Regulation Of Junctions Research Articles

Regulation of Tight Junctions by Sex Hormones in Goat Mammary Epithelial Cells.

Simple SummaryHow ovarian hormones affect goat lactation by regulating cell–cell junctions is still unclear. Through the in vivo and in vitro assays, we found that ovarian hormones could elevate cell–cell junction protein expression, which may affect the intercellular space and molecule transportation between the goat mammary epithelial cells. Our assessment suggests that ovarian hormones may affect goat milk production by regulating the cell–cell junction protein expression between mammary epithelial cells.The sex hormones of estrogen and progesterone (P4) play a vital role in mammary gland development and milk lactation in ruminants. The tight junction (TJ) between adjacent secretory epithelial cells is instrumental in establishing the mammary blood–milk barrier. However, whether estrogen and P4 exert their effect on mammary function via regulating TJ remain unclear. Here, to clarify the role of 17-β estradiol (E2) and P4 in the regulation of TJ in goat mammary gland, we first explored the relationships between the concentrations of E2, P4, and the protein expression of claudin-1, claudin-3, occludin, and ZO-1 during the mammary gland development in goat. Then, we further explored the mRNA and protein expression of claudin-1, claudin-3, occludin, and ZO-1 in the goat mammary epithelial cells (GMECs) in vitro under different concentrations of E2 and P4. The results demonstrated that the protein expression of claudin-1 decreased, but occludin and ZO-1 increased with the decline in E2 and P4 during the transition from pregnancy to lactation. In the in vitro studies, E2 exerted a positive effect on the mRNA expression of claudin-1, and accelerated the proteins’ expression of claudin-1 and ZO-1 in GMECs; P4 upregulated the mRNA expression of claudin-1, claudin-3, occludin, and ZO-1, and also improved the protein expression of claudin-1, claudin-3, and ZO-1 in the GMECs. The results demonstrated that E2 and P4 play an important role in regulating the expression of the mammary TJ components, which may ultimately affect the mammary gland development and milk lactation.

Identification of alveolar proteins associated with claudin‐18 using BioID

Tight junctions are protein complexes that form at sites of cell contact that function to regulate paracellular flow of small molecules, water, and ions. Distinct protein components contribute to the barrier function of tight junctions including the claudin family of transmembrane proteins and scaffolding proteins such as Zonula Occludens‐1(ZO‐1). Changes in tight junction morphology from the established smooth cobblestone patterning to a spiked junction are associated with an increase in barrier permeability (leak). Tight junction spike formation is associated with changes in claudin‐18 interacting partners, including ZO‐1. This suggests that protein‐claudin‐18 interactions are essential for tight junction morphology and therefore function. Using rat alveolar epithelial cells (rAECs), we used BioID to investigate the proximal claudin‐18 proteome. BioID uses a promiscuous biotinylating enzyme, BirA, which we fused to claudin‐18 to biotinylate proximal and interacting proteins. To determine that BirA‐claudin‐18 localized to tight junctions, rAECs were transduced with adenovirus to express BirA‐claudin‐18 and stained with claudin‐18 and streptavidin‐Cy3. This showed that the assay was functional and our chimera trafficked to tight junctions and colocalized to proximal proteins. To identify biotinylated proteins, we used tandem mass spectrometry (MS/MS) and identified 3771 protein matches within the BirA‐claudin‐18 sample. Proteins of interest were vetted, and a list of protein candidates were compiled based on peptide spectrum matches (PSMs) and known protein localization. Proteins of interest include retinoic acid and palladin, which are of specific interest because they are associated with a spike like structure found in the testis, the tubulobulbar complex, which we hypothesize behaves as a signaling platform in the testis and is comparable to tight junction spikes found in the lung. These findings show that BioID is a valid method for identification of proteins to claudin‐18, providing a discovery‐based approach to identify novel proteins that play a role in barrier function and tight junction regulation.

VE-cadherin enables trophoblast endovascular invasion and spiral artery remodeling during placental development.

