Tight Junction-associated Proteins Research Articles

MiR-185-5p / ATG101 axis alleviated intestinal barrier damage in intestinal ischemia reperfusion through autophagy

ObjectiveIntestinal ischemia-reperfusion (II/R) is a common pathological injury in clinic, and the systemic inflammatory response it causes will lead to multiple organ damage and functional failure. miR-185-5p has been reported to be a regulator of inflammatory response and autophagy, but whether it participates in the regulation of autophagy in II/R is still unclear. Therefore, we aimed to explore the mechanism of miR-185-5p regulating intestinal barrier injury in (II/R). MethodsCaco-2 cells was induced by oxygen-glucose deprivation/reoxygenation (OGD/R) to establish II/R model. The superior mesenteric artery of C57BL/6 mice was clamped for 45 min and then subjected to reperfusion for 4 h for the establishment of II/R mice model. miR-185-5p mimic, miR-185-5p inhibitor, pcDNA-autophagy-related 101 (ATG101) were respectively transfected into Caco-2 cells. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed to assess miR-185-5p expression. Western blot detected the level of ATG101 and tight junction-associated proteins ZO1, Occludin, E-cadherin, β-catenin, as well as autophagy markers ATG5, ATG12, LC3Ⅰ/Ⅱ, Beclin1 and SQSTM1. Transepithelial electrical resistance (TEER) values was detected by a resistance meter. FITC-Dextran was performed to measure cell permeability. 5-ethynyl-2’-deoxyuridine (EDU) staining measured cell proliferation. Transmission electron microscope was conducted to observe autophagosomes. Hematoxylin & eosin (H&E) staining observed the damage of mice intestinal. Immunohistochemistry (IHC) measured the percentage of ki67 positive cells. TdT-mediated dUTP nick-end labeling (TUNEL) assay assessed cell apoptosis in intestinal tissues of II/R. Dual-luciferase assay verified the targeting relationship between miR-185-5p and ATG101.Results miR-185-5p was overexpressed in OGD/R-induced Caco-2 cells and intestinal tissues of II/R mice. Knocking down miR-185-5p markedly promoted autophagy and TEER values, reduced cell permeability, and alleviated intestinal barrier damage. ATG101 was a target of miR-185-5p, and overexpression of ATG101 promoted autophagy and dampened OGD/R-induced intestinal barrier damage. Overexpression of miR-185-5p reversed the effect of overexpressed ATG101 on OGD/R-induced Caco-2 cells. ConclusionKnockdown of miR-185-5p enhanced autophagy and alleviated II/R intestinal barrier damage by targeting ATG101.

Cigarette smoke extract impairs gingival epithelial barrier function

We previously showed that junctional adhesion molecule 1 (JAM1) and coxsackievirus and adenovirus receptor (CXADR), tight junction-associated proteins, have important roles to maintain epithelial barrier function in gingival tissues. Smoking is considered to be a significant risk factor for periodontal disease. The present study was conducted to examine the effects of cigarette smoke extract (CSE) on JAM1 and CXADR in human gingival epithelial cells. CSE was found to cause translocation of JAM1 from the cellular surface to EGFR-positive endosomes, whereas CXADR did not. Using a three-dimensional multilayered gingival epithelial tissue model, CSE administration was found to increase permeability to lipopolysaccharide and peptidoglycan, whereas overexpression of JAM1 in the tissue model prevented penetration by those substrates. Furthermore, vitamin C increased JAM1 expression, and inhibited penetration of LPS and PGN induced by CSE. These findings strongly suggest that CSE disrupts gingival barrier function via dislocation of JAM1, thus allowing bacterial virulence factors to penetrate into subepithelial tissues. Furthermore, they indicate that vitamin C increases JAM1 expression and prevents disruption of gingival barrier function by CSE.

LncRNA446 Regulates Tight Junctions by Inhibiting the Ubiquitinated Degradation of Alix after Porcine Epidemic Diarrhea Virus Infection.

