquitous. Correlation of Tie2-pY992 and claudin-5 with VE-PTP polarization supports their collective interaction in the regulation of endothelial barrier function in the aorta, yet differences between the aorta and vena cava indicate additional flow-related determinants of permeability. Together, the results highlight new levels of endothelial cell functional mosaicism in the aorta and vena cava, where blood flow dynamics are well known to be heterogeneous.

Background: One of the most severe consequences of ageing is cognitive decline, which is associated with dysfunction of the brain microvasculature. Thus, repairing the brain vasculature could result in healthier brain function. Methods: To better understand the potential beneficial effect of endothelial progenitor cells (EPCs) in vascular repair, we studied the adhesion and integration of EPCs using the early embryonic mouse aorta–gonad–mesonephros – MAgEC 10.5 endothelial cell line. The EPC interaction with brain microvasculature was monitored ex vivo and in vivo using epifluorescence, laser confocal and two-photon microscopy in healthy young and old animals. The effects of senolysis, EPC activation and ischaemia (two-vessel occlusion model) were analysed in BALB/c and FVB/Ant: TgCAG-yfp_sb #27 mice. Results: MAgEC 10.5 cells rapidly adhered to brain microvasculature and some differentiated into mature endothelial cells (ECs). MAgEC 10.5-derived endothelial cells integrated into microvessels, established tight junctions and co-formed vessel lumens with pre-existing ECs within five days. Adhesion and integration were much weaker in aged mice, but were increased by depleting senescent cells using abt-263 or dasatinib plus quercetin. Furthermore, MAgEC 10.5 cell adhesion to and integration into brain vessels were increased by ischaemia and by pre-activating EPCs with TNFα. Conclusions: Combining progenitor cell therapy with senolytic therapy and the prior activation of EPCs are promising for improving EPC adhesion to and integration into the cerebral vasculature and could help rejuvenate the ageing brain. [ABSTRACT FROM AUTHOR]

Background: The use of conventional two-dimensional (2D) culture of the porcine intestinal epithelial cell (IEC) line IPEC-J2 in animal nutrition research has the disadvantage that IEC function is studied under unphysiological conditions, which limits the ability of transferring knowledge to the in vivo-situation. Thus, the aim of the present study was to establish a more convincing and meaningful three-dimensional (3D) culture of IPEC-J2 cells, which allows to study cell function in a more tissue-like environment, and to compare the effect of the endoplasmic reticulum (ER) stress inducer tunicamycin (TM) on ER stress indicators and the expression of tight junction proteins (TJP), inflammatory and apoptosis-related genes and the modulatory role of 1,25-dihydroxy-vitamin D3 (1,25D3) on these parameters in 2D and 3D cultures of IPEC-J2 cells. Results: A published protocol for 3D culture of Caco-2 cells was successfully adopted to IPEC-J2 cells as evident from fully differentiated 3D IPEC-J2 spheroids showing the characteristic spherical architecture with a single layer of IPEC-J2 cells surrounding a central lumen. Treatment of 2D IPEC-J2 cells and 3D IPEC-J2 spheroids with TM for 24 h markedly increased mRNA and/or protein levels of the ER stress target genes, heat shock protein family A (Hsp70) member 5 (HSPA5) and DNA damage inducible transcript 3 (DDIT3), whereas co-treatment with TM and 1,25D3 did not mitigate TM-induced ER stress in IPEC-J2 cells in the 2D and the 3D cell culture. In contrast, TM-induced expression of pro-inflammatory [interleukin-6 (IL6), IL8] and pro-apoptotic genes [BCL2 associated X, apoptosis regulator (BAX), caspase 3 (CASP3), CASP8] and genes encoding TJP [TJP1, claudin 1 (CLDN1), CLDN3, occludin (OCLN), cadherin 1 (CDH1), junctional adhesion molecule 1 (JAM1)] was reduced by co-treatment with TM and 1,25D3 in 3D IPEC-J2 spheroids but not in the 2D cell culture. Conclusions: The effect of 1,25D3 in the IPEC-J2 cell culture is dependent on the culture model applied. While 1,25D3 does not inhibit TM-induced expression of genes involved in inflammation, apoptosis and TJP in conventional 2D cultures of IPEC-J2 cells, TM-induced expression of these genes is abrogated by 1,25D3 in the more meaningful 3D IPEC-J2 cell culture model. [ABSTRACT FROM AUTHOR]

Background: Staphylococcus aureus (S. aureus) mastitis results in economic losses during dairy production. Understanding the biological progression of bovine S. aureus mastitis is vital for its prevention. Lipoteichoic acid is a key virulence factor of S. aureus (aLTA), but the main biological pathways involved in its effect on bovine mammary epithetionallial cells (Mac-T) apoptosis and necrosis have not been fully explored. Folic acid (FA) has anti-inflammatory and anti-apoptotic effects. However, the role of FA in mediating the effects of aLTA on apoptosis and necrosis remains unknown. Results: We found that low concentration of aLTA inhibited apoptosis and necrosis and that high concentration promoted the apoptosis and necrosis of Mac-T. FA pretreatment alleviated high concentration of aLTA induced apoptosis. Through transcriptomic analysis, we found that nuclear receptor subfamily 4 group A (NR4A), which alters the expression of downstream genes involved in apoptosis, proliferation, and inflammation, decreased under stimulation with a low concentration of aLTA and increased under stimulation with a high concentration of aLTA. Under stimulation with a high concentration of aLTA, the expression of the NR4A subfamily could be inhibited by FA. The results showed that aLTA may affect apoptosis and necrosis through the NR4A subfamily by targeting genes involved in bacterial invasion of epithelial cells, the IL-17 signaling pathway, DNA replication, longevity regulation, the cell cycle, and tight junction pathways. We further found that the expression trends of NR4A1 and the target genes of the NR4A subfamily (PTGS2, ESPL1, MCM5, and BUB1B) in the blood of healthy cows (Healthy), subclinical mastitis cows (SCM), and SCM supplemented with FA (SCM_FA) were consistent with those observed at the cellular level in this study. Conclusions: Our study revealed that low and high concentrations of aLTA have opposite effects on apoptosis and necrosis of Mac-T and that FA can alleviate the apoptosis induced by high concentration of aLTA. Transcriptome analysis revealed that the NR4A subfamily play a role in the ability of FA to alleviate the apoptosis and necrosis induced by high concentration of aLTA. [ABSTRACT FROM AUTHOR]

Neurovascular unit (NVU) inflammation via activation of glial cells and neuronal damage plays a critical role in neurodegenerative diseases. Though the exact mechanism of disease pathogenesis is not understood, certain biomarkers provide valuable insight into the disease pathogenesis, severity, progression and therapeutic efficacy. These markers can be used to assess pathophysiological status of brain cells including neurons, astrocytes, microglia, oligodendrocytes, specialized microvascular endothelial cells, pericytes, NVU, and blood-brain barrier (BBB) disruption. Damage or derangements in tight junction (TJ), adherens junction (AdJ), and gap junction (GJ) components of the BBB lead to increased permeability and neuroinflammation in various brain disorders including neurodegenerative disorders. Thus, neuroinflammatory markers can be evaluated in blood, cerebrospinal fluid (CSF), or brain tissues to determine neurological disease severity, progression, and therapeutic responsiveness. Chronic inflammation is common in age-related neurodegenerative disorders including Alzheimer's disease (AD), Parkinson's disease (PD), and dementia. Neurotrauma/traumatic brain injury (TBI) also leads to acute and chronic neuroinflammatory responses. The expression of some markers may also be altered many years or even decades before the onset of neurodegenerative disorders. In this review, we discuss markers of neuroinflammation, and neurodegeneration associated with acute and chronic brain disorders, especially those associated with neurovascular pathologies. These biomarkers can be evaluated in CSF, or brain tissues. Neurofilament light (NfL), ubiquitin C-terminal hydrolase-L1 (UCHL1), glial fibrillary acidic protein (GFAP), Ionized calcium-binding adaptor molecule 1 (Iba-1), transmembrane protein 119 (TMEM119), aquaporin, endothelin-1, and platelet-derived growth factor receptor beta (PDGFRβ) are some important neuroinflammatory markers. Recent BBB-on-a-chip modeling offers promising potential for providing an in-depth understanding of brain disorders and neurotherapeutics. Integration of these markers in clinical practice could potentially enhance early diagnosis, monitor disease progression, and improve therapeutic outcomes. [ABSTRACT FROM AUTHOR]

The genus Megalocytivirus, belonging to the family Iridoviridae, is one of the most detrimental virus groups to fish aquaculture. Megalocytivirus creates a virus-mock basement membrane (VMBM) on the surface of infected cells. This membrane provides attachment sites for lymphatic endothelial cells (LECs), disrupting fish's endothelial cell-extracellular matrix system. This disruption triggers injury to the vascular system and can result in death. Exploring the VMBM-cell interaction mechanism is crucial for uncovering the pathogenesis of Megalocytivirus and identifying therapeutic targets. Claudins, a class of tetra transmembrane proteins, play a key role in creating tight junctions between endothelial or epithelial cells. In this study, we demonstrated that the expression of Claudin2, a member of the Claudin family in fish, was significantly up-regulated by Megalocytivirus infection. Claudin2 was found in LECs attached to the surface of infected cells. It interacted with the VMBM viral components VP23R, VP08R, and VP33L at multiple binding sites through its two extracellular loops. However, it did not interact with the host basement membrane's nidogen. Therefore, Claudin2 is involved in the interaction of LEC with VMBM and plays a role in the disturbed distribution of extracellular matrix and endothelial cells in Megalocytivirus-infected fish tissues. This study aims to uncover the molecular mechanisms by which Megalocytivirus infection leads to pathological changes in the vascular system. [ABSTRACT FROM AUTHOR]

