Multilayered proteomics reveals that JAM-A promotes breast cancer progression via regulation of amino acid transporter LAT1.

Recent studies have shown that transmembrane-type tight junction proteins are upregulated in various cancers compared with their levels in normal tissues and are involved in cancer progression, suggesting that they are potential therapeutic targets. Here, we demonstrated the expression profile and a novel role of junctional adhesion molecule-A (JAM-A) in breast cancer. Immunohistochemistry of surgical specimens showed that JAM-A was highly expressed from carcinoma in situ lesions, as in other adenocarcinomas, with higher expression in invasive carcinomas. High expression of JAM-A contributed to malignant aspects such as lymph node metastasis and lymphatic involvement positivity. In breast cancer cells, JAM-A expression status affects malignant potentials including proliferation and migration. Multilayered proteomics revealed that JAM-A interacts with the amino acid transporter LAT1 in breast cancer cells. JAM-A regulates the expression of LAT1 and interacts with it on the whole cell membrane, leading to enhanced amino acid uptake to promote tumor growth. Double high expression of JAM-A and LAT1 predicts poor prognosis in patients with breast cancer. Of note, an antibody against an extracellular domain of JAM-A suppressed the proliferation of breast cancer cells. Our findings indicate the possibility of JAM-A-targeted therapy ideally combined with LAT1-targeted therapy as a new therapeutic strategy against breast cancer.

Low-molecular-weight heparin ameliorates intestinal barrier dysfunction in aged male rats via protection of tight junction proteins.

The intestinal barrier weakens and chronic gut inflammation occurs in old age, causing age-related illnesses. Recent research shows that low-molecular-weight heparin (LMWH), besides anticoagulation, also has anti-inflammatory and anti-apoptotic effects, protecting the intestinal barrier. This study aims to analyze the effect of LMWH on the intestinal barrier of old male rodents. This study assigned Sprague-Dawley male rats to four groups: young (3 months), young + LMWH, old (20 months), and old + LMWH. The LMWH groups received 1 mg/kg LMWH via subcutaneous injection for 7 days. Optical and transmission electron microscopy (TEM) were used to examine morphological changes in intestinal mucosa due to aging. Intestinal permeability was measured using fluorescein isothiocyanate (FITC)-dextran. ELISA kits were used to measure serum levels of IL-6 and IL-1ß, while Quantitative RT-PCR detected their mRNA levels in intestinal tissues. Western blotting and immunohistochemistry (IHC) evaluated the tight junction (TJ) protein levels such as occludin, zonula occludens-1 (ZO-1), and claudin-2. Western blotting assessed the expression of the apoptosis marker cleaved caspase 3, while IHC was used to detect LGR5+ intestinal stem cells. The intestinal permeability of aged rats was significantly higher than that of young rats, indicating significant differences. With age, the protein levels of occludin and ZO-1 decreased significantly, while the level of claudin-2 increased significantly. Meanwhile, our study found that the levels of IL-1ß and IL-6 increased significantly with age. LMWH intervention effectively alleviated age-related intestinal barrier dysfunction. In aged rats treated with LMWH, the expression of occludin and ZO-1 proteins in the intestine increased, while the expression of claudin-2 decreased. Furthermore, LMWH administration in aged rats resulted in a decrease in IL-1ß and IL-6 levels. LMWH also reduced age-related cleaved caspase3 expression, but IHC showed no difference in LGR5+ intestinal stem cells between groups. Research suggests that LMWH could potentially be a favorable therapeutic choice for age-related diseases associated with intestinal barrier dysfunction, by protecting TJ proteins, reducing inflammation, and apoptosis.

ZO-1 and IL-1RAP Phosphorylation: Potential Role in Mediated Brain-Gut Axis Dysregulation in Irritable Bowel Syndrome-like Stressed Mice.

