Tight Junction Proteins Research Articles

Tight Junctions and Cancer: Targeting Claudin-1 and Claudin-4 in Thyroid Pathologies.

Purpose: Claudins are tight junction proteins partaking in epithelial-mesenchymal transition and cancer progression. In this study, we investigated the expression patterns of claudin-1 and claudin-4 in thyroid pathologies, discussed their links with the pathogenesis of thyroid cancers, and reviewed the therapeutic potential of targeting claudins in cancers. Methods: The research group 162 cores of thyroid samples from patients (70 female and 11 male) diagnosed with thyroid adenoma, goiter, papillary, medullary, and anaplastic thyroid cancers. All samples were stained for the expression of claudin-1 and claudin-4, and the analysis of IHC was performed. Results: Goiter samples showed negative claudin-1 and mostly positive expression of claudin-4. Papillary thyroid cancer and thyroid adenoma showed positive expression of claudin-1, while claudin-4 was positive in papillary thyroid cancers, goiters, and adenomas. In The Cancer Genome Atlas cohort, claudin-1 and claudin-4 were overexpressed in papillary thyroid cancer compared to normal thyroid tissues. Patients with high claudin-1 expression had significantly lower 5-year overall survival than patients with low claudin-1 levels (86.75% vs. 98.65, respectively). In multivariate analysis, high claudin-1 expression (HR 7.91, CI 95% 1.79-35, p = 0.006) and advanced clinical stage remained statistically significant prognostic factors of poor prognosis in papillary thyroid cancer. Conclusions: The pattern of claudin-1 staining was pathology-specific and changed between cancers of different histology. This phenomenon may be associated with the different pathogenesis of thyroid cancers and early metastasis. The loss of claudin-1 and claudin-4 characterized more aggressive cancers. Several studies have shown the benefits of targeting claudins in cancers, but their implementation into clinical practice requires further trials.

Dietary Clostridium butyricum metabolites mitigated the disturbances in growth, immune response and gut health status of Ctenopharyngodon idella subjected to high cottonseed and rapeseed meal diet

Cottonseed meal and rapeseed meal exhibit a potential for fishmeal substitute in grass carp feed, while their excessive use contribute to growth decline and weakening immunity of aquatic animals. Clostridium butyricum metabolites (CBM) was recognized as a functional additive due to its antioxidant properties and maintenance of intestinal microbiota balance. CBM was added to a high of cottonseed and rapeseed meal diet to determine its effects on growth, immunity, and intestinal microbiota alterations of grass carp (Ctenopharyngodon idella) over 56 days. Eight hundred grass carp (mean weight, around 50 g) were randomized to five treatments and fed with the basic diet (CON), CBM0 diet (28 % cottonseed and 27 % rapeseed meal), and CBM diets (CBM0.5, CBM1, and CBM2, namely CBM0 diet supplemented with 500, 1000, and 2000 mg kg−1 CBM). The results indicated that compared to CBM0, The ingestion of 1000 mg kg−1 CBM diet by grass carp significantly promoted growth as measured by intestinal lipase activity, villus height, and muscle thickness. Moreover, accompanied by a decrease in intestine MDA content, and enhance antioxidant capacity by activating Keap1/Nrf2 signaling pathway to increase enzyme activities (SOD, CAT and T-AOC) and corresponding gene expression (mnsod, cat, gsto and gpx1) in the intestine of grass crap fed CBM1 diet. The dietary CBM1 diet increased serum levels of C3 and IgM, increased ACP activity and expression of the corresponding anti-inflammatory factors (tgf-β1 and il-15), and suppressed the expression of pro-inflammatory factors (tnf-α and il-12β), resulting in enhanced immunity. The dietary CBM1 diet up-regulates gene expression of tight junction proteins (zo-1, occludin, occludin7a and occludin-c), coupled with the decreases in DAO and D-lactate contents, implying that the decreased mucosal permeability could be observed in the gut. The dietary CBM1 diet largely altered the intestinal microbial community, especially reducing the relative abundance of intestinal pathogenic bacteria (Streptococcus and Actinomyces). And it significantly increased the content of short-chain fatty acids (acetic acid, butyric acid, isobutyric acid, propionic acid and isovaleric acid). Taken above, dietary CBM supplementation improved growth in grass carp and attenuated the intestinal oxidative stress, inflammation and microflora dysbacteriosis caused by high proportions of cottonseed and rapeseed meal diets.