Purpose of review: Cavernous malformations (CM) are vascular lesions in the brain and spinal cord, characterized by clusters of endothelial-lined caverns lacking proper tight junctions. These malformations may be discovered incidentally or present with symptoms such as headaches, focal neurologic deficits, or seizures, with or without hemorrhage. This review focuses on non-surgical management considerations important for women with CM, who face challenges related to pregnancy, exogenous hormone use, anticonvulsive therapy, bone health, and mental health. Recent findings: Emerging evidence suggests that both estrogen and progesterone may influence CM lesion behavior. Exogenous hormones, including those in oral contraceptives and oral hormone replacement therapy, indicate an elevated risk of symptomatic hemorrhage (SH) and may also influence seizure frequency and severity, particularly in women taking antiseizure medications (ASMs). Data suggest that the risk of CM hemorrhage during pregnancy is similar to the risk when not pregnant, although limitations to these studies will be reviewed. Summary: This review synthesizes the current literature on the interplay between estrogen and progesterone and CM lesion behavior, highlighting the importance of gender- and sex-specific factors in clinical decision-making. Special attention is given to the implications of exogenous hormone use, seizure management, and the psychological well-being of women with CM, underscoring the need for a multidisciplinary approach tailored to the unique needs of this patient population. [ABSTRACT FROM AUTHOR]

Ulcerative colitis (UC) is associated with the shedding of the gut mucus. Decellularized porcine small intestinal submucosa (SIS‐ECM) contains numerous active forms of extracellular matrix as well as growth factors. Herein, a mucus‐mimetic hydrogel (MAG) is designed as a barrier therapy by integrating SIS‐ECM with hyaluronic acid and epigallocatechin‐3‐gallate. MAG not only presented the viscoelastic behaviors with G' of 4000 Pa but also good thixotropy. Moreover, MAG as a physical shielding is effective to hinder E. coli invasion. Besides, the good repairing effect on the injured Caco‐2 cells is presented by MAG. When MAG as a biomimetic mucus is rectally administrated to colitis mouse, it alleviated effectively the colitis symptoms and improved the colonic shorting. Moreover, expression of IL‐1β, IL‐6, and TNF‐α at the laminae propria is substantially inhibited by MAG. Besides, the colonic epitheliums are well rearranged, and the tight junction proteins (ZO‐1, Occludin‐1, Claudin‐5) between them are greatly upregulated after MAG treatment. Meanwhile, the mucus secretion of goblets cells is effectively repaired by MAG treatment. The repairing mechanism of intestinal mucosa barriers is associated with promoting the proliferation and differentiation of intestinal stem cells. Collectively, MAG might be a promising strategy of barrier therapy for UC. [ABSTRACT FROM AUTHOR]

Neurodegenerative diseases are often accompanied by neuroinflammation and impairment of the blood-brain barrier (BBB) mediated by activated glial cells through their release of proinflammatory molecules. To study the effects of glial cells on mouse brain endothelial cells (mBECs), we developed an in vitro BBB model with inflammation by preactivating mixed glial cells (MGCs) with lipopolysaccharide (LPS) before co-culturing with mBECs to study the influence of molecules released by activated MGCs. The response of the mBECs to activated MGCs was compared to direct stimulation with LPS. The cytokine profile of activated MGCs was analyzed together with their effects on the mBEC's integrity, expression of tight junction proteins, adhesion molecules, and BBB-specific transport proteins. Stimulation of MGCs significantly upregulated mRNA expression and secretion of several pro-inflammatory cytokines. Co-culturing mBECs with pre-stimulated MGCs significantly affected the barrier integrity of mBECs similar to direct stimulation with LPS. The gene expression levels of tight junction proteins were unaltered, but tight junction proteins revealed rearrangements with respect to subcellular distribution. Compared to direct stimulation with LPS, the expression of cell-adhesion molecules was significantly increased when mBECs were co-cultured with prestimulated MGCs and thus pre-activating MGCs transforms mBECs into a proinflammatory phenotype. [ABSTRACT FROM AUTHOR]

Integrity of epithelial and endothelial cell barriers is of critical importance for health, barrier disruption is a hallmark of numerous diseases, of which many are driven by carbonyl stressors such as methylglyoxal (MG). Carnosine and anserine exert some MG-quenching activity, but the impact of these and of other histidine containing dipeptides on cell barrier integrity has not been explored in detail. In human proximal tubular (HK-2) and umbilical vein endothelial (HUVEC) cells, exposure to 200 µM MG decreased transepithelial resistance (TER), i.e. increased ionic permeability and permeability for 4-, 10- and 70-kDa dextran, membrane zonula occludens (ZO-1) abundance was reduced, methylglyoxal 5-hydro-5-methylimidazolones (MG-H1) formation was increased. Carnosine, balenine (ß-ala-1methyl-histidine) and anserine (ß-ala-3-methyl-histidine) ameliorated MG-induced reduction of TER in both cell types. Incubation with histidine, 1-/3-methylhistidine, but not with ß-alanine alone, restored TER, although to a lower extent than the corresponding dipeptides. Carnosine and anserine normalized transport and membrane ZO-1 abundance. Aminoguanidine, a well-described MG-quencher, did not mitigate MG-induced loss of TER. Our results show that the effects of the dipeptides on epithelial and endothelial resistance and junction function depend on the methylation status of histidine and are not exclusively explained by their quenching activity. [ABSTRACT FROM AUTHOR]

Formation of fluid-filled lumina by epithelial tissues is essential for organ development. How cells control the hydraulic and cortical forces to control lumen morphology is not well understood. Here, we quantified the mechanical role of tight junctions in lumen formation using MDCK-II cysts. We found that the paracellular ion barrier formed by claudin receptors is not required for the hydraulic inflation of a lumen. However, the depletion of the zonula occludens scaffold resulted in lumen collapse and folding of apical membranes. Combining quantitative measurements of hydrostatic lumen pressure and junctional tension with modeling enabled us to explain lumen morphologies from the pressure-tension force balance. Tight junctions promote lumen inflation by decreasing cortical tension via the inhibition of myosin. In addition, our results suggest that excess apical area contributes to lumen opening. Overall, we provide a mechanical understanding of how epithelial cells use tight junctions to modulate tissue and lumen shape. [Display omitted] • Depletion of ZO scaffold causes lumen collapse and apical membrane folding • The claudin permeability barrier is not necessary for hydraulic lumen inflation • Tight junctions promote lumen inflation by decreasing cortical tension • Lumen shape controlled by pressure-tension force balance and apical area constraints Mukenhirn et al. show that epithelial cells use tight junctions to control lumen shape, and the depletion of the tight junction scaffold leads to lumen collapse and increased cortical tension. Claudin-depleted MDCK-II cysts maintain lumen pressure and shape despite high ion permeability. [ABSTRACT FROM AUTHOR]

Gastrointestinal disease is a leading cause of death in mature horses. A lack of in vitro modeling has impeded the development of novel therapeutics. The objectives of this study were to develop and further characterize a small intestinal monolayer cell culture derived from equine jejunum including establishing normal measurements of intestinal permeability and restitution. Three-dimensional enteroids, derived from postmortem sampling of equine jejunum, were utilized to develop confluent epithelial monolayers. The presence of differentiated intestinal epithelial cell types and tight junctions were confirmed using histology, reverse transcription PCR (RT-PCR), RNAscope, protein immunofluorescence and transmission electron microscopy. Transepithelial resistance (TER) and macromolecule flux were assessed as measurements of paracellular and transcellular permeability. Scratch assays were utilized to model and assess intestinal restitution. Monolayer cell cultures reached 100% confluency by ~5–7 days. Equine jejunum monolayers were confirmed as epithelial in origin, with identification of differentiated intestinal epithelial cell types and evidence of tight junction proteins. Function of the intestinal barrier was supported by acquisition of physiologically normal TER values (179.9 ± 33.7 ohms*cm2) and limited macromolecule flux (22 ± 8.8% at 60 min). Additionally, following a scratch wound, epithelial cell monolayers migrated to close gap defects within 24 h. In conclusion, this study describes the development of a novel intestinal epithelial monolayer cell culture for equine jejunum, and provides evidence of intestinal epithelial cell differentiation, formation of physiologically relevant barrier function and use as a model of intestinal restitution to test potential therapeutics for equine colic. [ABSTRACT FROM AUTHOR]