Porcine epidemic diarrhea (PED) is a highly contagious disease, caused by porcine epidemic diarrhea virus (PEDV), which causes huge economic losses. Tight junction-associated proteins play an important role during virus infection; therefore, maintaining their integrity may be a new strategy for the prevention and treatment of PEDV. Long noncoding RNAs (lncRNAs) participate in numerous cellular functional activities, yet whether and how they regulate the intestinal barrier against viral infection remains to be elucidated. Here, we established a standard system for evaluating intestinal barrier integrity and then determined the differentially expressed lncRNAs between PEDV-infected and healthy piglets by lncRNA-seq. A total of 111 differentially expressed lncRNAs were screened, and lncRNA446 was identified due to significantly higher expression after PEDV infection. Using IPEC-J2 cells and intestinal organoids as in vitro models, we demonstrated that knockdown of lncRNA446 resulted in increased replication of PEDV, with further damage to intestinal permeability and tight junctions. Mechanistically, RNA pulldown and an RNA immunoprecipitation (RIP) assay showed that lncRNA446 directly binds to ALG-2-interacting protein X (Alix), and lncRNA446 inhibits ubiquitinated degradation of Alix mediated by TRIM25. Furthermore, Alix could bind to ZO1 and occludin and restore the expression level of the PEDV M gene and TJ proteins after lncRNA446 knockdown. Additionally, Alix knockdown and overexpression affects PEDV infection in IPEC-J2 cells. Collectively, our findings indicate that lncRNA446, by inhibiting the ubiquitinated degradation of Alix after PEDV infection, is involved in tight junction regulation. This study provides new insights into the mechanisms of intestinal barrier resistance and damage repair triggered by coronavirus. IMPORTANCE Porcine epidemic diarrhea is an acute, highly contagious enteric viral disease severely affecting the pig industry, for which current vaccines are inefficient due to the high variability of PEDV. Because PEDV infection can lead to severe injury of the intestinal epithelial barrier, which is the first line of defense, a better understanding of the related mechanisms may facilitate the development of new strategies for the prevention and treatment of PED. Here, we demonstrate that the lncRNA446 directly binds one core component of the actomyosin-tight junction complex named Alix and inhibits its ubiquitinated degradation. Functionally, the lncRNA446/Alix axis can regulate the integrity of tight junctions and potentially repair intestinal barrier injury after PEDV infection.

High expression of MARVELD3 as a potential prognostic biomarker for oral squamous cell carcinoma

Objectives: Tight junction-associated marvel proteins (TAMP) is a transmembrane protein whose members are associated with tight junctions between cells and epithelial remodeling. MARVEL domain containing 3 (MARVELD3) is one of the members of the TAMP. MARVELD3, as a novel tight junction protein involved in bicellular tight junction assembly, has attracted growing attention in the field of oncology. This study aimed to investigate the prognostic role of MARVELD3 and to determine how it functions in tumorigenesis in oral squamous cell carcinoma (OSCC), thus providing additional data to help the guidance of clinical practice. Materials and Methods: RNA-seq data and relevant clinical information were obtained from TCGA. Bioinformatics means used in this study included differential gene expression analysis, KM survival curve analysis, univariate and multivariate Cox regression analyses, nomogram analysis, ROC curve analysis, methylation level analysis, gene function enrichment analysis, and immune cell infiltration analysis. Results: MARVELD3 was significantly higher expressed in OSCC tissue than in normal tissue, and the overall survival of the high expression group was significantly lower than that of the normal group. Univariate and multivariate Cox regression analyses showed that MARVELD3 could serve as an independent contributing factor to poor OSCC prognosis. The nomograms and ROC curves supported the results above. Its expression was negatively correlated with DNA methylation sites. Analysis of PPI networking and gene functional enrichment showed that MARVELD3 was involved in the functional activities of DNA and RNA and was associated with immune cell infiltration. Conclusion: The high expression of MARVELD3 is associated with poor prognosis in OSCC, and MARVELD3 could be recognized as a novel independent prognostic factor for OSCC.

Dexmedetomidine post-conditioning protects blood-brain barrier integrity by modulating microglia/macrophage polarization via inhibiting NF-κB signaling pathway in intracerebral hemorrhage.

