Proteins Zonula Occludens-1 Research Articles

Evaluation of Tight Junction Integrity in Brain Endothelial Cells Using Confocal Microscopy.

The blood-brain barrier (BBB) is a highly complex and dynamic microvascular barrier that protects the brain parenchyma from the entry of pathogens, toxins, and other macromolecules and is a critical structure that helps to maintain brain homeostasis. The BBB is formed mainly by brain capillary endothelial cells and perivascular astrocytes and pericytes. One of the primary properties of the BBB is a tight regulation of paracellular permeability due to the presence of tight junctional complexes (also, adherens and gap junctions) between the neighboring microvascular endothelial cells. Alterations in the assembly of the tight junctions impair BBB properties, particularly influenced barrier integrity and permeability. The tight junctions of the BBB are mainly composed of proteins including claudins, occludin, and zonula occludens-1 (ZO-1). Zonula occludens-1 binds to the actin cytoskeleton, and its localization provides valuable information on the status of BBB integrity and permeability. Immunofluorescence localization of ZO-1 and/or other tight junction proteins is a reliable indicator of barrier integrity and permeability in microvascular endothelial cells. In microvascular endothelial cells, f-actin stress fiber formation significantly influences the rate and size of the inter-endothelial cell gap that form as cells retract from their borders. Rhodamine phalloidin is a popular conjugate used as a fluorescent label for f-actin. Herein, we describe the procedures for ZO-1 immunofluorescence and f-actin labeling followed by confocal microscopic imaging to determine barrier integrity and tight junction organization in brain microvascular endothelial cells in vitro.

Determination of Tight Junction Integrity in Brain Endothelial Cells Based on Tight Junction Protein Expression.

Blood-brain barrier (BBB) dysfunction and hyperpermeability that occurs following traumatic and ischemic insults lead to various downstream ill effects such as cerebral edema and elevation of intracranial pressure. The inter-endothelial tight junctions that consist of tight junction proteins are critical regulators of BBB dysfunctions and hyperpermeability. The major tight junction-associated proteins of the BBB are occludin, claudins, and junctional adhesion molecules that are intracellularly linked to the adaptor protein zonula occludens-1 (ZO-1). Quantitative measurement of tight junction-associated proteins provides valuable insight into barrier integrity and mechanisms that regulate microvascular hyperpermeability. Western blot analysis is a commonly used method to separate and identify proteins in a mixture using gel electrophoresis. Understanding the changes in the expression of one or more of these proteins is critical to evaluating barrier integrity and permeability in health and disease. Furthermore, studying them will provide insight into the associated downstream signaling pathways and evaluation of therapeutic approaches for regulating BBB permeability. Herein, we have described the protocol for immunoblot analysis of ZO-1 as an indicator of tight junction integrity in brain microvascular endothelial cells.

Plant-derived squalene supplementation improves growth performance and alleviates acute oxidative stress-induced growth retardation and intestinal damage in piglets

Piglets are particularly susceptible to oxidative stress, which causes inferior growth performance and intestinal damage. Squalene (SQ), a natural bioactive substance enriched in shark liver oil, shows excellent antioxidant properties and can currently be obtained at a low cost from deodorizer distillate during the production of plant oil. This study aimed to evaluate the effects of plant-derived SQ supplementation on the growth performance of piglets and explore the beneficial roles of SQ against oxidative stress and intestinal injury in diquat-challenged piglets. Forty piglets were randomly divided into five groups and fed a basal diet supplemented with SQ at 0, 500, 1000, or 2000 mg/kg for 5 weeks. Acute oxidative stress was induced in the piglets with diquat (10 mg/kg BW) at the fourth week of the experiment, followed by a 7-d recovery period. Results showed that before the diquat challenge, SQ supplementation significantly improved growth performance (average daily gain and feed conversion ratio) and serum antioxidant status, and after the diquat challenge, SQ supplementation significantly mitigated diquat-induced growth arrest, intestinal villous atrophy, intestinal epithelial cell apoptosis, intestinal hyperpermeability, and deficiency of intestinal epithelial tight junction proteins (zonula occludens-1, occludin, and claudin-3). Under oxidative stress induced by diquat, SQ supplementation consistently improved the antioxidant status of the small intestine, liver, and muscle. In vitro, SQ was shown to alleviate hydrogen peroxide (H2O2)-induced increase of the levels of intracellular reactive oxygen species and apoptosis of porcine intestinal epithelial cells. Taken together, SQ supplementation improves growth performance and effectively alleviates acute oxidative stress-induced growth retardation and intestinal injury via improving antioxidant capacity in piglets. Our findings may provide an efficient strategy for alleviating oxidative stress-induced inferior growth performance and intestinal damage in piglets.

