Candidate proteins interacting with cytoskeleton in cells from the basal airway epithelium in vitro.

Introduction: The cytoskeleton consists of actin, microtubules, septins, and intermediate filaments and, in most cells, is anchored to an extracellular matrix. Each cell has a unique arrangement of this network and readjusts it from time to time. To investigate the regulation of these reorganizations, we identified interactors from extracts of four cultured lines representing basal cells from the airway epithelium. Methods: After immunoprecipitation with an antibody against keratin 17, samples were processed by liquid chromatography and tandem mass spectrometry. Samples not undergoing antibody-mediated capture were processed in parallel. Results: The main keratins of basal cells, namely, Krt14 (type I) and Krt5 (type II), constituted 67% of the total keratin recovered. Several other intermediate filament proteins, nestin, lamin-B1, and prelamin A/C, were present but not enriched upon immunoprecipitation. Although the class of armadillo-repeat proteins was represented by beta-catenin1 and plakoglobin, other desmosome plaque constituents were absent. Large cytolinkers were represented by the spectraplakin, microtubule-actin cross-linking factor (Macf1), which was enriched by immunoprecipitation, and the plakin, plectin, which was not enriched. Subunits of actin filaments and microtubules, along with numerous proteins associated with them, were recovered in both immunoprecipitated samples and those lacking the capture step. Coefficients of determination were computed based on abundance. The actin-associated proteins, alpha-spectrin and brain-specific angiogenesis inhibitor (Baiaip2l), were modestly correlated with keratin abundance but highly correlated with one another and with the keratin-binding protein, annexin A2. This interaction network resembled the pedestal formed by pathogenic Escherichia coli. Microtubule-associated proteins, dynamin 1-like protein and cytoplasmic dynein 1 heavy chain (Dync1h1), were enriched by immunoprecipitation, suggesting association with keratins, whereas kinesin-1 heavy chain and microtubule-associated protein retinitis pigmentosa 1 (EB1), were not enriched. Dync1h1 abundance was negatively correlated with that of all the septins, suggesting resemblance to a known antagonistic septin-dynein 1 relationship on microtubules. Conclusion: The cell lines showed remarkable uniformity with respect to the candidates interacting with cytoskeleton. The alpha-spectrin-Baiap2l network may link actin filaments to keratin precursor particles. A smaller interaction network centered on Dync1h1 was negatively correlated with all spectrin-Baiap2l constituents, suggesting that it and its binding partners are excluded from the pedestal-like domain.

Xiaoyankangjun tablet alleviates dextran sulfate sodium-induced colitis in mice by regulating gut microbiota and JAK2/STAT3 pathway.

Xiaoyankangjun tablet (XYKJP) is a traditional Chinese medicine formulation used to treat intestinal disorders in clinical practice. However, the specific therapeutic mechanism of action of XYKJP in colitis has not yet been elucidated. This study aimed to reveal the multifaceted mechanisms of action of XYKJP in treating colitis. The model established based on DSS-induced colitis in C57BL/6 mice was employed to estimate the effect of XYKJP on colitis, which was then followed by histological assessment, 16S rRNA sequencing, RT-qPCR, ELISA, and Western blot. XYKJP alleviated the symptoms of DSS-induced colitis mainly by reducing oxidative stress, inflammatory responses, and intestinal mucosal repair in colitis tissues. In addition, XYKJP regulated the intestinal flora by increasing the relative abundance of Akkermansia and Bifidobacterium and reducing the relative abundance of Coriobacteriaceae_UCG-002. Mechanistically, XYKJP increased the content of short-chain fatty acids (SCFAs) in the feces, particularly propanoic acid and butyric acid, activated their specific receptor GPR43/41, furthermore activated the Nrf2/HO-1 pathway, and suppressed the JAK2/STAT3 pathway. XYKJP significantly alleviated the symptoms of experimental colitis and functioned synergistically by regulating the intestinal flora, increasing the production of SCFAs, and activating their specific receptors, thereby repressing oxidative stress and inflammation.

Microbiome and metabolome analyses reveal significant alterations of gut microbiota and bile acid metabolism in ETEC-challenged weaned piglets by dietary berberine supplementation.