Intracerebral hemorrhage (ICH) is one of the most devastating forms of stroke. Dexmedetomidine (DEX) has shown certain neuroprotective roles in ICH. Nevertheless, the details concerning the underlying molecular mechanism of DEX’s protective effects still need further elucidation. Herein, a model of ICH was established. The rats were randomly divided into the sham group, the ICH group, and the ICH + DEX group. Neurological outcomes, neuronal injury, and apoptosis were evaluated. Brain water content, Evans blue extravasation, and the expression of tight junction-associated proteins were also detected to assess the blood-brain barrier (BBB) integrity. Subsequently, the microglia/macrophage polarization state and inflammatory cytokine levels were observed. To further explore the underlying mechanism, NF-κB signaling pathway-associated proteins were detected. The results showed that DEX exerted neuroprotective effects against ICH-induced neurological deficits. DEX significantly increased the numbers of the surviving neurons and ameliorated neuronal cell loss and apoptosis in ICH. The rats that received the DEX displayed a lower level of brain water content and EB extravasation, moreover, ZO-1, occludin, and claudin-5 were markedly increased by DEX. Additionally, DEX facilitated M2 microglia/macrophage polarization, the M1-associated markers were reduced by DEX, while the M2-associated identification significantly increased. We found that DEX dramatically diminished pro-inflammatory cytokines expression, simultaneously promoting anti-inflammatory cytokines expression. DEX inhibited nuclear translocation of NF-κB in ICH rats. Our data suggest that DEX post-conditioning protects BBB integrity by modulating microglia/macrophage polarization via inhibiting the NF-κB signaling pathway in ICH.

Effect Of Treadmill Running On Gut Microbiota And Alzheimer’S Disease Neuropathology In Triple Transgenic Mice

The brain-gut-microbiota axis is involved in neurodegenerative disorders such as Alzheimer’s disease (AD). The therapeutic roles of exercise training against AD pathology have been well-established in animal studies. The findings from recent studies show that exercise training induces symbiotic changes in the gut microbiota. PURPOSE: To the effect of exercise training on the gut microbiota and AD neuropathology in triple transgenic (3xTg)-AD mice. METHODS: At 3 months of age, 11 wild type (WT) and 22 transgenic mice were randomly assigned to either WT control (WT, n = 11) or AD control (AD, n = 11) or AD exercise (AD-EXE, n = 11). AD-EXE mice were subjected to treadmill running at a moderate intensity with a frequency of 5 days per week for 20 weeks. Morris water maze (MWM) was conducted to evaluate cognition and memory, and fecal samples were collected to assess the gut environment. Blood and brain samples were collected for biochemical and histological assays, respectively. RESULTS: After 20-week-treadmill running, AD+EXE mice had better MWM-based cognitive performance (p < 0.05) compared to AD mice. AD+EXE mice were characterized by increased Akkermansiaceae and Bacteriodaceae species in conjunction with decreased Clostridaceae and Erysipelotrichaceae species compared to AD mice. In the affected regions of the brain, AD+EXE mice had attenuations in selective loss of tight junction-associated blood barrier proteins (i.e., claudin and ZO1), AD neuropathology (i.e., amyloid precursor protein, β-site APP cleaving enzyme 1, p-tau), neuroinflammation and apoptosis in conjunction with enhanced neurogenesis (i.e., bran-derived neurotrophic factor), better synaptic stability/plasticity and enhanced antioxidation (i.e., anti-oxidant superoxide dismutase, and catalase) compared to AD mice. CONCLUSIONS: The current findings of the study suggested that treadmill running induced changes in the gut microbiota composition and attenuated AD neuropathology in the 3xTg-AD mice. Yet, the exact mechanism(s) by which the brain-gut-microbiota axis leads to the therapeutic effects of treadmill exercise on AD neuropathology remain to be further investigated. This work was supported by the Ministry of Education of the Republic of Korea and the National Research Foundation of Korea (NRF-2020R1A2C1009365).

The effects of cold exposure on the ileum mechanical barrier in mice and its mechanisms

Objective: To study the mechanisms of cold exposure mediated ileum mechanical barrier injury in mice. Methods: Twenty mice were randomly divided into the control and cold exposure groups. Both the control and cold exposure groups were placed in the climate room with (24±2)℃ and 40% humidity. The mice in the cold exposure group were moved to the climate room at (4±2)℃ every day for 3 hours for three consecutive weeks. Three weeks later, the ileum tissues of mice were collected. Changes in ileum tissue structure were observed by hematoxylin-eosin staining and Masson staining. The related protein expression levels of the tight junction, inflammatory cytokines, and the NF-κB pathway were detected by Western blot. Results: Compared with the control group, the circular muscle layer of the ileum in cold exposed mice became thin, a large number of inflammatory cells infiltrated, the length of villi became short, the depth of recess was increased, and tissue fibrosis appeared. The expression levels of ideal tight junction-associated proteins in cold exposed mice were decreased significantly (P＜0.05), while the protein expression levels of IL-1β, IL-6 and phosphorescent p65 were increased significantly (P＜0.05). Conclusion: Cold exposure can damage the tight junction of the mouse ileum, destroy the integrity of the mechanical barrier and activate the NF-κB signaling pathway to promote the occurrence of the inflammatory response.