Soloxolone Methyl Reduces the Stimulatory Effect of Leptin on the Aggressive Phenotype of Murine Neuro2a Neuroblastoma Cells via the MAPK/ERK1/2 Pathway.

Despite the proven tumorigenic effect of leptin on epithelial-derived cancers, its impact on the aggressiveness of neural crest-derived cancers, notably neuroblastoma, remains largely unexplored. In our study, for the first time, transcriptome analysis of neuroblastoma tissue demonstrated that the level of leptin is elevated in neuroblastoma patients along with the severity of the disease and is inversely correlated with patient survival. The treatment of murine Neuro2a neuroblastoma cells with leptin significantly stimulated their proliferation and motility and reduced cell adhesion, thus rendering the phenotype of neuroblastoma cells more aggressive. Given the proven efficacy of cyanoenone-bearing semisynthetic triterpenoids in inhibiting the growth of neuroblastoma and preventing obesity in vivo, the effect of soloxolone methyl (SM) on leptin-stimulated Neuro2a cells was further investigated. We found that SM effectively abolished leptin-induced proliferation of Neuro2a cells by inducing G1/S cell cycle arrest and restored their adhesiveness to extracellular matrix (ECM) proteins to near control levels through the upregulation of vimentin, zonula occludens protein 1 (ZO-1), cell adhesion molecule L1 (L1cam), and neural cell adhesion molecule 1 (Ncam1). Moreover, SM significantly suppressed the leptin-associated phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and ribosomal protein S6 kinase A1 (p90RSK), which are key kinases that ensure the survival and proliferation of cancer cells. Further molecular modeling studies demonstrated that the inhibitory effect of SM on the mitogen-activated protein kinase (MAPK)/ERK1/2 signaling pathway can be mediated by its direct interaction with ERK2 and its upstream regulators, son of sevenless homolog 1 (SOS) and mitogen-activated protein kinase kinase 1 (MEK1). Taken together, our findings in murine Neuro2a cells provide novel evidence of the stimulatory effect of leptin on the aggressiveness of neuroblastoma, which requires further detailed studies in human neuroblastoma cells and relevant animal models. The obtained results indicate that SM can be considered a promising drug candidate capable of reducing the impact of adipokines on tumor progression.

Bioconversion of feather waste into bioactive nutrients in water by Bacillus licheniformis WHU.

Feathers become hazardous pollutants when deposited directly into the environment. The rapid expansion of the poultry industry has significantly increased feather waste, necessitating the development of new ways to degrade and utilize feathers. This study investigated the ability of Bacillus licheniformis WHU to digest intact chicken feathers in water. The results indicated that yields of free amino acids, bioactive peptides, and keratin-derived nano-/micro-particles were improved in bacteria- versus purified keratinase-derived feather hydrolysate. Bacteria-derived feather hydrolysate supplementation induced health benefits in mice, including significantly increased intestinal villus height and zonula occludens-1 protein expression, as well as increased secretory immunoglobulin A levels in the intestinal mucosa and superoxide dismutase activity in serum. Additionally, feather hydrolysate supplementation modulated the mouse gut microbiota, reflected by increased relative abundance of probiotics such as Lactobacillus spp., decreased relative abundance of Proteobacteria at the phylum level and pathogens such as Staphylococcus spp., and increased Bacteroidota/Firmicutes ratio. This study developed a simple, cost-effective method to degrade feathers by B. licheniformis WHU digestion, yielding a hydrolysate that can be directly used as a bioactive nutrient resource. The study findings have applications in the livestock, poultry, and aquaculture industries, which have high demands for cheap protein. KEY POINTS: • Bacillus licheniformis could degrade intact feather in water. • The resulting feather hydrolysate shows prebiotic effects on mouse.

MiR-10b-5p rescues leaky gut linked with gastrointestinal dysmotility and diabetes.

