Tight Junction Proteins Claudin-1 Research Articles

Genome analysis of Bifidobacterium adolescentis and investigation of its effects on inflammation and intestinal barrier function

Numerous studies have confirmed that gut microbiota is a key driver in the occurrence and progression of inflammatory bowel disease (IBD). Based on the bacterial collection constructed in our previous studies, we founded that Bifidobacterium adolescentis AF91-08b2A has the potential beneficial function. We designed cohort studies, genomic studies and animal experiments to further explore the probiotic function of Bifidobacterium adolescentis AF91-08b2A and its therapeutic effect on IBD. The depletion of B. adolescentis in individuals with IBD suggested its significance for intestinal health. Genomic analysis highlighted the probiotic attributes of B. adolescentis AF91-08b2A, including resistance to antibiotics and stress, and metabolic pathways related to energy and carbohydrate metabolism, which are likely to enhance its therapeutic efficacy. In DSS-induced mice colitis model, the strain significantly enhanced the disease activity index (DAI), curbed weight loss, and attenuated colonic damage. It effectively modulated the immune response by reducing the levels of pro-inflammatory cytokines such as IL-6, IL-1β, IL-17A, IFN-γ, and TNF-α, while promoting the secretion of anti-inflammatory cytokines like IL-4, IL-10, and TGF-β1. The restoration of tight junction proteins ZO-1, occludin, and claudin-2 by B. adolescentis AF91-08b2A demonstrated its capacity to safeguard the intestinal epithelial barrier. Collectively, our findings indicate B. adolescentis AF91-08b2A as a valuable therapeutic option for UC, with its multifaceted approach to reducing inflammation and fortifying the intestinal barrier.

Claudin 18.2: An attractive marker in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDA) is a highly aggressive tumor with limited treatment options. Zolbetuximab, a monoclonal antibody against the tight junction protein Claudin 18.2 has recently been developed. At present, few and conflicting data have been reported regarding the clinical-pathological features of Claudin 18.2 expression in PDA. The present study investigated the expression of Claudin 18.2 in histological samples from PDA patients with the aim of verifying its utility as a therapeutic biomarker. Claudin 18.2 immunoreactivity was assessed by immunohistochemical staining on 70 formalin-fixed, paraffin-embedded PDA specimens (28 surgical specimens and 42 fine needle aspiration biopsies). The results obtained were associated with the clinicopathological characteristics and the survival rate of patients. Claudin 18.2 was detected only in neoplastic cells, not in normal pancreatic tissue. Claudin 18.2 was positive in 50% of samples and a higher expression was associated with well- and moderately-differentiated tumors and lymph node-negative status. The high expression of Claudin 18.2 in neoplastic tissue and absence in normal cells suggested that this protein had an attractive role in PDA as both a diagnostic and a prognostic-therapeutic marker. High expression of Claudin 18.2 in neoplastic tissue was associated with more favorable prognostic parameters and the high percentage of positive samples obtained suggests that Zolbetuximab may be suitable for a large number of patients.

Dietary purslane (Portulaca oleracea L.) leaf powder maintains growth and intestinal health in Oreochromis niloticus under chronic water-borne cadmium exposure by strengthening the gut barriers, modulating the intestinal nutrient transporters, and relieving oxidative stress.

High cadmium (Cd) concentrations pose a threat to aquatic life globally. This study examined the efficiency of adding purslane (Portulaca oleracea L.) leaf powder (PLP) to Oreochromis niloticus diets on Cd's negative effects. PLP was analyzed using high-performance liquid chromatography, and its main constituents were gallic acid, chlorogenic acid, and pyrocatechol. Nile tilapia (180 fish, 34.5 ± 0.5 g) were divided into four groups in triplicate. A basal diet was given to the control group. The PLP group received a basal diet containing 10 g PLP/kg diet. The Cd group was exposed to 50 µg/L water. The Cd + PLP group was exposed to Cd and fed diets containing PLP. Results showed that PLP significantly rescued Cd-induced effects. PLP improved fish survival, feed conversion ratio, and growth retardation caused by Cd. PLP also restored decreased activities of lipase, trypsin, and amylase in the intestine. Furthermore, PLP corrected disturbances in leptin and growth hormone levels induced by Cd. PLP mitigated pathological alterations, replenished antioxidants (SOD, CAT, and GSH), and reduced lipid peroxidation in the intestinal tissues. PLP supplementation depleted significant Cd accumulation in the intestine and muscles. Additionally, PLP corrected altered expressions of tight junction proteins (zo-1, zo-2, and claudin-4) and nutrient transporters (glut-1, slc15a2, slc26a6, and slc4a4) in Cd-exposed fish. Conclusively, PLP shows promise as a dietary supplement to mitigate Cd's harmful impacts on fish growth. Its antioxidant activity and regulation of intestinal tight junction proteins and nutrient transporters contribute to its effectiveness. PLP supplementation holds the potential for reducing the detrimental effects of Cd in aquaculture.

