Proteins Claudin-1 Research Articles

Invasive micropapillary carcinoma of the breast and invasive breast carcinoma of no special type: a comparison of claudin proteins’ expression and its impact on survival

Invasive micropapillary carcinoma of the breast is characterized by clusters of cells presenting with inverted polarity. Although the apico–basal polarity is a fundamental property of the epithelium, the biological alterations leading to the inside-out pattern observed in invasive micropapillary carcinoma (IMPC) remain mostly unknown. The regulation of tight junctions in polarity formation and maintenance is acknowledged. By using immunohistochemistry, we have analysed claudin-1, -3, -4, and -7 tight junction proteins expression and their prognostic value on IMPCs and compared them to invasive breast carcinomas of no special type (IBC-NST) tumors. Our cohort consisted of 37 IMPCs, 36 IBC-NST and 9 mixed IMPC/IBC-NST tumors. Two scoring systems were used to quantify protein expression: a 4-tier scoring system and the H-score method. Distant metastasis free survival (DMFS) intervals and overal survival (OS) data were used for prognosis evaluation. The analysed samples were characterized mainly by low or no claudin-1 expression whereas claudins-3, -4 and -7 showed variable positivity. We have found no significant differences in claudin-3 and -4 protein expression between IMPC and IBC-NST groups with either scoring methods, however high claudin-7 expression was found in significantly more IMPCs than IBC-NST tumors according to the H-score system (p = 0.02). The 4-tier scoring method revealed association of claudin-7 expression with molecular tumor subtypes (p = 0.001). IMPC and IBC-NST tumors did not show difference in DMFS (p = 0.70). In the analysis of pure IMPC and IBC-NST tumors, positive/high claudin-4 protein expression was significantly associated with shorter DMFS (p = 0.02/p = 0.008, respectively according to the two scoring methods). Claudin-3 and claudin-7 expression showed no association with DMFS or OS. Changes in epithelial polarity seem not to be related to claudin-1, -3, and -4 expression. Increased claudin-4 expression may have a role in breast cancer progression.

Dietary sanguinarine supplementation improves the growth performance and intestinal immunity of broilers

Dietary sanguinarine (SAN) can enhance the growth performance of poultry and livestock, but the regulatory mechanism of the SAN monomer on intestinal homeostasis and how it promotes growth performance has not yet been clarified. In this study, 200 chickens were divided into four groups and fed different doses of SAN (0, 0.225, 0.75, 2.25mg/kg) for transcriptome and microbiota analysis. The data showed that different doses of SAN supplementation increased the feed conversion rate (FCR) of 22 to 42 d old and 1 to 42 d old broilers (P < 0.01), and 0.225 mg/kg SAN reduced the contents of alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CREA) and blood urea nitrogen (BUN) in serum (P < 0.01). Dietary SAN increased the villus height and the villus height/crypt depth (V/C) ratio in the ileum (P < 0.01). The levels of tight junction proteins (zonula occludens-1, occludin and claudin-1) were up-regulated in the ileum and cecum (P < 0.01) and the levels of immunoglobulin (Ig) A, IgM, IgG, interleukin (IL)-4, IL-10 and interferon (IFN)-γ were up-regulated in the serum and ileum (P < 0.01). RNA-seq analysis revealed 385 differentially expressed genes (DEGs) (|log2 fold change| ≥ 1, FDR < 0.05) between the SAN group and CON group. Kyoto Encyclopedia of Genes and Genomes pathway analysis showed 15 pathways mostly associated with the immune system. Additionally, the reverse transcription-PCR results showed that the relative mRNA expression of β-defensin and mucin 2 were up-regulated (P < 0.01) and Toll-like receptor (TLR) 2 and TLR 4 mRNA were down-regulated by SAN (P < 0.01), which was consistent with the transcriptomic analysis. Western blot analysis also showed that SAN reduced the expression of inflammatory proteins such as TLR4, nuclear factor-kappa B and IL-1β in the ileum (P < 0.01). In addition, at the genus level, SAN significantly increased the relative abundance of bacteria (Bacteroides, unclassified_f__Lachnospiraceae, Lactobacillus and Romboutsia) involved in acetate and butyrate production in the cecum, which are associated with enhanced intestinal immune function and maintaining intestinal health. In conclusion, SAN ameliorates the growth performance of broilers, enhances intestinal immune function, regulates the structure of microbiota and maintains intestinal health.