Diabetes has substantive co-occurrence with disorders of gut-brain interactions (DGBIs). The pathophysiological and molecular mechanisms linking diabetes and DGBIs are unclear. MicroRNAs (miRNAs) are key regulators of diabetes and gut dysmotility. We investigated whether impaired gut barrier function is regulated by a key miRNA, miR-10b-5p,linking diabetesand gut dysmotility. We created a new mouse line using the Mb3Cas12a/Mb3Cpf1 endonuclease todeletemir-10b globally. Loss of function studies in the mir-10b knockout (KO) mice were conducted to characterize diabetes, gut dysmotility, and gut barrier dysfunction phenotypes in these mice. Gain of function studieswere conducted by injecting these mir-10b KO mice with a miR-10b-5p mimic. Further, we performed miRNA-sequencing analysis from colonic mucosa from mir-10bKO, wild type,and miR-10b-5p mimic injected mice to confirm (1) deficiency of miR-10b-5p in KO mice, and (2) restoration of miR-10b-5p after the mimic injection. Congenital loss of mir-10b in mice led to the development of hyperglycemia, gut dysmotility, and gut barrier dysfunction. Gut permeability was increased, but expression of the tight junction protein Zonula occludens-1 was reducedin the colon of mir-10b KO mice. Patients with diabetes or constipation- predominant irritable bowel syndrome, a known DGBI that is linked to leaky gut, had significantly reduced miR-10b-5p expression. Injection of a miR-10b-5p mimic in mir-10b KO mice rescued these molecular alterations and phenotypes. Our study uncovered a potential pathophysiologic mechanism of gut barrier dysfunction that links both the diabetes and gut dysmotility phenotypes in mice lacking miR-10b-5p. Treatment with a miR-10b-5p mimic reversed the leaky gut, diabetic, and gut dysmotility phenotypes, highlighting the translational potential of the miR-10b-5p mimic.

Effects of Anticoccidial Vaccination and Taraxacum officinale Extract on the Growth Performance, Biochemical Parameters, Immunity, and Intestinal Morphology of Eimeria-Challenged Chickens.

A total of 160 Ross 308 male chickens were used in a 2 × 2 factorial design to examine the effects of anticoccidial vaccination (ACV; lack or 1× dose recommended by the manufacturer) and dietary supplementation with Taraxacum officinale (dandelion) extract (DE; with or without) on growth performance, immunity, biochemical parameters, and intestinal morphology in broiler chickens challenged with Eimeria spp. At 20 days of age, all birds were challenged with a 25× dose of ACV, including Eimeria acervulina, E. maxima, E. mitis, and E. tenella. No interaction between ACV and DE was observed in terms of growth performance. Vaccinated birds showed increased feed intake (FI) and feed conversion ratio (FCR) during the 11-20 day period. Meanwhile, DE supplementation led to decreased FI and body weight gain (BWG) during the 1-10 day period. ACV effectively induced immunity against Eimeria, as evidenced by reduced oocyst shedding and less intestinal lesions, decreased levels of pro-inflammatory interleukin-6, and improved BWG during both the post infection (PI) period (21-35 days) and the entire growth period. DE supplementation lowered FCR and increased BWG during the 35-42 day period, increased the concentration of butyric acid in the cecal digesta, and lowered oocyst shedding PI. In vaccinated birds, DE elevated levels of plasma total protein and immunoglobulin M, and influenced tight junction proteins zonula occludens-1 and claudin-3, indicating a more robust epithelial barrier. DE also lowered alanine aminotransferase activity in unvaccinated birds. Both ACV and DE independently improved intestinal morphology in the jejunum, decreasing crypt depth and increasing the villus height-to-crypt ratio. These findings suggest that both ACV and DE could be effective strategies for managing coccidiosis in broiler chickens.

Curcumin reduces blood-nerve barrier abnormalities and cytotoxicity to endothelial cells and pericytes induced by cisplatin.