Junctional adhesion molecule-A deletion increases phagocytosis and improves survival in a murine model of sepsis

Expression of the tight junction–associated protein junctional adhesion molecule-A (JAM-A) is increased in sepsis, although the significance of this is unknown. Here, we show that septic JAM-A –/– mice have increased gut permeability, yet paradoxically have decreased bacteremia and systemic TNF and IL-1β expression. Survival is improved in JAM-A–/– mice. However, intestine-specific JAM-A–/– deletion does not alter mortality, suggesting that the mortality benefit conferred in mice lacking JAM-A is independent of the intestine. Septic JAM-A–/– mice have increased numbers of splenic CD44hiCD4+ T cells, decreased frequency of TNF+CD4+ cells, and elevated frequency of IL-2+CD4+ cells. Septic JAM-A–/– mice have increased numbers of B cells in mesenteric lymph nodes with elevated serum IgA and intraepithelial lymphocyte IgA production. JAM-A–/– × RAG–/– mice have improved survival compared with RAG–/– mice and identical mortality as WT mice. Gut neutrophil infiltration and neutrophil phagocytosis are increased in JAM-A–/– mice, while septic JAM-A–/– mice depleted of neutrophils lose their survival advantage. Therefore, increased bacterial clearance via neutrophils and an altered systemic inflammatory response with increased opsonizing IgA produced through the adaptive immune system results in improved survival in septic JAM-A–/– mice. JAM-A may be a therapeutic target in sepsis via immune mechanisms not related to its role in permeability.

Oridonin attenuates dextran sulfate sodium‑induced ulcerative colitis in mice via the Sirt1/NF‑κB/p53 pathway.

Ulcerative colitis (UC) is a serious chronic inflammatory bowel disease. Oridonin (Ori) has anti-inflammatory, antibacterial and antitumor activities. The current study aimed to investigate the regulatory role of Ori in UC. BALB/C mice were induced to form a model of UC using dextran sulfate sodium (DSS), after which UC mice received high-(Ori-H) and low-doses of Ori (Ori-L). Subsequently, the length of the colon was measured and hematoxylin and, eosin staining was performed to detect colonic injury. Western blot analysis was performed to detect expression level in tight junction-associated proteins in murine colon tissue. Additionally, myeloperoxidase activity and inflammatory factor concentration were detected in colon tissue using ELISA. TUNEL and western blot assays were also performed to detect cell apoptosis, and the expression level of Sirt1/NF-κB/p53 pathway-related proteins was also determined using western blot analysis. The results revealed that Ori ameliorated clinical symptoms and pathological lesions in mice with DSS-induced UC. Furthermore, Ori protected the integrity of the colonic mucosal barrier, reduced the inflammatory response and decreased oxidative stress levels in mice with DSS-induced UC. Ori treatment also inhibited intestinal mucosal cell apoptosis. These effects may have occurred via the Sirtuin-1/NF-κB/p53 pathway. In conclusion, Ori treatment inhibited DSS-induced inflammatory response, oxidative stress and intestinal mucosal apoptosis in UC mice.

Hyperglycemia Aggravates Blood-Brain Barrier Disruption Following Diffuse Axonal Injury by Increasing the Levels of Inflammatory Mediators through the PPARγ/Caveolin-1/TLR4 Pathway.