Background and Objectives: Irritable Bowel Syndrome (IBS) is a common gastrointestinal disorder often exacerbated by stress, influencing the brain-gut axis (BGA). BGA dysregulation, disrupted intestinal barrier function, altered visceral sensitivity and immune imbalance defects underlying IBS pathogenesis have been emphasized in recent investigations. Phosphoproteomics reveals unique phosphorylation details resulting from environmental stress. Here, we employ phosphoproteomics to explore the molecular mechanisms underlying IBS-like symptoms, mainly focusing on the role of ZO-1 and IL-1RAP phosphorylation. Materials and Methods: Morris water maze (MWM) was used to evaluate memory function for single prolonged stress (SPS). To assess visceral hypersensitivity of IBS-like symptoms, use the Abdominal withdrawal reflex (AWR). Colonic bead expulsion and defecation were used to determine fecal characteristics of the IBS-like symptoms. Then, we applied a phosphoproteomic approach to BGA research to discover the molecular mechanisms underlying the process of visceral hypersensitivity in IBS-like mice following SPS. ZO-1, p-S179-ZO1, IL-1RAP, p-S566-IL-1RAP and GFAP levels in BGA were measured by western blotting, immunofluorescence staining, and enzyme-linked immunosorbent assay to validate phosphorylation quantification. Fluorescein isothiocyanate-dextran 4000 and electron-microscopy were performed to observe the structure and function of the intestinal epithelial barrier. Results: The SPS group showed changes in learning and memory ability. SPS exposure affects visceral hypersensitivity, increased fecal water content, and significant diarrheal symptoms. Phosphoproteomic analysis displayed that p-S179-ZO1 and p-S566-IL-1RAP were significantly differentially expressed following SPS. In addition, p-S179-ZO1 was reduced in mice's DRG, colon, small intestine, spinal and hippocampus and intestinal epithelial permeability was increased. GFAP, IL-1ß and p-S566-IL-1RAP were also increased at the same levels in the BGA. And IL-1ß showed no significant difference was observed in serum. Our findings reveal substantial alterations in ZO-1 and IL-1RAP phosphorylation, correlating with increased epithelial permeability and immune imbalance. Conclusions: Overall, decreased p-S179-ZO1 and increased p-S566-IL-1RAP on the BGA result in changes to tight junction structure, compromising the structure and function of the intestinal epithelial barrier and exacerbating immune imbalance in IBS-like stressed mice.

Gallic acid attenuates LPS-induced inflammation in Caco-2 cells by suppressing the activation of the NF-κB/MAPK signaling pathway.

Inflammatory bowel disease (IBD) is a chronic inflammatory disease characterized by intestinal barrier dysfunction, inflammatory synergistic effects and excessive tissue injury. Gallic acid (GA) is renowned for its remarkable biological activity, encompassing anti-inflammatory and antioxidant properties. However, the underlying mechanisms by which GA protects against intestinal inflammation have not been fully elucidated. The aim of this study is to investigate the effect of GA on the inflammation of a lipopolysaccharide (LPS)-stimulated human colon carcinoma cell line (Caco-2) and on the intestinal barrier dysfunction, and explore the underlying molecular mechanism involved. Our findings demonstrate that 5 µg/mL GA restores the downregulation of the mRNA and protein levels of Claudin-1, Occludin, and ZO-1 and decreases the expressions of inflammatory factors such as IL-6, IL-1ß and TNF-α induced by LPS. In addition, GA exhibits a protective effect by reducing the LPS-enhanced early and late apoptotic ratios, downregulating the mRNA levels of pro-apoptotic factors ( Bax, Bad, Caspase-3, Caspase-8, and Caspase-9), and upregulating the mRNA levels of anti-apoptotic factor Bcl-2 in Caco-2 cells. GA also reduces the levels of reactive oxygen species increased by LPS and restores the activity of antioxidant enzymes, namely, superoxide dismutase and catalase, as well as the level of glutathione. More importantly, GA exerts its anti-inflammatory effects by inhibiting the LPS-induced phosphorylation of key signaling molecules in the NF-κB/MAPK pathway, including p65, IκB-α, p38, JNK, and ERK, in Caco-2 cells. Overall, our findings show that GA increases the expressions of tight junction proteins, reduces cell apoptosis, relieves oxidative stress and suppresses the activation of the NF-κB/MAPK pathway to reduce LPS-induced intestinal inflammation in Caco-2 cells, indicating that GA has potential as a therapeutic agent for intestinal inflammation.