This study mainly investigated the effects of berberine (BBR) on the bile acid metabolism in gut-liver axis and the microbial community in large intestine of weaned piglets challenged with enterotoxigenic Escherichia coli (ETEC) by microbiome and metabolome analyses. Sixty-four piglets were randomly assigned to four groups including Control group, BBR group, ETEC group, and BBR + ETEC group. Dietary BBR supplementation upregulated the colonic mRNA expression of Occludin, Claudin-5, trefoil factor 3 (TFF3), and interleukin (IL)-10, and downregulated colonic IL-1ß and IL-8 mRNA expression in piglets challenged with ETEC K88 (p < 0.05). The hepatic non-targeted metabolome results showed that dietary BBR supplementation enriched the metabolic pathways of primary bile acid biosynthesis, tricarboxylic acid cycle, and taurine metabolism. The hepatic targeted metabolome analyses showed that BBR treatment increased the hepatic concentrations of taurocholic acid (TCA) and taurochenodeoxycholic acid (TDCA), but decreased the hepatic cholic acid (CA) concentration (p < 0.05). Further intestinal targeted metabolome analyses indicated that the deoxycholic acid (DCA), hyocholic acid (HCA), 7-ketodeoxycholic acid (7-KDCA), and the unconjugated bile acid concentrations in ileal mucosa was decreased by dietary BBR treatment (p < 0.05). Additionally, BBR treatment significantly upregulated the hepatic holesterol 7 α-hydroxylase (CYP7A1) and sterol 27-hydroxylase (CYP27A1) mRNA expression, and upregulated the ileal mRNA expression of farnesoid X receptor (FXR) and apical sodium-dependent bile acid transporter (ASBT) as well as the colonic mRNA expression of FXR, fibroblast growth factor19 (FGF19), takeda G protein-coupled receptor 5 (TGR5) and organic solute transporters beta (OST-ß) in piglets (p < 0.05). Moreover, the microbiome analysis showed that BBR significantly altered the composition and diversity of colonic and cecal microbiota community, with the abundances of Firmicutes (phylum), and Lactobacillus and Megasphaera (genus) significantly increased in the large intestine of piglets (p < 0.05). Spearman correlation analysis showed that the relative abundances of Megasphaera (genus) were positively correlated with Claudin-5, Occludin, TFF3, and hepatic TCDCA concentration, but negatively correlated with hepatic CA and glycocholic acid (GCA) concentration (p < 0.05). Moreover, the relative abundances of Firmicute (phylum) and Lactobacillus (genus) were positively correlated with hepatic TCDCA concentration (p < 0.05). Collectively, dietary BBR supplementation could regulate the gut microbiota and bile acid metabolism through modulation of gut-liver axis, and attenuate the decreased intestinal tight junction expression caused by ETEC, which might help maintain intestinal homeostasis in weaned piglets.

Immune-mediated disruption of the blood-brain barrier after intracerebral hemorrhage: Insights and potential therapeutic targets.

AIMS: Intracerebral hemorrhage (ICH) is a condition that arises due to the rupture of cerebral blood vessels, leading to the flow of blood into the brain tissue. One of the pathological alterations that occurs during an acute ICH is an impairment of the blood-brain barrier (BBB), which leads to severe perihematomal edema and an immune response. DISCUSSION: A complex interplay between the cells of the BBB, for example, pericytes, astrocytes, and brain endothelial cells, with resident and infiltrating immune cells, such as microglia, monocytes, neutrophils, T lymphocytes, and others accounts for both damaging and protective mechanisms at the BBB following ICH. However, the precise immunological influence of BBB disruption has yet to be richly ascertained, especially at various stages of ICH. CONCLUSION: This review summarizes the changes in different cell types and molecular components of the BBB associated with immune-inflammatory responses during ICH. Furthermore, it highlights promising immunoregulatory therapies to protect the integrity of the BBB after ICH. By offering a comprehensive understanding of the mechanisms behind BBB damage linked to cellular and molecular immunoinflammatory responses after ICH, this article aimed to accelerate the identification of potential therapeutic targets and expedite further translational research.

Intestinal barrier permeability: the influence of gut microbiota, nutrition, and exercise.