Hyperglycemia aggravates brain damage after diffuse axonal injury (DAI), but the underlying mechanisms are not fully defined. In this study, we aimed to investigate a possible role for hyperglycemia in the disruption of blood-brain barrier (BBB) integrity in a rat model of DAI and the underlying mechanisms. Accordingly, 50% glucose was intraperitoneally injected after DAI to establish the hyperglycemia model. Hyperglycemia treatment aggravated neurological impairment and axonal injury, increased cell apoptosis and glial activation, and promoted the release of inflammatory factors, including TNF-α, IL-1β, and IL-6. It also exacerbated BBB disruption and decreased the expression of tight junction-associated proteins, including ZO-1, claudin-5, and occludin-1, whereas the PPARγ agonist rosiglitazone (RSG) had the opposite effects. An in vitro BBB model was established by a monolayer of human microvascular endothelial cells (HBMECs). Hyperglycemia induction worsened the loss of BBB integrity induced by oxygen and glucose deprivation (OGD) by increasing the release of inflammatory factors and decreasing the expression of tight junction-associated proteins. Hyperglycemia further reduced the expression of PPARγ and caveolin-1, which significantly decreased after DAI and OGD. Hyperglycemia also further increased the expression of toll-like receptor 4 (TLR4), which significantly increased after OGD. Subsequently, the PPARγ agonist RSG increased caveolin-1 expression and decreased TLR4 expression and inflammatory factor levels. In contrast, caveolin-1 siRNA abrogated the protective effects of RSG in the in vitro BBB model of hyperglycemia by increasing TLR4 and Myd88 expression and the levels of inflammatory factors, including TNF-α, IL-1β, and IL-6. Collectively, we demonstrated that hyperglycemia was involved in mediating secondary injury after DAI by disrupting BBB integrity by inducing inflammation through the PPARγ/caveolin-1/TLR4 pathway.

Hypericumsampsonii attenuates inflammation in mice with ulcerative colitis via regulation of PDE4/PKA/CREB signaling pathway

Ethnopharmacological relevanceHypericum sampsonii Hance (Yuanbaocao), a traditional herbal medicine with various pharmacological properties, is traditionally used to treat diarrhea and enteritis in China for hundreds of years. Investigations have uncovered its anti-inflammatory effects and corresponding bioactive constituents in H. sampsonii, however, the mechanisms of action for the treatment of enteritis are still unclear. Aims of the studyThis study aims to investigate the therapeutic effects and molecular mechanisms of H. sampsonii in a dextran sulfate sodium (DSS)-induced ulcerative colitis (UC) mice model. Materials and methodsThe major ingredients of the ethyl acetate extract (HS) in H. sampsonii were analyzed by UPLC-QTOF-MS. The inflammatory state of UC mice was caused by 3% DSS once daily for seven days. During DSS treatment, the mice in the positive drug group and the other three groups were orally administered 5-ASA (positive control) or HS daily. After treatment with HS or 5-ASA for a week, colonic pathological observation and the molecular biological index were performed for therapeutic evaluation, including visual inspection in the length and weight of colons and spleens, pathological morphology by hematoxylin and eosin (HE) staining, determination of oxidative markers, inflammatory cytokines and tumor necrosis factor-alpha (TNF-α) levels in colonic tissues as well as spleen index. Gene expression levels of inflammatory cytokines, antioxidant enzymes and PDE4 were detected using kits and PCR, while the expression of colonic tight junction proteins and relative signals of PKA/CREB signaling pathway were analyzed by Western blot. ResultsThe main components in HS were found to be polycyclic polyprenylated acylphloroglucinols (PPAPs). HS distinctly alleviated DSS-stimulated UC-like lesions symptoms as evidenced by a significant recovery from body weight, colon lengths, and histological injuries of colons. HS reduced the accumulation of pro-inflammatory cytokines and improved the mRNA level of IL-10. Simultaneously, the colonic mRNA expression levels of IL-1β, IL-17, iNOS and COX-2 were all significantly suppressed by HS in a dose-dependent manner. Furthermore, HS restored the protein expression of tight junction-associated protein (ZO-1 and occluding). Besides, HS significantly inhibited the protein level of PDE4 and decreased the expressions of PKA and phosphorylated CREB. ConclusionThis is the first work about main composition and anti-UC effect of Hypericum sampsonii Hance. For the first time, this study reveals HS is not toxic in a single dose and exert significantly protective effect in DSS-colitis mice. The underlying mechanisms may involve the improvement to inflammatory status, the protection for intestinal barrier function, the inhibition of PDE4, and the activation of PKA/CREB signaling pathway. This study provided an experimental basis for the traditional application of H. sampsonii Hance in the treatment of diarrhea and dysentery.

Tight junction proteins occludin and ZO-1 as regulators of epithelial proliferation and survival.