Network Analysis of Gut Microbial Communities Reveals Key Reason for Quercetin Protects against Colitis

As one of the most representative natural products among flavonoids, quercetin (QUE) has been reported to exhibit beneficial effects on gut health in recent years. In this study, we utilized a dextran sulfate sodium (DSS)-induced colitis mice model to explore the protective effects and underlying mechanisms of QUE on colitis. Our data demonstrated that QUE oral gavage administration significantly ameliorates the symptoms and histopathological changes associated with colitis. Additionally, the concentration of mucin-2, the number of goblet cells, and the expression of tight junction proteins (such as ZO-1, Occludin, and Claudin-1) were all found to be increased. Furthermore, QUE treatment regulated the levels of inflammatory cytokines and macrophage polarization, as well as the oxidative stress-related pathway (Nrf2/HO-1) and associated enzymes. Additionally, 16S rDNA sequencing revealed that QUE treatment rebalances the alterations in colon microbiota composition (inlcuding Bacteroidaceae, Bacteroides, and Odoribacter) in DSS-induced colitis mice. The analysis of network dynamics reveals a significant correlation between gut microbial communities and microenvironmental factors associated with inflammation and oxidative stress, in conjunction with the previously mentioned findings. Collectively, our results suggest that QUE has the potential to treat colitis by maintaining the mucosal barrier, modulating inflammation, and reducing oxidation stress, which may depend on the reversal of gut microbiota dysbiosis.

Effect of Probiotics on Improving Intestinal Mucosal Permeability and Inflammation after Surgery.

We explored the mechanisms underlying the improvement of postoperative ileus (POI) following probiotic pretreatment. We assessed intestinal permeability, inflammation, tight junction (TJ) protein expression in the gut epithelium, and plasma interleukin (IL)-17 levels in a guinea pig model of POI. Guinea pigs were divided into control, POI, and probiotic groups. The POI and probiotic groups underwent surgery, but the probiotic group received probiotics before the procedure. The ileum and proximal colon were harvested. Intestinal permeability was measured via horseradish peroxidase permeability. Inflammation was evaluated via leukocyte count in the intestinal wall muscle layer, and calprotectin expression in each intestinal wall layer was analyzed immunohistochemically. TJ proteins were analyzed using immunohistochemical staining, and plasma IL-17 levels were measured using an enzyme-linked immunosorbent assay. The POI group exhibited increased intestinal permeability and inflammation, whereas probiotic pretreatment reduced the extent of these POI-induced changes. Probiotics restored the expression of TJ proteins occludin and zonula occludens-1 in the proximal colon, which were increased in the POI group. Calprotectin expression significantly increased in the muscle layer of the POI group and was downregulated in the probiotic group; however, no distinct differences were observed between the mucosal and submucosal layers. Plasma IL-17 levels did not significantly differ among the groups. Probiotic pretreatment may relieve POI by reducing intestinal permeability and inflammation and TJ protein expression in the gut epithelium. These findings suggest a potential therapeutic approach for POI management.

Aging alters the expression of trophic factors and tight junction proteins in the mouse choroid plexus

BackgroundThe choroid plexus (CP) is an understudied tissue in the central nervous system and is primarily implicated in cerebrospinal fluid (CSF) production. CP also produces numerous neurotrophic factors (NTF) which circulate to different brain regions. Regulation of NTFs in the CP during natural aging is largely unknown. Here, we investigated the age and gender-specific transcription of NTFs along with the changes in the tight junctional proteins (TJPs) and the water channel protein Aquaporin (AQP1).MethodsMale and female mice were used for our study. Age-related transcriptional changes were analyzed using quantitative PCR at three different time points: mature adult, middle-aged, and aged. Transcriptional changes during aging were further confirmed with digital droplet PCR. Additionally, we used immunohistochemical analysis (IHC) for the evaluation of in vivo protein expression. We further investigated the cellular phenotype of these NTFS, TJP, and water channel proteins in the mouse CP by co-labeling them with the classical vascular marker, Isolectin B4, and epithelial cell marker, Plectin.ResultsAging significantly altered NTF gene expression in the CP. Brain-derived neurotrophic factor (BDNF), Midkine (MDK), VGF, Insulin-like growth factor (IGF1), IGF2, Klotho (KL), Erythropoietin (EPO), and its receptor (EPOR) were reduced in the aged CP of males and females. Vascular endothelial growth factor (VEGF) transcription was gender-specific; in males, gene expression was unchanged in the aged CP, while females showed an age-dependent reduction. Age-dependent changes in VEGF localization were evident, from vasculature to epithelial cells. IGF2 and klotho localized in the basolateral membrane of the CP and showed an age-dependent reduction in epithelial cells. Water channel protein AQP1 localized in the tip of epithelial cells and showed an age-related reduction in mRNA and protein levels. TJP’s JAM, CLAUDIN1, CLAUDIN2 and CLAUDIN5 were reduced in aged mice.ConclusionsOur study highlights transcriptional level changes in the CP during aging. The age-related transcriptional changes exhibit similarities as well as gene-specific differences in the CP of males and females. Altered transcription of the water channel protein AQP1 and TJPs could be involved in reduced CSF production during aging. Importantly, reduction in the neurotrophic factors and longevity factor Klotho can play a role in regulating brain aging.Graphical