The intestinal wall is a selectively permeable barrier between the content of the intestinal lumen and the internal environment of the body. Disturbances of intestinal wall permeability can potentially lead to unwanted activation of the enteric immune system due to excessive contact with gut microbiota and its components, and the development of endotoxemia, when the level of bacterial lipopolysaccharides increases in the blood, causing chronic low-intensity inflammation. In this review, the following aspects are covered: the structure of the intestinal wall barrier; the influence of the gut microbiota on the permeability of the intestinal wall via the regulation of functioning of tight junction proteins, synthesis/degradation of mucus and antioxidant effects; the molecular mechanisms of activation of the pro-inflammatory response caused by bacterial invasion through the TLR4-induced TIRAP/MyD88 and TRAM/TRIF signaling cascades; the influence of nutrition on intestinal permeability, and the influence of exercise with an emphasis on exercise-induced heat stress and hypoxia. Overall, this review provides some insight into how to prevent excessive intestinal barrier permeability and the associated inflammatory processes involved in many if not most pathologies. Some diets and physical exercise are supposed to be non-pharmacological approaches to maintain the integrity of intestinal barrier function and provide its efficient operation. However, at an early age, the increased intestinal permeability has a hormetic effect and contributes to the development of the immune system.

Effects of Hermetia illucens larvae meal and astaxanthin on intestinal histology and expression of tight junction proteins in weaned piglets.

The weaning phase in piglets causes significant physiological stress, disrupts intestinal integrity and reduces productivity, necessitating strategies to improve intestinal health and nutrient absorption. While current research highlights the role of diet in mitigating these adverse effects, identifying effective dietary supplements remains a challenge. This study evaluated the effects of Hermetia illucens (HI) larvae meal and astaxanthin (AST) on the intestinal histology of weaned piglets. In a controlled experiment, 48 weaned piglets were divided into six groups and received varying levels of HI larval meal (2.5% and 5%) and AST in their diets. The methodology involved comprehensive histological examinations of the small intestine, assessing absorption area, villi elongation, crypt depth, goblet cells, enterocytes and expression of ileal tight junction (TJ) proteins. The study found that HI larval meal significantly improved nutrient absorption in the jejunum and ileum (p < 0.001), thereby enhancing feed conversion. AST supplementation increased the number of enterocytes (p < 0.001). Both HI larval meal and AST positively affected intestinal morphology and function, increasing muscularis muscle mass and villi elongation (p < 0.001 and p < 0.05, respectively). The 2.5% HI meal improved the villi length to crypt depth ratio and slightly increased the goblet cell count (both p < 0.05). Ki-67 antibody analysis showed increased cell proliferation in the duodenal and jejunal crypts, particularly with the 2.5% HI meal (p < 0.001). Insect meal did not affect TJ protein expression, indicating that it had no effect on intestinal permeability. These findings suggest that HI larval meal and AST can enhance the intestinal wellness and productivity of weaned piglets.

Breaking Barriers in Functional Dyspepsia: A Systematic Review and Meta-analysis on Duodenal Tight Junction Protein Expression.

Background/Aims: Disruptions in tight junction (TJ) protein expression leading to duodenal epithelial barrier impairment may contribute to increased intestinal permeability, potentially playing a role in functional dyspepsia (FD) pathophysiology. Currently published studies evaluated the role of several TJ proteins in FD patients with inconsistent results. Therefore, we conducted this systematic review and metaanalysis to evaluate the duodenal mucosal expression of several TJ proteins in FD. Methods: We performed a systematic electronic search on PubMed, EMBASE, and Scopus using predefined keywords. Diagnosis of FD by Rome III or Rome IV criteria was considered acceptable. Full articles satisfying our inclusion and exclusion criteria were included. The principal summary outcome was the mean difference of several TJ proteins in FD patients and control subjects. Results: A total of 8 and 5 studies were included in our qualitative and quantitative synthesis, respectively, with a total population of 666 participants, out of which 420 were FD patients. No significant differences were observed between FD patients and controls in the expression of claudin-1 (-0.102 [95% CI, -0.303, 0.099]), claudin-2 (0.161 [95% CI, -0.134, 0.456)], claudin-3 (0.278 [95% CI, -0.280, 0.837]), claudin-4 (0.045 [95% CI, -0.264, 0.354]), ZO-1 (-0.221 [95% CI, -0.683, 0.241]), ZO-2 (-0.070 [95% CI, -0.147, 0.007]), ZO-3 (-0.129 [95% CI, -0.376, 0.118]), ß-catenin (-0.135 [95% CI, -0.484, 0.214]), E-cadherin (-0.083 [95% CI, -0.229, 0.063]), and occludin (-0.158 [95% CI, -0.409, 0.093]). Conclusions: The expressions of all evaluated proteins including claudin-1, claudin-2, claudin-3, claudin-4, ZO-1, ZO-2, ZO-3, ß-catenin, E-cadherin, and occludin did not significantly differ between FD patients and controls. However, due to the limited number of included studies, results should be interpreted with caution.