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

Objective: Endothelial dysfunction plays an important role in the pathogenesis of chronic kidney disease. Prostacyclin (PGI2), an endothelial cell‐produced endogenous prostaglandin, plays a crucial role in maintaining endothelial function. However, its effects on intestinal microcirculation and barrier function are not fully understood. We hypothesized that PGI2 improves intestinal microcirculation and barrier function via endothelial protective effects. Methods: ICR and ICGN (a spontaneous nephrotic model) mice were used in this study. Intestinal microcirculation was visualized in vivo to investigate PGI2 effects. Beraprost served as PGI2. PGI2 administration spanned 4 weeks, following which we assessed its influence on intestinal endothelial, intestinal barrier, and renal functions. Results: We visualized intestinal microcirculation and endothelial glycocalyx in the intestinal blood vessels. Beraprost administration induced a 1.2‐fold dilatation of the vascular diameter of the small intestine. Intestinal blood flow in ICGN mice was significantly reduced compared that in ICR mice but improved with beraprost administration. ICGN mice exhibited reduced serum albumin levels, decreased ambulation, an imbalance in intestinal reactive oxygen species (ROS)/nitric oxide (NO), and impaired tight junctions; all were ameliorated by beraprost administration. Conclusions: Beraprost improves intestinal microcirculation and barrier function by ameliorating ROS/NO imbalances, thereby reducing physical inactivity during renal failure. [ABSTRACT FROM AUTHOR]

Acute respiratory distress syndrome (ARDS) is a severe lung condition without targeted therapy that is characterized by the disruption of epithelial and endothelial barriers. The role of the tight junction protein occludin in the pathogenesis of this disease is unknown, although it has previously been deemed redundant in some tissues. The aim of the present study is to determine whether occludin is required for lung function by controlling alveolar barrier integrity in mouse models. Immunofluorescence staining of lungs from ARDS patients revealed a significant decrease in occludin expression compared to controls. Gene delivery of shRNA against occludin in the mouse lung reduced occludin levels and induced lung injury, as assessed by wet-to-dry-ratio, histology, and cellularity and protein content of bronchial alveolar lavage fluid. Conversely, gene delivery of an occludin-expressing plasmid increased occludin expression and dampened endotoxin-induced lung injury. In primary rat alveolar epithelial cells, occludin levels were positively correlated with barrier integrity, as well as membrane localization of claudin-18, another tight junction protein. Collectively, our data demonstrate that occludin plays a significant role in alveolar barrier function and that targeting occludin may provide a new therapeutic approach for ARDS. [ABSTRACT FROM AUTHOR]

It is currently impossible to perform an evidence-based risk assessment for medication use during breastfeeding. The ConcePTION project aims to provide information about the use of medicines during lactation. The study aimed to develop and characterize an in vitro model of the blood–milk barrier to determine the extent of the milk transfer of xenobiotics, relying on either on human mammary epithelial cells (hMECs) or immortalized cell lines derived from breast tissue. The hMECs were cultured and characterized for epithelial markers; further, the ability to form an epithelial barrier was investigated. Drug transporter functionality in the cultured hMECs was analyzed with specific probe substrates. The hMECs showed an epithelial morphology and the expression of epithelial markers and tight junctions. They formed a reproducible tight barrier with a transepithelial electrical resistance greater than 400 Ωcm2, unlike immortalized cell lines. Different levels of mRNA expression were detected for 81 genes of membrane transporters. Functional assays showed no evidence for the transporter-mediated secretion of medicines across the hMECs. Nevertheless, the hMEC-based in vitro model covered a 50-fold range of permeability values, differentiating between passive transcellular and paracellular-mediated transport. The cultured hMECs proved to be a promising in vitro model for biorelevance; the wide characterization of hMECs makes them useful for studying medicine partitioning in milk. [ABSTRACT FROM AUTHOR]

Rutin is a flavonoid compound with potential for treating Alzheimer's disease, preventing brain damage, mitigating cerebral ischemia–reperfusion injury, and exhibiting anti-glioblastoma activity. However, its efficacy is limited by its low solubility, poor bioavailability, and limited permeability across the blood–brain barrier (BBB). To enhance the bioavailability and brain-targeting ability of Rutin, transferrin-modified Rutin liposome (Tf-Rutin-Lip) was developed using liposomes as a delivery system. Rutin liposomes were prepared using the thin-film dispersion method, and the preparation conditions were optimized using the response surface methodology. Then, transferrin (Tf) was incorporated into the liposomes through covalent modification, yielding Tf-Rutin liposomes. The toxicity of these liposomes on bEnd.3 cells, as well as their impact on the tight junctions of these cells, was rigorously evaluated. Additionally, in vitro and in vivo experiments were conducted to validate the brain-targeting efficacy of the Tf-Rutin liposomes. A susceptible detection method was developed to characterize the pharmacokinetics of Tf-Rutin-Lip further. The optimized conditions for the preparation of Tf-Rutin-Lip were determined as follows: a lipid-to-cholesterol ratio of 4.63:1, a drug-to-lipid ratio of 1:45.84, a preparation temperature of 42.7 °C, a hydration volume of 20 mL, a sonication time of 10 min, a surfactant concentration of 80 mg/mL, a DSPE-MPEG-2000 concentration of 5%, and a DSPE-PEG2000-COOH to DSPE-MPEG-2000 molar ratio of 10%. The liposomes did not affect the cell activity of bEnd.3 cells at 24 h and did not disrupt the tight junction of the blood–brain barrier. Tf-modified liposomes were taken up by bEnd.3 cells, which, in turn, passed through the BBB, thus improving liposomal brain targeting. Furthermore, the results of pharmacokinetic experiments showed that the Cmax, AUC0-∞, AUC0-t, MRT0-∞, and t1/2 of Tf-Rutin-Lip increased 1.99-fold, 2.77-fold, 2.58-fold, 1.26-fold, and 1.19-fold compared to those of free Rutin solution, respectively. These findings suggest that Tf-Rutin-Lip is brain-targeted and may enhance the efficacy of Rutin in the treatment of brain disorders. [ABSTRACT FROM AUTHOR]

Preeclampsia (preE) is a hypertensive disorder in pregnancies. It is the third leading cause of mortality among pregnant women and fetuses worldwide, and there is much we have yet to learn about its pathophysiology. One complication includes cerebral edema, which causes a breach of the blood–brain barrier (BBB). Urinary marinobufagenin (MBG) is elevated in a preE rat model prior to developing hypertension and proteinuria. We investigated what effect MBG has on the endothelial cell permeability of the BBB. Human brain microvascular endothelial cells (HBMECs) were utilized to examine the permeability caused by MBG. The phosphorylation of ERK1/2, Jnk, p38, and Src was evaluated after the treatment with MBG. Apoptosis was evaluated by examining caspase 3/7. MBG ≥ 1 nM inhibited the proliferation of HBMECs by 46–50%. MBG induced monolayer permeability, causing a decrease in the phosphorylation of ERK1/2 and the activated phosphorylation of Jnk, p38, and Src. MBG increased the caspase 3/7 expression, indicating the activation of apoptosis. Apoptotic signaling or the disruption of endothelia tight junction proteins was not observed when using the p38 inhibitor as a pretreatment in MBG-treated cells. The MBG-induced enhancement of the HBMEC monolayer permeability occurs by the downregulation of ERK1/2, the activation of Jnk, p38, Src, and apoptosis, resulting in the cleavage of tight junction proteins, and are attenuated by p38 inhibition. [ABSTRACT FROM AUTHOR]

Human milk oligosaccharides promote the growth and adhesion of Bifidobacteria , thus exerting multiple biological functions on intestinal epithelial cells. Bacterial surface proteins play an important role in bacterial–host intestinal epithelial interactions. In this study, we aimed to investigate the effects of surface proteins extracted from Bifidobacterium bifidum DNG6 (B. bifidum DNG6) consuming 2′-fucosyllactose (2′-FL) on Caco-2 cells monolayer barrier injury induced by lipopolysaccharide, compared with lactose and galacto-oligosaccharides. Our results indicated that 2′-FL may promote the surface proteins of B. bifidum DNG6 to improve intestinal barrier injury by positively regulating the NF-κB signaling pathway, reducing inflammation (TNF-α reduced by 50.34%, IL-6 reduced by 22.83%, IL-1β reduced by 37.91%, and IL-10 increased by 63.47%) and strengthening tight junction proteins (ZO,1 2.39×; claudin,1 2.79×; and occluding, 4.70×). The findings of this study indicate that 2′-FL can further regulate intestinal barrier damage by promoting the alteration of B. bifidum DNG6 surface proteins. The findings of this research will also provide theoretical support for the development of synbiotic formulations. The list of standard abbreviations for JDS is available at adsa.org/jds-abbreviations-24. Nonstandard abbreviations are available in the Notes. [Display omitted] [ABSTRACT FROM AUTHOR]

The article discusses the use of mesenchymal stromal cells (MSCs) in limiting house dust mite extract-induced mixed granulocytic lung inflammation in an experimental asthma model. The study found that intranasal administration of adipose tissue-derived MSCs reduced lung leukocyte infiltration, eosinophil and neutrophil counts, and altered gene expression related to inflammation. Additionally, MSCs were effective in reducing subepithelial collagen deposition and changing T effector cell responses. Further research is needed to fully understand the long-term effects of MSC transplantation in non-T2-driven asthmatic inflammation. [Extracted from the article]