Ligilactobacillus salivarius LZZAY01 accelerated autophagy and apoptosis in colon cancer cells and improved gut microbiota in CAC mice.

Colorectal cancer (CRC) is one of the malignant tumors globally, with high morbidity and mortality rates. The mainstay treatment of CRC includes surgery, radiotherapy, and chemotherapy. However, these treatments are associated with a high recurrence rate, poor prognosis, and highly toxic side effects. The probiotics have the potential to prevent CRC, and they display a favorable safety performance. Probiotics could provide a potential strategy to prevent and treat CRC. The impact of LZZAY01 on cancer cell lines CT-26, HCT-116, and SW-620 was evaluated by conducting cytotoxicity and clonogenicity tests. A model of colitis-associated cancer (CAC) was established in C57BL/6j mice following induction with AOM/DSS. The levels of autophagy and apoptosis proteins, tight junction proteins, and inflammatory factors were detected by western blotting, immunofluorescence assay, and enzyme-linked immunosorbent assay. High-throughput sequencing of gut 16S rRNA was performed to analyze the abundance and diversity of the gut microbiome. LZZAY01, a new strain of Ligilactobacillus salivarius, was certified by an evolutionary tree and average nucleotide identity. LZZAY01 enhanced autophagy and apoptosis in CT-26, HCT-116, and SW-620 cell lines. It preserved the integrity of the intestinal barrier by regulating the tight junction protein ZO-1 and claudin-1. The tumor necrosis factor-α and interleukin-6 were reduced by LZZAY01. The abundance and diversity of the intestinal microbiota were enhanced, especially the beneficial bacterial species maintaining the balance of the intestinal flora such as Bifidobacterium and Lactobacillus. L. salivarius LZZAY01 improved CAC via suppressing the growth of colon cancer cells, promoting autophagy and apoptosis, enhancing intestinal tight junctions, reducing intestinal barrier degradation, modifying the gut microbiota abundance, and decreasing inflammatory reactions.IMPORTANCEAlthough similar probiotics have been shown to have anticancer potential in colorectal cancer (CRC), there is a paucity of research related to the preventive function of probiotics against CRC. And there are fewer studies about the mechanism of probiotics' preventive effects on CRC. The regulation of tumor cell proliferation and apoptosis by the active ingredients of probiotics may be one of the mechanisms of their prevention of CRC. In this study, we explored the effects of L. salivarius LZZAY01 on autophagy and apoptosis of colon cancer cells in vitro and in vivo and proposed a possible mechanism for the prevention of CRC by probiotics.

L-Carnitine Improves Muscle Nutrient Metabolism and Intestinal Health in High-Fat-Fed Carp (Cyprinus carpio).

L-Carnitine is widely recognized for its involvement in lipid metabolism, but its effects on muscle quality and gut health in carp have not been well studied. The research aimed to investigate how L-carnitine influences muscle quality and intestinal health in high-fat-fed carp. The study was separated into four groups that received either the standard diet, a high-fat diet (HFD), or a HFD supplemented with 500 mg/kg L-carnitine (LLC), or a HFD supplemented with 1000 mg/kg L-carnitine (HLC) for 56 days. L-Carnitine was found to significantly reduce blood lipid levels. In addition, L-carnitine increased the crude protein content and decreased the crude fat content of high-fat-fed carp muscle while improving muscle fiber morphology and muscle quality. L-Carnitine increased the expression of genes related to intestinal tight junction proteins (claudin-2, occludin, and zo-1), improved the expression of genes related to intestinal inflammation, and enhanced the physical barrier function and organization of the intestine. Analysis of intestinal flora and intestinal metabolites showed that L-carnitine increased the diversity of the intestinal flora, increased the abundance of Cetobacterium, and influenced intestinal levels of bile acids, arachidonic acid, and tryptophan-related metabolites. In conclusion, supplementation with 1000 mg/kg L-carnitine improved muscle quality and intestinal health significantly in high-fat-fed carp by regulating muscle nutrient metabolism and intestinal flora.