During formation of the mammalian placenta, trophoblasts invade the maternal decidua and remodel spiral arteries to bring maternal blood into the placenta. This process, known as endovascular invasion, is thought to involve the adoption of functional characteristics of vascular endothelial cells (ECs) by trophoblasts. The genetic and molecular basis of endovascular invasion remains poorly defined, however, and whether trophoblasts utilize specialized endothelial proteins in an analogous manner to create vascular channels remains untested. Vascular endothelial (VE-)cadherin is a homotypic adhesion protein that is expressed selectively by ECs in which it enables formation of tight vessels and regulation of EC junctions. VE-cadherin is also expressed in invasive trophoblasts and is a prime candidate for a molecular mechanism of endovascular invasion by those cells. Here, we show that VE-cadherin is required for trophoblast migration and endovascular invasion into the maternal decidua in the mouse. VE-cadherin deficiency results in loss of spiral artery remodeling that leads to decreased flow of maternal blood into the placenta, fetal growth restriction, and death. These studies identify a non-endothelial role for VE-cadherin in trophoblasts during placental development and suggest that endothelial proteins may play functionally unique roles in trophoblasts that do not simply mimic those in ECs.

In vitro effects of 5-Hydroxy-L-tryptophan supplementation on primary bovine mammary epithelial cell gene expression under thermoneutral or heat shock conditions

Serotonin (5-HT) is an autocrine-paracrine molecule within the mammary gland regulating homeostasis during lactation and triggering involution after milk stasis. Exposure of dairy cows to hyperthermia during the dry period alters mammary gland involution processes leading to reduced subsequent yields. Herein, primary bovine mammary epithelial cells (pBMEC) under thermoneutral (TN, 37 °C) or heat shock (HS, 41.5 °C) conditions were cultured with either 0, 50, 200, or 500 μM 5-Hydroxy-L-tryptophan (5-HTP; 5-HT precursor) for 8-, 12- or 24-h. Expression of 95 genes involved in 5-HT signaling, involution and tight junction regulation were evaluated using a Multiplex RT-qPCR BioMark Dynamic Array Circuit. Different sets of genes were impacted by 5-HTP or temperature, or by their interaction. All 5-HT signaling genes were downregulated after 8-h of HS and then upregulated after 12-h, relative to TN. After 24-h, apoptosis related gene, FASLG, was upregulated by all doses except TN-200 μM 5-HTP, and cell survival gene, FOXO3, was upregulated by HS-50, 200 and 500 μM 5-HTP, suggesting 5-HTP involvement in cell turnover under HS. Supplementing 5-HTP at various concentrations in vitro to pBMEC modulates the expression of genes that might aid in promoting epithelial cell turn-over during involution in dairy cattle under hyperthermia.

Review article: therapeutic targets for the pharmacologic management of coeliac disease—the future beyond a gluten‐free diet

Coeliac disease (CeD) is an immune-mediated small bowel enteropathy resulting from dietary gluten exposure. Presently, the only effective treatment is adoption of a gluten-free diet (GFD), although strict adherence is challenging to maintain, and inadvertent gluten exposures are inevitable for most patients. Hence, there is substantial interest in drug development in CeD and multiple novel therapies are under investigation. To review existing and upcoming clinical trial programmes for pharmacologic agents for CeD. A narrative review was performed, informed by a search of MEDLINE, Embase, the Cochrane CENTRAL Library and clinicaltrials.gov. We summarise the pathophysiology of CeD and the specific steps that are potentially amenable to pharmacologic treatment. We evaluate the evidence supporting existing and future drug targets, including trials of peptidases, gluten sequestrants, tight junction regulators, anti-transglutaminase 2 therapies, immune tolerizing agents, advanced biologics and small molecules, and microbiome-targeted strategies. We highlight unique considerations for conducting CeD trials, including identifying appropriate study populations, assessing results in the context of a gluten challenge, and interpreting CeD-specific clinical and histologic outcomes. Understanding these factors is crucial for accurately appraising the evidence. Finally, we outline what the future of CeD therapy may hold with the introduction of pharmacotherapies. There is a need for pharmacologic options for CeD, either used adjunctively with a GFD for accidental or intentional gluten exposures or for refractory disease. Multiple promising agents are in development, and these trials are likely to lead to approvals for the first generation of pharmacologic agents for CeD within the next 5years.