Mechanosensitive recruitment of Vinculin maintains junction integrity and barrier function at epithelial tricellular junctions

Apical cell-cell junctions, including adherens junctions and tight junctions, adhere epithelial cells to one another and regulate selective permeability at both bicellular junctions and tricellular junctions (TCJs). Although several specialized proteins are known to localize at TCJs, it remains unclear how actomyosin-mediated tension transmission at TCJs contributes to the maintenance of junction integrity and barrier function at these sites. Here, utilizing the embryonic epithelium of gastrula-stage Xenopus laevis embryos, we define a mechanism by which the mechanosensitive protein Vinculin helps anchor the actomyosin network at TCJs, thus maintaining TCJ integrity and barrier function. Using an optogenetic approach to acutely increase junctional tension, we find that Vinculin is mechanosensitively recruited to apical junctions immediately surrounding TCJs. In Vinculin knockdown (KD) embryos, junctional actomyosin intensity is decreased and becomes disorganized at TCJs. Using fluorescence recovery after photobleaching (FRAP), we show that Vinculin KD reduces actin stability at TCJs and destabilizes Angulin-1, a key tricellular tight junction protein involved in regulating barrier function at TCJs. When Vinculin KD embryos are subjected to increased tension, TCJ integrity is not maintained, filamentous actin (F-actin) morphology at TCJs is disrupted, and breaks in the signal of the tight junction protein ZO-1 signal are detected. Finally, using a live imaging barrier assay, we detect increased barrier leaks at TCJs in Vinculin KD embryos. Together, our findings show that Vinculin-mediated actomyosin organization is required to maintain junction integrity and barrier function at TCJs and reveal new information about the interplay between adhesion and barrier function at TCJs.

NX210c drug candidate peptide strengthens mouse and human blood-brain barriers

BackgroundAlterations of blood-brain barrier (BBB) and blood-spinal cord barrier have been documented in various animal models of neurodegenerative diseases and in patients. Correlations of these alterations with functional deficits suggest that repairing barriers integrity may represent a disease-modifying approach to prevent neuroinflammation and neurodegeneration induced by the extravasation of blood components into the parenchyma. Here, we screened the effect of a subcommissural organ-spondin-derived peptide (NX210c), known to promote functional recovery in several models of neurological disorders, on BBB integrity in vitro and in vivo.MethodsIn vitro, bEnd.3 endothelial cell (EC) monolayers and two different primary human BBB models containing EC, astrocytes and pericytes, in static and microfluidic conditions, were treated with NX210c (1-100 µM), or its vehicle, for 4 h and up to 5 days. Tight junction (TJ) protein levels, permeability to dextrans and transendothelial electrical resistance (TEER) were evaluated. In vivo, young and old mice (3- and 21-month-old, respectively) were treated daily intraperitoneally with NX210c at 10 mg/kg or its vehicle for 5 days and their brains collected at day 6 to measure TJ protein levels by immunohistochemistry.ResultsNX210c induced an increase in claudin-5 protein expression after 24-h and 72-h treatments in mouse EC. Occludin level was also increased after a 24-h treatment. Accordingly, NX210c decreased by half the permeability of EC to a 40-kDa FITC-dextran and increased TEER. In the human static BBB model, NX210c increased by ∼ 25% the TEER from 3 to 5 days. NX210c also increased TEER in the human 3D dynamic BBB model after 4 h, which was associated with a reduced permeability to a 4-kDa FITC-dextran. In line with in vitro results, after only 5 days of daily treatments in mice, NX210c restored aging-induced reduction of claudin-5 and occludin levels in the hippocampus, and also in the cortex for occludin.ConclusionsIn summary, we have gathered preclinical data showing the capacity of NX210c to strengthen BBB integrity. Through this property, NX210c holds great promises of being a disease-modifying treatment for several neurological disorders with high unmet medical needs.

Naringenin alleviates intestinal ischemia/reperfusion injury by inhibiting ferroptosis via targeting YAP/STAT3 signaling axis