To study the therapeutic effect and mechanism of Shaoyao Gancao Decoction (SGD) on ulcerative colitis (UC) mice based on the perspective of intestinal barrier, and this study provides a new consultation for the clinical application of SGD. The chemical composition of SGD was characterized by HPLC. The UC mouse model was constructed by 3 % dextran sodium sulfate (DSS), which were randomly divided into the model group (DSS), the positive drug group (5-ASA), the Shaoyao group (SYD), Gancao group (GCD), and the Shaoyao Gancao Decoction group (SGD) at low, medium, and high dosages, respectively. The effects of each drug treatment group on UC were evaluated by the rate of body weight loss, disease activity index (DAI), colon length, spleen index, histopathological evaluations, and the levels of serum inflammatory factors (IL-1β, IL-6, IL-10, IL-21, and TNF-α). The goblet cell was observed by Alcian blue/periodic acid-Schiff (AB/PAS) straining, ELISA was used to detect the content of LPS in serum, and Western blot was used to detect the changes in the expression of tight junction proteins ZO-1, occludin, and the pathway proteins TLR4 and NF-κBp65 in the colonic tissues, to explore the protective effect of SGD on the intestinal barrier of UC mice. The vivo absorption process of the main active ingredients in the SG, SY and GC groups was determined by LC-MS. The contents of albiflorin, paeoniflorin, liquiritin apioside, liquiritin and glycyrrhetinic acid were 6.1227 mg/g, 20.8993 mg/g, 4.0054 mg/g, 3.6140 mg/g and 8.2515 mg/g, respectively. Compared with DSS group, SGD reduced weight loss(P<0.01) and DAI scores(P<0.05), prevented colon shortening(P<0.01), and ameliorated histopathological damage of the colon in UC mice(P<0.01). SGD also protected the intestinal barrier to alleviate UC by significantly reducing serum LPS and inflammatory factor levels, altering the number of goblet cells, promoting tight junction proteins (ZO-1 and occludin) and decreasing the expression of TLR4 and NF-κB in colonic tissues. Pharmacokinetic results showed that there was no significant difference in C max , AUC 0-t (μg/L. h) and T max of albiflorin and paeoniflorin between the SY and SG groups, the T max was within 1 h; the AUC 0-t (μg/L. h) of liquiritin and glycyrrhizic acid were about 1.6 and 1.9 times higher in the SG group compared to the GC group, respectively. The C max , T max and AUC 0-t (μg/L. h) of glycyrrhizinic acid were significantly reduced to 0.73, 0.68 and 0.68 times of that of the GC group. SGD may have a therapeutic effect on DSS-induced UC mice by repairing the damaged intestinal barrier through the TLR4/NF-κB pathway. The combination of Shaoyao and Gancao increased the absorption of liquiritin and glycyrrhizic acid in vivo. The combination of Shaoyao and Gancao could promote the absorption of Gancao, and that the pairing of the two herbs could have a synergistic effect. • SGD repaired intestinal barrier damage of ulcerative colitis by inhibiting the TLR4/ NF-κB signaling pathway. • SGD promoted the absorption of the active ingredients of Gancao and provides a synergistic effect. • SGD classic prescription has been used for many intestinal diseases, and SGD for UC has been reported rarely. [ABSTRACT FROM AUTHOR]

A long-term exposure to cigarette smoke (CS) alters the integrity of airway epithelial barrier, contributes to lung dysfunction, and elicits the expression and activity of lung cathepsin S (CatS), a cysteine protease that participates in the remodeling of connective tissue and cell junctions. Here, we observed that a short-term (4 days) exposure of mice to CS increased the expression and activity of CatS, while the expression level of zonula occludens 1 (ZO-1), an epithelial tight junction protein that stabilizes barrier assembly, was reduced in lung tissue lysates. Present data support that proteolytically active CatS may contribute to the defect of ZO-1 in CS-exposed mice. [ABSTRACT FROM AUTHOR]

The weaning phase in piglets causes significant physiological stress, disrupts intestinal integrity and reduces productivity, necessitating strategies to improve intestinal health and nutrient absorption. While current research highlights the role of diet in mitigating these adverse effects, identifying effective dietary supplements remains a challenge. This study evaluated the effects of Hermetia illucens (HI) larvae meal and astaxanthin (AST) on the intestinal histology of weaned piglets. In a controlled experiment, 48 weaned piglets were divided into six groups and received varying levels of HI larval meal (2.5% and 5%) and AST in their diets. The methodology involved comprehensive histological examinations of the small intestine, assessing absorption area, villi elongation, crypt depth, goblet cells, enterocytes and expression of ileal tight junction (TJ) proteins. The study found that HI larval meal significantly improved nutrient absorption in the jejunum and ileum (p < 0.001), thereby enhancing feed conversion. AST supplementation increased the number of enterocytes (p < 0.001). Both HI larval meal and AST positively affected intestinal morphology and function, increasing muscularis muscle mass and villi elongation (p < 0.001 and p < 0.05, respectively). The 2.5% HI meal improved the villi length to crypt depth ratio and slightly increased the goblet cell count (both p < 0.05). Ki‐67 antibody analysis showed increased cell proliferation in the duodenal and jejunal crypts, particularly with the 2.5% HI meal (p < 0.001). Insect meal did not affect TJ protein expression, indicating that it had no effect on intestinal permeability. These findings suggest that HI larval meal and AST can enhance the intestinal wellness and productivity of weaned piglets. [ABSTRACT FROM AUTHOR]

Background: Glioblastoma multiforme (GBM) is an aggressive tumor, difficult to treat pharmacologically because of the blood-brain barrier (BBB), which is rich in ATP-binding cassette (ABC) transporters and tight junction (TJ) proteins. The BBB is disrupted within GBM bulk, but it is competent in brain-adjacent-to-tumor areas, where eventual GBM foci can trigger tumor relapse. How GBM cells influence the permeability of BBB is poorly investigated. Methods: To clarify this point, we co-cultured human BBB models with 3 patient-derived GBM cells, after separating from each tumor the stem cell/neurosphere (SC/NS) and the differentiated/adherent cell (AC) components. Also, we set up cultures of BBB cells with the conditioned medium of NS or AC, enriched or depleted of IL-6. Extracellular cytokines were measured by protein arrays and ELISA. The intracellular signaling in BBB cells was measured by immunoblotting, in the presence of STAT3 pharmacological inhibitor or specific PROTAC. The competence of BBB was evaluated by permeability assays and TEER measurement. Results: The presence of GBM cells or their conditioned medium increased the permeability to doxorubicin, mitoxantrone and dextran-70, decreased TEER, down-regulated ABC transporters and TJ proteins at the transcriptional level. These effects were higher with AC or their medium than with NS. The secretome analysis identified IL-6 as significantly more produced by AC than by NS. Notably, AC-conditioned medium treated with an IL-6 neutralizing antibody reduced the BBB permeability to NS levels, while NS-conditioned medium enriched with IL-6 increased BBB permeability to AC levels. Mechanistically, IL-6 released by AC GBM cells activated STAT3 in BBB cells. In turn, STAT3 down-regulated ABC transporter and TJ expression, increased permeability and decreased TEER. The same effects were obtained in BBB cells treated with STA-21, a pharmacological inhibitor of STAT3, or with a PROTAC targeting STAT3. Conclusions: Our work demonstrates for the first time that the degree of GBM differentiation influences BBB permeability. The crosstalk between GBM cells that release IL-6 and BBB cells that respond by activating STAT3, controls the expression of ABC transporters and TJ proteins on BBB. These results may pave the way for novel therapeutic tools to tune BBB permeability and improve drug delivery to GBM. [ABSTRACT FROM AUTHOR]

Porphyromonas gingivalis (P. gingivalis), the main pathogen responsible for periodontitis, is linked to systemic disorders via the oral-gut axis. Short-chain fatty acids (SCFAs) are vital for gut health, but their role in P. gingivalis-induced gut disorders remains unclear. This study utilized metabolomics and 16 S rRNA sequencing to explore gut microbiota and SCFAs levels in P. gingivalis-induced periodontitis mouse models. Significant changes were observed in gut, including a reduction in SCFAs-producing bacteria, such as Lactobacillus, Ligilactobacillus, Allobucalum, and a notable decrease in Firmicutes and Actinobacteriota. The intestinal permeability tests and histological analyses revealed that periodontitis led to epithelial inflammation, reduced mucin secretion, and compromised gut barrier integrity. In vitro experiments with Caco-2 cells co-cultured with P. gingivalis showed that the bacterium disrupted cellular junctions by impairing autophagy, specifically through the ATG5-LC3 pathway, leading to decreased expression of tight junction proteins and reduced SCFA absorption. Remarkably, rapamycin treatment both in vitro and in vivo restored gut barrier function by enhancing autophagy, increasing tight junction protein expression, and promoting SCFAs absorption via MCT1 and SMCT1, alongside GPR43/GPR109a pathway activation. These findings reveal autophagy's novel role in regulating SCFAs metabolism in P. gingivalis-induced gut dysbiosis, offering insights for preventing and treating periodontitis-related systemic diseases. [ABSTRACT FROM AUTHOR]

Cell junctions, including anchoring, occluding and communicating junctions, play an indispensable role in the structural and functional organization of multicellular tissues, including in liver. Specifically, hepatic cell junctions mediate intercellular adhesion and communication between liver cells. The establishment of the hepatic cell junction network is a prerequisite for normal liver functioning. Hepatic cell junctions indeed support liver‐specific features and control essential aspects of the hepatic life cycle. This review paper summarizes the role of cell junctions and their components in relation to liver physiology, thereby also discussing their involvement in hepatic dysfunctionality, including liver disease and toxicity. [ABSTRACT FROM AUTHOR]