MiR-1301-3p Inhibits Epithelial-Mesenchymal Transition via Targeting RhoA in Pancreatic Cancer.

Micro(mi)RNAs play an essential role in the epithelial-mesenchymal transition (EMT) process in human cancers. This study aimed to uncover the regulatory mechanism of miR-1301-3p on EMT in pancreatic cancer (PC). The miRNA profilings from Gene Expression Omnibus data sets (GSE31568, GSE41372, and GSE32688) demonstrated the downregulation of miR-1301-3p in PC tissues, which was validated with 72 paired PC tissue samples through qRT-PCR detection. The low level of miR-1301-3p was associated with a poor prognosis for PC patients from the PC cohort of The Cancer Genome Atlas and the validation cohort. Gene Ontology analyses indicated that the target genes of miR-1301-3p were involved in cell cycle and adherent junction regulation. In vitro assays revealed that miR-1301-3p suppressed the proliferation and migration abilities of PC cells. Western blotting and luciferase reporter assays suggested that miR-1301-3p inhibited RhoA expression by targeting its 3′-untranslated region; RhoA upregulated N-cadherin and vimentin levels; however, it downregulated the E-cadherin level. In conclusion, our study showed that miR-1301-3p could serve as a prognostic biomarker for PC and suppress PC cell malignancy by targeting the RhoA-induced EMT process.

Collagen-targeted tumor-specific transepithelial penetration enhancer mediated intravesical chemoimmunotherapy for non-muscle-invasive bladder cancer

Intravesical instillation of chemotherapeutics or immune-stimulating agents could reduce the recurrence rate of non-muscle-invasive bladder cancer (NMIBC) after transurethral resection of the bladder tumors. Its efficacy, however, remains to be improved due to the bladder epithelial barrier. Although certain transmucosal delivery carriers are able to enhance the transepithelial penetration of intravesical agents, they could hardly differentiate carcinoma and adjacent normal tissues of the bladder wall. Here, we reported polyethylene glycol (PEG) & glutaraldehyde co-modified fluorinated chitosan (PGFCS) as a collagen-targeted transepithelial penetration enhancer, which could create a tumor-targeted adhesive interface by the aldehyde-selective reaction with collagen amines enriched in the tumor, thus opening the transepithelial-delivery barrier at the tumor site though the fluorinated-chitosan-mediated tight junction regulation. Interestingly, with the help of PGFCS pre-treatment, intravesical instillation of chemotherapeutics pirabucin (THP) combined with immune stimulating agent interleukin-12 could trigger potent antitumor chemoimmunotherapeutic responses in destructing orthotopic bladder tumors and inhibiting cancer recurrence. Our work presents a unique type of tumor-specific transepithelial penetration enhancer, which shows great potential for safe and effective intravesical instillation of NMIBC.

Structural changes of Gap junction visualized in simulation utilizing a double membrane

Gap junctions are ubiquitous throughout the body, being present in all but one human tissue. They are essential for many body functions including, fetal development, cardiac muscle contraction, and functional neural networks. Numerous pathologies, such as wound healing, ocular cataracts, and heart disease have been linked to Gap junctions. However, there is still much that is unknown regarding Gap junction regulation and internalization. We are looking at the structural changes in Gap junction models using a Cx26/Cx43 hybrid structure caused by phosphorylation, ubiquitylation, and interaction of key residues with regulatory proteins MAPK, PKC, and Nedd4-E3-Ub-ligase.