Epithelial cells are the first line of mucosal defense. In the intestine, a single layer of epithelial cells must establish a selectively permeable barrier that supports nutrient absorption and waste secretion while preventing the leakage of potentially harmful luminal materials. Key to this is the tight junction, which seals the paracellular space and prevents unrestricted leakage. The tight junction is a protein complex established by interactions between members of the claudin, zonula occludens, and tight junction-associated MARVEL protein (TAMP) families. Claudins form the characteristic tight junction strands seen by freeze-fracture microscopy and create paracellular channels, but the functions of ZO-1 and occludin, founding members of the zonula occludens and TAMP families, respectively, are less well defined. Recent studies have revealed that these proteins have essential noncanonical (nonbarrier) functions that allow them to regulate epithelial apoptosis and proliferation, facilitate viral entry, and organize specialized epithelial structures. Surprisingly, neither is required for intestinal barrier function or overall health in the absence of exogenous stressors. Here, we provide a brief overview of ZO-1 and occludin canonical (barrier-related) functions, and a more detailed examination of their noncanonical functions.

MarvelD3 Is Upregulated in Ulcerative Colitis and Has Attenuating Effects during Colitis Indirectly Stabilizing the Intestinal Barrier.

In inflammatory bowel disease (IBD), the impaired intestinal barrier is mainly characterized by changes in tight junction protein expression. The functional role of the tight junction-associated MARVEL protein MARVELD3 (MD3) in IBD is yet unknown. (i) In colon biopsies from IBD patients we analyzed MD3 expression and (ii) in human colon HT-29/B6 cells we studied the signaling pathways of different IBD-relevant cytokines. (iii) We generated a mouse model with intestinal overexpression of MD3 and investigated functional effects of MD3 upregulation. Colitis, graded by the disease activity index, was induced by dextran sodium sulfate (DSS) and the intestinal barrier was characterized electrophysiologically. MD3 was upregulated in human ulcerative colitis and MD3 expression could be increased in HT-29/B6 cells by IL-13 via the IL13Rα1/STAT pathway. In mice DSS colitis, MD3 overexpression had an ameliorating, protective effect. It was not based on direct enhancement of paracellular barrier properties, but rather on regulatory mechanisms not solved yet in detail. However, as MD3 is involved in regulatory functions such as proliferation and cell survival, we conclude that the protective effects are hardly targeting the intestinal barrier directly but are based on regulatory processes supporting stabilization of the intestinal barrier.

TLR4 Deletion Improves Cognitive Brain Function and Structure in Aged Mice

Toll-like receptor-4 (TLR4), a member of the TLR family, plays a key role in inflammation-related diseases of the nervous system. TLR4 knockout mice are widely used in various neurological disease studies, and there is a clear correlation between inflammation and behavior. Therefore, elucidating the effect of TLR4 on neurobehavioral function is essential, and the related mechanisms need to be explored. Male TLR4 knockout (TLR4−/−) and wild-type (TLR4+/+) mice of different ages (4, 8, and 16 months) were used for behavioral experiments. Synaptic spine, blood–brain barrier (BBB) integrity, memory regulatory proteins, cortical blood flow, and inflammatory factor examinations were also conducted to explore the possible mechanism by which TLR4 works. Here, we found that compared with 16-m-old TLR4+/+ mice, age-matched TLR4−/− mice had better learning and memory abilities, increased expression of neuronal synaptic spines, and increased memory-related regulatory proteins in the hippocampus. TLR4 knockout significantly attenuated the fear response in 16-m-old mice. The TLR4−/− mice also had better blood–brain barrier integrity, increased expression of tight junction-associated proteins, increased cerebral cortical blood flow and reduced proinflammatory cytokine expression in the cortex and cerebrospinal fluid. Our results suggest that TLR4 deletion ameliorates significant neurobehavioral dysfunction during the aging stage, as well as multiple abnormalities in brain function and structure due to alterations in tight junction-associated proteins and inflammatory factors.

Phillygenin Protects the Intestinal Barrier from Dysfunction via let-7b Signaling Pathway and Regulation of Intestinal Microbiota.