BackgroundIntestinal ischemia/reperfusion injury (IRI) is a significant clinical emergency, and investigating novel therapeutic approaches and understanding their underlying mechanisms is essential for improving patient outcomes. Naringenin (Nar), a flavanone present in tomatoes and citrus fruits, is frequently consumed in the human diet and recognized for having immunomodulatory, anti-inflammatory, and antioxidant properties. Despite Nar being able to alleviate intestinal IRI, the exact molecular mechanisms remain elusive. PurposeTo investigate Nar's protective properties on intestinal IRI and elucidate the mechanisms, a comprehensive approach that combines network pharmacology analysis with experimental verification in vitro and in vivo was adopted. MethodsThe oxygen-glucose deprivation/reoxygenation (OGD/R) model in IEC-6 cells and a murine model of intestinal IRI were used. Nar's effects on intestinal IRI were assessed through histological analysis using H&E staining and tight junction (TJ) protein expression. Ferroptosis-related parameters, including iron levels, superoxide dismutase (SOD), glutathione (GSH), reactive oxygen species (ROS), malondialdehyde (MDA), and mitochondrial morphology, were analyzed. Network pharmacology was utilized to predict the pathways through which Nar exerts its anti-ferroptosis effects. Further mechanistic insights were obtained through si-RNA transfection, YAP inhibitor (verteporfin, VP) treatment, ferroptosis inhibitor (Ferrostatin-1) and ferroptosis inducer (Erastin) application, co-immunoprecipitation (Co-IP) and Western blotting. ResultsOur results revealed that pretreatment with Nar significantly mitigated intestinal tissue damage and improved gut barrier function, as evidenced by increased TJ proteins (ZO-1 and Occludin). Nar reduced iron, MDA, and ROS, while it increased GSH and SOD levels. Additionally, Nar alleviated mitochondrial damage in mice. Nar treatment increased GPX4 and SLC7A11, while decreasing ACSL4 levels both in vivo and in vitro. Network pharmacology analysis suggested that Nar may target the Hippo signaling pathway. Notably, YAP, a key transcriptional co-activator within the Hippo pathway, was downregulated in intestinal IRI mice and OGD/R-induced IEC-6 cells. Nar pretreatment activated YAP, thereby augmenting anti-ferroptosis effects. The inhibition of YAP activation by VP or YAP knockdown increased p-STAT3 expression, thereby diminishing Nar's efficacy. Co-IP and immunofluorescence studies confirmed the interaction between YAP and STAT3. ConclusionThis study shows that Nar can inhibit ferroptosis in intestinal IRI via activating YAP, which in turn suppresses STAT3 phosphorylation, thereby unveiling a novel mechanism and supporting Nar's potential to be a promising therapeutic agent for intestinal IRI.

Prenatal exposure to low doses of benzophenone-3 elicits disruption of cortical vasculature in fetuses through perturbations in Wnt/β-catenin signaling correlating with depression-like behavior in offspring mice

Benzophenone-3 (BP-3), commonly used in personal care products, is routinely detected in environmental and human matrices. Evidence delineates a correlation between gestational BP-3 exposure and emotional and social disorders in children and adolescents. However, sensitive target cells and the mode of action underlying the early responses to environmentally relevant level of BP-3 exposure remain unclear. In this study, 0.3 and 3 mg/kg of BP-3 were administered to pregnant mice. Compared with the control group, the cortical blood vessel development process manifested the highest susceptibility to BP-3 exposure using transcriptomic sequencing at embryonic day 14 (E14). Notably, the diminution in vascular density and tight junction proteins presence was observed in the fetal cortex at E14, concomitant with the suppressed transcriptional activity of genes essential to angiogenesis and barrier formation. Strikingly, the investigation revealed that BP-3 exposure impeded vascular sprouting in aortic ring explants and neuroendothelial migration, implicating the Wnt/β-catenin signaling pathway. Moreover, BP-3 exposure compromised perivascular neural stem cell differentiation. Cortical vascular injury correlated with the exhibition of depression-like behavior in four-week postnatal progeny. These insights underscore the cerebrovasculature as an early sensitive target for low doses of BP-3 exposure, fostering the development of biomarkers and the establishment of the adverse outcome pathway framework for BP-3 hazard evaluation.

Investigating the modulatory effects of lactoferrin on depressed rats through 16S rDNA gene sequencing and LC-MS metabolomics analysis.

Lactoferrin is a natural multifunctional glycoprotein with potential antidepressant-like effects. However, the mechanism of its antidepressant effect has not been explored from the perspective of gut flora metabolism. Therefore, we employed both 16S rDNA gene sequencing and LC-MS metabolomics analysis to investigate the regulatory effects and mechanisms of lactoferrin in a rat model of depression. After one week of acclimatization, twenty-four 7-week-old male Sprague-Dawley rats were randomly and equally assigned into three groups: the control group, the model group, and the lactoferrin intervention group. The control group rats were housed under standard conditions, while the rats in the model and lactoferrin intervention groups were individually housed and exposed to chronic unpredictable mild stress for 44days simultaneously. The lactoferrin intervention group was provided with water containing 2% lactoferrin (2g/100ml). Behavioural tests were conducted at week 7. Upon completion of the behavioral tests, the rats were anesthetized with isoflurane, humanely euthanized using a rat guillotine, and tissue samples were collected for further experiments. The results indicated that lactoferrin intervention led to an increase in sucrose solution consumption, horizontal movement distance, number of cross platforms, and residence time in the target quadrant. Additionally, it resulted in an increase in jejunal tight junction protein ZO-1 expression and a suppression of serum expression of inflammatory factors, Lipopolysaccharide and Diamine oxidase. In summary, lactoferrin can regulate the metabolic disorder of intestinal flora, reduce intestinal permeability, and further regulate the metabolic balance of hippocampal tissues through the microbiota-gut-brain axis. This process ultimately alleviates the depression-like behavior in rats.