Lactobacillus rhamnosus GG and Tannic Acid Synergistically Promote the Gut Barrier Integrity in a Rat Model of Experimental Diarrhea via Selective Immunomodulatory Cytokine Targeting.

SCOPE: Diarrhea is a common health issue that contributes to a significant annual death rate among children and the elderly worldwide. The anti-diarrheal activity of Lactobacillus rhamnosus GG (LGG) and tannic acid (TA), alone or combined, is examined, in addition to their effect on intestinal barrier integrity. METHODS AND RESULTS: Fifty-six adult male Wistar rats are randomly assigned into seven groups: control, LGG alone, TA alone, diarrhea model, diarrhea+LGG, diarrhea+TA, and diarrhea+LGG+TA-treated groups. Diarrhea is induced by high-lactose diet (HLD) consumption. LGG (1x109 CFU/rat) and TA (100 mg Kg-1 d-1) were given orally 4 days after HLD feeding and continued for 10 days. Ileum specimens are processed for biochemical analysis of the local intestinal cytokines, polymerase chain reaction (PCR), and histological study. Also, immunohistochemistry-based identification of Proliferating Cell Nuclear Antigen (PCNA) and zonula occludens 1 (ZO-1) is performed. Compared to the diarrhea model group, both treatments maintain the intestinal mucosal structure and proliferative activity and preserve ZO-1 expression, with the combination group showing the maximal effect. However, LGG-treated diarrheic rats show a remarkable decrease in the intestinal tissue concentrations of tumor necrosis factor-alpha (TNF-α) and nuclear factor Kappa beta (NF-κB); meanwhile, TA treatment leads to a selective decrease of interferon-gamma (INF-γ) and transforming growth factor-beta (TGF-ß1). CONCLUSION: Individual LGG and TA treatments significantly alleviate diarrhea, probably through a selective immunomodulatory cytokine-dependent mechanism, while the combination of both synergistically maintains the intestinal mucosa by keeping the intestinal epithelial barrier function and regenerative capability.

The Basic Requirement of Tight Junction Proteins in Blood-Brain Barrier Function and Their Role in Pathologies.

This review addresses the role of tight junction proteins at the blood-brain barrier (BBB). Their expression is described, and their role in physiological and pathological processes at the BBB is discussed. Based on this, new approaches are depicted for paracellular drug delivery and diagnostics in the treatment of cerebral diseases. Recent data provide convincing evidence that, in addition to its impairment in the course of diseases, the BBB could be involved in the aetiology of CNS disorders. Further progress will be expected based on new insights in tight junction protein structure and in their involvement in signalling pathways.

Flagellin conjugated Per a 10 and its T cell peptides attenuate airway inflammation and restore cellular function.

Adjuvants were used to modulate response towards relevant immune cells. The present study aims to investigate FlaA-conjugated Per a 10 and T cell peptides in amelioration of allergic airway disease in mice. Mice given Per a 10 showed allergic features with higher cellular infiltration, IgE, Th-2 cytokines and alarmins. Fusion protein treatment reduced lung inflammation (p < 0.0001) and cellular infiltrates (p < 0.001) with higher IgG2a/IgE indicating resolution of disease. Immunotherapy with FPT1 and FPT3 reduces IL-4, IL-5 and IL-13 levels (p < 0.0001) with a fourfold increase in IFN-γ secretion in BALF. FPT1- and FPT3-treated mice have increased IL-10 and TGF-ß levels (p < 0.001) with CD4+Foxp3+ T cells (p < 0.01) indicating Treg response. There was enhanced expression of claudin-1 (1.7-fold) and occludin (fourfold) in lungs of FPT1- and FPT3-treated mice with reduced TSLP (p < 0.01) and IL-33 (p < 0.0001) secretion in BALF indicating recovery of epithelial function. Peptide-conjugated FlaA proteins showed protective immunity in mice and have potential for immunotherapy with restoration of cellular function.

FAM189A2 plays a tumour suppressor role in lung adenocarcinoma by influencing cell apoptosis, CXCR4 expression and tight junction proteins.