Reconstitution of the full transmembrane cadherin-catenin complex

The dynamic regulation of epithelial adherens junctions relies on all components of the E-cadherin-catenin complex. Previously, the complexes have been partially reconstituted and composed only of α-catenin, β-catenin, and the E-cadherin cytoplasmic domain. However, p120-catenin and the full-length E-cadherin including the extracellular, transmembrane, and intra-cellular domains are vital to the understanding of the relationship between extracellular adhesion and intracellular signaling. Here, we reconstitute the complete and full-length cadherin-catenin complex, including full-length E-cadherin, α-catenin, β-catenin, and p120-catenin, into nanodiscs. We are able to observe the cadherin in nanodiscs by cryo-EM. We also reconstitute α-catenin, β-catenin, and p120-catenin with the E-cadherin cytoplasmic tail alone in order to analyze the affinities of their binding interactions. We find that p120-catenin does not associate strongly with α- or β-catenin and binds much more transiently to the cadherin cytoplasmic tail than does β-catenin. Overall, this work creates many new possibilities for biochemical studies understanding transmembrane signaling of cadherins and the role of p120-catenin in adhesion activation.

AJUBA and WTIP can compete with LIMD1 for junctional localization and LATS regulation.

Each of the three mammalian Ajuba family proteins, AJUBA, LIMD1 and WTIP, exhibit tension-dependent localization to adherens junctions, and can associate with Lats kinases. However, only LIMD1 has been directly demonstrated to directly regulate Lats activity in vivo. To assess the relationship of LIMD1 to AJUBA and WTIP, and the potential contributions of AJUBA and WTIP to Lats regulation, we examined the consequences of over-expressing AJUBA and WTIP in MCF10A cells. Over-expression of either AJUBA or WTIP reduced junctional localization of LIMD1, implying that these proteins can compete for binding to adherens junctions. This over-expression also reduced junctional localization of LATS1, implying that AJUBA or WTIP are unable to efficiently recruit Lats kinases to adherens junctions. This over-expression was also associated with increased YAP1 phosphorylation and decreased YAP1 nuclear localization, consistent with increased Lats kinase activity. These observations indicate that AJUBA and WTIP compete with LIMD1 for association with adherens junctions but have activities distinct from LIMD1 in Hippo pathway regulation. They further suggest that the ability of Ajuba family proteins to associate with Lats kinases in solution is not sufficient to enable regulation in vivo, and that tumor suppressor activities of AJUBA and WTIP could stem in part from competition with LIMD1 for regulation of Lats kinases at cell junctions.

Efferent neurons control hearing sensitivity and protect hearing from noise through the regulation of gap junctions between cochlear supporting cells.

It is critical for hearing that the descending cochlear efferent system provides a negative feedback to hair cells to regulate hearing sensitivity and protect hearing from noise. The medial olivocochlear (MOC) efferent nerves project to outer hair cells (OHCs) to regulate OHC electromotility, which is an active cochlear amplifier and can increase hearing sensitivity. Here, we report that the MOC efferent nerves also could innervate supporting cells (SCs) in the vicinity of OHCs to regulate hearing sensitivity. MOC nerve fibers are cholinergic, and acetylcholine (ACh) is a primary neurotransmitter. Immunofluorescent staining showed that MOC nerve endings, presynaptic vesicular acetylcholine transporters (VAChTs), and postsynaptic ACh receptors were visible at SCs and in the SC area. Application of ACh in SCs could evoke a typical inward current and reduce gap junctions (GJs) between them, which consequently enhanced the direct effect of ACh on OHCs to shift but not eliminate OHC electromotility. This indirect, GJ-mediated inhibition had a long-lasting influence. In vivo experiments further demonstrated that deficiency of this GJ-mediated efferent pathway decreased the regulation of active cochlear amplification and compromised the protection against noise. In particular, distortion product otoacoustic emission (DPOAE) showed a delayed reduction after noise exposure. Our findings reveal a new pathway for the MOC efferent system via innervating SCs to control active cochlear amplification and hearing sensitivity. These data also suggest that this SC GJ-mediated efferent pathway may play a critical role in long-term efferent inhibition and is required for protection of hearing from noise trauma.NEW & NOTEWORTHY The cochlear efferent system provides a negative feedback to control hair cell activity and hearing sensitivity and plays a critical role in noise protection. We reveal a new efferent control pathway in which medial olivocochlear efferent fibers have innervations with cochlear supporting cells to control their gap junctions, therefore regulating outer hair cell electromotility and hearing sensitivity. This supporting cell gap junction-mediated efferent control pathway is required for the protection of hearing from noise.