The study investigates the positive effects of phillygenin on intestinal tight junction via the let-7b signaling pathway and the regulation of intestinal microbiota. The expression levels of tight junction proteins are determined through PCR and Western blot. DSS-induced mice colitis is used to verify the protective effects of phillygenin on intestinal barrier and tight junction. Fecal microbiota transplantation is used to verify the role intestinal microbiota. let-7b is detected in the colon tissues of patients with acute stercoral obstruction. Phillygenin could promote the expression of occludin, which might be inhibited by let-7b inhibitor. DSS-induced mice colitis showed that phillygenin could lower the colonic permeability and maintain the tight junction-associated proteins. The effects of phillygenin could be deprived by anti-let-7b and rescued by FMT of normal intestinal microbiota. Clinical samples verified a lower level of let-7b in stercoral obstruction patients. Phillygenin could protect the intestinal barrier from dysfunction via the signaling pathway of let-7b by regulating intestinal microbiota.

Donepezil ameliorates oxygen‑glucose deprivation/reoxygenation‑induced cardiac microvascular endothelial cell dysfunction through PARP1/NF‑κB signaling.

Ischemia/reperfusion (I/R) injury is a serious clinical condition characterized by high morbidity and mortality rates. Donepezil plays a neuroprotective role in I/R-associated diseases. The aim of the present study was to investigate the role and the potential mechanism of action of donepezil in I/R-induced myocardial microvascular endothelial cell dysfunction. An I/R model was simulated using oxygen-glucose deprivation/reoxygenation (OGD/R) injury in human cardiac microvascular endothelial cells (CMECs). Cell viability and lactate dehydrogenase release were examined following treatment with donepezil. Commercial kits were used to evaluate cell apoptosis, cell permeability and caspase-3 activity. The expression levels of apoptosis-associated proteins, as well as proteins found in tight junctions or involved in the poly(ADP-ribose) polymerase 1 (PARP1)/NF-κB pathway, were measured using western blotting. These parameters were also examined following PARP1 overexpression. The results demonstrated that donepezil increased cell viability and reduced toxicity in OGD/R-treated CMECs. The apoptotic rate, caspase-3 activity and protein expression levels of Bax and cleaved caspase-3 were significantly reduced following donepezil treatment, which was accompanied by Bcl-2 upregulation. Moreover, cell permeability was notably reduced, coupled with a marked increase in the expression of tight junction-associated proteins. The expression levels of proteins related to PARP1/NF-κB signaling were significantly downregulated in CMECs following donepezil treatment. However, the protective effects of donepezil on OGD/R-induced CMEC injury were reversed following PARP1 overexpression. In conclusion, donepezil suppressed OGD/R-induced CMEC dysfunction via PARP1/NF-κB signaling. This finding provided insight into the mechanism underlying myocardial I/R injury.

P016 Colonoids derived from UC patients retain higher transcriptional response to inflammatory triggers in-vitro, even after several passages

Abstract Background Ulcerative colitis (UC) patients show high variations in disease phenotype and response to therapy. Intestinal epithelia play a key role in UC pathogenesis, and therefore patient-derived epithelial model to study this variation are warranted. Few studies suggest that colonoids derived from UC patients retain some disease-related transcriptional and epigenetic changes, but they also raise questions regarding their persistence in culture. Here, we aimed to characterize if UC epithelial dysfunctions and response to inflammatory signals retained in colonoid culture. Methods Rectal biopsies were used to generate epithelial organoids (colonoids) that were grown through 5–10 passages using L-WRN conditioned medium. Media and total RNA were collected for measuring CXCL1 secretion using ELISA and qPCR, respectively. Results 3 control and 5 UC patients-derived colonoids were generated. Median age was 17 years and 75% were females. Control patients were evaluated for loose stool, abdominal pain, and anemia, but had normal rectal mucosa endoscopically and histologically. UC included newly diagnosed and established patients. Colonoids were allowed to differentiate for 48 hours before applying different inflammatory cocktails to induce inflammation for 24 h with IFNγ+LPS, IFNγ+TNFα, and TNFα+LPS (20 ng/ml each trigger). UC organoids secreted significant higher levels of CXCL1 as detected in the media at baseline (p=0.02) and with IFNy+TNFa (p=0.01), but no difference was noted with TNFa+LPS (Fig. 1). In addition, CXCL1 mRNA showed significant higher levels after inflammatory triggering in UC vs. controls (Fig. 1) with IFNγ +LPS, (p=0.02), IFNγ+TNFα (p=0.003), and TNFα+LPS (p=0.016). Similarly, several other genes showed significant and substantial induction in UC organoids vs. controls, in some or all the inflammatory triggers (Fig. 2). The tight junction-associated protein, ZO1 showed substantially higher induction in UC compared to controls with the 3 inflammatory cocktails [IFNγ +LPS, (p=0.03), IFNγ+TNFα (p=0.02), and TNFα+LPS (p=0.005)]. Significantly higher expression in UC organoid was further noted in comparison to control in IL8 levels upon IFNγ +LPS (p=0.002), in interferon inducible IDO1 with IFNγ+TNFα (p=0.001), and in goblet associated MUC2 (p=0.04) and bacterial sensing DUOX2 (p=0.001) with TNFα+LPS. Conclusion Our data suggest that UC colonoids retain some inflammatory response activity in-vitro even after several passages. We show that UC organoids secrete higher level of CXCL1. Additionally, we note higher transcriptional response to different inflammatory signals in UC organoids potentially implying an “immune” epithelial transcriptional memory that may contribute to UC chronicity.