Surface Proteins of Bifidobacterium Bifidum DNG6 Growing in 2'-FL Alleviating LPS-Induced Intestinal Barrier Injury in vitro.

Human milk oligosaccharides (HMOs) 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 aim 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 (Lac) and galacto-oligosaccharides (GOS). 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 to 50.34%, IL-6 reduced to 22.83%, IL-1ß reduced to 37.91%, and IL-10 increased to 63.47%)and strengthening tight junction (ZO-1 2.39 times, Claudin-1 2.79 times, and Occludin 4.70 times). The findings of this study indicate that 2'-FL can further regulate intestinal barrier damage by promoting the alteration of B. bifidum DNG6 surface protein. The findings of this research will also provide theoretical support for the development of synbiotic formulations.

Nerve growth factor alleviates arsenic-induced testicular injury by enhancing the function of Sertoli cells.

Sertoli cells (SCs) maintain testicular homeostasis and promote spermatogenesis by forming the blood-testis barrier (BTB) and secreting growth factors. The pro-proliferative and anti-apoptotic effects of nerve growth factor (NGF) on SCs have been proved previously. It is still unclear whether the damage effect of arsenic on testis is related to the inhibition of NGF expression, and whether NGF can mitigate arsenic-induced testicular damage by decreasing the damage of SCs induced by arsenic. Here, the lower expression of NGF in testes of arsenic exposed mice (freely drinking water containing 15 mg/l of NaAsO2) was observed through detection of Western blot and Real-time PCR. Subsequently, hematoxylin and eosin (HE) staining, Evans blue staining and transmission electron microscopy were used to evaluate the pathology, BTB permeability and tight junction integrity in testes of control mice, arsenic exposed mice (freely drinking water containing 15 mg/l of NaAsO2) and arsenic + NGF treated mice (freely drinking water containing 15 mg/l of NaAsO2 + intraperitoneal injection with 30 µg/kg of NGF), respectively. Evidently, spermatogenic tubule epithelial cells in testis of arsenic exposed mice were disordered and the number of cell layers was reduced, accompanied by increased permeability and damaged integrity of the tight junction in BTB, but these changes were less obvious in testes of mice treated with arsenic + NGF. In addition, the sperm count, motility and malformation rate of mice treated with arsenic + NGF were also improved. On the basis of the above experiments, the viability and apoptosis of primary cultured SCs treated with arsenic (10 µM NaAsO2) or arsenic + NGF (10 µM NaAsO2 + 100 ng/mL NGF) were detected by Cell counting kit-8 (CCK8) and transferase-mediated DUTP-biotin nick end labeling (TUNEL) staining, respectively. It is found that NGF ameliorated the decline of growth activity and the increase of apoptosis in arsenic-induced SCs. This remarkable biological effect that NGF inhibited the increase of Bax expression and the decrease of Bcl-2 expression in arsenic-induced SCs was also determined by western blot and Real-time PCR. Moreover, the decrease in transmembrane resistance (TEER) and the expression of tight junction proteins ZO-1 and occludin was mitigated in SCs induced by arsenic due to NGF treatment. In conclusion, the above results confirmed that NGF could ameliorate the injury effects of arsenic on testis, which might be related to the function of NGF to inhibit arsenic-induced SCs injury.

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

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

High carbohydrate diet decreases microbial diversity and increases IL-1ß levels in mice colon.