First Responders to Hyperosmotic Stress in Murine Astrocytes: Connexin 43 Gap Junctions Are Subject to an Immediate Ultrastructural Reorganization.

Simple SummaryGap junctions are intercellular channels that provide the means for direct transport of small molecules, ions, and water between connected cells. With these functions, gap junctions are essential for the maintenance of astrocytic homeostasis and of particular importance in the context of pathophysiological disbalances. These include the hyperosmolar hyperglycemic syndrome or the pathology after brain trauma. We demonstrate that short-term hyperosmolarity reduces intercellular communication via gap junctions. These functional changes coincide with the transformation of gap junction ultrastructure as evidenced by freeze-fracture replica immunolabeling and transmission electron microscopy. The hyperosmolarity-induced immediate changes in the ultrastructural assembly of connexons, the protein constituents of gap junction channels, have not been described in astrocytes before and are revealing the coherence of structure and function in gap junctions. Phosphorylation of Connexin 43, the main gap junction protein in astrocytes, at amino acid 368 (Serine) might link the two.In a short-term model of hyperosmotic stress, primary murine astrocytes were stimulated with a hyperosmolar sucrose solution for five minutes. Astrocytic gap junctions, which are mainly composed of Connexin (Cx) 43, displayed immediate ultrastructural changes, demonstrated by freeze–fracture replica immunogold labeling: their area, perimeter, and distance of intramembrane particles increased, whereas particle numbers per area decreased. Ultrastructural changes were, however, not accompanied by changes in Cx43 mRNA expression. In contrast, transcription of the gap junction regulator zonula occludens (ZO) protein 1 significantly increased, whereas its protein expression was unaffected. Phosphorylation of Serine (S) 368 of the Cx43 C–terminus has previously been associated with gap junction disassembly and reduction in gap junction communication. Hyperosmolar sucrose treatment led to enhanced phosphorylation of Cx43S368 and was accompanied by inhibition of gap junctional intercellular communication, demonstrated by a scrape loading-dye transfer assay. Taken together, Cx43 gap junctions are fast reacting elements in response to hyperosmolar challenges and can therefore be considered as one of the first responders to hyperosmolarity. In this process, phosphorylation of Cx43S368 was associated with disassembly of gap junctions and inhibition of their function. Thus, modulation of the gap junction assembly might represent a target in the treatment of brain edema or trauma.

Pathogenic mechanisms of preeclampsia with severe features implied by the plasma exosomal mirna profile

ABSTRACT Preeclampsia is a complication of pregnancy characterized by high blood pressure and organ damage after 20 gestational weeks. It is associated with high maternal and fetal morbidity and mortality. However, at present, there is no effective prevention or treatment for this condition. Previous studies have revealed that plasma exosomal mirnas from pregnant women with preeclampsia could serve as biomarkers of pathogenic factors. However, the roles of plasma exosomal mirnas in preeclampsia with severe features (sPE), which is associated with poorer pregnancy outcomes, remain unknown. Thus, the aims of this study were to characterize plasma exosomal miRNAs in sPE and explore the related pathogenic mechanisms using bioinformatic analysis. Plasma exosomes were isolated using a mirVana RNA isolation kit. the exosomal miRNAs were detected using high-throughput sequencing and the mirnas related to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and gene ontology (GO) terms were analyzed using the clusterprofiler package of R. Fifteen miRNAs exhibited increased expression and fourteen miRNAs exhibited reduced expression in plasma exosomes from women with sPE as compared to normal pregnant women. Further, gene set enrichment analysis revealed that the differentially expressed plasma exosomal miRNAs were related to the stress response and cell junction regulation, among others. In summary, this study is the first to identify the differentially expressed plasma exosomal miRNAs in sPE. These findings highlight promising pathogenesis mechanisms underlying preeclampsia.