Indole-3-propionic acid enhances growth performance and reduces diarrhea via modulating redox status and intestinal inflammation in weaned piglets

Indole-3-propionic acid (IPA) has anti-inflammatory properties, which can be beneficial for weaned piglets with underdeveloped immune systems. The study explores the impact of IPA supplementation on growth performance, oxidative stress, and inflammation response in weaned piglets. In experiment (Exp.) 1, ninety weaned piglets were divided into six groups (five replicates per group, three pigs per replicate), with each group receiving a basal diet with varying amounts of IPA (0, 50, 100, 200, 400, or 600 mg/kg) for 42 d. Piglets fed the diets with 50, 100, and 200 mg/kg of IPA exhibited reduced feed conversion ratios (F: G) compared to the control piglets (P = 0.035). Notably, 50 and 100 mg/kg IPA treatments significantly reduced diarrhea incidence and serum interleukin (IL)-6 content (P < 0.05). Conversely, a high dosage of 600 mg/kg IPA led to increased serum contents of tumor necrosis factor (TNF)-α, and IL-6 (P < 0.05). Optimal antioxidant benefits were observed at 100 mg/kg IPA supplementation, which significantly reduced malondialdehyde levels while enhancing serum total antioxidant capacity and total superoxide dismutase activity on d 14 (P < 0.05). Exp. 2 investigated the effects of IPA on lipopolysaccharide (LPS) challenge in weaned piglets. The study consisted of 32 weaned piglets allocated into 4 groups, with 8 replicates per group and 1 piglet per replicate: a control group, a LPS challenge group, a LPS challenge group supplemented with 100 mg/kg IPA, and a group supplemented with 100 mg/kg IPA alone. Upon administration of LPS or saline injection, the results indicated that dietary IPA supplementation in challenged piglets enhanced villus height: crypt depth, modulated IL-8 and IL-22 mRNA relative expression, and increased the tight junction protein claudin-1 mRNA relative expression in the intestinal mucosa (P < 0.05). These findings suggest that dietary supplementation of IPA at specific concentrations significantly improves growth performance, reduces diarrhea incidence, and mitigates inflammation and oxidative stress in weaned piglets. It may be concluded that incorporating IPA into the diet of weaned piglets can effectively improve their health and development.

Sustained immune activation and impaired epithelial barrier integrity in the ectocervix of women with chronic HIV infection.

Chronic systemic immune activation significantly influences human immunodeficiency virus (HIV) disease progression. Despite evidence of a pro-inflammatory environment in the genital tract of HIV-infected women, comprehensive investigations into cervical tissue from this region remain limited. Similarly, the consequences of chronic HIV infection on the integrity of the female genital epithelium are poorly understood, despite its importance in HIV transmission and replication. Ectocervical biopsies were obtained from HIV-seropositive (n = 14) and HIV-seronegative (n = 47) female Kenyan sex workers. RNA sequencing and bioimage analysis of epithelial junction proteins (E-cadherin, desmoglein-1, claudin-1, and zonula occludens-1) were conducted, along with CD4 staining. RNA sequencing revealed upregulation of immunoregulatory genes in HIV-seropositive women, primarily associated with heightened T cell activity and interferon signaling, which further correlated with plasma viral load. Transcription factor analysis confirmed the upregulation of pro-inflammatory transcription factors, such as RELA, NFKB1, and IKZF3, which facilitates HIV persistence in T cells. Conversely, genes and pathways associated with epithelial barrier function and structure were downregulated in the context of HIV. Digital bioimage analysis corroborated these findings, revealing significant disruption of various epithelial junction proteins in ectocervical tissues of the HIV-seropositive women. Thus, chronic HIV infection associated with ectocervical inflammation, characterized by induced T cell responses and interferon signaling, coupled with epithelial disruption. These alterations may influence HIV transmission and heighten susceptibility to other sexually transmitted infections. These findings prompt exploration of therapeutic interventions to address HIV-related complications and mitigate the risk of sexually transmitted infection transmission.

Inflammation-Induced Claudin-2 Upregulation Limits Pancreatitis Progression by Enhancing Tight Junction-Controlled Pancreatic Ductal Transport.