Western diet is known to contribute to intestinal dysbiosis and the progression of inflammation. Although the Turkish diet has different macronutrient contents, the intestinal inflammatory disease incidences in Türkiye are comparable to Western countries. Thus, we hypothesized that high carbohydrate diets also contribute to inflammation of the colon. We compared diets with different macronutrient compositions and investigated their effects on colonic microbiota, cytokine, histology, and tight junction protein levels. High carbohydrate diet caused the lowest microbial diversity and is accompanied by the highest expression of interleukin-1ß and claudin-1. A low carbohydrate diet with zero fiber resulted in the lowest inflammatory markers as well as the lowest occludin and claudin levels. Overall, our results indicate that carbohydrate and fiber contents of the diets are important contributors to colon health.

Mast cell activation triggered by SARS-CoV-2 causes inflammation in brain microvascular endothelial cells and microglia.

SARS-CoV-2-induced excessive inflammation in brain leads to damage of blood-brain barrier, hypoxic-ischemic injury, and neuron degeneration. The production of inflammatory cytokines by brain microvascular endothelial cells and microglia is reported to be critically associated with the brain pathology of COVID-19 patients. However, the cellular mechanisms for SARS-CoV-2-inducing activation of brain cells and the subsequent neuroinflammation remain to be fully delineated. Our research, along with others', has recently demonstrated that SARS-CoV-2-induced accumulation and activation of mast cells (MCs) in mouse lung could further induce inflammatory cytokines and consequent lung damages. Intracerebral MCs activation and their cross talk with other brain cells could induce neuroinflammation that play important roles in neurodegenerative diseases including virus-induced neuro-pathophysiology. In this study, we investigated the role of MC activation in SARS-CoV-2-induced neuroinflammation. We found that (1) SARS-CoV-2 infection triggered MC accumulation in the cerebrovascular region of mice; (2) spike/RBD (receptor-binding domain) protein-triggered MC activation induced inflammatory factors in human brain microvascular endothelial cells and microglia; (3) MC activation and degranulation destroyed the tight junction proteins in brain microvascular endothelial cells and induced the activation and proliferation of microglia. These findings reveal a cellular mechanism of SARS-CoV-2-induced neuroinflammation.

Lycopene Ameliorated DSS-Induced Colitis by Improving Epithelial Barrier Functions and Inhibiting the Escherichia coli Adhesion in Mice.

Adherent-invasive Escherichia coli plays an important role in the pathogenesis of inflammatory bowel disease. Blocking the adhesion of E. coli to intestinal epithelial cells appears to be useful for attenuating inflammatory bowel disease. Lycopene has been reported to have anti-inflammatory and antimicrobial activities. The aim of this study was to test the intervention effect of lycopene on colitis in mice and to investigate the possible mechanism through which lycopene affects the adhesion of E. coli to intestinal epithelial cells. Lycopene (12 mg/kg BW) attenuated dextran sulfate sodium (DSS)-induced colitis, decreased the proportion of E. coli, and activated the NLR family pyrin domain containing 12 and inactivated nuclear factor kappa B pathways. Furthermore, lycopene inhibited the adhesion of E. coli O157:H7 to Caco-2 cells by blocking the interaction between E. coli O157:H7 and integrin ß1. Lycopene ameliorated DSS-induced colitis by improving epithelial barrier functions and inhibiting E. coli adhesion. Overall, these results show that lycopene may be a promising component for the prevention and treatment of colitis.

The Use of Polysaccharide AOP30 from the Rhizome of Alpinia officinarum Hance to Alleviate Lipopolysaccharide-Induced Intestinal Epithelial Barrier Dysfunction and Inflammation via the TLR4/NfκB Signaling Pathway in Caco-2 Cell Monolayers.