Cisplatin is a platinum-based antineoplastic agent used to treat cancers of solid organs. Neuropathy is one of its major side effects, necessitating dose reduction or cessation. Previous studies suggested that cisplatin causes microvascular toxicity, including pericyte detachment. This study aimed to clarify whether these alterations occurred in the blood-nerve barrier (BNB) of capillaries after cisplatin treatment. Electron microscopic analysis of rat sciatic nerves with cisplatin neuropathy showed increased frequency and severity of pericyte detachment. Moreover, the vascular basement membrane did not tightly encircle around the endothelial cells and pericytes. Cultured human umbilical vein endothelial cells and human brain vascular pericytes showed reduced viability, increased caspase-3 activity and enhanced oxidative stress following cisplatin treatment. In addition, cisplatin decreased transendothelial electrical resistance (TEER) and the expression of the tight junction proteins occludin and zonula occludens-1. Curcumin, a polyphenol found in the root of Curcuma longa, had favourable effects on cisplatin neuropathy in previous work. Therefore, curcumin was tested to determine whether it had any effect on these abnormalities. Curcumin alleviated pericyte detachment, cytotoxicity, oxidative stress, TEER reduction and tight junction protein expression. These data indicate that cisplatin causes BNB disruption in the nerves and might result in neuropathy. Curcumin might improve neuropathy via the restoration of BNB. Whether alterations in the BNB occur and curcumin is effective in patients with cisplatin neuropathy remain to be investigated.

Gemcitabine enhances pharmacokinetic exposure of the major components of Danggui Buxue Decoction in rat via the promotion of intestinal permeability and down-regulation of CYP3A for combination treatment of non-small cell lung cancer

Context Danggui Buxue Decoction (DBD), a traditional Chinese medicine formula, has the potential to enhance the antitumor effect of gemcitabine in non-small cell lung cancer (NSCLC) treatment by increasing gemcitabine’s active metabolites. However, whether gemcitabine affects the pharmacokinetics of DBD’s major components remains unclear. Objective This study evaluates the herb–drug interaction between DBD’s major components and gemcitabine and validates the underlying pharmacokinetic mechanism. Materials and methods The pharmacokinetics of 3.6 g/kg DBD with and without a single-dose administration of 50 mg/kg gemcitabine was investigated in Sprague-Dawley rats. The effects of gemcitabine on intestinal permeability, hepatic microsomal enzymes in rat tissues, and CYP3A overexpressing HepG2 cells were determined using western blot analysis. Results The combination of gemcitabine significantly altered the pharmacokinetic profiles of DBD’s major components in rats. The C max and AUC of calycosin-7-O-β-d-glucoside notably increased through sodium-glucose transporter 1 (SGLT-1) expression promotion. The AUC of ligustilide and ferulic acid was also significantly elevated with the elimination half-life (t 1/2) prolonged by 2.4-fold and 7.8-fold, respectively, by down-regulating hepatic CYP3A, tight junction proteins zonula occludens-1 (ZO-1) and occludin expression. Discussion and conclusions Gemcitabine could modulate the pharmacokinetics of DBD’s major components by increasing intestinal permeability, enhancing transporter expression, and down-regulating CYP3A. These findings provide critical information for clinical research on DBD as an adjuvant for NSCLC with gemcitabine and help make potential dosage adjustments more scientifically and rationally.

Potential mechanisms of different methylation degrees of pectin driving intestinal microbiota and their metabolites to modulate intestinal health of Micropterus salmoides

Four diets containing 8 % cellulose, low methyl-esterified pectin (LMP), high methyl-esterified pectin (HMP) and MMP (half LMP and half HMP) were designed to evaluate the potential mechanisms by which different esterification degrees of pectin drive intestinal microbiota and their metabolites modulating the intestinal health of Micropterus salmoides. The results showed that both dietary LMP and HMP consistently upregulated intestinal zonula occludens protein 1 (Zo-1), Caludin-1, and Caludin-4, and downregulated intestinal tumor necrosis factor-alpha (TNF-α), interleukin-8 (IL-8), and interleukin-1 beta (IL-1β) gene expression (P < 0.05). Dietary HMP separately upregulated intestinal Occludin, nuclear factor erythroid2-related factor 2 (Nrf2), B-cell lymphoma-2 (Bcl-2), and Bcl-2 associated agonist of cell death (BAD) gene expression, as well as the digesta propionate content, OTUs, Sobs, Shannon, Chao, and ACE indices (P < 0.05), whereas dietary LMP decreased digesta arginine, 4-aminobutyric, L-tyrosine, and phenylalanine contents (P < 0.05). Moreover, dietary HMP decreased plasma lipopolysaccharide and d-lactic acid contents and increased intestinal superoxide dismutase and glutathione peroxidase activities and immunoglobulin (Ig) receptor and IgM levels (P < 0.05). Collectively, dietary HMP improves intestinal health by increasing intestinal flora α-diversity and enhancing intestinal mechanical barrier, anti-inflammatory, antioxidant, and immune functions. On the contrary, the interference of dietary LMP with butyrate, tyrosine, arginine, and 4-aminobutyric acid metabolism is the main reason for its detrimental effects on intestinal health.