Selenium nanoparticles affect chicken offspring's intestinal health better than other selenium sources

This study aimed to assess the effects of maternal diets containing various selenium (Se) sources on the intestinal mucosal function in the jejunum of chicken offspring. A total of 630, 18-wk-old Hy-Line Grey hens and 70 18-wk-old Hy-Line Grey breeders were randomly allocated into 7 groups, with 5 replicates in each group (18 hens and tow roosters). After 4 wk of Se depletion, the birds were fed either a nonsupplemented basal diet (control) or the same basal diet supplemented with 0.15 mg/kg selenium nanoparticles (Nano-Se), 0.30 mg/kg Nano-Se, 0.30 mg/kg selenocysteine (Sec), 0.30 mg/kg sodium selenite (SS), 0.30 mg/kg selenomethionine (SeMet), or 0.15 mg/kg Nano-Se + 0.15 mg/kg Sec, for 8 wk. Frtilized eggs were collected and incubated during the final week of the experiment. Jejunal tissues from embryonic d 18 and the hatch day were collected for analysis, and the 7-d survival rate of the offspring was recorded. Compared to the control, the maternal diet of 0.30 mg/kg Nano-Se, 0.30 mg/kg Sec, and 0.30 mg/kg SeMet significantly increased the survival of 7-day-old offspring (P < 0.05). The maternal diet supplemented with 0.30 mg/kg Nano-Se significantly increased intestinal villus height and the villus height/crypt depth ratio in chicks at embryonic d 18 and in 1-day-old (P < 0.05). The maternal diet containing 0.30 mg/kg Nano-Se and Sec increased the mRNA expression levels of tight junction proteins in 1-day-old offspring (P < 0.05). Supplemental 0.30 mg/kg Nano-Se significantly increased the mRNA expression levels of marker genes in intestinal enteroendocrine, stem, and Paneth cells (P < 0.05). In 1-day-old chicks, the number of intestinal goblet cells, as well as the mRNA expression levels of intestinal mucin2 (Muc2) and goblet cell differentiation factors (Spdef and C-myc), were the highest in diets supplemented with 0.30 mg/kg Nano-Se. Moreover, the expression levels of intestinal Muc2 and Spdef in chicks at embryonic d 18 was the highest with 0.30 mg/kg Nano-Se supplementation (P < 0.05). Supplementing with 0.30 mg/kg Nano-Se significantly reduced reactive oxygen species levels and decreased the mRNA expression levels of apoptosis-related genes in 1-day-old chicks (P < 0.05). Additionally, 0.30 mg/kg Nano-Se supplementation significantly down-regulated NLRP3 pathway gene expression in 1-day-old chicks (P < 0.05). In conclusion, maternal dietary supplementation with Nano-Se improved jejunal microarchitecture, antioxidant levels, and the expression of tight-junction protein in chicken offspring along with supporting goblet cell development by inhibiting the NLRP3 signaling pathway.

Medium- and Long-Term Immune Responses in the Small Intestine in Piglets from Oral Vaccination against Escherichia coli.

Post-weaning stress, together with Escherichia coli, are two of the key factors in the occurrence of post-weaning diarrhea. There are different commercial vaccines that induce immunity at the local or systemic level, improving farm health and avoiding economic losses in the pork industry. That is why the objective of this study was to evaluate the effect of an oral enterotoxigenic E. coli F4/F18 vaccine on immunity and intestinal integrity in the middle and long term after inoculation. The gene expression of the biomarkers indicative of cellular infiltration (calprotectin, CAL), tight junction proteins (occludin, OCL; zonulin, ZON; and claudin, CLA) and a panel of proinflammatory (interleukins, IL: IL1α, IL1β, IL6, IL8, IL12p35 and IL12p40; interferons, IFN: IFNα and IFNγ; and tumoral necrosis factor, TNF: TNFα) and anti-inflammatory mediator cytokines (TGFβ and IL10) were analyzed, as well as histomorphology in jejunum and ileum, the cell density of goblet cells, intraepithelial lymphocytes and IgA-producing cells. Differences were observed in ZON, CLA and CAL, with greater gene expression in observed in vaccinated piglets at 42 days post vaccination (dpv) in the ileum. Regarding the expression of cytokines, the vaccinated animals showed significant differences in IL1α, IL6, IL12p35, IL12p40, IFNα, IFNγ, TNFα and TGFβ at 42 dpv in the jejunum or ileum. The villi showed greater height in the vaccinated piglets and the ratio between villus height and crypt depth was significantly greater in the vaccinated group in the jejunum at 84 dpv. The count of IgA-producing cells shows higher values for the unvaccinated group in the ileum, while intraepithelial lymphocytes show a significant increase in both jejunum and ileum in vaccinated piglets. We can conclude that oral vaccination against E. coli produces an evident effect, which manifests itself even in the middle and long term after the challenge, including immune response, decrease in antimicrobials usage, better histological structure in intestine and the improvement of performance.