Nobiletin-rich kososan, a Kampo formula, prevents the onset of apathy-like behavior and neuroinflammation in sickness behavior mouse model induced by increasing doses of lipopolysaccharide.

Infectious diseases are often concomitant with symptoms of lassitude and emotional disturbances, including depression, the so-called sickness behavior. Kososan, a Kampo (traditional Japanese herbal) formula, has been clinically used for depressive mood, with demonstrated efficacy in stress-induced depressive-like behavior mouse models. Additionally, our previous study has shown that nobiletin-rich kososan (NKS) prevents aging-related depressive-like behaviors and neuroinflammation in mice. Here, we examined whether NKS alleviates depressive-like behavior and neuroinflammation in a mouse model of sickness behavior induced by lipopolysaccharide (LPS). Repeated oral administration of NKS and the positive control antidepressant paroxetine (Paro) significantly prevented this behavior. NKS and Paro significantly increased the anti-inflammatory milieu in the hippocampus and prefrontal cortex (PFC), as well as brain microglia, of LPS-injected mice. The expression of the vascular tight junction protein claudin-5 was also significantly increased by the treatment with NKS, but not with Paro, in the hippocampus and PFC of LPS-injected mice. In vitro analysis using brain microvascular endothelial cells (BMVECs) showed that incubation with 5% serum derived from mice orally administered NKS resulted in a significant increase in the expression of anti-inflammatory heme oxygenase 1 as well as autophagic flux markers. Moreover, the claudin-5 levels in BMVECs were also increased under LPS-stimulated conditions. These results suggest that NKS exerts prophylactic effects against the LPS-induced apathy-like behavior, partly mediated by the increase in the anti-inflammatory milieu and in the levels of tight junction proteins in the brain. This study provides scientific evidence supporting the potential efficacy of NKS in preventing post-infection depression.

Proteoglycan 4 (Lubricin) and regulation of xanthine oxidase in synovial macrophage as a mechanism of controlling synovitis

BackgroundSynovial macrophages (SMs) are important effectors of joint health and disease. A novel Cx3CR1 + TREM2 + SM population expressing the tight junction protein claudin-5, was recently discovered in synovial lining. Ablation of these SMs was associated with onset of arthritis. Proteoglycan 4 (PRG4) is a mucinous glycoprotein that fulfills lubricating and homeostatic roles in the joint. The aim of this work is to study the role of PRG4 in modulating synovitis in the context of SM homeostasis and assess the contribution of xanthine oxidase (XO)-hypoxia inducible factor alpha (HIF-1a) axis to this regulation.MethodsWe used Prg4FrtloxP/FrtloxP;R26FlpoER/+, a novel transgenic mouse, where the Prg4Frt allele normally expresses the PRG4 protein and was designed to flank the first two exons of Prg4 with a flippase recognition target and “LOXP” sites. Inducing flippase activity with tamoxifen (TAM) inactivates the Frt allele and thus creates a conditional knockout state. We studied anti-inflammatory SMs and XO by quantitative immunohistochemistry, isolated RNA and studied immune pathway activations by multiplexed assays and isolated SMs and studied PRG4 signaling dysfunction in relation to glycolytic switching due to pro-inflammatory activation. Prg4 inactivated mice were treated with oral febuxostat, a specific XO inhibitor, and quantification of Cx3CR1 + TREM2 + SMs, XO immunostaining and synovitis assessment were conducted.ResultsPrg4 inactivation induced Cx3CR1 + TREM2 + SM loss (p < 0.001) and upregulated glycolysis and innate immune pathways in the synovium. In isolated SMs, Xdh (p < 0.01) and Hif1a (p < 0.05) were upregulated. Pro-inflammatory activation of SMs was evident by enhanced glycolytic flux and XO-generated reactive oxygen species (ROS). Febuxostat reduced glycolytic flux (p < 0.001) and HIF-1a levels (p < 0.0001) in SMs. Febuxostat also reduced systemic inflammation (p < 0.001), synovial hyperplasia (p < 0.001) and preserved Cx3CR1 + TREM2 + SMs (p < 0.0001) in synovia of Prg4 inactivated mice.ConclusionsPRG4 is a biologically significant modulator of synovial homeostasis via inhibition of XO expression and downstream HIF-1a activation. PRG4 signaling is anti-inflammatory and promotes synovial homeostasis in chronic synovitis, where direct XO inhibition is potentially therapeutic in chronic synovitis.