Gut microbiome-based thiamine metabolism contributes to the protective effect of one acidic polysaccharide from Selaginella uncinata (Desv.) Spring against inflammatory bowel disease

Inflammatory bowel disease (IBD) is a serious disorder, and exploration of active compounds to treat it is necessary. An acidic polysaccharide named SUSP-4 was purified from Selaginella uncinata (Desv.) Spring, which contained galacturonic acid, galactose, xylose, arabinose, and rhamnose with the main chain structure of →4)-α-d-GalAp-(1→ and →6)-β-d-Galp-(1→ and the branched structure of →5)-α-l-Araf-(1→ . Animal experiments showed that compared with Model group, SUSP-4 significantly improved body weight status, disease activity index (DAI), colonic shortening, and histopathological damage, and elevated occludin and zonula occludens protein 1 (ZO-1) expression in mice induced by dextran sulfate sodium salt (DSS). 16S ribosomal RNA (rRNA) sequencing indicated that SUSP-4 markedly downregulated the level of Akkermansia and Alistipes. Metabolomics results confirmed that SUSP-4 obviously elevated thiamine levels compared with Model mice by adjusting thiamine metabolism, which was further confirmed by a targeted metabolism study. Fecal transplantation experiments showed that SUSP-4 exerted an anti-IBD effect by altering the intestinal flora in mice. A mechanistic study showed that SUSP-4 markedly inhibited macrophage activation by decreasing the levels of phospho-nuclear factor kappa-B (p-NF-κB) and cyclooxygenase-2 (COX-2) and elevating NF-E2-related factor 2 (Nrf2) levels compared with Model group. In conclusion, SUSP-4 affected thiamine metabolism by regulating Akkermania and inhibited macrophage activation to adjust NF-κB/Nrf2/COX-2-mediated inflammation and oxidative stress against IBD. This is the first time that plant polysaccharides have been shown to affect thiamine metabolism against IBD, showing great potential for in-depth research and development applications.

Expression of Zonula Occludens-1 and Claudin-1 Proteins in Japanese Quails Testis

Expression of Zonula Occludens-1 and Claudin-1 Proteins in Japanese Quails Testis

TNF-α evokes blood-brain barrier dysfunction through activation of Rho-kinase and neurokinin 1 receptor

Ischaemic stroke, accompanied by neuroinflammation, impairs blood–brain barrier (BBB) integrity through a complex mechanism involving activation of both RhoA/Rho kinase/myosin light chain-2 and neurokinin 1 receptor (NK1R). Using an in vitro model of human BBB composed of brain microvascular endothelial cells (BMEC), astrocytes and pericytes, this study examined the potential contributions of these elements to BBB damage induced by elevated availability of pro-inflammatory cytokine, TNF-α. Treatment of human BMECs with TNF-α significantly enhanced RhoA activity and the protein expressions of Rho kinase and phosphorylated Ser19MLC-2 while decreasing that of NK1R. Pharmacological inhibition of Rho kinase by Y-27632 and NK1R by CP96345 neutralised the disruptive effects of TNF-α on BBB integrity and function as ascertained by reversal of decreases in transendothelial electrical resistance and increases in paracellular flux of low molecular weight permeability marker, sodium fluorescein, respectively. Suppression of RhoA activation, mitigation of actin stress fibre formation and restoration of plasma membrane localisation of tight junction protein zonula occludens-1 appeared to contribute to the barrier-protective effects of both Y-27632 and CP96345. Attenuation of TNF-α-mediated increases in NK1R protein expression in BMEC by Y-27632 suggests that RhoA/Rho kinase pathway acts upstream to NK1R. In conclusion, specific inhibition of Rho kinase in cerebrovascular conditions, accompanied by excessive release of pro-inflammatory cytokine TNF-α, helps preserve endothelial cell morphology and inter-endothelial cell barrier formation and may serve as an important therapeutic target.