Alpinia officinarum Hance is rich in carbohydrates and is flavored by natives. The polysaccharide fraction 30 is purified from the rhizome of A. officinarum Hance (AOP30) and shows excellent immunoregulatory ability when administered to regulate immunity. However, the effect of AOP30 on the intestinal epithelial barrier is not well understood. Therefore, the aim of this study is to investigate the protective effect of AOP30 on the intestinal epithelial barrier using a lipopolysaccharide (LPS)-induced intestinal epithelial barrier dysfunction model and further explore its underlying mechanisms. Cytotoxicity, transepithelial electrical resistance (TEER) values, and Fluorescein isothiocyanate (FITC)-dextran flux are measured. Simultaneously, the protein and mRNA levels of tight junction (TJ) proteins, including zonula occludens-1 (ZO-1), Occludin, and Claudin-1, are determined using Western blotting and reverse-transcription quantitative polymerase chain reaction methods, respectively. The results indicate that AOP30 restores the LPS-induced decrease in the TEER value and cell viability. Furthermore, it increases the mRNA and protein expression of ZO-1, Occludin, and Claudin-1. Notably, ZO-1 is the primary tight junction protein altered in response to LPS-induced intestinal epithelial dysfunction. Additionally, AOP30 downregulates the production of TNFα via the Toll-like receptor 4 (TLR4)/NF-κB signaling pathway. Collectively, the findings of this study indicate that AOP30 can be developed as a functional food ingredient or natural therapeutic agent for addressing intestinal epithelial barrier dysfunction. It sheds light on the role of AOP30 in improving intestinal epithelial function.

[Corrigendum] Long non­coding RNA NEAT1 promotes ovarian cancer cell invasion and migration by interacting with miR­1321 and regulating tight junction protein 3 expression.

Subsequently to the publication of the above article, an interested reader drew to the authors' attention that, for the cell invasion and migration assay images shown for the A2780 cell line in Figs. 1 and Fig. 3 on p. 3433 and 3435 respectively, the same data panel had apparently been selected to show the results of the si­NEAT1 experiment in Fig. 1 and the si­TJP3 experiment in Fig. 3. After having re­examined their original data, the authors have realized that the image correctly shown for Fig. 1 was inadvertently copied across to Fig. 3. The corrected version of Fig. 3, now correctly showing the data for the si­TJP3 experiment with the A2780 cell line, is shown on the next page. Note that this error did not significantly affect the results or the conclusions reported in this paper. All the authors agree to the publication of this Corrigendum, are grateful to the Editor of Molecular Medicine Reports for allowing them the opportunity to correct this error, and apologize to the readership for any inconvenience caused. [Molecular Medicine Reports 22: 3429­3439, 2020; DOI: 10.3892/mmr.2020.11428].

TJP1 suppresses trophoblast cell invasion by expressing E2F8 in the human placenta.

Adequate extravillous trophoblast (EVT) invasion into the maternal decidua is important for human placental development. We identified that E2F transcription factor 8 (E2F8) suppresses EVT invasion, and that tight junction protein-1 (TJP1) is a potential downstream target gene of E2F8. We investigated the role of TJP1 in the human placenta and regulation of TJP1 expression by E2F8. TJP1 expression decreased in E2F8 knockdown HTR-8/SVneo cells. TJP1 and E2F8 were co-expressed in villi in the first-trimester placenta and in EVTs and villi in the third-trimester placenta. TJP1 was significantly increased in the pre-eclamptic compared with control placenta. TJP1 knockdown increased the invasion of HTR-8/SVneo cells, while TJP1 overexpression inhibited cell invasion. Halo-E2F8 overexpression significantly increased TJP1 expression and TJP1 transcription compared with control placenta. Our findings suggest that E2F8 promotes TJP1 transcription, and that TJP1 expression by E2F8 inhibits EVT invasion. TJP1 and E2F8 may be related to pre-eclampsia pathogenesis.

Characterization of Blood-Brain Barrier Opening Induced by Transcranial Histotripsy in Murine Brains.