Pancreatitis is an inflammatory disease of the pancreas that can arise due to various factors, including environmental risks such as diet, alcohol, and smoking, as well as genetic predispositions. In some cases, pancreatitis may progress and become chronic, leading to irreversible damage and impaired pancreatic function. Genome-wide association studies (GWAS) have identified polymorphisms at the X-linked CLDN2 locus as risk factors for both sporadic and alcohol-related chronic pancreatitis. CLDN2 encodes claudin-2 (CLDN2), a paracellular cation-selective channel localized at tight junctions and expressed in the pancreas and other secretory organs. However, whether and how CLDN2 may modify pancreatitis susceptibility remains poorly understood. We aimed to clarify the potential role of CLDN2 in the onset and progression of pancreatitis. We employed multiple methodologies to examine the role of CLDN2 in human pancreatic tissue, caerulein-induced experimental pancreatitis mouse model, and pancreatic ductal epithelial organoids. In both human chronic pancreatitis tissues and caerulein-induced experimental pancreatitis, CLDN2 protein was significantly upregulated in pancreatic ductal epithelial cells. Our studies using pancreatic ductal epithelial organoids and mice demonstrated the inflammatory cytokine IFNγ upregulates claudin-2 expression at both RNA and protein levels. Following caerulein treatment, Ifng KO mice had diminished upregulation of CLDN2 relative to WT mice, indicating that caerulein-induced claudin-2 expression is partially driven by IFNγ. Functionally, Cldn2 knockout mice developed more severe caerulein-induced experimental pancreatitis, indicating CLDN2 plays a protective role in pancreatitis development. Pancreatic ductal epithelial organoid-based studies demonstrated that CLDN2 is critical for sodium-dependent water transport and necessary for cAMP-driven, CFTR-dependent fluid secretion. These findings suggest that functional crosstalk between CLDN2 and CFTR is essential for fluid transport in pancreatic ductal epithelium, which may protect against pancreatitis by adjusting pancreatic ductal secretion to prevent worsening autodigestion and inflammation. In conclusion, our studies suggest CLDN2 upregulation during pancreatitis may play a protective role in limiting disease development, and decreased CLDN2 function may increase pancreatitis severity. These results point to the possibility of modulating pancreatic ductal CLDN2 function as an approach for therapeutic intervention of pancreatitis.

Effects of organic acid blends on the growth performance, intestinal morphology, microbiota, and serum lipid parameters of broiler chickens

Organic acids have emerged as promising alternatives to antibiotic growth promoters in poultry production. The present study was conducted to determine the effects of organic acids blends v.i.z. Acidapure liquid and Acidapure powder supplementation on the growth performance, gut health, gut microbiota, and serum lipid profile of broiler chickens. A total of 120-day-old chicks with similar live body weights were randomly divided into four groups. Each group was further divided into 3 replicates, and each further divided into three replicates of ten bird. The birds in Group 1 (T1) were fed a basal diet supplemented with plain drinking water, those in Group 2 (T2) received basal feed supplemented with Acidapure powder (1 kg/MT feed) and plain drinking water, those in Group 3 (T3) received basal feed supplemented with Acidapure liquid in the drinking water (0.2 ml/l water), and those in Group 4 (T4) received basal feed supplemented with Acidapure powder (1 kg/MT feed) and Acidapure liquid in the drinking water (0.2 ml/l water). Acidapure powder and Acidapure liquid were added to the feed and water of the broilers from 0–42 days of life. The results showed that compared with the control (T1), supplementation with Acidapure powder and liquid in broiler chickens for 42 days increased (P < 0.05) ABW and ADG and reduced FCR in the treatment groups (T2, T3 and T4). At d 21 and 42, all forms of Acidapure supplement increased the VH and CD in the jejunum and ileum and reduced the pH of the ileum. Compared with the control (T1), the combination of Acidapure powder and liquid (T4) increased the gene expression of the tight junction proteins Claudin-1 and Zona Occludense 1 (ZO-1). Compared with the control, Acidapure supplementation reduced the cecal coliform count and total viable count (TVC) and decreased the serum cholesterol and triglyceride levels. In conclusion, Acidapure, as a blend of organic acids, effectively enhances the growth performance and gut health of broilers, making it a viable and safe alternative to traditional antimicrobial growth promoters.

Decursin protects against DSS-induced experimental colitis in mice by inhibiting the cGAS-STING signaling pathway.

While studies have shown that Angelica gigas Nakai (A. gigas) can alleviate ulcerative colitis in mice, the therapeutic role of its main active ingredient, decursin, is uncertain. Therefore, we aimed to investigate the protective effect and mechanism of decursin against inflammatory bowel disease (IBD) in vivo using mice. IBD was simulated via induction with 3% dextran sodium sulfate (DSS), with or without daily treatment with decursin (10 mg/kg or 20 mg/kg) or 5-amino salicylic acid (5-ASA; 100 mg/kg) for 14 days. Mice were weighed and monitored daily for disease activity index (DAI) scoring. Colon tissues were collected for histopathological staining analysis, and serum was collected for ELISA measurement of proinflammatory cytokines. Western blotting was employed to analyze colonic expression levels of the tight junction-related proteins ZO-1, Occludin, and Claudin 1, as well as cGAS-STING signaling pathway-associated proteins. The expression levels of major proteins were verified using immunohistochemistry and immunofluorescence. Compared with the control group, DSS-induced mice showed decreased body weight, increased DAI scores, shortening of the colon, disrupted colon tissue structure, increased serum levels of proinflammatory cytokines, increased expression of factors involved in activating the cGAS-STING signaling pathway, and reduced expression of ZO-1, Occludin, and Claudin 1. Under decursin treatment, the pathological state of IBD was less severe, proinflammatory factors were downregulated, and activation of the cGAS-STING signaling pathway was inhibited. Our findings indicate that decursin helps restore the intestinal mucosal barrier and prevents activation of the cGAS-STING signaling cascade, alleviating experimental IBD in mice.