Wuzi Yanzong administration alleviates Sertoli cell injury by recovering AKT/mTOR-mediated autophagy and the mTORC1–mTROC2 balance in aging-induced testicular dysfunction

Ethnopharmacological relevanceWuzi Yanzong Prescription (WZ), a classic traditional Chinese medicine formula, has the properties of kidney nourishing and essence strengthening, and it is widely used to treat male infertility with a long history. Sertoli cells are injured with aging, resulting in testicular dysfunction, and WZ effectively rejuvenates the age-related decline of testicular function. However, whether the therapeutic effects of WZ on aging-related testicular dysfunction are dependent on the restoration of Sertoli cell function remains unclear. Aim of the studyIn a mouse model of natural aging, we explored the protective effects of WZ and its potential mechanisms. Materials and methodsFifteen-month-old C57BL/6 mice were randomized to receive either standard diet or WZ (2 and 8 g/kg) for 3 months. Meanwhile, 10 1-month-old mice were considered the adult control group and received standard diet for 3 months. The testis and epididymis were rapidly collected, and the sperm quality, testicular histology, Sertoli cell numbers, tight junction (TJ) ultrastructure, and blood–testis barrier-associated protein expression and localization were assessed. ResultsWZ significantly increased sperm concentration and sperm viability, improved the degenerative histomorphology and elevated the seminiferous epithelium height. Furthermore, WZ increased the number of Sertoli cells, restored the ultrastructure of the Sertoli cell TJ, and upregulated the expression of TJ-associated proteins (zonula occludens-1 and Claudin11), ectoplasm specialized-associated proteins (N-Cadherin, E-Cadherin and β-Catenin), and gap junction-associated protein (connexin 43), but did not affect the expression of Occludin and cytoskeletal protein (Vimentin). In addition, WZ did not change the localization of zonula occludens-1 and β-Catenin in aged testis. Moreover, WZ increased the expression of autophagy-associated proteins (light chain 3 beta and autophagy related 5) and decreased the expression of p62, phosphorylated mammalian target of rapamycin, and phosphorylated AKT in Sertoli cells. Finally, we found that WZ attenuated mTOR complex 1 (mTORC1) activity and upregulated mTORC2 activity, as evidenced by inhibition of the expression of the regulatory-associated protein of mTOR, phosphorylated p70 S6K, and phosphorylated ribosomal protein s6 and enhancement of the expression of Rictor in the Sertoli cells of aging mice. ConclusionsWZ improves the injury of Sertoli cells by restoring AKT/mTOR-mediated autophagy and the mTORC1–mTROC2 balance in Sertoli cells during aging. Our findings provide a new mechanism of WZ in the treatment of aging-induced testicular dysfunction.

Human Milk Exosomes Induce ZO-1 Expression via Inhibition of REDD1 Expression in Human Intestinal Epithelial Cells.

Human milk exosomes (HMEs) enhance intestinal barrier function and contribute to an improvement in inflammation and mucosal injury, such as necrotizing enteritis (NEC), in infants. Here, we aimed to elucidate the intracellular factors involved in HME-induced expression of zonula occludens-1 (ZO-1), a tight junction protein, in Caco-2 human intestinal epithelial cells. HME treatment for 72 h significantly increased transepithelial electrical resistance in these cells. The mean ZO-1 protein levels in cells treated with HME for 72 h were significantly higher than those in the control cells. The mRNA and protein levels of regulated in development and DNA damage response 1 (REDD1) in HME-treated cells were significantly lower than those in the control cells. Although HME treatment did not increase the mechanistic target of rapamycin (mTOR) level in Caco-2 cells, it significantly increased the phosphorylated mTOR (p-mTOR) level and p-mTOR/mTOR ratio. The ZO-1 protein levels in cells treated with an inducer of REDD1, cobalt chloride (CoCl2) alone were significantly lower than those in the control cells. However, ZO-1 protein levels in cells co-treated with HME and CoCl2 were significantly higher than those in cells treated with CoCl2 alone. Additionally, REDD1 protein levels in cells treated with CoCl2 alone were significantly higher than those in the control cells. However, REDD1 protein levels in cells co-treated with HME and CoCl2 were significantly lower than those in cells treated with CoCl2 alone. This HME-mediated effect may contribute to the development of barrier function in the infant intestine and protect infants from diseases.