OBJECTIVE: Transcranial histotripsy has shown promise as a non-invasive neurosurgical tool, as it has the ability to treat a wide range of locations in the brain without overheating the skull. One important effect of histotripsy in the brain is the blood-brain barrier (BBB) opening (BBBO) at the ablation site, but there is a knowledge gap concerning the extent of histotripsy-induced BBBO. Here we describe induction of BBBO by transcranial histotripsy and use of magnetic resonance imaging (MRI) and histology to quantify changes in BBBO at the periphery of the histotripsy ablation zone over time in the healthy mouse brain. METHODS: An eight-element, 1 MHz histotripsy transducer with a focal distance of 32.5 mm was used to treat the brains of 23 healthy female BL6 mice. T1-gadolinium (T1-Gd) MR images were acquired immediately following histotripsy treatment and during each of the subsequent 4 wk to quantify the size and intensity of BBB leakage. RESULTS: The T1-Gd MRI results revealed that the hyperintense BBBO volume increased over the first week and subsided gradually over the following 3 wk. Histology revealed complete loss of tight junction proteins and blood vessels in the center of the ablation region immediately after histotripsy, partial recovery in the periphery of the ablation zone 1 wk following histotripsy and near-complete recovery of tight junction complex after 4 wk. CONCLUSION: These results provide the first evidence of transcranial histotripsy-induced BBBO and repair at the periphery of the ablation zone.

Intestinal Permeability, Irritable Bowel Syndrome with Constipation, and the Role of Sodium-Hydrogen Exchanger Isoform 3 (NHE3).

Increased intestinal permeability has been identified as one of the many pathophysiological factors associated with the development of irritable bowel syndrome (IBS), a common disorder of gut-brain interaction. The layer of epithelial cells that lines the intestine is permeable to a limited degree, and the amount of paracellular permeability is tightly controlled to enable the absorption of ions, nutrients, and water from the lumen. Increased intestinal permeability to macromolecules can be triggered by a variety of insults, including infections, toxins from food poisoning, or allergens, which in turn cause an inflammatory response and are associated with abdominal pain in patients with IBS. This review article discusses increased intestinal permeability in IBS, focusing on IBS with constipation (IBS-C) through the lens of a patient case with a reported prior diagnosis of "leaky gut syndrome" upon initial contact with a gastrointestinal specialist. We review advantages and disadvantages of several methods of measuring intestinal permeability in patients and discuss when measuring intestinal permeability is appropriate in the therapeutic journey of patients with IBS-C. Furthermore, we discuss a possible mechanism of restoring the intestinal barrier to its healthy state through altering intracellular pH by inhibiting sodium-hydrogen exchanger isoform 3 (NHE3). Tenapanor is a minimally absorbed, small-molecule inhibitor of NHE3 that has been approved by the US Food and Drug Administration for the treatment of IBS-C in adults. Preclinical studies showed that tenapanor may restore the intestinal barrier in IBS-C by affecting the conformation of tight junction proteins via NHE3 inhibition to block the paracellular transport of macromolecules from the intestinal lumen. Testing for increased permeability in patients with IBS-C who experience abdominal pain may help inform the choice of therapeutics and alter patients' misconceptions about "leaky gut syndrome".

Effects of oregano and/or rosemary extracts on growth performance, digestive enzyme activities, cecal bacteria, tight junction proteins, and antioxidants-related genes in heat-stressed broiler chickens.

The study examined the impact of adding oregano extract and/or rosemary to broiler diets to counteract the growth inhibition caused by heat stress (HS). It also investigated the effects on the activity of digestive enzymes, microbiological composition, and the expression of antioxidant and tight junction-related proteins. Three hundred- and fifty-day-old male broilers, were randomly assigned to 7 treatment groups, with each group comprising 5 replicates, and each replicate containing 10 chicks in a cage. The diets were: 1) a basal diet, 2) a diet supplemented with 50 mg/kg of rosemary, 3) a diet supplemented with 100 mg/kg of rosemary, 4) a diet supplemented with 50 mg/kg of oregano, 5) a diet supplemented with 100 mg/kg of oregano, 6) a combination diet containing 50 mg/kg each of rosemary and oregano, and 7) a combination diet containing 100 mg/kg each of rosemary and oregano. Dietary oregano extract enhanced the growth and feed utilization of heat-stressed birds, especially at a concentration of 50 mg/kg. Moreover, oregano extract improved jejunal protease and amylase activities. The extracts of rosemary and oregano significantly reduced IgG and IgM levels. Dietary 50 mg oregano extract significantly upregulated intestinal integrity-related genes including jejunal CLDNI, ZO-1, ZO-2, and MUC2. Dietary 50 mg oregano extract significantly downregulated hepatic NADPH oxidase 4 (NOX4) and nitric oxide synthase 2 (NOS2) expressions. Our results suggest that incorporating oregano leaf extract into the diet at a concentration of 50 mg/kg improves the growth performance of broilers exposed to heat stress. This improvement could be attributed to enhanced gut health and the modulation of genes associated with oxidative stress and tight junction proteins.