Brain-targeted autoimmunity is strongly associated with Long COVID and its chronic fatigue syndrome as well as its affective symptoms

IntroductionAutoimmune responses contribute to the pathophysiology of Long COVID, affective symptoms and myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). ObjectivesTo examine whether Long COVID, and its accompanying affective symptoms and CFS are associated with immunoglobulin (Ig)A/IgM/IgG directed at neuronal proteins including myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), synapsin, α + β-tubulin, neurofilament protein (NFP), cerebellar protein-2 (CP2), and the blood–brain-barrier-brain-damage (BBD) proteins claudin-5 and S100B. MethodsIgA/IgM/IgG to the above neuronal proteins, human herpes virus-6 (HHV-6) and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) were measured in 90 Long COVID patients and 90 healthy controls, while C-reactive protein (CRP), and advanced oxidation protein products (AOPP) in association with affective and CFS ratings were additionally assessed in a subgroup thereof. ResultsLong COVID is associated with significant increases in IgG directed at tubulin (IgG-tubulin), MBP, MOG and synapsin; IgM-MBP, MOG, CP2, synapsin and BBD; and IgA-CP2 and synapsin. IgM-SARS-CoV-2 and IgM-HHV-6 antibody titers were significantly correlated with IgA/IgG/IgM-tubulin and -CP2, IgG/IgM-BBD, IgM-MOG, IgA/IgM-NFP, and IgG/IgM-synapsin. Binary logistic regression analysis shows that IgM-MBP and IgG-MBP are the best predictors of Long COVID. Multiple regression analysis shows that IgG-MOG, CRP and AOPP explain together 41.7 % of the variance in the severity of CFS. Neural network analysis shows that IgM-synapsin, IgA-MBP, IgG-MOG, IgA-synapsin, IgA-CP2, IgG-MBP and CRP are the most important predictors of affective symptoms due to Long COVID with a predictive accuracy of r = 0.801. ConclusionBrain-targeted autoimmunity contributes significantly to the pathogenesis of Long COVID and the severity of its physio-affective phenome.

Study repair function of mucin-2 on the tight junction protein of uterine epithelial cells under bacterial endotoxins

To analysis repair function of mucin-2(MUC2) and glycoprotein particles on the tight junction protein of uterus under bacterial endotoxins. In this experiment, we showed that the thicker mucus layer of the uterus is used to prevent the translocation of endotoxin at 21d postdelivery. When endotoxin acts on the uterus to thin its mucous layer, the cells in the lamina propria of the uterus secrete a large number of glycoprotein particles at 27d postdelivery. Due to a significantly decrease in the expression of glycosyltransferase, the glycoprotein particles are incompletely glycosylation MUC2, which can interact with the cell membrane and are released in large quantities in the form of exocytosis. These glycoprotein particles can significantly repair tight junction proteins in the inter-cellular space and significantly increase the expression of Claudin-1, JAM (Junction adhesion molecule-A), E-cadherin, ZO-1(Zonula occludens-1) and desmosome proteins after endotoxin treatment. The results of the present study show that endotoxins can thin the uterine mucus layer and accelerate the release of incompletely glycosylated MUC2 from lamina propria cells. In inter-cellular spaces, MUC2 can increase its expression levels and distribution area to repair the tight junction structure of cells with larger gaps. Further strengthening of the barrier prevents endotoxin translocation by repairing the tight junction structure of uterine epithelial cells.

Axial heterogeneity of superficial proximal tubule paracellular transport in mice.