Transmembrane proteins play key roles in the development of lung cancer. The family with sequence similarity 189 member A2 (FAM189A2) gene encodes a transmembrane structural protein, yet its involvement in lung adenocarcinoma remains largely unexplored. This study elucidated its role in lung adenocarcinoma and its possible molecular mechanism. Our findings revealed diminished expression levels of FAM189A2 in LUAD tissues. Additionally, the activity of LUAD cells was significantly inhibited by overexpression of FAM189A2. Following FAM189A2 overexpression, the expression of OCLN and TJP2 was upregulated in LUAD cells, while CXCR4 expression experiences a notable decrease. Moreover, the coimmunoprecipitation experiment confirmed the direct interaction between FAM189A2 and CXCR4. The infiltration levels of T cells (CD4+ memory resting, CD8+, regulatory), NK cells, B memory cells, endothelial cells and cancer-associated fibroblasts were significantly correlated with FAM189A2 expression. These results indicate FAM189A2 may act as a tumour suppressor in LUAD through tight junction protein (TJP) and CXCR4 regulation. Moreover, FAM189A2 is significantly correlated with the immune microenvironment of LUAD, which may be involved in prognosis and immunotherapeutic efficacy.

The protective effects of Axitinib on blood-brain barrier dysfunction and ischemia-reperfusion injury in acute ischemic stroke.

BACKGROUND AND PURPOSE: The pathophysiological features of acute ischemic stroke (AIS) often involve dysfunction of the blood-brain barrier (BBB), characterized by the degradation of tight junction proteins (Tjs) leading to increased permeability. This dysfunction can exacerbate cerebral injury and contribute to severe complications. The permeability of the BBB fluctuates during different stages of AIS and is influenced by various factors. Developing effective therapies to restore BBB function remains a significant challenge in AIS treatment. High levels of vascular endothelial growth factor (VEGF) in the early stages of AIS have been shown to worsen BBB breakdown and stroke progression. Our study aimed to investigate the protective effects of the VEGF receptor inhibitor Axitinib on BBB dysfunction and cerebral ischemia/reperfusion-induced injury. METHODS: BEnd3 cell exposed to oxygen-glucose deprivation (OGD) model was constructed to estimate pharmacological activity of Axitinib (400 ng/ml) on anti-apoptosis and pathological barrier function recovery. In vivo, rats were subjected to a 1 h transient middle cerebral artery occlusion and 23 h reperfusion (tMCAO/R) to investigate the permeability of BBB and cerebral tissue damage. Axitinib was administered through the tail vein at the beginning of reperfusion. BBB integrity was assessed by Evans blue leakage and the expression levels of Tjs claudin-5 and occludin. RESULTS: Our research revealed that co-incubation with Axitinib enhanced the cell viability of OGD-insulted bEnd3 cells, decreased LDH leakage rate, and suppressed the expression of apoptosis-related proteins cytochrome C and Bax. Axitinib also mitigated the damage to Tjs and facilitated the restoration of transepithelial electrical resistance in OGD-insulted bEnd.3 cells. In vivo, Axitinib administration reduced intracerebral Evans blue leakage and up-regulated the expression of Tjs in the penumbra brain tissue in tMCAO/R rats. Notably, 10 mg/kg Axitinib exerted a significant anti-ischemic effect by decreasing cerebral infarct volume and brain edema volume, improving neurological function, and reducing pro-inflammatory cytokines IL-6 and TNF-α in the brain. CONCLUSIONS: Our study highlights Axitinib as a potent protectant of blood-brain barrier function, capable of promoting pathological blood-brain barrier recovery through VEGF inhibition and increased expression of tight junction proteins in AIS. This suggests that VEGF antagonism within the first 24 h post-stroke could be a novel therapeutic approach to enhance blood-brain barrier function and mitigate ischemia-reperfusion injury.

Oligomeric Tau-induced oxidative damage and functional alterations in cerebral endothelial cells: Role of RhoA/ROCK signaling pathway.