Humans can be exposed to per- and polyfluoroalkyl substances (PFASs) via many exposure routes, including diet, which may lead to several adverse health effects. So far, little is known about PFAS transport across the human intestinal barrier. In the current study, we aimed to assess the transport of 5 PFASs (PFOS, PFOA, PFNA, PFHxS and HFPO-DA) in a human induced pluripotent stem cell (hiPSC)-derived intestinal epithelial cell (IEC) model. This model was extensively characterized and compared with the widely applied human colonic adenocarcinoma cell line Caco-2 and a human primary IEC-based model, described to most closely resemble in vivo tissue. The hiPSC-derived IEC layers demonstrated polarized monolayers with tight junctions and a mucus layer. The monolayers consisted of enterocytes, stem cells, goblet cells, enteroendocrine cells, and Paneth cells that are also present in native tissue. Transcriptomics analysis revealed distinct differences in gene expression profiles, where the hiPSC-derived IECs showed the highest expression of intestinal tissue-specific genes relative to the primary IEC-based model and the Caco-2 cells clustered closer to the primary IEC-based model than the hiPSC-derived IECs. The order of PFAS transport was largely similar between the models and the apparent permeability (Papp) values of PFAS in apical to basolateral direction in the hiPSC-derived IEC model were in the following order: PFHxS > PFOA > HFPO-DA > PFNA > PFOS. In conclusion, the hiPSC-derived IEC model highly resembles human intestinal physiology and is therefore a promising novel in vitro model to study transport of chemicals across the intestinal barrier for risk assessment of chemicals. [ABSTRACT FROM AUTHOR]

Proteins and peptides have been increasingly developed as pharmaceuticals owing to their high potency and low side effects. However, their administration routes are confined to injections, such as intra-muscular and intra-venous injections, making patient compliance a challenge. Hence, non-injectable delivery systems are crucial to expanding the clinical use of proteins and peptides. In this context, two choline-based ionic liquids (ILs), namely, choline geranic acid ([Ch][Ger]) and choline citric acid ([Ch][Cit]), have been identified as promising agents for enhancing the permeation and prolonging the retention time of glucagon (GC) after intra-nasal administration. Notably, intra-nasal delivery of GC via ILs (GC/ILs) elicited rapid and smooth reversal of acute hypoglycaemia without leading to rebound hyperglycaemia in type 1 diabetic rats subjected to insulin induction. In addition, ILs could improve the transcellular transport of GC through electrostatic interaction. ILs could also transiently open inter-cellular tight junctions transiently to facilitate the paracellular transport of GC. Safety tests indicated that continuous intra-nasal delivery of ILs led to reversible changes, such as epithelial cell inflammation, goblet cell overgrowth, and impacts on the distribution of nasal cilia. However, these changes could be alleviated by the innate self-repair ability of mucosal epithelial cells. This study highlights the considerable potential of ILs for long-term nasal delivery of biomacromolecules. [Display omitted] • Intra-nasal delivery of glucagon in ionic liquids (GC/ILs) for acute hypoglycaemia rescue • ILs improved the stability and solubility of GC through hydrogen bonding interactions. • ILs enhanced the paracellular transport of GC as a novel transient permeation enhancer. • The water content of ILs significantly affected the nasal retention and efficacy of GC/ILs. • The long-term regimen of nasal delivery of GC/ILs showed a favourable safety profile. [ABSTRACT FROM AUTHOR]

Background: Altered gut metabolites, especially short-chain fatty acids (SCFAs), in feces and plasma are observed in patients with Parkinson's disease (PD). Objective: We aimed to investigate the colonic expression of two SCFA receptors, free fatty acid receptor (FFAR)2 and FFAR3, and gut barrier integrity in patients with PD and correlations with clinical severity. Methods: In this retrospective study, colonic biopsy specimens were collected from 37 PD patients and 34 unaffected controls. Of this cohort, 31 participants (14 PD, 17 controls) underwent a series of colon biopsies. Colonic expression of FFAR2, FFAR3, and the tight junction marker ZO-1 were assayed by immunofluorescence staining. The You Only Look Once (version 8, YOLOv8) algorithm was used for automated detection and segmentation of immunostaining signal. PD motor function was assessed with the Movement Disorder Society (MDS)-Unified Parkinson's Disease Rating Scale (UPDRS), and constipation was assessed using Rome-IV criteria. Results: Compared with controls, PD patients had significantly lower colonic expression of ZO-1 (p < 0.01) and FFAR2 (p = 0.01). On serial biopsy, colonic expression of FFAR2 and FFAR3 was reduced in the pre-motor stage before PD diagnosis (both p < 0.01). MDS-UPDRS motor scores did not correlate with colonic marker levels. Constipation severity negatively correlated with colonic ZO-1 levels (r = -0.49, p = 0.02). Conclusions: Colonic expression of ZO-1 and FFAR2 is lower in PD patients compared with unaffected controls, and FFAR2 and FFAR3 levels decline in the pre-motor stage of PD. Our findings implicate a leaky gut phenomenon in PD and reinforce that gut metabolites may contribute to the process of PD. [ABSTRACT FROM AUTHOR]

The consumption of fructose is increasing day by day. Understanding the impact of increasing fructose consumption on the small intestine is crucial since the small intestine processes fructose into glucose. ∆9-Tetrahydrocannabinol (THC), a key cannabinoid, interacts with CB1 and CB2 receptors in the gastrointestinal tract, potentially mitigating inflammation. Therefore, this study aimed to investigate the effects of the high-fructose diet (HFD) on the jejunum of rats and the role of THC consumption in reversing these effects. Experiments were conducted on Sprague–Dawley rats, with the experimental groups as follows: control (C), HFD, THC, and HFD + THC. The HFD group received a 10% fructose solution in drinking water for 12 weeks. THC groups were administered 1.5 mg/kg/day of THC intraperitoneally for the last four weeks. Following sacrification, the jejunum was evaluated for mucus secretion capacity. IL-6, JNK, CB2 and PCNA expressions were assessed through immunohistochemical analysis and the ultrastructural alterations via transmission electron microscopy. The results showed that fructose consumption did not cause weight gain but triggered inflammation in the jejunum, disrupted the cell proliferation balance, and increased mucus secretion in rats. Conversely, THC treatment displayed suppressed inflammation and improved cell proliferation balance caused by HFD. Ultrastructural examinations showed that the zonula occludens structures deteriorated in the HFD group, along with desmosome shrinkage. Mitochondria were found to be increased due to THC application following HFD. In conclusion, the findings of this research reveal the therapeutic potential of THC in reversing HFD-related alterations and provide valuable insights for clinical application. [ABSTRACT FROM AUTHOR]

The prolonged exposure to arsenic results in intestinal barrier dysfunction, which is strongly concerned with detrimental processes such as oxidative stress and the inflammatory response. Ferulic acid (FA), as a phenolic acid, possesses the capability to mitigate arsenic‐induced liver damage and cardiotoxic effects dependent on inhibition of oxidative stress and inflammatory responses. FA can mitigate testicular tissue damage and alveolar epithelial dysfunction, the mechanism of which may rely on nuclear factor erythroid 2‐related factor 2/heme oxygenase 1 (Nrf2/HO‐1) activation and nuclear factor‐kappa B (NF‐κB) pathway blocking. Based on the antioxidant and anti‐inflammatory properties of FA, we speculated that FA might have the potential to inhibit arsenic‐induced intestinal damage. To confirm this scientific hypothesis, mice exposed to sodium arsenite were treated with FA to observe colonic histopathology and TJ protein levels, and oxidative stress and TJ protein levels in Caco‐2 cells exposed to sodium arsenite were assessed after FA intervention. In addition, molecular levels of NF‐κB and Nrf2/HO‐1 pathway in colon and Caco‐2 cells were also detected. As shown in our data, FA inhibited arsenic‐induced colon injury, which was reflected in the improvement of mucosal integrity, the decrease of down‐regulated expression of tight junction (TJ) proteins (Claudin‐1, Occludin, and ZO‐1) and the inhibition of oxidative stress. Similarly, treatment with FA attenuated the inhibitory effect of arsenic on TJ protein expression in Caco‐2 cells. In addition to suppressing the activation of NF‐κB pathway, FA retrieved the activation of Nrf2/HO‐1 pathway in colon and intestinal epithelial cells induced by arsenic. In summary, our findings propose that FA has the potential to mitigate arsenic‐induced intestinal damage by preserving the integrity of intestinal epithelial TJs and suppressing oxidative stress. These results lay the groundwork for the potential use of FA in treating colon injuries caused by arsenic. [ABSTRACT FROM AUTHOR]

Oleanolic acid (OA) is a naturally occurring pentacyclic triterpene compound that has been reported to cause cholestatic liver injury. However, the regulation and pathogenic role of bile acids in OA‐induced development of cholestatic liver injury remains largely unclear. Farnesoid X receptor (FXR) is a metabolic nuclear receptor that plays an important role in bile acid homeostasis in the liver by regulating efflux transporters bile salt export pump (BSEP) and multidrug resistance‐associated protein 2 (MRP2). The aim of this study was to investigate the effect of OA on hepatocyte tight junction function and determine the role of FXR, BSEP, and MRP2 in the mechanism of impairment of transport of bile acids induced by OA. Both in vivo and in vitro models were used to characterize the OA‐induced liver injury. The liquid chromatography–tandem mass spectrometry (LC‐MS) was employed to characterize the efflux function of the transporters, and the results showed that OA caused a blockage of bile acids efflux. OA treatment resulted in decreased expression levels of the tight junction proteins zonula occludens‐1 and occludin. Immunofluorescence results showed that OA treatment significantly reduced the number of bile ducts and the immunofluorescence intensity. Pretreatment with agonists of FXR and MRP2, respectively, in animal experiments attenuated OA‐induced liver injury, while pretreatment with inhibitors of BSEP and MRP2 further aggravated OA‐induced liver injury. These results suggest that OA inhibits FXR‐mediated BSEP and MRP2, leading to impaired bile acid efflux and disruption of tight junctions between liver cells, resulting in liver damage. Oleanolic acid (OA) is a naturally occurring pentacyclic triterpene compound that has been reported to cause cholestatic liver injury. In this study, OA inhibits FXR‐mediated BSEP and MRP2, leading to impaired bile acid efflux and disruption of tight junctions between liver cells, resulting in liver damage. Activation of FXR and MRP2 mitigated OA‐induced liver injury, while inhibition of BSEP and MRP2 further aggravated OA‐induced liver injury. [ABSTRACT FROM AUTHOR]