A considerable amount of NaCl reabsorption in proximal tubules (PTs) occurs via the paracellular transport regulated by the tight junction proteins claudins (Cldns). However, the paracellular transport properties in mouse superficial PTs remain unclear. We characterized these properties in superficial PT S1-S3 segments from mice expressing [wild-type (WT, WTS1-WTS3)] or lacking claudin-2 [knockout (KO, KOS1-KOS3)]. We isolated and perfused segments with symmetrical solutions in the presence of bath ouabain and measured the diffusion potential upon changing the salt composition of the lumen or bath. Based on the diffusion potential corrected for the liquid junction potential (dVT), we calculated the paracellular Na+ over Cl- permeability (PNa/PCl) ratio. The PNa/PCl values upon reducing luminal NaCl averaged 1.27, 1.04, and 0.85 in WTS1, WTS2, and WTS3 and 0.34, 0.55, and 0.80 in KOS1, KOS2, and KOS3, respectively. The dVT values exhibited a symmetrical response to bidirectional NaCl concentration gradients in WTS1-WTS3 and KOS1-KOS3. WTS1 and WTS3 were monovalent cation-selective, with WTS1 demonstrating stronger cation selectivity. The order of permeabilities relative to Cl- was K+ > Rb+ > Na+ > Li+, whereas both KOS1 and KOS3 exhibited monovalent cation selectivity loss and consequently enhanced anion selectivity, especially in KOS1. Protamine addition to the lumen and bath similarly decreased PNa/PCl values upon reduced luminal NaCl in the order of WTS1 > WTS3 > KOS3 > KOS1. Therefore, this study presents evidence of axial heterogeneity in paracellular transport across superficial PTs in mice.

Acceptable daily intake of aspartame aggravates enteritis pathology and systemic inflammation in colitis mouse model.

In this study, a dextran sodium sulfate-induced ulcerative colitis model in C57BL/6 mice was used to explore the effect of acceptable daily intake (ADI) of aspartame on inflammation in colonic tissues. The effects of aspartame on the inflammatory state of the colon in mice were comprehensively evaluated by comparing the body weight, colon length/colon length index, splenic index, disease activity index (DAI) score, histological activity index (HAI) score, the expression levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, claudin-3, and occludin, the infiltration characteristics of macrophage and neutrophil and the composition of the gut microbiota in the control group, aspartame group, ulcerative colitis model group, and aspartame + ulcerative colitis group. We demonstrated that, in a mouse model of dextran sulfate-induced ulcerative colitis, ADI of aspartame caused a significant decrease in body weight, colon length/colon length index, DAI scores, and expression levels of the proteins claudin-3 and occludin. Moreover, ADI of aspartame caused an increase in the splenic index, HAI scores, the levels of proinflammatory factors TNF-α, IL-1β, and IL-6 both in intestinal tissue and serum and infiltration of macrophages and neutrophils in colon tissues. These results showed that ADI of aspartame promoted intestinal pathology and systemic inflammation in a mouse model of colitis.

Endothelial insulin-like growth factor-1 signaling regulates vascular barrier function and atherogenesis

Abstract Background Progressive deposition of cholesterol in the arterial wall characterizes atherosclerosis, which underpins most cases of myocardial infarction and stroke. Insulin-like growth factor-1 (IGF-1) is a hormone that regulates systemic growth and metabolism and possesses anti-atherosclerotic properties. However, the role of IGF-1 in the endothelium remains unexplored. Purpose To study the effect of increased endothelial IGF-1 receptor (IGF-1R) expression on atherogenesis using in vivo and in vitro models. Methods We generated atherosclerosis prone ApoE-/- transgenic mice with endothelium restricted overexpression of human IGF-1R (hIGFREO-ApoE-/-) or signalling defective K1003R mutant IGF-1R (mIGFREO-ApoE-/-). ApoE-/- littermates were controls. Following 12 weeks of western diet, we assessed atherosclerosis, systemic metabolism, leukocyte homeostasis and vascular permeability using histology, flow cytometry, metabolic testing, bone marrow transplantation and in vivo perfusion with either VE-Cadherin, Evans blue or BODIPY-LDL. For in vitro studies, human umbilical vein endothelial cells were transduced with custom-made lentivirus particles allowing doxycyline-inducible wild-type or mutant K1003R IGF-1R overexpression; with transduced cells not exposed to doxycycline as controls. Confluent cells were exposed to 100mM hydrogen peroxide to model the permeability-inducing conditions observed in atherosclerosis. In vitro, we assessed junctional proteins, FITC-dextran permeability and transcellular BODIPY-LDL uptake. Results We show that mice with endothelial overexpression of IGF-1R have less atherosclerosis, arterial cholesterol uptake, and vascular leakage of multiple organs. Conversely, mice with endothelial overexpression of signalling defective IGF-1R did not exhibit these phenomena. We found no differences in systemic metabolism or growth. In complementary in vitro studies, overexpressing wildtype IGF-1R altered the localization of some tight junction proteins (VE-Cadherin and Claudin 5) and reduced leakage across human endothelial monolayers; this was not observed with signalling defective IGF-1R. Moreover, it reduced endothelial cell internalization of cholesterol-rich lipoproteins and increased the association of these particles with clathrin, but not caveolin-1, both of which participate in vesicular uptake of lipoproteins. Conclusion Overall, our findings indicate that endothelial IGF-1 signalling modulates both para- and trans-cellular vascular barrier function. This is particularly relevant to the phenomenon of atherosclerosis and suggests that increased vascular IGF-1 signalling reduces atherosclerosis. Beyond this, our data are potentially relevant to many disease processes associated with altered vascular barrier function. This discovery could lead to novel therapeutics to normalise vascular barrier function.