Despite of yet unknown mechanism, microvascular deposition of oligomeric Tau (oTau) has been implicated in alteration of the Blood-Brain Barrier (BBB) function in Alzheimer's disease (AD) brains. In this study, we employed an in vitro BBB model using primary mouse cerebral endothelial cells (CECs) to investigate the mechanism underlying the effects of oTau on BBB function. We found that exposing CECs to oTau induced oxidative stress through NADPH oxidase, increased oxidative damage to proteins, decreased proteasome activity, and expressions of tight junction (TJ) proteins including occludin, zonula occludens-1 (ZO-1) and claudin-5. These effects were suppressed by the pretreatment with Fasudil, a RhoA/ROCK signaling inhibitor. Consistent with the biochemical alterations, we found that exposing the basolateral side of CECs to oTau in the BBB model disrupted the integrity of the BBB, as indicated by an increase in FITC-dextran transport across the model, and a decrease in trans endothelial electrical resistance (TEER). oTau also increased the transmigration of peripheral blood mononuclear cells (PBMCs) in the BBB model. These functional alterations in the BBB induced by oTau were also suppressed by Fasudil. Taken together, our findings suggest that targeting the RhoA/ROCK pathway can be a potential therapeutic strategy to maintain BBB function in AD.

Microbe-Derived Antioxidants Protect IPEC-1 Cells from H2O2-Induced Oxidative Stress, Inflammation and Tight Junction Protein Disruption via Activating the Nrf2 Pathway to Inhibit the ROS/NLRP3/IL-1ß Signaling Pathway.

Oxidative stress can induce inflammation and tight junction disruption in enterocytes. The initiation of inflammation is thought to commence with the activation of the ROS/NLRP3/IL-1ß signaling pathway, marking a crucial starting point in the process. In our previous studies, we found that microbe-derived antioxidants (MAs) showed significant potential in enhancing both antioxidant capabilities and anti-inflammatory effects. The main aim of this research was to investigate the ability of MAs to protect cells from oxidative stress caused by H2O2, to reduce inflammatory responses, and to maintain the integrity of tight junction proteins by modulating the ROS/NLRP3/IL-1ß signaling pathway. IPEC-1 cells (1 × 104 cells/well) were initially exposed to 100 mg/L of MAs for 12 h, after which they were subjected to 1 mM H2O2 treatment for 1 h. We utilized small interfering RNA (siRNA) to inhibit the expression of NLRP3 and Nrf2. Inflammatory factors such as IL-1ß and antioxidant enzyme activity levels were detected by ELISA. Oxidative stress marker ROS was examined by fluorescence analysis. The NLRP3/IL-1ß signaling pathway, Nrf2/HO-1 signaling pathway and tight junction proteins (ZO-1 and Occludin) were detected by RT-qPCR or Western blotting. In our research, it was observed that MA treatment effectively suppressed the notable increase in H2O2-induced inflammatory markers (TNF-α, IL-1ß, and IL-18), decreased ROS accumulation, mitigated the expression of NLRP3, ASC, and caspase-1, and promoted the expression of ZO-1 and Occludin. After silencing the NLRP3 gene with siRNA, the protective influence of MAs was observed to be linked with the NLRP3 inflammasome. Additional investigations demonstrated that the treatment with MAs triggered the activation of Nrf2, facilitating its translocation into the nucleus. This process resulted in a notable upregulation of Nrf2, NQO1, and HO-1 expression, along with the initiation of the Nrf2-HO-1 signaling pathway. Consequently, there was an enhancement in the activities of antioxidant enzymes like SOD, GSH-Px, and CAT, which effectively mitigated the accumulation of ROS, thereby ameliorating the oxidative stress state. The antioxidant effectiveness of MAs was additionally heightened in the presence of SFN, an activator of Nrf2. The antioxidant and anti-inflammatory functions of MAs and their role in regulating intestinal epithelial tight junction protein disruption were significantly affected after siRNA knockdown of the Nrf2 gene. These findings suggest that MAs have the potential to reduce H2O2-triggered oxidative stress, inflammation, and disruption of intestinal epithelial tight junction proteins in IPEC-1 cells. This reduction is achieved by blocking the ROS/NLRP3/IL-1ß signaling pathway through the activation of the Nrf2 pathway.

Efficacy of Laurus nobilis L. for Tight Junction Protein Imbalance in Leaky Gut Syndrome.

Laurus nobilis L. (LNL) belongs to the evergreen Lauraceae family. It is native to the Mediterranean and widely distributed in the southern United States, Europe, and the Middle East. LNL is rich in active ingredients of the sesquiterpene lactone series and has been reported to have antioxidant, anti-inflammatory, and anticancer effects. And parthenolide, known as a sesquiterpene lactone-based compound, inhibits the activation of lipopolysaccharide-binding protein (LBP), which is a major trigger for leaky gut syndrome. However, the effectiveness of LNL in improving the state of increased intestinal permeability has not yet been reported. Therefore, we demonstrated the efficacy of LNL, which is known to be rich in parthenolide, in improving intestinal permeability induced by IL-13. We investigated the improvement in permeability and analyzed major tight junction proteins (TJs), permeability-related mechanisms, weight and disease activity indices, and corresponding cytokine mechanisms. LNL maintained TJs homeostasis and clinical improvement by reducing increased claudin-2 through the inhibition of IL-13/STAT6 activation in TJ-damaged conditions. These results are expected to be effective in preventing leaky gut syndrome through the TJ balance and to further improve intestinal-related diseases, such as inflammatory bowel disease.