Maresin-like 1 Ameliorates Neuropathology of Alzheimer's Disease in Brains of a Transgenic Mouse Model.

(1) Background: Impeded resolution of inflammation contributes substantially to the pathogenesis of Alzheimer's disease (AD); consequently, resolving inflammation is pivotal to the amelioration of AD pathology. This can potentially be achieved by the treatment with specialized pro-resolving lipid mediators (SPMs), which should resolve neuroinflammation in brains. (2) Methods: Here, we report the histological effects of long-term treatment with an SPM, maresin-like 1 (MarL1), on AD pathogenesis in a transgenic 5xFAD mouse model. (3) Results: MarL1 treatment reduced Aβ overload, curbed the loss of neurons in brains especially cholinergic neurons associated with cleaved-caspase-3-associated apoptotic degeneration, reduced microgliosis and the pro-inflammatory M1 polarization of microglia, curbed the AD-associated decline in anti-inflammatory Iba1+Arg-1+-M2 microglia, inhibited phenotypic switching to pro-inflammatory N1 neutrophils, promoted the blood-brain barrier-associated tight-junction protein claudin-5 and decreased neutrophil leakage in 5xFAD brains, and induced the switch of neutrophils toward the inflammation-resolving N2 phenotype. (4) Conclusions: Long-term administration of MarL1 mitigates AD-related neuropathogenesis in brains by curbing neuroinflammation and neurodegeneration, based on the histological results. These findings provide preclinical leads and mechanistic insights for the development of MarL1 into an effective modality to ameliorate AD pathogenesis.

Lactobacillus acidophilus 6074 Fermented Jujube Juice Ameliorated DSS-induced Colitis via Repairing Intestinal Barrier, Modulating Inflammatory Factors, and Gut Microbiota.

Lactobacillus acidophilus L. acidophilus Lactobacillus, Bifidobacterium, and Akkermansia, This study aimed to explore the ameliorative effects and underlying mechanisms of oral administration Lactobacillus acidophilus 6074 fermented jujube juice (LAFJ) on dextran sulfate sodium (DSS)-induced colitis in mice. In this study, jujube juice was used as a substrate and fermented by L. acidophilus 6074 to investigate its effects on gut microbiota, intestinal barrier function, oxidative stress, inflammatory factors, and short-chain fatty acids (SCFAs) in mice with colitis and to reveal its potential mechanism for alleviating colitis. The results demonstrated that fermentation caused significant changes in the nutrients and nonnutrients of jujube juice, mainly in organic acids (malic acid, lactic acid, citric acid, and succinic acid) and free amino acids (Thr, Met, Ser, Ile, and Lys). High-dose LAFJ (20 mL/kg/day) significantly reduced the disease activity index (DAI), improved histopathological morphology, and increased colon length in colitis mice. LAFJ alleviated colon damage and preserved the integrity of the colonic mucosal barrier by promoting the expression of colonic tight junction proteins occludin, claudin-1, and zonula occluden-1 (ZO-1). Furthermore, LAFJ inhibited the production of proinflammatory factors and attenuated oxidative stress. Gut microbiota of mice revealed that LAFJ increased beneficial bacteria such as Lactobacillus, Bifidobacterium, and Akkermansia, promoted the production of SCFAs, and inhibited the growth of harmful microorganisms. Overall, LAFJ could reshape and restore gut microbiota imbalance caused by intestinal inflammation and alleviate the development of colitis, which may become a novel dietary intervention.

Blood-brain barrier dysfunction in multiple system atrophy: A human postmortem study.