FMT and TCM to treat diarrhoeal irritable bowel syndrome with induced spleen deficiency syndrome- microbiomic and metabolomic insights

BackgroundDiarrheal irritable bowel syndrome (IBS-D) is a functional bowel disease with diarrhea, and can be associated with common spleen deficiency syndrome of the prevelent traditional Chinese medicine (TCM) syndrome. Fecal microbiota transplantation (FMT) could help treating IBS-D, but may provide variable effects. Our study evaluated the efficacy of TCM- shenling Baizhu decoction and FMT in treating IBS-D with spleen deficiency syndrome, with significant implications on gut microbiome and serum metabolites.MethodsThe new borne rats were procured from SPF facility and separated as healthy (1 group) and IBS-D model ( 3 groups) rats were prepared articially using mother’s separation and senna leaf treatment. 2 groups of IBS-D models were further treated with TCM- shenling Baizhu decoction and FMT. The efficacy was evaluated by defecation frequency, bristol stool score, and intestinal tight junction proteins (occludin-1 and claudin-1) expression. Microbiomic analysis was conducted using 16 S rRNA sequencing and bioinformatics tools. Metabolomics were detected in sera of rats by LC-MS and annotated by using KEGG database.ResultsSignificant increment in occludin-1 and claudin-1 protein expression alleviated the diarrheal severity in IBS-D rats (P < 0.05) after treatment with FMT and TCM. FMT and TCM altered the gut microbiota and regulated the tryptophan metabolism, steroid hormone biosynthesis and glycerophospholipid metabolism of IBS-D rats with spleen deficiency syndrome.The microbial abundance were changed in each case e.g., Monoglobus, Dubosiella, and Akkermansia and othe metabolic profiles.ConclusionFMT and TCM treatment improved the intestinal barrier function by regulating gut microbiota and improved metabolic pathways in IBS-D with spleen deficiency syndrome.

Effects of hyperglycemia on airway epithelial barrier function in WT and CF 16HBE cells

Cystic fibrosis related diabetes (CFRD), the main co-morbidity in cystic fibrosis (CF), is associated with higher rates of lung function decline. We hypothesize that airway epithelial barrier function is impaired in CF and is further exacerbated under hyperglycemia, worsening pulmonary outcomes. Using 16HBE cells, we studied the effects of hyperglycemia in airway epithelial barrier function. Results show increased paracellular dye flux in CF cells in response to insulin under hyperglycemia. Gene expression experiments identified claudin-4 (CLDN4) as a key tight junction protein dysregulated in CF cells. CLDN4 protein localization by confocal microscopy showed that CLDN4 was tightly localized at tight junctions in WT cells, which did not change under hyperglycemia. ln contrast, CLDN4 was less well-localized in CF cells at normal glucose and localization was worsened under hyperglycemia. Treatment with highly effective modulator compounds (ETI) reversed this trend, and CFTR rescue was not affected by insulin or hyperglycemia. Bulk RNA sequencing showed differences in transcriptional responses in CF compared to WT cells under normal or high glucose, highlighting promising targets for future investigation. One of these targets is protein tyrosine phosphatase receptor type G (PTPRG), which has been previously found to play a role in defective Akt signaling and insulin resistance.