Expression and Localization Profiles of Tight Junction Proteins in Immune Cells Depend on Their Activation Status.

The ability of the immune system to combat pathogens relies on processes like antigen sampling by dendritic cells and macrophages migrating through endo- and epithelia or penetrating them with their dendrites. In addition, other immune cell subtypes also migrate through the epithelium after activation. For paracellular migration, interactions with tight junctions (TJs) are necessary, and previous studies reported TJ protein expression in several immune cells. Our investigation aimed to characterize, in more detail, the expression profiles of TJ proteins in different immune cells in both naïve and activated states. The mRNA expression analysis revealed distinct expression patterns for TJ proteins, with notable changes, mainly increases, upon activation. At the protein level, LSR appeared predominant, being constitutively present in naïve cell membranes, suggesting roles as a crucial interaction partner. Binding experiments suggested the presence of claudins in the membrane only after stimulation, and claudin-8 translocation to the membrane occurred after stimulation. Our findings suggest a dynamic TJ protein expression in immune cells, implicating diverse functions in response to stimulation, like interaction with TJ proteins or regulatory roles. While further analysis is needed to elucidate the precise roles of TJ proteins, our findings indicate important non-canonical functions of TJ proteins in immune response.

Metformin suppresses NFE2L1 pathway activation to inhibit gap junction beta protein expression in NSCLC.

OBJECTIVE: Non-small-cell lung cancer (NSCLC) is a deadly form of cancer that exhibits extensive intercellular communication which contributed to chemoradiotherapy resistance. Recent evidence suggests that arrange of key proteins are involved in lung cancer progression, including gap junction proteins (GJPs). METHODS AND RESULTS: In this study, we examined the expression patterns of GJPs in NSCLC, uncovering that both gap junction protein, beta 2 (GJB2) and gap junction protein, beta 2 (GJB3) are increased in LUAD and LUSC. We observed a correlation between the upregulation of GJB2, GJB3 in clinical samples and a worse prognosis in patients with NSCLC. By examining the mechanics, we additionally discovered that nuclear factor erythroid-2-related factor 1 (NFE2L1) had the capability to enhance the expression of connexin26 and connexin 31 in the NSCLC cell line A549. In addition, the use of metformin was discovered to cause significant downregulation of gap junction protein, betas (GJBs) by limiting the presence of NFE2L1 in the cytoplasm. CONCLUSION: This emphasizes the potential of targeting GJBs as a viable treatment approach for NSCLC patients receiving metformin.

Effects of dietary energy levels on microorganisms and short-chain fatty acids of rumen and tight junction proteins in Honghe Yellow cattle.

This study was conducted to investigate the effects of dietary energy levels on microorganisms and short-chain fatty acids (SCFAs) of rumen and the expression of tight junction proteins in Honghe Yellow cattle. A total of fifteen male Honghe Yellow cattle were randomly divided into three treatments (five replicates per treatment), consisting of formulated energy concentrations of 5.90 MJ/kg (high-energy diet, group H), 5.60 MJ/kg (medium-energy diet, group M) and 5.30 MJ/kg (low-energy diet, group L). The results showed that compared with group H, the expression of Claudin-1 in rumen epithelium of groups M and L was increased, but the expression of ZO-1 was decreased (p < 0.05). Moreover, compared with group H, group M down-regulated the expression of Occludin and Claudin-1 in the brain (p < 0.05). For rumen bacteria, the dominant phyla included Bacteroidetes and Firmicutes, the abundance of Actinobacteriota in groups M and L was significantly increased compared with group H (p < 0.05). At the genus level, the relative abundance of Corynebacterium, Eubacterium_nodatum_group and Neisseraceae in groups M and L was significantly decreased compared with group H (p < 0.05). For rumen fungi, the dominant phyla included Basidiomycota, Ascomycota and Neocariastigomycota, the relative abundance of Ascomycetes was significantly higher than that of groups M and L compared with group H (p < 0.05). At the genus level, the relative abundance of Neocelimastigaceae and Myceliophthora in groups M and L was significantly reduced compared with group H (p < 0.05). Furthermore, the expression of Claudin-1 in rumen epithelium was significantly positively correlated with Actinobacteriota, Corynebacterium and Neisseriaceae. The expression of ZO-1 in the spinal cord was significantly positively correlated with Myceliophthora. The expression of Occludin in brain was positively correlated with valerate content (p < 0.05). In summary, dietary energy levels affected the rumen microbiota of Honghe Yellow cattle. The expression of Claudin-1 in rumen epithelium and the total SCFAs concentration were increased with decreasing dietary energy levels, but the expression of Claudin-1 in brain and ZO-1 in the spinal cord were reduced with decreasing dietary energy levels. Meanwhile, the rumen microbiota and SCFAs were significantly correlated with the expression of TJP.