Inhibiting PI3K/Akt-Signaling Pathway Improves Neurobehavior Changes in Anti-NMDAR Encephalitis Mice by Ameliorating Blood–Brain Barrier Disruption and Neuronal Damage

The disruption of the blood–brain barrier (BBB) is hypothesized to be involved in the progression of anti-N-methyl-d-aspartate receptor (NMDAR) encephalitis, but its mechanism is still unclear. Recently, the phosphatidylinositol 3-kinase (PI3K)/threonine kinase (Akt) pathway is involved in the regulation of the BBB in various diseases. This study is aimed to investigate the mechanism of BBB damage and neurobehavior changes in anti-NMDAR encephalitis mice. Female C57BL/6J mice were actively immunized to establish an anti-NMDAR encephalitis mouse model and evaluate the neurobehavior changes of mice. To study its potential mechanism, LY294002 (PI3K inhibitor, 8 mg/kg) and Recilisib (PI3K agonist, 10 mg/kg) were treated by intraperitoneal injection, respectively. Anti-NMDAR encephalitis mice showed neurological deficits, increased BBB permeability, open endothelial tight junctions (TJs), and decreased expression of TJ-related proteins zonula occludens (ZO)-1 and Claudin-5. However, administration of PI3K inhibitor significantly reduced the expression of p-PI3K and p-Akt, improved neurobehavior function, decreased BBB permeability, and upregulated the expressions of ZO-1 and Claudin-5. Furthermore, PI3K inhibition reversed the decline of NMDAR NR1 in the membranes of hippocampal neurons, which reduced the loss of neuron-specific nucleoprotein (NeuN) and microtubule-associated protein 2 (MAP2). In contrast, administration of the PI3K agonist Recilisib showed a tendency to exacerbate BBB breakdown and neurological deficits. Our results showed that the activation of PI3K/Akt, along with the changes in TJ-related proteins ZO-1 and Claudin-5, may be closely related to BBB damage and neurobehavior changes in anti-NMDAR encephalitis mice. PI3K inhibition attenuates BBB disruption and neuronal damage in mice, thereby improving neurobehavior.

Comparison study of protective effects of porcine bile acids and sheep bile acids against heat stress in chickens.

Heat stress (HS) is known to exert negative effects on the poultry and breeding industry, resulting in severe economic losses. Bile acids (BAs), an important component of bile, play a crucial role in improving the production performance of livestock and poultry, alleviating stress injury, and ensuring the health of livestock and poultry. At present, porcine BAs are widely used because of their therapeutic effects on HS; however, it remains unclear whether the same effects are exerted by sheep BAs, which are different from porcine BAs and have different compositions. In this study, we compared the anti-HS effects of porcine BAs and sheep BAs in the diet by establishing an HS model of chicks and investigating the chicken performance, HS-related genes' expression, oxidative stress markers, jejunal histoarchitecture, inflammatory cytokines' expression, jejunal SIgA concentration, and cecal bacterial flora. The results showed that the addition of sheep BAs to the diet increased the average daily weight gain and the feed conversion ratio of chicks. Under HS, sheep BAs was more effective than porcine BAs in improving the activities of LDH and ALT in serum and the content / activity of MDA, SOD and GSH in serum and tissue, reducing the mRNA expression of heat shock proteins (HSP60, HSP70, and HSP90) in the liver and jejunum, and improving the histological structure and the expression of tight junction proteins (Occludin and ZO-1), and enriching intestinal bacterial flora. However, porcine BAs was significantly lower than sheep BAs in reducing the mRNA expression of inflammatory factors (IL-6, IL-1 β and TNF- α). The effect of sheep BAs was more significant in alleviating HS injury in chicks than porcine BAs, suggesting that sheep BAs have great potential as new feed nutrition and health additive to improve poultry production performance and prevent HS. This article is protected by copyright. All rights reserved.