Blood-brain barrier-restricted translocation of Toxoplasma gondii from cortical capillaries.

The cellular barriers of the central nervous system proficiently protect the brain parenchyma from infectious insults. Yet, the single-celled parasite Toxoplasma gondii commonly causes latent cerebral infection in humans and other vertebrates. Here, we addressed the role of the cerebral vasculature in the passage of T. gondii to the brain parenchyma. Shortly after inoculation in mice, parasites mainly localized to cortical capillaries, in preference over post-capillary venules, cortical arterioles or meningeal and choroidal vessels. Early invasion to the parenchyma (days 1-5) occurred in absence of a measurable increase in blood-brain barrier (BBB) permeability, perivascular leukocyte cuffs or hemorrhage. However, sparse focalized permeability elevations were detected adjacently to replicative parasite foci. Further, T. gondii triggered inflammatory responses in cortical microvessels and endothelium. Pro- and anti-inflammatory treatments of mice with LPS and hydrocortisone, respectively, impacted BBB permeability and parasite loads in the brain parenchyma. Finally, pharmacological inhibition or Cre/loxP conditional knockout of endothelial focal adhesion kinase (FAK), a BBB intercellular junction regulator, facilitated parasite translocation to the brain parenchyma. The data reveal that the initial passage of T. gondii to the central nervous system occurs principally across cortical capillaries. The integrity of the microvascular BBB restricts parasite transit, which conversely is exacerbated by the inflammatory response.

E3 ubiquitin ligase ASB17 is required for spermiation in mice.

BackgroundA major goal of spermiation is to degrade the apical ectoplasmic specialization (ES) junction between Sertoli cells and elongating spermatids in preparation for the eventual disengagement of spermatids into the lumen. E3 ubiquitin ligases mediate the process of ubiquitination and the subsequent proteasomal degradation, but their specific role during spermiation remains largely unexplored.MethodsAnkyrin repeat and SOCS box protein 17 (Asb17)-knockout mice were generated via a CRISPR/Cas9 approach. Epididymal sperm parameters were assessed by a computer-assisted sperm analysis (CASA) system, and morphological analysis of testicular tissues were performed based on histological and immunostaining staining, and transmission electron microscopy (TEM). The interactions between ASB17 and Espin (ESPN) were predicted by HawkDock server and validated through protein pull-down and immunoprecipitation assays.ResultsWe report that ASB17, an E3 ligase, is required for the completion of spermiation and that mice lacking Asb17 are oligozoospermic owing to spermiation failure. ASB17-deficient mice are fertile; however, spermatids exhibit a disorganized ES junction, resulting in retention within the seminiferous epithelium. Mechanistically, ASB17 deficiency leads to excess accumulation of ESPN, an actin-binding essential structural component of the ES. We determined that ASB17 regulates the removal of the ES through ubiquitin mediated protein degradation of ESPN.ConclusionsIn summary, our study describes a role for ASB17 in the regulation of cell-cell junctions between germ cells and somatic cells in the testis. These findings establish a novel mechanism for the regulatory role of E3 ligases during spermatogenesis.