High-fat diet led to testicular inflammation and ferroptosis via dysbiosis of gut microbes

The disorder of gut microbiota has negative impact on male reproductive, and testicular damage is associated with obesity. However, the detailed mechanism of gut microbiota on the obesity-induced testis injury are still unknown. Therefore, we constructed a mouse model to investigate the effects of obesity on testis injury. In this study, we found that HFD-induced obesity could disorder gut microbiota homeostasis, which increased the abundance of Brevundimonas, Desulfovibrionaceae_unclassified and Ralstonia, ultimately leading to the overproduction of lipopolysaccharides (LPS). Meanwhile, HFD-feeding promoted intestinal permeability via inhibiting expression of tight junction proteins (ZO-1, Occludin and Claudin) and reducing excretion of mucus, leading to translocation of LPS. The over-accumulation of LPS in the bloodstream triggered an inflammatory response by activating TLR4/NF-κB pathway in testis. On the other hand, the gut microbiota produced-LPS also could induce ferroptosis in testis, as reflected by enhancing iron content and lipid peroxidation (MDA), as well as decreasing ferroptosis-related proteins, including GPX4, FTH1 and SLC1A11. Moreover, inhibition of LPS ligand (TLR4) with Resatorvid (TAK-242) alleviated obesity-induced testis injury through suppression of inflammation and ferroptosis. In conclusion, this study provides novel insights into the underlying mechanisms of obesity–related testis injury induced by gut microbiota disorder via the gut–testis axis, thus offering potential targets to counteract obesity-induced male reproductive disorder.

Esculetin Combats Multidrug-Resistant Salmonella Infection and Ameliorates Intestinal Dysfunction via the Nrf2 Pathway.

The increasing incidence of multidrug-resistant (MDR) Salmonella enterica serovar Typhimurium (S. Tm), known for causing invasive enteric infections, presents a significant public health challenge. Given the diminishing efficacy of existing antibiotics, it is imperative to explore novel alternatives for the treatment of MDR S. Tm infections. Here, we identified esculetin (EST), a natural coumarin abundant in dietary foods and herbs, as a compound exhibiting broad-spectrum antibacterial properties against a range of MDR bacteria. Our findings demonstrate that EST effectively inhibited the proliferation and expansion of MDR S. Tm in both in vitro experiments and animal models. Specifically, EST significantly downregulated the type 3 secretion system-1 (T3SS-1) virulence expression of MDR S. Tm, thereby preventing its invasion into intestinal epithelial cells. In S. Tm-infected mice, we observed cecal injury characterized by the upregulation of inflammatory cytokines, a reduction in goblet cell numbers, a decreased expression of tight junction proteins, and microbial dysbiosis. Conversely, EST treatment ameliorated these pathological changes induced by S. Tm infection and reduced oxidative stress by activating the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway, thereby improving intestinal barrier function. These results suggest that dietary coumarins or a targeted plant-based diet may offer a promising strategy to counteract MDR bacteria-induced enteric diseases.

Recombinant filaggrin-2 improves skin barrier function and attenuates ultraviolet B (UVB) irradiation-induced epidermal barrier disruption

The integrity of the skin barrier is essential for maintaining skin health, with the stratum corneum and filaggrin 2 (FLG-2) playing a key role. FLG-2 deficiency or mutation has been linked to diseases such as atopic dermatitis, while external stressors such as ultraviolet B (UVB) radiation further damage the epidermal barrier. This study investigated the effects of recombinant filaggrin (rFLG) on skin barrier function and UVB induced epidermal destruction. Cell experiments showed that 10 μg/mL of rFLG could increase the mobility of HaCaT cells from 20 % to 42 %, increase the epithelial resistance (TEER) value by about 2 times, and up-regulate the tight junction associated protein by about 2 times. In mouse models of UVB-induced epidermal barrier destruction, rFLG at concentrations of 0.5, 1, and 2 mg/mL showed effective cell uptake and skin penetration, alleviating erythema, and reducing skin thickness in mice by 1.5–3 times. Among them, 2 mg/mL of rFLG treatment restored the expression of tight junction proteins (LOR, ZO-1, and caspase-14), reduced collagen degradation, and reduced oxidative stress by normalizing serum hydroxyproline and superoxide dismutase levels. In addition, 2 mg/mL of rFLG inhibited UVB-induced upregulation of matrix metalloproteinases (MMP-3 and MMP-9) and reduced pro-inflammatory factors (IL-10, IL-1α, IL-6, and TNF-α) and apoptotic markers (P38, Bax, and Bcl-2) to normal levels. These findings suggested that rFLG effectively enhanced skin barrier integrity and mitigated UVB-induced epidermal barrier destruction, highlighting its potential as a therapeutic agent for diseases associated with skin barrier dysfunction.