TIPE2 aggravates experimental colitis and disrupts intestinal epithelial barrier integrity by activating JAK2/STAT3/SOCS3 signal pathway

Ulcerative colitis (UC) is a chronic relapsing and progressive inflammatory disease of the colon. TIPE2 is a negative regulator of innate and adaptive immunity that maintains immune homeostasis. We found that TIPE2 was highly expressed in mucosa of mice with colitis. However, the role of TIPE2 in colitis remains unclear. We induced colitis in mice with dextran sulfate sodium (DSS) and treated them with TIPE2, and investigated the inflammatory activity of the colon in vivo by cytokines detection and histopathological analyses. We also measured inflammatory alteration and tight junctions induced by DSS in vitro. The results demonstrated that administration of TIPE2 promoted the severity of colitis in mice and human colon epithelial cells. Furthermore, TIPE2 aggravated intestinal epithelial barrier dysfunction by decreasing the expression of the tight junction proteins Occludin, Claudin-1 and ZO-1. In addition, TIPE2 exacerbated intestinal inflammatory response by inhibiting the expression of SOCS3, remarkably activating JAK2/STAT3 signaling pathway, and increasing the translocation of phosphorylated STAT3 into the nucleus. Silencing of TIPE2 attenuated the DSS-induced activation of JAK2/STAT3, thereby rescuing epithelial inflammatory injury and restoring barrier dysfunction. These results indicate that TIPE2 augments experimental colitis and disrupted the integrity of the intestinal epithelial barrier by activating the JAK2/STAT3/SOCS3 signaling pathway.

Glucocorticoid-Induced Leucine Zipper Protein and Yeast-Extracted Compound Alleviate Colitis and Reduce Fungal Dysbiosis

Inflammatory bowel diseases (IBD) have a complex, poorly understood pathogenesis and lack long-lasting effective treatments. Recent research suggests that intestinal fungal dysbiosis may play a role in IBD development. This study investigates the effects of the glucocorticoid-induced leucine zipper protein (GILZp)”, known for its protective role in gut mucosa, and a yeast extract (Py) with prebiotic properties, either alone or combined, in DSS-induced colitis. Both treatments alleviated symptoms via overlapping or distinct mechanisms. In particular, they reduced the transcription levels of pro-inflammatory cytokines IL-1β and TNF-α, as well as the expression of the tight junction protein Claudin-2. Additionally, GILZp increased MUC2 transcription, while Py reduced IL-12p40 and IL-6 levels. Notably, both treatments were effective in restoring the intestinal burden of clinically important Candida and related species. Intestinal mycobiome analysis revealed that they were able to reduce colitis-associated fungal dysbiosis, and this effect was mainly the result of a decreased abundance of the Meyerozima genus, which was dominant in colitic mice. Overall, our results suggest that combined treatment regimens with GILZp and Py could represent a new strategy for the treatment of IBD by targeting multiple mechanisms, including the fungal dysbiosis.

Improvement effect and mechanism of XuanFuDaiZhe tang on rats with diarrheal irritable bowel syndrome induced by colorectal dilation

Ethnopharmacological relevanceXuanFuDaiZhe Tang (XFDZT) is used in traditional Chinese medicine (TCM) to treat diarrhoea-predominant irritable bowel syndrome (IBS-D). Our laboratory has demonstrated that XFDZT remarkably improves various gastrointestinal motility disorders in animal models. However, previous studies have only focused on one or several protein targets without systematically investigating dynamic changes and protein interrelations. Aim of the studyTo explore the mechanisms underlying the therapeutic action of XFDZT in IBS-D using a network pharmacology approach and in vivo experiments. Materials and methodsThe active compounds of XFDZT were selected from TCM Systems Pharmacology and TCM Integrated databases, and potential targets were identified using the Swiss Target Prediction databases. Targets related to IBS-D were mined from the DisGeNet, Drug Bank, and Therapeutic Target databases. The intersecting protein–protein interactions (PPIs) of the drug–disease crossover genes were analysed, and a central PPI network was constructed using the STRING database and Cytoscape 3.7.2. Following Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, a gene pathway network was constructed to identify key target genes and pathways. Using haematoxylin and eosin staining and western blotting, we validated how XFDZT controls water expression in the body to treat IBS-D infection. ResultsFirst, the results showed that XFDZT contained 1037 active ingredients and 1458 corresponding targets. After intersecting the 252 IBS targets, 108 targets were identified. The main targets of XFDZT were albumin, aquaporins such as AQP1 and AQP3, calmodulin, and the cellular enzyme CYP2C9. GO and KEGG enrichment predicted that the action pathways were the neuroactive ligand–receptor interaction, calcium signalling pathway, serotonergic synapse signalling pathway, cGMP–PKG signalling pathway, cAMP signalling pathway, and MLCK–MLC signalling pathway. Second, an IBS-D rat model was constructed using colorectal dilation (CRD). CRD can significantly induce IBS-D symptoms such as diarrhoea, abdominal pain, and anxiety and depression-like behaviour in rats. XFDZT (10, 20, and 40 g/kg) administered for 14, 21, and 28 days significantly reversed these changes in IBS-D rats in a time- and dose-dependent manner, suggesting that XFDZT significantly improved IBS-D. Finally, the mechanism by which XFDZT improves IBS-D was explored from the perspective of AQPs, tight junction proteins, and motility-related proteins in colon tissue. Compared with the control group, the protein expression of AQP1, AQP3, and AQP8 in the colon tissue of IBS-D rats was significantly downregulated, whereas the protein expression of AQP7 was significantly upregulated. The expression of tight junction-related proteins claudin-1, occludin, and ZO-1 in colon tissue was significantly downregulated, whereas the expression of motility-related proteins p-MLC, MLC, MLCK, and CaM in colon tissue was significantly upregulated, suggesting that IBS-D rats had AQP disorders, epithelial intercellular connections, and motility in colon tissue. The above changes were significantly reversed by XFDZT administration (5, 10, and 20 g/kg) for 14 days. ConclusionXFDZT significantly improved diarrhoea, abdominal pain, anxiety, and depression in IBS-D rats, and its mechanism of action may be related to the regulation of AQPs, tight junction proteins, and the MLCK–MLC pathway. This study provided a pharmacological experimental basis for the development of XFDZT as a novel drug for the treatment of IBS-D.