Regulation of blood\u2013brain barrier integrity by microbiome-associated methylamines and cognition by trimethylamine N-oxide

BackgroundCommunication between the gut microbiota and the brain is primarily mediated via soluble microbe-derived metabolites, but the details of this pathway remain poorly defined. Methylamines produced by microbial metabolism of dietary choline and l-carnitine have received attention due to their proposed association with vascular disease, but their effects upon the cerebrovascular circulation have hitherto not been studied.ResultsHere, we use an integrated in vitro/in vivo approach to show that physiologically relevant concentrations of the dietary methylamine trimethylamine N-oxide (TMAO) enhanced blood-brain barrier (BBB) integrity and protected it from inflammatory insult, acting through the tight junction regulator annexin A1. In contrast, the TMAO precursor trimethylamine (TMA) impaired BBB function and disrupted tight junction integrity. Moreover, we show that long-term exposure to TMAO protects murine cognitive function from inflammatory challenge, acting to limit astrocyte and microglial reactivity in a brain region-specific manner.ConclusionOur findings demonstrate the mechanisms through which microbiome-associated methylamines directly interact with the mammalian BBB, with consequences for cerebrovascular and cognitive function.24wuPkhfjSm7S4zaEcHM79Video abstract

Effects of larazotide acetate, a tight junction regulator, on the liver and intestinal damage in acute liver failure in rats.

The epithelial cells are the strongest determinants of the physical intestinal barrier. Tight junctions (TJs) hold the epithelial cells together and allow for selective paracellular permeability. Larazotide acetate (LA) is a synthetic octapeptide that reduces TJ permeability by blocking zonulin receptors. In this study, we aimed to investigate the effects of LA, a TJ regulator, on the liver and intestinal histology in the model of acute liver failure (ALF) in rats. The thioacetamide (TAA) group received intraperitoneal (ip) injections of 300mg/kg TAA for 3days. The TAA+LA(dw) (drinking water) group received prophylactic 0.01mg/mL LA orally for 7days before the first dose of TAA. The LA(dw) group received 0.01mg/mL LA orally. The TAA + LA(g) (gavage) group received prophylactic 0.01mg/mL LA via oral gavage for 7 days before the first dose of TAA. The LA(g) group received 0.01mg/mL LA via oral gavage. While liver tissue was evaluated only with light microscopy, intestinal samples were examined with light and electron microscopy. Serum ammonia, AST, and ALT levels in the TAA group were significantly higher than in control groups (all p < 0.01). Serum ALT levels in the TAA + LA(dw) group were significantly lower than in the TAA group (p < 0.05). However, serum ammonia and ALT levels did not differ between the TAA and other groups. Serious liver damage in the TAA group was accompanied by marked intestinal damage. There was no significant difference between the TAA and TAA + LA(dw) groups and TAA and TAA + LA(g) groups for liver damage scores. However, intestinal damage scores significantly decreased in the TAA + LA(dw) group compared to the TAA group. In the TAA + LA(dw) group, fusion occurred between the surface epithelial cells of neighboring villi and connecting regions formed as epithelial bridges between the villi. Our findings suggest that LA reduced intestinal damage by acting on TJs in the TAA-induced ALF model in rats.

A joint analysis using exome and transcriptome data identifies candidate polymorphisms and genes involved with umbilical hernia in pigs

BackgroundUmbilical Hernia (UH) is characterized by the passage of part of the intestine through the umbilical canal forming the herniary sac. There are several potential causes that can lead to the umbilical hernia such as bacterial infections, management conditions and genetic factors. Since the genetic components involved with UH are poorly understood, this study aimed to identify polymorphisms and genes associated with the manifestation of umbilical hernia in pigs using exome and transcriptome sequencing in a case and control design.ResultsIn the exome sequencing, 119 variants located in 58 genes were identified differing between normal and UH-affected pigs, and in the umbilical ring transcriptome, 46 variants were identified, located in 27 genes. Comparing the two methodologies, we obtained 34 concordant variants between the exome and transcriptome analyses, which were located in 17 genes, distributed in 64 biological processes (BP). Among the BP involved with UH it is possible to highlight cell adhesion, cell junction regulation, embryonic morphogenesis, ion transport, muscle contraction, within others.ConclusionsWe have generated the first exome sequencing related to normal and umbilical hernia-affected pigs, which allowed us to identify several variants possibly involved with this disorder. Many of those variants present in the DNA were confirmed with the RNA-Seq results. The combination of both exome and transcriptome sequencing approaches allowed us to better understand the complex molecular mechanisms underlying UH in pigs and possibly in other mammals, including humans. Some variants found in genes and other regulatory regions are highlighted as strong candidates to the development of UH in pigs and should be further investigated.