Melatonin Improves Vasogenic Edema via Inhibition to Water Channel Aquaporin-4 (AQP4) and Metalloproteinase-9 (MMP-9) Following Permanent Focal Cerebral Ischemia.

Background: The efficacy of melatonin in reducing vasogenic and cytotoxic edema was investigated using a model of permanent middle cerebral artery occlusion (pMCAO). Methods: Rats underwent pMCAO, followed by intravenous administration of either melatonin (5 mg/kg) or a vehicle 10 min post-insult. Brain infarction and edema were assessed, and Western blot analyses were conducted to examine the expression levels of aquaporin-4 (AQP4), metalloproteinase-9 (MMP-9), and the neurovascular tight-junction protein ZO-1 upon sacrifice. The permeability of the blood-brain barrier (BBB) was measured using spectrophotometric quantification of Evans blue dye leakage. Results: Compared to controls, melatonin-treated rats exhibited a significant reduction in infarct volume by 26.9% and showed improved neurobehavioral outcomes (p < 0.05 for both). Melatonin treatment also led to decreased Evans blue dye extravasation and brain edema (p < 0.05 for both), along with lower expression levels of AQP4 and MMP-9 proteins and better preservation of ZO-1 protein (p < 0.05 for all). Conclusions: Therefore, melatonin offers neuroprotection against brain swelling induced by ischemia, possibly through its modulation of AQP4 and MMP-9 activities in glial cells and the extracellular matrix (ECM) during the early phase of ischemic injury.

Intestinal Epithelial PTPN2 Limits Pathobiont Colonization by Immune-Directed Antimicrobial Responses.

Loss of activity of the inflammatory bowel disease (IBD) susceptibility gene, protein tyrosine phosphatase non-receptor type 2 (PTPN2), is associated with altered microbiome composition in both human subjects and mice. Further, expansion of the bacterial pathobiont, adherent-invasive E. coli (AIEC), is strongly linked to IBD pathogenesis. The mechanism by which intestinal epithelial cells (IEC) maintain equilibrium between commensal microbiota and immune cells to restrict invading pathobionts is poorly understood. Here, we investigated the role of IEC-specific PTPN2 in regulating AIEC colonization. Tamoxifen-inducible, intestinal epithelial cell-specific Ptpn2 knockout mice (Ptpn2 ΔIEC) and control Ptpn2 fl/fl mice were infected with either non-invasive E. coli K12, or fluorescent-tagged mAIEC (mAIECred) for four consecutive days or administered PBS. Subsequently, bacterial colonization in mouse tissues was quantified. mRNA and protein expression were assayed in intestinal epithelial cells (IECs) or whole tissue lysates by PCR and Western blot. Tissue cytokine expression was determined by ELISA. Intestinal barrier function was determined by in vivo administration of 4 kDa FITC-dextran (FD4) or 70kDa Rhodamine-B dextran (RD70) fluorescent probes. Confocal microscopy was used to determine the localization of tight-junction proteins. Ptpn2 ΔIEC mice exhibited increased mAIECred - but not K12 - bacterial load in the distal colon compared to infected Ptpn2 fl/fl mice. The higher susceptibility to mAIECred infection was associated with altered levels of antimicrobial peptide (AMPs). Ileal RNA expression of the alpha-defensin AMPs, Defa5 and Defa6, as well as MMP7, was significantly lower in Ptpn2 ΔIEC vs. Ptpn2 fl/fl mice, after mAIECred but not K12 infection. Further, we observed increased tight junction-regulated permeability determined by elevated in vivo FD4 but not RD70 permeability in Ptpn2 ΔIEC-K12 mice compared to their respective controls. This effect was further exacerbated in Ptpn2 ΔIEC mAIEC-infected mice. Further, Ptpn2 ΔIEC mice displayed lower IL-22, IL-6, IL-17A cytokine expression post mAIEC infection compared to Ptpn2 fl/fl controls. Recombinant IL-22 reversed the FD4 permeability defect and reduced bacterial burden in Ptpn2 ΔIEC mice post mAIEC challenge. Our findings highlight that intestinal epithelial PTPN2 is crucial for mucosal immunity and gut homeostasis by promoting anti-bacterial defense mechanisms involving coordinated epithelial-immune responses to restrict pathobiont colonization.