APPLICATION OF IN VITRO MODELS: INCORPORATING BLOOD BRAIN BARRIER MODELS WITH GLIOMA, AND THE USE OF MICROflUIDICS

Abstract AIMS The blood brain barrier (BBB) presents one of the main obstacles for the development of novel glioblastoma treatments. The various transporters and enzymes within the BBB reduce the disposition of novel therapies into the brain. There is a need for an in vitro BBB-glioma model with proven efficacy of predicting CNS delivery at earlier stages in the drug discovery pipeline. METHOD A BBB-glioma lab-on-a-chip model could enable rapid identification of novel therapies that can cross the BBB at earlier stages of the drug development process, before going to clinical trials. The application of microfluidic technology allows for the detection of soluble tissue-derived factors and the assessment of the levels of soluble markers released from malignant tissue. Adding ‘flow’ to the model, is more reflective of the dynamic flow present at the BBB. RESULTS Validation studies of the current BBB model and converting this model over to a lab-on-a-chip model have shown the presence of an endothelial cell barrier and the tight junction proteins Zo-1 and Claudin-1, through western blotting, immunocytochemistry, and permeability experiments using varying molecular weights of FitC-dextran. These validation studies have been completed using brain endothelial monolayers on polycarbonate transwell inserts, with the immortalised cell line HCMEC/d3, and the short-term culture cells HBMEC. Also, validation studies using a co-culture of endothelial cells, astrocytes and pericytes have begun, using the same techniques to show a barrier has formed, on both the current BBB model and the lab-on-a-chip model. CONCLUSION The aim of this study is to combine the use of an all-human BBB-glioma model with lab-on-a-chip technology to produce a high throughput model to test novel therapies at an earlier stage. The lab-on-a-chip model will be validated for the purpose of measuring permeability and drug response and to investigate the expression of novel targets verses expression in conventional models.

Capsaicin attenuates the effect of inflammatory cytokines in a HaCaT cell model for basal keratinocytes.

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

Metformin modulates the TXNIP-NLRP3-GSDMD pathway to improve diabetic bladder dysfunction

To validate the therapeutic efficacy of metformin on diabetic bladder dysfunction (DBD) and further elucidate whether the TXNIP-NLRP3-GSDMD axis serves as a target for metformin in ameliorating DBD. C57BL/6J mice were induced with diet-induced obesity by being fed a high-fat diet (HFD) for 16 weeks. After establishing the model, the mice were treated with metformin for 4 weeks, and their glucose metabolism-related parameters were assessed. Urine spot assays and urodynamic measurements were conducted to reflect the bladder function and urinary behavior in mice, while histological examination was performed to observe morphological changes. Western blot analysis was employed to measure the expression levels of pyroptotic factors such as TXNIP, NLRP3, GSDMD, and tight junction proteins. Metformin treatment significantly improved glucose tolerance and insulin sensitivity in mice. Moreover, it showed promise in decreasing urinary spot occurrence, reducing urination frequency, alleviating non-voiding contractions, and stabilizing peak urinary pressure. Following metformin therapy, mice displayed restored epithelial fold structure, increased thickness of the muscular layer, substantial decrease in muscle fiber content, notably reduced levels of TXNIP and GSDMD proteins in the metformin-treated group compared to the DBD group, and restored expression of tight junction proteins Zo-1, Claudin-1, and Occludin. Metformin ameliorates urothelial cells damage in DBD mice by inhibiting TXNIP generation and reducing NLRP3 and GSDMD production.