Gualou Xiebai Decoction ameliorates increased Caco-2 monolayer permeability induced by bile acids via tight junction regulation, oxidative stress suppression and apoptosis reduction.

Gualou Xiebai Decoction (GXD), a classic prescription, is widely used to dealing with inflammatory diseases in China for thousands of years. Abnormal metabolic state of bile acids (BAs) is confirmed to cause intestinal epithelial barrier dysfunction. In preliminary work, we observed that GXD could decrease intestinal permeability in hyperlipidemia mice. The present study aimed to explore the protective effect of GXD on intestinal mucosa in vitro. Caco-2 cell monolayer permeability among different groups was determined by measuring the concentrations of FITC-dextran in the lower compartments and transepithelial electrical resistance (TEER). Meanwhile, mRNA and protein expressions of tight junctions (TJs) were investigated. Generation of intracellular reactive oxygen species (ROS) and the ratio of cell apoptosis induced by BAs were assessed by fluorescence probe and flow cytometry. GXD was shown to keep the cell monolayer in low permeable status, increase TEER and mRNA and protein expressions of occludin (Ocln) and zonula occluden 2 (ZO2) remarkably in cells challenged with cholic acid (CA), deoxycholic acid (DCA) and glycocholic acid (GCA). However, no significant effects were uncovered against the pathological effects of taurocholic acid (TCA). Meanwhile, generation of ROS and increased levels of apoptotic cells caused by CA, DCA and GCA were dramatically decreased by GXD, which were not observed on TCA. GXD could significantly attenuate intestinal barrier dysfunction induced by BAs via TJs regulation, oxidative stress suppression and cell apoptosis decrease, but such effects and behind mechanisms differed among different kinds of BAs.

Metallothionein 2 activation by pravastatin reinforces epithelial integrity and ameliorates radiation-induced enteropathy.

BackgroundRadiotherapy or accidental exposure to ionizing radiation causes severe damage of healthy intestinal tissues. Intestinal barrier function is highly sensitive to ionizing radiation, and loss of epithelial integrity results in mucosal inflammation, bacterial translocation, and endotoxemia. Few studies have of epithelial integrity as a therapeutic target to treat radiation toxicity. Here, we examined the effects of pravastatin (PS) and the molecular mechanisms underlying epithelial integrity on radiation-induced enteropathy.MethodsThe radio-mitigative effects of PS were evaluated in a minipig model by quantifying clinical symptoms, and performing histological and serological analyses and mRNA sequencing in intestinal tissues. To evaluate the role of intercellular junctions on radiation damage, we used tight junction regulator and metallothionein 2 (MT2) as treatments in a mouse model of radiation-induced enteropathy. Caco-2 monolayers were used to examine functional epithelial integrityand intercellular junction expression.FindingUsing a minipig model of pharmaceutical oral bioavailability, we found that PS mitigated acute radiation-induced enteropathy. PS-treated irradiated minipigs had mild clinical symptoms, lower intestinal inflammation and endotoxin levels, and improved gastrointestinal integrity, compared with control group animals. The results of mRNA sequencing analysis indicated that PS treatment markedly influenced intercellular junctions by inhibiting p38 MAPK signaling in the irradiated intestinal epithelium. The PS-regulated gene MT2 improved the epithelial barrier via enhancement of intercellular junctions in radiation-induced enteropathy.InterpretationPS regulated epithelial integrity by modulating MT2 in radiation-damaged epithelial cells. These findings suggested that maintenance of epithelial integrity is a novel therapeutic target for treatment of radiation-induced gastrointestinal damage.FundingAs stated in the Acknowledgments