Tas2R143 regulates the expression of the Blood-Testis Barrier tight junction protein in TM4 cells through the NF-κB signaling pathway.

Although bitter receptors, known as Tas2Rs, have been identified in the testes and mature sperm, their expression in testicular Sertoli cells (SCs) and their role in recognizing harmful substances to maintain the immune microenvironment remain unknown. To explore their potential function in spermatogenesis, this study utilized TM4 cells and discovered the high expression of the bitter receptor Tas2R143 in the cells. Interestingly, when the Tas2R143 gene was knocked down for 24 and 48 h, there was a significant downregulation (P < 0.05) in the expression of tight junction proteins (occludin and ZO-1) and NF-κB. Additionally, Western blot results demonstrated that the siRNA-133+NF-κB co-treatment group displayed a significant downregulation (P < 0.05) in the expression of occludin and ZO-1 compared to both the siRNA-133 transfection group and the NF-κB inhibitors treatment group. These findings suggest that Tas2R143 likely regulates the expression of occludin and ZO-1 through the NF-κB signaling pathway and provides a theoretical basis for studying the regulatory mechanism of bitter receptors in the reproductive system, aiming to attract attention to the chemical perception mechanism of spermatogenesis.

Mechanisms of Peitu Yimu acupuncture in repairing intestinal mucosal barrier by regulating CRF/CRFR1 pathway in diarrhea-predominant irritable bowel syndrome rats. / 培土抑木针法调节CRF/CRFR1é€šè·¯ä¿®å¤è ¹æ³»åž‹è‚ æ˜“æ¿€ç»¼åˆå¾å¤§é¼ è‚ é»è†œå±éšœçš„æœºåˆ¶ç ”ç©¶.

OBJECTIVES: To investigate the effect of Peitu Yimu(strengthening spleen and soothing liver) acupuncture on intestinal mucosal barrier function and corticotropin-releasing factor (CRF)/CRF receptor 1 (CRFR1) pathway in rats with diarrhea-predominant irritable bowel syndrome (IBS-D), so as to explore its underlying mechanism in alleviating IBS-D. METHODS: Forty female SD rats were randomly divided into blank, model, electroacupuncture (EA), and agonist groups, with 10 rats in each group. Except for the blank group, rats in the other groups were given folium sennae infusion by gavage combined with chronic unpredictable mild stress to establish IBS-D model. Rats in the EA group received acupuncture at "Tianshu"(ST25) and EA at "Zusanli"(ST36) and "Taichong"(LR3) (2 Hz/15 Hz) on one side for 20 min, with the side chosen alternately every other day, for 14 days after modeling. Rats in the agonist group received acupuncture 30 min after intravenous injection of CRFR1 agonist urocortin, with the same manipulation method and time as the EA group. Before and after intervention, visceral pain threshold and stool Bristol scores were measured. Elevated plus maze test and open field test were used to detect anxiety and depression like behavior of rats. ELISA was used to detect the contents of CRF and CRFR1 in rats serum. Immunohistochemistry was used to detect the positive expressions of CRF, CRFR1, zonula occludens protein 1(ZO-1), occlusal protein(Occludin), and closure protein 1 (Claudin-1) in colon tissue. RESULTS: Compared with the blank group, the visceral pain threshold, open arm time percentage (OT%), total distance of movement in the open field test, and positive expression of ZO-1, Occludin, and Claudin-1 in colon were decreased (P<0.01, P<0.05), while Bristol stool scores, serum CRF and CRFR1 contents, and positive expressions of CRF and CRFR1 in colon were increased (P<0.01) in the model group. After intervention and compared with the model group, the visceral pain threshold, OT%, total distance of movement in the open field test, and positive expressions of ZO-1, Occludin, and Claudin-1 in colon were increased (P<0.05, P<0.01), while Bristol stool scores, serum CRF and CRFR1 contents, and positive expressions of CRF and CRFR1 in colon were decreased (P<0.01) in the EA group；the Bristol stool scores, serum CRF content, and CRF positive expression in colon were significantly decreased in the agonist group (P<0.01). CONCLUSIONS: Peitu Yimu acupuncture can significantly improve visceral hypersensitivity and anxiety-depression state in IBS-D rats. Its mechanism may be related to the inhibition of CRF/CRFR1 pathway and restoration of intestinal tight junction protein expressions.