Reperfusion after cerebral ischemia leads to secondary damage to the nervous system, called cerebral ischemia/reperfusion injury (CIRI). The blood–brain barrier (BBB) consists of endothelial cells and tight junction (TJ) proteins, and its disruption aggravates CIRI. Two GSE datasets identified Tissue Factor Pathway Inhibitor 2 (TFPI2) as a differentially upregulated gene (Log2FC > 1, p < 0.01) in the cerebral cortex of ischemic rats, and TFPI2 affects angiogenesis of endothelial cells. Moreover, genes (c-Jun, c-Fos, FosL1) encoding subunits of Activator Protein-1 (AP-1), a transcription factor involved in IRI, were highly expressed in ischemic samples. Thus, the effects of the AP-1/TFPI2 axis on CIRI were explored. We determined increased TFPI2 expression in the cerebral cortex of rats receiving middle cerebral artery occlusion (MCAO) for 90 min and reperfusion (R) for 48 h. Then AAV2-shTFPI2 particles (5 × 1010 vg) were injected into the right lateral ventricle of rats 3 weeks before MCAO/R. TFPI2 knockdown decreased infarct size and neuronal injury in ischemic rats. It improved BBB integrity, demonstrated by reduced FITC-dextran leakage in brain tissues of MCAO/R-operated rats. Furthermore, it increased the expression of TJ proteins (Occludin, Claudin-5, TJP-1) in the cerebral cortex of rats with CIRI. Consistently, we found that TFPI2 knockdown mitigated cell damage in mouse endothelial bEND.3 cells with oxygen and glucose deprivation (ODG) for 6 h and reoxygenation (R) for 18 h (OGD/R) treatment. High co-expression of c-Jun and c-Fos significantly elevated TFPI2 promoter activity. c-Jun knockdown inhibited TFPI2 expression in OGD/R-treated bEND.3 cell. Collectively, our findings demonstrate that inhibition of the AP-1/TFPI2 axis alleviates CIRI. [ABSTRACT FROM AUTHOR]

Introduction: Tight junctions (TJs) and their constituent proteins play pivotal roles in cellular physiology and anatomy by establishing functional boundaries within and between neighboring cells. While the involvement of TJ proteins, such as claudins, in cancer is extensively studied, studies highlighting their interaction with immune system are still meager. Studies indicate that alterations in cytokines and immune cell populations can affect TJ proteins, compromising TJ barrier function and exacerbating pro-inflammatory conditions, potentially leading to epithelial cell malignancy. Disrupted TJs in established tumors may foster a pro-tumor immune microenvironment, facilitating tumor progression, invasion, epithelial-to-mesenchymal transition and metastasis. Although previous literature contains many studies describing the involvement of TJs in pathogenesis of malignancies their role in modulating the immune microenvironment of tumors is just beginning to be unleashed. Areas covered: This article for the first time attempts to discern the importance of interaction between TJs and immune microenvironment in malignancies. To achieve the above aim a thorough search of databases like PubMed and Google Scholar was conducted to identify the recent and relevant articles on the topic. Expert opinion: Breaking the vicious cycle of dysbiosis/infections/chemical/carcinogen-induced inflammation-TJ remodeling-malignancy-TJ dysregulation-more inflammation can be used as a strategy to complement the effect of immunotherapies in various malignancies. [ABSTRACT FROM AUTHOR]

In patients with rheumatoid arthritis (RA), intestinal flora imbalance and butyrate metabolism disorders precede clinical arthritis and are associated with the pathogenesis of RA. This imbalance can alter the immunology and intestinal permeability of the intestinal mucosa, leading to damage to the intestinal barrier. In this context, bacteria and their metabolites can enter the bloodstream and reach the distant target tissues of the host, resulting in local inflammation and aggravating arthritis. Additionally, arthritis is also exacerbated by bone destruction and immune tolerance due to disturbed differentiation of osteoclasts and adaptive immune cells. Of note, butyrate is a metabolite of intestinal flora, which not only locally inhibits intestinal immunity and targets zonulin and tight junction proteins to alleviate intestinal barrier-mediated arthritis but also inhibits osteoclasts and autoantibodies and balances the immune responses of T and B lymphocytes throughout the body to repress bone erosion and inflammation. Therefore, butyrate is a key intermediate linking intestinal flora to the host. As a result, restoring the butyrate-producing capacity of intestinal flora and using exogenous butyrate are potential therapeutic strategies for RA in the future. [ABSTRACT FROM AUTHOR]

Acute respiratory distress syndrome (ARDS) is a severe lung disease characterized by significant hypoxemia, which impairs the oxygen supply necessary for optimal lung function. This study aimed to investigate the effects of sodium propionate (SP), the primary end product of intestinal flora fermentation of dietary fiber, on lipopolysaccharide (LPS)-induced ARDS in rats. The rats were treated with SP, after which the lung wet/dry ratio, arterial partial oxygen pressure (PaO2), levels of pro- and anti-inflammatory cytokines, tight junction proteins ZO-1 and Occludin, as well as LC3 and phosphorylated PI3K (p-PI3K)/p-AKT/p-mTOR protein levels, were measured. Additionally, histopathological analysis was conducted. The results indicated that SP effectively alleviated arterial hypoxemia in rats and mitigated the pathological damage to both intestinal and lung tissues caused by LPS. Notably, SP significantly reduced the levels of inflammatory factors TNF-α and IL-6 in the blood and bronchoalveolar lavage fluid (BALF) of ARDS rats, while increasing the concentration of the anti-inflammatory factor IL-10. Furthermore, SP inhibited the activation of the PI3K/AKT/mTOR signaling pathway and enhanced the LC3II/LC3I ratio in lung tissue. Therefore, SP may improve LPS-induced ARDS in rats by inhibiting the activation of the PI3K/AKT/mTOR signaling pathway, promoting autophagy, decreasing the production and release of inflammatory markers, and reducing alveolar epithelial damage. [ABSTRACT FROM AUTHOR]

Background: Lactiplantibacillus species are extensively studied for their ability to regulate host immune responses and functional therapeutic potentials. Nevertheless, there is a lack of understanding on the mechanisms of interactions with the hosts during immunoregulatory activities. Methods: Two Lactiplantibacillus plantarum strains MKMB01 and MKMB02 were tested for probiotic potential following Indian Council of Medical Research (ICMR) guidelines. Human colorectal adenocarcinoma cells such as HT-29, caco-2, and human monocytic cell THP-1 were also used to study the potential of MKMB01 and MKMB02 in regulating the host immune response when challenged with enteric pathogen Salmonella enterica typhimurium. Cells were pre-treated with MKMB01 and MKMB02 for 4 h and then stimulated with Salmonella. qRT-PCR and ELISA were used to analyze the genes and protein expression. Confocal microscopy and field emission scanning electron microscopy (FESEM) were used to visualize the effects. An Agilent Seahorse XF analyzer was used to determine real-time mitochondrial functioning. Results: Both probiotic strains could defend against Salmonella by maintaining gut integrity via expressing tight junction proteins (TJPs), MUC-2, and toll-like receptors (TLRs) negative regulators such as single Ig IL-1-related receptor (SIGIRR), toll-interacting protein (Tollip), interleukin-1 receptor-associated kinase (IRAK)-M, A20, and anti-inflammatory transforming growth factor-β and interleukin-10. Both strains also downregulated the expression of pro-inflammatory cytokines/chemokines interleukin-1β, monocyte chemoattractant protein (MCP)-1, tumor necrosis factor-alpha (TNF-α), interleukin 6, and nitric oxide (NO). Moreover, TNF-α sheddase protein, a disintegrin and metalloproteinase domain 17 (ADAM17), and its regulator iRhom2 were downregulated by both strains. Moreover, the bacteria also ameliorated Salmonella-induced mitochondrial dysfunction by restoring bioenergetic profiles, such as non-mitochondrial respiration, spare respiratory capacity (SRC), basal respiration, adenosine triphosphate (ATP) production, and maximal respiration. Conclusions: MKMB01 and MKMB02 can reduce pathogen-induced gut-associated disorders and therefore should be further explored for their probiotic potential. [ABSTRACT FROM AUTHOR]