Multiple system atrophy (MSA) is a rare neurodegenerative disease characterized by an accumulation of phosphorylated α-synuclein (p-αsyn) in oligodendrocytes in the form of glial cytoplasmic inclusions (GCIs). In MSA, not only mature oligodendrocytes but also oligodendrocyte precursor cells (OPCs) are affected. The latter play an important role in remyelination by differentiating into mature oligodendrocytes, as well as maintaining the blood-brain barrier (BBB) by promoting the expression of tight junction proteins. We have hypothesized that in MSA, the BBB is impaired as a result of aberrant interactions between affected OPCs and the cerebral vasculature. To verify this hypothesis, we conducted a neuropathological examination of postmortem brains from MSA patients and control subjects, focusing on the primary motor area, one of the main regions affected in MSA. Using double immunofluorescence, we quantified the expression of tight junction protein claudin-5 in capillary endothelial cells and found that it was significantly lower in MSA than in controls in both the gray matter and white matter. Furthermore, a significantly higher amount of fibrinogen was extravasated into the brain parenchyma in MSA patients than in controls. In addition, leakage of IgG was detected almost specifically in MSA brain parenchyma, as visualized in three dimensions by combining techniques of chemical tissue clearing and light sheet microscopy. Finally, we confirmed accumulation of p-αsyn-positive GCIs along the cerebral vasculature within OPCs. These results suggest that BBB dysfunction and associated fibrinogen extravasation are constant findings in MSA, presumably triggered by the deposition of p-αsyn in perivascular OPCs.

Effects of graded yeast cell wall supplementation on growth performance, immunity and intestinal development of broiler chickens raised in floor pens for 42 days.

This study was conducted to evaluate the effects of dietary supplementation of a novel soluble yeast cell wall (YCW) on growth performance, gut health, intestinal morphology, and immune response in broiler chickens for 42 days. A total of 480 one-day-old Cobb 500 male broilers were randomly assigned to four treatments with six replicates and each replicate of twenty broiler chickens: a control group (CON) without feed additive supplementation, and three groups supplemented with YCW at 0.025 % (YCW1), 0.050 % (YCW2), and 0.100 % (YCW3). Results showed that 0.025 % and 0.100 % YCW supplementation significantly increased (P < 0.05) final body weight (BW) and overall body weight gain (BWG) while reducing overall feed conversion ratio (FCR) compared to the CON group. The YCW supplementation also improved (P < 0.05) the balance of gut microbiota by increasing beneficial bacteria (Lactobacillus) and decreasing Salmonella, a potential foodborne pathogen in humans in the ceca. Although intestinal morphology was not significantly affected, YCW supplementation numerically increased the villus height: crypt depth ratio (VH:CD) compared to the CON group. Furthermore, YCW reduced the mRNA expression of pro-inflammatory cytokines (IL-1β and INF-γ) and tight junction protein claudin-1 (CLDN-1) (P < 0.05), suggesting balanced immune response and improved intestinal barrier function. In conclusion, the supplementation of soluble YCW in broiler diets positively influenced growth performance, gut microbiota composition, and immune response, demonstrating its potential as a viable alternative to antibiotics for improving broilers' health.

Oral colon-retentive inulin gels protect against radiation-induced hematopoietic and gastrointestinal injury by improving gut homeostasis.

Ionizing radiation-induced injury often occurs in nuclear accidents or large-dose radiotherapy, leading to acute radiation syndromes characterized by hematopoietic and gastrointestinal injuries even to death. However, current radioprotective drugs are only used in hospitals with unavoidable side effects. Here, we heated the aqueous solution of inulin, a polysaccharide dietary fiber, forming colon-retentive gel as a radiation protector in radiotherapy. Mouse models were established after 60Co γ-ray irradiation of the total body or abdomen. Inulin gels were orally administered to the mice every day from 3 days pre-radiation to 3 days post-radiation. The hematopoietic system was well protected with good blood cell recovery and cell proliferation in the femur and spleen. Oral inulin gels increased the relative abundances of key commensal microorganisms including f_Lachnospiraceae, Akkermansia, Blautia, and short-chain fatty acids (SCFAs) metabolites. The secretion of the anti-inflammation cytokines IL-22 and IL-10 in the intestinal cells also increased. Similarly, the expression of the tight junction proteins claudin-1 and occludin in the gut mucosa was affected. In an orthotopic murine colorectal cancer model, oral inulin gels followed by 10-Gy abdomen radiation improved the radiotherapy efficiency with low attenuated radiation injury. Taken the data together, these results suggest that oral inulin gels are a bioactive material against ionizing radiation-induced injury.