Phyllanthus emblica Fruit Improves Obesity by Reducing Appetite and Enhancing Mucosal Homeostasis via the Gut Microbiota-Brain-Liver Axis in HFD-Induced Leptin-Resistant Rats.

The impact of leptin resistance on intestinal mucosal barrier integrity, appetite regulation, and hepatic lipid metabolism through the microbiota-gut-brain-liver axis has yet to be determined. Water extract of Phyllanthus emblica L. fruit (WEPE) and its bioactive compound gallic acid (GA) effectively alleviated methylglyoxal (MG)-triggered leptin resistance in vitro. Therefore, this study investigated how WEPE and GA intervention relieve leptin resistance-associated dysfunction in the intestinal mucosa, appetite, and lipid accumulation through the microbiota-gut-brain-liver axis in high-fat diet (HFD)-fed rats. The results showed that WEPE and GA significantly reduced tissues (jejunum, brain, and liver) MG-evoked leptin resistance, malondialdehyde (MDA), proinflammatory cytokines, SOCS3, orexigenic neuropeptides, and lipid accumulation through increasing leptin receptor, tight junction proteins, antimicrobial peptides, anorexigenic neuropeptides, excretion of fecal triglyceride (TG), and short-chain fatty acids (SCFAs) via a positive correlation with the Allobaculum and Bifidobacterium microbiota. These novel findings suggest that WEPE holds the potential as a functional food ingredient for alleviating obesity and its complications.

Roles of astrocytic sonic hedgehog production and its signal for regulation of the blood-brain barrier permeability.

Sonic hedgehog (Shh) is a secreted glycopeptide belonging to the hedgehog family that is essential for morphogenesis during embryonic development. The Shh signal is mediated by two membrane proteins, Patched-1 (Ptch-1) and Smoothened (Smo), following the activation of transcription factors such as Gli. Shh decreases the permeability of the blood-brain barrier (BBB) and plays a key role in its function. In the damaged brain, BBB function is remarkably disrupted. The BBB disruption causes brain edema and neuroinflammation resulting from the extravasation of serum components and the infiltration of inflammatory cells into the cerebral parenchyma. Multiple studies have suggested that astrocyte is a source of Shh and that astrocytic Shh production is increased in the damaged brain. In various experimental animal models of acute brain injury, Shh or Shh signal activators alleviate BBB disruption by increasing tight junction proteins in endothelial cells. Furthermore, activation of astrocytic Shh signaling reduces reactive astrogliosis, neuroinflammation, and increases the production of vascular protective factors, which alleviates BBB disruption in the damaged brain. These findings suggest that astrocytic Shh and Shh signaling protect BBB function in the damaged brain and that target drugs for Shh signaling are expected to be novel therapeutic drugs for acute brain injuries.