REV-ERBα agonist SR10067 attenuates Th2 cytokine-mediated barrier dysfunction in human bronchial epithelial cells.

Allergens and Th2 cytokines affect the homeostatic environment in the airways, leading to increased mucus production by goblet cells associated with altered adherens junctional complex (AJC) and tight junction (TJ) proteins responsible for maintaining epithelial barrier function. Circadian clock-dependent regulatory mechanisms such as inflammation and epithelial barrier function are gaining more attention due to their therapeutic potential against allergic inflammatory lung diseases. Currently, there are no studies to support whether REV-ERBα activation can attenuate Th2 cytokine-induced epithelial barrier dysfunction in human bronchial epithelial cells. We hypothesized that Th2 cytokine-induced epithelial barrier dysfunction may be protected by activating REV-ERBα. Treatment with Th2 cytokines or HDM significantly reduced the cell impedance, as confirmed by transepithelial electrical resistance (TEER). However, pre-treatment with SR10067 attenuated Th2 cytokine-induced barrier dysfunction, such as decreased permeability, improved TEER, localization of AJC and TJ proteins, and mRNA and protein levels of selected epithelial barrier and circadian clock targets. Overall, we showed for the first time that REV-ERBα activation regulates altered epithelial barrier function that may have direct implications for the treatment of asthma and other allergic diseases.

Junctional adhesion molecular 3 (JAM3) is a novel tumor suppressor and improves the prognosis in breast cancer brain metastases via the TGF-β/Smad signal pathway.

Breast cancer brain metastasis (BCBM) is a deadly clinical problem, and the exact underlying mechanisms remain elusive. Junctional adhesion molecule (JAM), a tight junction protein, is a key negative regulator of cancer cell invasion and metastasis. Junction adhesion molecule 3 (JAM3) expression in breast cancer was analyzed using bioinformatics methods and confirmed by PCR, western blotting, and immunofluorescence (IF) in cell lines. The effects of exogenous expression of JAM3 using lentiviral vectors on invasion, adhesion, and apoptosis were verified using transwell assays and flow cytometry. Differentially expressed genes (DEGs) were detected by RNA sequencing and verified by q‒PCR and Western blotting. The effect of JAM3 silencing using siRNA was assessed by an adhesion assay. Kaplan‒Meier analysis was applied to calculate the impact of JAM3 expression and classic clinicopathologic characteristics on survival. Bioinformatics analysis revealed that JAM3 expression was reduced in BCBM. Exogenous expression of JAM3 minimizes the ability of breast cancer cells to invade and adhere and promotes their apoptosis. Silencing JAM3 results in morphology changes and the recovery of invasion and adhesion to ECMs, and the TGF-β/Smad signaling pathway may be involved. JAM3 predicts less metastasis and good survival in patients with BCBM. Statistical analysis of BCBM samples detected by immunohistochemistry (IHC) and the associated clinicopathological characteristics revealed that low levels of JAM3 expression and high levels of TNF-β1 are linked to the clinical progression of both primary and metastatic breast tumors. Kaplan-Meier analysis revealed that a high expression level of JAM3 was associated with longer survival. JAM3 can serve as a key negative regulator of breast cancer cell invasion, apoptosis, and brain metastasis, possibly through the TGF/Smad signaling pathway. JAM3 is anticipated to be a promising biomarker for the diagnosis and prognosis of breast cancer.

Remote ischemic conditioning slows blood-retinal barrier damage in type 1 diabetic rats

Diabetic retinopathy (DR) is one of the major complications of diabetes and can cause severe visual impairment. Blood-retina barrier (BRB) destruction resulted from chronic hyperglycemia underlines its major pathological process. However, current treatments have limited efficacy and may even cause serious complications. Remote ischemic conditioning (RIC), through repeated transient mechanical occlusion of limb blood vessels, has been confirmed to promote blood–brain barrier integrity after stroke, but its role in BRB disruption has not been elucidated. This study aimed to investigate the protective effects of RIC on the BRB in diabetic rats and its potential mechanisms. 48 Sprague-Dawley rats were randomly assigned to the Sham group, Sham + RIC group, diabetes mellitus (DM) group and DM+RIC group. The diabetic model was successfully induced by intraperitoneal injection of streptozotocin. RIC treatment was administered daily and lasted for 9 weeks. In functional analysis, RIC improved the retinal function based on electroretinogram data and reduced the leakage of BRB in diabetic rats. In proteomic analysis, tight junction pathway was enriched after RIC treatment, in which Patj gene was significantly increased. We also found that RIC increased mRNA levels of Patj, claudin-1 and zonula occludens (ZO)-1, protein expression of claudin-1 when compared with diabetic models. In conclusion, RIC slowed BRB damage in diabetic rats, which may be related to the preservation of tight junction proteins. RIC may be a promising protective strategy for the treatment of DR.