Contact inhibition of proliferation is a critical cell density control mechanism governed by the Hippo signalling pathway. The biochemical signalling underlying cell density‐dependent cues regulating Hippo signalling and its downstream effectors, YAP, remains poorly understood. Here, we reveal that the tight junction protein ZO‐2 is required for the contact‐mediated inhibition of proliferation. We additionally determined that the well‐established molecular players of this process, namely Hippo kinase LATS1 and YAP, are regulated by ZO‐2 and that the scaffolding function of ZO‐2 promotes the interaction with and phosphorylation of YAP by LATS1. Mechanistically, YAP is phosphorylated when ZO‐2 brings LATS1 and YAP together via its SH3 and PDZ domains, respectively, subsequently leading to the cytoplasmic retention and inactivation of YAP. In conclusion, we demonstrate that ZO‐2 maintains Hippo signalling pathway activation by promoting the stability of LATS1 to inactivate YAP. [ABSTRACT FROM AUTHOR]

Ciliopathies are a diverse group of disorders resulting from abnormalities in the development or function of multiple organs. While significant research has clarified the role of the primary cilium in transducing numerous signalling pathways, elucidating causes of neuronal and skeletal development disorders, the origins of other ciliopathy-related conditions, such as hepatic fibrocystic diseases, remain elusive. Additionally, attempts to correlate specific ciliary proteins with distinct phenotypes have been largely unsuccessful due to the variable and overlapping symptoms of ciliopathies. This study aims to elucidate the extraciliary roles of the protein B9D2 in the development of biliary dysgenesis, a condition present in Meckel-Gruber and Joubert syndromes caused by mutations in this protein. Traditionally, B9D2 is known for its role at the transition zone of the primary cilium in the transduction of signalling pathways notably Wingless and Hedgehog. Our work demonstrates that before ciliogenesis occurs, B9D2 is crucial for the maturation and maintenance of tight junctions ensuring epithelial barrier tightness and appropriate biliary lumen formation. This study provides new insights into the mechanisms underlying biliary dysgenesis in hepatic ciliopathies, suggesting that further exploration of the non-ciliary functions of proteins involved in ciliopathies could lead to a better understanding and treatment of these complex disorders. [ABSTRACT FROM AUTHOR]

Ulcerative colitis (UC) is a debilitating inflammatory bowel disease characterized by intestinal inflammation, barrier dysfunction, and dysbiosis, with limited treatment options available. This study systematically investigates the therapeutic potential of a synbiotic composed of galactooligosaccharides (GOS) and Limosilactobacillus reuteri in a murine model of colitis, revealing that GOS and L. reuteri synergistically protect against intestinal inflammation and barrier dysfunction by promoting the synthesis of pentadecanoic acid, an odd-chain fatty acid, from Bacteroides acidifaciens. Notably, the synbiotic, B. acidifaciens, and pentadecanoic acid are each capable of suppressing intestinal inflammation and enhancing tight junction by inhibiting NF-κB activation. Furthermore, similar reduction in B. acidifaciens and pentadecanoic acid levels are also observed in the feces from both human UC patients and lipopolysaccharide-induced intestinal inflammation in pigs. Our findings elucidate the protective mechanism of the synbiotic and highlight its therapeutic potential, along with B. acidifaciens and pentadecanoic acid, for UC and other intestinal inflammatory disorders. Here, Yujun Wu and colleagues report that a synbiotic composed of galactooligosaccharides and Limosilactobacillus reuteri alleviates gut inflammation in animals by enriching pentadecanoic acid synthesis from Bacteroides acidifaciens, showing potential for treating ulcerative colitis. [ABSTRACT FROM AUTHOR]

Sepsis is a global health challenge marked by limited clinical options and high mortality rates. AMP-activated protein kinase (AMPK) is a cellular energy sensor that mediates multiple crucial metabolic pathways that may be an attractive therapeutic target in sepsis. Pre-clinical experimental studies have demonstrated that pharmacological activation of AMPK can offer multiple potential benefits during sepsis, including anti-inflammatory effects, induction of autophagy, promotion of mitochondrial biogenesis, enhanced phagocytosis, antimicrobial properties, and regulation of tight junction assembly. This review aims to discuss the existing evidence supporting the therapeutic potential of AMPK activation in sepsis management. [ABSTRACT FROM AUTHOR]

Introduction: The resolution of the skin's inflammatory response is only possible if its barrier function is restored. TRPV1 channel activation plays an important role during inflammation but the effect of this activation on the skin barrier under inflammatory conditions has not been clarified. We hypothesize that it could potentially aid the keratinocyte barrier by reducing inflammatory cytokine release and promoting tight junction development. Methods: To explore the role of TRPV1 activation in inflammation, we designed and optimized an in vitro model of keratinocytes with basal epidermal layer characteristics using HaCaT cells and TNFα to induce inflammation. Results: TNFα increased the gene expression of tight junction protein claudin 1 (CLDN1) by at least 2.60 ± 0.16-fold, in a concentration-dependent manner, over a 48 h period. The administration of a capsaicin pre-treatment reduced the CLDN1 expression to 1.51 ± 0.16-fold during the first 6 h after TNFα induction, whereas IL-8 cytokine release was reduced 0.64 ± 0.17-fold. After 48 h, CLDN1 protein levels increased by a factor of 6.57 ± 1.39 compared to cells only treated with TNFα. Discussion: These results suggest that activation of TRPV1 by capsaicin can potentiate the increase in CLDN1 expression and CLDN1 protein synthesis induced by TNFα in cultured keratinocytes, while reducing the release of IL-8. [ABSTRACT FROM AUTHOR]

Background: Brain microglia and infiltrating monocyte-derived macrophages are vital in preserving blood vessel integrity after stroke. Understanding mechanisms that induce immune cells to adopt vascular-protective phenotypes may hasten the development of stroke treatments. IL-33 is a potent chemokine released from damaged cells, such as CNS glia after stroke. The activation of IL-33/ST2 signaling has been shown to promote neuronal viability and white matter integrity after ischemic stroke. The impact of IL-33/ST2 on blood-brain barrier (BBB) integrity, however, remains unknown. The current study fills this gap and reveals a critical role of IL-33/ST2 signaling in macrophage-mediated BBB protection after stroke. Methods: Transient middle cerebral artery occlusion (tMCAO) was performed to induce ischemic stroke in wildtype (WT) versus ST2 knockout (KO) male mice. IL-33 was applied intranasally to tMCAO mice with or without dietary PLX5622 to deplete microglia/macrophages. ST2 KO versus WT bone marrow or macrophage cell transplantations were used to test the involvement of ST2+ macrophages in BBB integrity. Macrophages were cocultured in transwells with brain endothelial cells (ECs) after oxygen-glucose deprivation (OGD) to test potential direct effects of IL33-treated macrophages on the BBB in vitro. Results: The ST2 receptor was expressed in brain ECs, microglia, and infiltrating macrophages. Global KO of ST2 led to more IgG extravasation and loss of ZO-1 in cerebral microvessels 3 days post-tMCAO. Intranasal IL-33 administration reduced BBB leakage and infarct severity in microglia/macrophage competent mice, but not in microglia/macrophage depleted mice. Worse BBB injury was observed after tMCAO in chimeric WT mice reconstituted with ST2 KO bone marrow, and in WT mice whose monocytes were replaced by ST2 KO monocytes. Macrophages treated with IL-33 reduced in vitro barrier leakage and maintained tight junction integrity after OGD. In contrast, IL-33 exerted minimal direct effects on the endothelial barrier in the absence of macrophages. IL-33-treated macrophages demonstrated transcriptional upregulation of an array of protective factors, suggesting a shift towards favorable phenotypes. Conclusion: Our results demonstrate that early-stage IL-33/ST2 signaling in infiltrating macrophages reduces the extent of acute BBB disruption after stroke. Intranasal IL-33 administration may represent a new strategy to reduce BBB leakage and infarct severity. [ABSTRACT FROM AUTHOR]

Giardia duodenalis, an important zoonotic protozoan parasite, adheres to host intestinal epithelial cells (IECs) via the ventral disc and causes giardiasis characterized mainly by diarrhea. To date, it remains elusive how excretory-secretory products (ESPs) of Giardia enter IECs and how the cells respond to the entry. Herein, we initially demonstrated that ESPs evoked IEC endocytosis in vitro. We indicated that ESPs contributed vitally in triggering intrinsic apoptosis, pro-inflammatory responses, tight junction (TJ) protein expressional changes, and autophagy in IECs. Endocytosis was further proven to be implicated in those ESPs-triggered IEC responses. Ten predicted virulent excretory-secretory proteins of G. duodenalis were investigated for their capability to activate clathrin/caveolin-mediated endocytosis (CME/CavME) in IECs. Pyridoxamine 5'-phosphate oxidase (PNPO) was confirmed to be an important contributor. PNPO was subsequently verified as a vital promoter in the induction of giardiasis-related IEC apoptosis, inflammation, and TJ protein downregulation. Most importantly, this process seemed to be involved majorly in PNPO-evoked CME pathway, rather than CavME. Collectively, this study identified Giardia ESPs, notably PNPO, as potentially important pathogenic factors during noninvasive infection. It was also noteworthy that ESPs-evoked endocytosis might play a role in triggering giardiasis-inducing cellular regulation. These findings would deepen our understanding about the role of ESPs, notably PNPO, in the pathogenesis of giardiasis and the potential attributed endocytosis mechanism. [ABSTRACT FROM AUTHOR]