Endothelial lincRNA-p21 alleviates cerebral ischemia/reperfusion injury by maintaining blood-brain barrier integrity.

Blood-brain barrier (BBB) disruption is increasingly recognized as an early contributor to the pathophysiology of cerebral ischemia/reperfusion (I/R) injury, and is also a key event in triggering secondary damage to the central nervous system. Recently, long non-coding RNA (lncRNA) have been found to be associated with ischemic stroke. However, the roles of lncRNA in BBB homeostasis remain largely unknown. Here, we report that long intergenic non-coding RNA-p21 (lincRNA-p21) was the most significantly down-regulated lncRNA in human brain microvascular endothelial cells (HBMECs) after oxygen and glucose deprivation/reoxygenation (OGD/R) treatment among candidate lncRNA, which were both sensitive to hypoxia and involved in atherosclerosis. Exogenous brain-endothelium-specific overexpression of lincRNA-p21 could alleviate BBB disruption, diminish infarction volume and attenuate motor function deficits in middle cerebral artery occlusion/reperfusion (MCAO/R) mice. Further results showed that lincRNA-p21 was critical to maintain BBB integrity by inhibiting the degradation of junction proteins under MCAO/R and OGD/R conditions. Specifically, lincRNA-p21 could inhibit autophagy-dependent degradation of occludin by activating PI3K/AKT/mTOR signaling pathway. Besides, lincRNA-p21 could inhibit VE-cadherin degradation by binding with miR-101-3p. Together, we identify that lincRNA-p21 is critical for BBB integrity maintenance, and endothelial lincRNA-p21 overexpression could alleviate cerebral I/R injury in mice, pointing to a potential strategy to treat cerebral I/R injury.

High-altitude hypoxia promotes BRD4-mediated activation of the Wnt/ß-catenin pathway and disruption of intestinal barrier.

Hypobaric hypoxia, commonly experienced at elevated altitudes, presents significant physiological challenges. Our investigation is centered on the impact of the bromodomain protein 4 (BRD4) under these conditions, especially its interaction with the Wnt/ß-Catenin pathway and resultant effects on glycolytic inflammation and intestinal barrier stability. By combining transcriptome sequencing with bioinformatics, we identified BRD4's key role in hypoxia-related intestinal anomalies. Clinical parameters of altitude sickness patients, including serum BRD4 levels, inflammatory markers, and barrier integrity metrics, were scrutinized. In vitro studies using CCD 841 CoN cells depicted expression changes in BRD4, Interleukin (IL)-1ß, IL-6, and ß-Catenin. Transepithelial electrical resistance (TEER) and FD4 analyses assessed barrier resilience. Hypoxia-induced mouse models, analyzed via H&E staining and Western blot, provided insights into barrier and protein alterations. Under hypoxic conditions, marked BRD4 expression variations emerged. Elevated serum BRD4 in patients coincided with intensified Wnt signaling, inflammation, and barrier deterioration. In vitro, findings showed hypoxia-induced upregulation of BRD4 and inflammatory markers but a decline in Occludin and ZO1, affecting barrier strength-effects mitigated by BRD4 inhibition. Mouse models echoed these patterns, linking BRD4 upregulation in hypoxia to barrier perturbations. Hypobaric hypoxia-induced BRD4 upregulation disrupts the Wnt/ß-Catenin signaling, sparking glycolysis-fueled inflammation and weakening intestinal tight junctions and barrier degradation.