Vascular Development of Fetal and Postnatal Neocortex of the Pig, the European Wild Boar Susscrofa.

The development of the brain's vascular system is a predominantly prenatal process in mammalian species and is required for neurogenesis and further brain development. Our recent work on fetal pig has revealed that many neurodevelopmental processes start well before birth and proceed rapidly reaching near-mature status already around birth. Here, we analyzed the development of neocortical vasculature from embryonic day (E) 45 onward (gestation in pig lasts 114 days) using qualitative and quantitative image analyses and protein blots. In all cortical layers, vessel volume from total brain volume at E100 resembled that of a postnatal day (P) 30 piglet. Endothelial cells expressed the tight junction protein claudin-5 from E45 onward. GFAP+ and AQP4+ astrocytes, PDGFRβ+ pericytes, and α-SMA+ smooth muscle cells are detectable near vessels at E60 suggesting an early assembly of blood-brain barrier components. The vascular system in the visual cortex is advanced before birth with an almost mature pattern at E100. Findings were confirmed by blots that showed a steady increase of expression of tight junction and angiogenesis-related proteins (claudin-5, occludin, VE-cadherin, PECAM-1/CD31) from E65 onward until P90. The expression profile was similar in visual and somatosensory cortex. Together, we report a rapid maturation of the vascular system in pig cortex.

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.

Celecoxib and rofecoxib have different effects on small intestinal ischemia/reperfusion injury in rats.

Intestinal ischemia/reperfusion (I/R) injury is associated with high mortality and there is an unmet need for novel therapies. The intestinal expression of cyclooxygenase-2 (COX-2) increases rapidly after mesenteric I/R, but it is still a question of debate whether selective COX-2 inhibitors can mitigate I/R-induced gut injury. Here we aimed to compare the effect of celecoxib and rofecoxib, two selective COX-2 inhibitors, on intestinal I/R-induced injury in rats. Wistar rats were treated with celecoxib (10 and 100mg/kg), rofecoxib (5 and 50mg/kg), or vehicle for 8 days via gavage and then were subjected to sham operation or mesenteric I/R. Small intestinal inflammation and tissue damage were assessed by histology and quantification of inflammatory and tight junction proteins. The intestinal activity of COX enzymes was determined by a COX activity assay. The higher dose of celecoxib reduced the I/R-associated increase in inflammatory mediators (myeloperoxidase, pentraxin 3, COX-2, interleukin-1β) and loss of tight junction proteins (claudin-1, occludin), whereas the lower dose of celecoxib was only marginally effective. However, even high-dose celecoxib failed to prevent the histological injury of the mucosa. In contrast to celecoxib, rofecoxib did not affect intestinal inflammation and injury at any of the tested doses. Neither celecoxib nor rofecoxib affected the I/R-induced changes of HO-1 and PPAR-γ, known off-targets of COX-inhibitors, but celecoxib increased the I/R-induced elevation of Bax/Bcl-2, a marker of apoptosis, whereas rofecoxib reduced the elevation of phospho-Akt. Importantly, high-dose celecoxib, but not rofecoxib, has already reduced intestinal COX-1 activity. Our study provides evidence for the higher anti-inflammatory efficacy of celecoxib compared to rofecoxib in mesenteric I/R injury, which is likely due to its lower selectivity for COX-2. However, even high-dose celecoxib was unable to reduce the mucosal damage. Our results suggest that selective COX-2 inhibitors have only limited therapeutic value in intestinal I/R injury.

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 [knockout (KO, KOS1-KOS3)] claudin-2. 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.NEW & NOTEWORTHY Research on isolated perfused S2 segments of proximal tubules in mice, both expressing and lacking claudin-2, indicates that claudin-2 forms leaky monovalent cation-selective paracellular channels within the tight junctions of proximal tubules. This study characterized the paracellular transport properties in isolated and perfused superficial proximal tubule S1-S3 segments in both groups of mice. The findings demonstrate, for the first time, functional heterogeneity in the paracellular pathway along the axis of the superficial proximal tubules.

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.

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.