Apical cell-cell junctions, including adherens junctions and tight junctions, adhere epithelial cells to one another and regulate selective permeability at both bicellular junctions and tricellular junctions (TCJs). Although several specialized proteins are known to localize at TCJs, it remains unclear how actomyosin-mediated tension transmission at TCJs contributes to the maintenance of junction integrity and barrier function at these sites. Here, utilizing the embryonic epithelium of gastrula-stage Xenopus laevis embryos, we define a mechanism by which the mechanosensitive protein Vinculin helps anchor the actomyosin network at TCJs, thus maintaining TCJ integrity and barrier function. Using an optogenetic approach to acutely increase junctional tension, we find that Vinculin is mechanosensitively recruited to apical junctions immediately surrounding TCJs. In Vinculin knockdown (KD) embryos, junctional actomyosin intensity is decreased and becomes disorganized at TCJs. Using fluorescence recovery after photobleaching (FRAP), we show that Vinculin KD reduces actin stability at TCJs and destabilizes Angulin-1, a key tricellular tight junction protein involved in regulating barrier function at TCJs. When Vinculin KD embryos are subjected to increased tension, TCJ integrity is not maintained, filamentous actin (F-actin) morphology at TCJs is disrupted, and breaks in the signal of the tight junction protein ZO-1 signal are detected. Finally, using a live imaging barrier assay, we detect increased barrier leaks at TCJs in Vinculin KD embryos. Together, our findings show that Vinculin-mediated actomyosin organization is required to maintain junction integrity and barrier function at TCJs and reveal new information about the interplay between adhesion and barrier function at TCJs. [Display omitted] • Vinculin is mechanosensitively recruited to tricellular junctions • Vinculin's actin-binding function is needed for tricellular actomyosin organization • Vinculin reinforces tricellular junction integrity in response to increased tension • Vinculin is required to maintain barrier function at tricellular junctions Epithelial tricellular junctions are vulnerable to disruption. Here, van den Goor et al. reveal that Vinculin is mechanosensitively recruited to vertebrate tricellular junctions, where it regulates actomyosin organization and barrier function. Vinculin reinforces tricellular junction integrity when epithelia experience increased mechanical force. [ABSTRACT FROM AUTHOR]

Background: RNA editing, especially A-to-I editing sites, is a common RNA modification critical for stem cell differentiation, muscle development, and disease occurrence. Unveiling comprehensive RNA A-to-I editing events associated with myogenesis of the skeletal muscle satellite cells (MuSCs) is essential for extending our knowledge of the mechanism underpinning muscle development. Results: A total of 9,632 RNA editing sites (RESs) were screened in the myoblasts (GM), myocytes (DM1), and myotubes (DM5) samples. Among these sites, 4,559 A-to-I edits were classified and further analyzed. There were 3,266 A-to-I sites in the protein-coding region, out of which 113 missense sites recoded protein. Notably, five A-to-I sites in the 3' UTR of four genes (TRAF6, NALF1, SLC38A1, ENSCHIG00000019092) altered their targeted miRNAs. Furthermore, a total of 370 A-to-I sites with different editing levels were detected, including FBN1, MYH10, GSK3B, CSNK1D, and PRKACB genes. These genes were predominantly enriched in the cytoskeleton in muscle cells, the hippo signaling pathway, and the tight junction. Furthermore, we identified 14 hub genes (TUFM, GSK3B, JAK2, RPSA, YARS1, CDH2, PRKACB, RUNX1, NOTCH2, CDC23, VCP, FBN1, RARS1, MEF2C) that potentially related to muscle development. Additionally, 123 stage-specific A-to-I editing sites were identified, with 43 sites in GM, 25 in DM1, and 55 in DM5 samples. These stage-specific edited genes significantly enriched essential biological pathways, including the cell cycle, oocyte meiosis, motor proteins, and hedgehog signaling pathway. Conclusion: We systematically identified the RNA editing events in proliferating and differentiating goat MuSCs, which was crucial for expanding our understanding of the regulatory mechanisms of muscle development. [ABSTRACT FROM AUTHOR]

Background: Sepsis is defined as a life‐threatening disease. Autophagy and the microbiome are increasingly connected with sepsis. The aim of this study was to investigate the protective effect of autophagy and the possible mechanisms. Methods: The septic rat model was established by cecal ligation perforation (CLP). Rapamycin (Rap), 3‐methyladenine (3‐MA), and chloroquine (CQ) were administered to interfere autophagy. Western blot (WB) was used to detect the expression of key proteins in autophagy. Hematoxylin and eosin (H&E) staining and enzyme‐linked immunosorbent assays (ELISAs) were used to identify the effect of autophagy on various organs. 16S ribosomal RNA gene sequencing was used to analyze the changes of the gut microbiota. Results: Rap significantly upregulated the expression of key autophagy proteins, and 3‐MA reduced the relative expression compared to the CLP group. The autophagic flux showed a corresponding trend. Interestingly, the autophagy inducer significantly decreased the mortality and the lipopolysaccharide (LPS) level in serum compared with the CLP group. Autophagy activation significantly improves the inflammatory response in sepsis. Histopathological sections showed that CLP destroyed the tight junctions between ileal epithelial cells, while autophagy induction reversed the damage. The sequencing results showed that autophagy activation increased the alpha diversity and alterted the composition and structure of gut microbiota. The abundance of Proteobacteria was markedly decreased in the Rap group, whereas Bacteroidetes was notably increased compared with the CLP group. Additionally, the protective effect of autophagy further changed the biomarkers in the microbial community. The top 35 functions in each sample were analyzed to obtain 18 genes including RNA synthesis, ATP binding and transport, chromosome assignment, osmotic polysaccharide transport, transcytosis, and methylation. Conclusion: Autophagy is able to improve inflammation and may directly or indirectly regulate the microbiota of septic rats. Autophagy may be an important target for future clinical interventions in the treatment of sepsis. [ABSTRACT FROM AUTHOR]

The blood-brain barrier (BBB) is a critical interface that maintains the central nervous system homeostasis by controlling the exchange of substances between the blood and the brain. Disruption of the BBB plays a vital role in the development of neuroinflammation and neurological dysfunction in sepsis, but the mechanisms by which the BBB becomes disrupted during sepsis are not well understood. Here, we induced endotoxemia, a major type of sepsis, in mice by intraperitoneal injection of lipopolysaccharide (LPS). LPS acutely increased BBB permeability, activated microglia, and heightened inflammatory responses in brain endothelium and parenchyma. Concurrently, LPS or proinflammatory cytokines activated the NF-κB pathway, inhibiting Wnt/β-catenin signaling in brain endothelial cells in vitro and in vivo. Cell culture study revealed that NF-κB p65 directly interacted with β-catenin to suppress Wnt/β-catenin signaling. Pharmacological NF-κB pathway inhibition restored brain endothelial Wnt/β-catenin signaling activity and mitigated BBB disruption and neuroinflammation in septic mice. Furthermore, genetic or pharmacological activation of brain endothelial Wnt/β-catenin signaling substantially alleviated LPS-induced BBB leakage and neuroinflammation, while endothelial conditional ablation of the Wnt7a/7b co-receptor Gpr124 exacerbated the BBB leakage caused by LPS. Mechanistically, Wnt/β-catenin signaling activation rectified the reduced expression levels of tight junction protein ZO-1 and transcytosis suppressor Mfsd2a in brain endothelial cells of mice with endotoxemia, inhibiting both paracellular and transcellular permeability of the BBB. Our findings demonstrate that endotoxemia-associated systemic inflammation decreases endothelial Wnt/β-catenin signaling through activating NF-κB pathway, resulting in acute BBB disruption and neuroinflammation. Targeting the endothelial Wnt/β-catenin signaling may offer a promising therapeutic strategy for preserving BBB integrity and treating neurological dysfunction in sepsis. [ABSTRACT FROM AUTHOR]

Background and Purpose Experimental Approach Key Results Conclusions and Implications Intracranial atherosclerosis is one of the most common causes of ischaemic stroke. However, there is a substantial knowledge gap on the development of intracranial atherosclerosis. Intracranial arteries are characterized by an upregulation of tight junctions between endothelial cells, which control endothelial permeability. We investigated the role of N6‐methyladenosine (m6A), a common RNA modification, on endothelial integrity, focusing on the pro‐atherogenic microRNA miR‐494‐3p and tight junction proteins TJP1 and PECAM1.We assessed the m6A landscape, along with the expression of miR‐494‐3p, TJP1 and PECAM1 in postmortem human vertebral arteries (VA), internal carotid arteries (ICA), and middle cerebral arteries (MCA) with various stages of intimal thickening and plaque formation. The interactions between m6A‐modified miR‐494‐3p mimics, TJP1 and PECAM1, were investigated in vitro using primary human (brain) endothelial cells.Increased m6A expression was observed in the luminal lining of atherosclerosis‐affected VAs, accompanied by reduced TJP1 and PECAM1, but not VE‐cadherin, expression. Colocalization of m6A and miR‐494‐3p in the luminal lining of VA plaques was confirmed, indicating m6A methylation of miR‐494‐3p in intracranial atherosclerosis. Moreover, site‐specific m6A‐modification of miR‐494‐3p led to repression specifically of TJP1 protein expression at cell–cell junctions of brain microvascular endothelial cells, while unmodified miR‐494‐3p showed no effect.This study highlights increasing m6A levels during intracranial atherogenesis. Increases in m6A‐miR‐494‐3p contribute to the observed decreased TJP1 expression in endothelial cell–cell junctions. This is likely to have a negative effect on endothelial integrity and may thus accelerate intracranial atherosclerosis progression. [ABSTRACT FROM AUTHOR]