SRT1720 ALLEVIATES ENDOTHELIAL CRIF1 DEFICIENCY-INDUCED CARDIAC FUNCTION

Objective: CR6-interacting factor1 (CRIF1) is a mitochondrial inner membrane and plays an important role in peptide synthesis and oxidative phosphorylation. Cardiomyocyte-specific deletion of CRIF1 led to an exaggerated decline in cardiac function and impaired cardiomyopathy. However, whether endothelial deletion of CRIF1 influences cardiac function has not yet been fully determined. Design and method: We developed an endothelial cell-specific CRIF1 deletion mouse and observed cardiac function in vivo. Results: Our results showed that endothelial deletion of CRIF1 increased heart-to-body weight ratio, enhanced lethality, and lowered fractional shortening of the left ventricle, which contributed to severe cardiac dysfunction. Furthermore, we observed severe mitochondrial dysfunction, decreased ATP level, inflammation, and enhanced oxidative stress in CRIF1 KO mice heart tissues, along with reduced SIRT1 expression. The SIRT1 activator SRT1720 attenuated cardiac dysfunction by activating endothelial nitric oxide synthase, reducing oxidative stress, inhibiting inflammation, and endothelial junction associated protein Zonula occludens-1 in CRIF1 KO mice. Conclusions: Therefore, our results suggest that endothelial mitochondrial OXPHOS dysfunction plays an essential role in maintaining cardiac function, and SIRT1 activator could be a promising therapeutic strategy for endothelial dysfunction-induced cardiac dysfunction.

Bifidobacterium bifidum E3 Combined with Bifidobacterium longum subsp. infantis E4 Improves LPS-Induced Intestinal Injury by Inhibiting the TLR4/NF-κB and MAPK Signaling Pathways In Vivo.

Changes in the functions of the intestinal barrier occur in parallel with the development of sepsis. The protection by Bifidobacterium strains (BB, BL, BB12, and BLBB) was evaluated in mice injected with lipopolysaccharide (LPS). The results revealed an increase in the ratio of ileal villus length to crypt depth in the BLBB group compared with that in the LPS group, as were the number of IgA+ plasma, CD4+/CD8+ T, and dendritic cells. The levels of diamine oxidase (DAO) and d-lactic acid in the serum were lessened in the BLBB group after LPS injection compared with that in the LPS group. In addition, the BLBB group exhibited an increased expression level of tight junction proteins (zonula occludens-1, occludin, and claudin-1), mucin (MUC2) mRNA, reduced NFκB/MAPK signaling pathways, and decreased expression levels of inflammatory cytokines (IL-1β, IL-6, and TNF-α). The BLBB group enriched the relative abundance of Muribaculaceae, Lachnospiraceae_NK4A136_group, Clostridia_Ucg-014, and Alistipes, resulting in an increase in strains producing short-chain fatty acids. Furthermore, the BLBB group leads to higher levels of deoxycholic acid and biosynthesized linoleate. This study suggested that the BLBB group could enhance the capacity of the intestinal barrier and intestinal mucosal immunity, reduce intestinal inflammation, and improve the composition of gut microbiota. Bifidobacterium bifidum E3 combined with Bifidobacterium longum subsp. infantis E4 may thus serve as a probiotic against the intestinal injury caused by LPS.

Extracellular ATP accelerates cell death and decreases tight junction protein ZO-1 in hypoxic cochlear strial marginal cells in neonatal rats

In the cochlea, extracellular ATP (eATP) plays an important role in both physiological and pathological processes, but its role in the hypoxic cochlea remains unclear. The present study aims to investigate the relationship between eATP and hypoxic marginal cells (MCs) in the stria vascularis in cochlea. Combining various methodologies, we found that eATP accelerates cell death and decreases tight junction protein zonula occludens-1 (ZO-1) in hypoxic MCs. Flow cytometry and western blot analyses revealed an increase in apoptosis levels and suppression of autophagy, indicating that eATP causes additional cell death by increasing the apoptosis of hypoxic MCs. Given that autophagy inhibits apoptosis to protect MCs under hypoxia, apoptosis is probably enchanced by suppressing autophagy. Interleukin-33(IL-33)/suppression of tumorigenicity-2(ST-2)/matrix metalloprotein 9(MMP9) pathway activation was also observed during the process. Further experiments involving the use of additional IL-33 protein and an MMP9 inhibitor indicated that this pathway is responsible for the damage to the ZO-1 protein in hypoxic MCs. Our study revealed an adverse effect of eATP on the survival and ZO-1 protein expression of hypoxic MCs, as well as the underlying mechanism.