Netrin-1 Ameliorates Postoperative Delirium-Like Behavior in Aged Mice by Suppressing Neuroinflammation and Restoring Impaired Blood-Brain Barrier Permeability.

Postoperative delirium (POD) is a common and serious postoperative complication in elderly patients, and its underlying mechanism is elusive and without effective therapy at present. In recent years, the neuroinflammatory hypothesis has been developed in the pathogenesis of POD, in which the damaged blood-brain barrier (BBB) plays an important role. Netrin-1 (NTN-1), an axonal guidance molecule, has been reported to have strong inflammatory regulatory and neuroprotective effects. We applied NTN-1 (45 μg/kg) to aged mice using a POD model with a simple laparotomy to assess their systemic inflammation and neuroinflammation by detecting interleukin-6 (IL-6), interleukin-10 (IL-10), and high mobility group box chromosomal protein-1 (HMGB-1) levels. We also assessed the reactive states of microglia and the permeability of the BBB by detecting cell junction proteins and the leakage of dextran. We found that a single dose of NTN-1 prophylaxis decreased the expression of IL-6 and HMGB-1 and upregulated the expression of IL-10 in the peripheral blood, hippocampus, and prefrontal cortex. Nerin-1 reduced the activation of microglial cells in the hippocampus and prefrontal cortex and improved POD-like behavior. NTN-1 also attenuated the anesthesia/surgery-induced increase in BBB permeability by upregulating the expression of tight junction-associated proteins such as ZO-1, claudin-5, and occludin. These findings confirm the anti-inflammatory and BBB protective effects of NTN-1 in an inflammatory environment in vivo and provide better insights into the pathophysiology and potential treatment of POD.

Recreating gut-liver axis during NAFLD onset by using a Caco-2/HepG2 co-culture system

Nonalcoholic fatty liver disease (NAFLD) onset and its progression towards nonalcoholic steatohepatitis (NASH) features increased intestinal permeability and leaky gut, thereby favoring the escape of endotoxin [lipopolysaccharides (LPS)] from the gut to the liver. The aim of this study was to resemble the crosstalk between intestine and liver during NAFLD by using an in vitro model of co-culture system. Enterocytes (Caco-2) were seeded on Transwell filters (pore size: 0.4 μm) and cultured for 21 days to constitute a confluent monolayer, and then they were co-cultivated with hepatocytes (HepG2) for an additional 24 h. Caco-2 on the apical chamber were exposed to LPS and/or a mixture of palmitic and oleic acid (PAOA) for 24 h. FITC-4000 dextrans (FD4) permeability across Caco-2 monolayer was increased by the treatment of Caco-2 cells with PAOA and LPS, consistently with tight junction-associated proteins reduction. Caco-2 exposure to PAOA/LPS promoted ApoB, triglyceride (TG), and free fatty acid secretion in basolateral media. In turn, HepG2 co-cultured with Caco-2 exposed to LPS, PAOA, or both accumulated lipid droplets and increased intracellular TG content. Likewise, Caco-2 released pro-inflammatory cytokines in basolateral media. These events triggered endoplasmic reticulum (ER) and oxidative stress, enhancing reactive oxygen species (ROS), H2O2, aldehyde derivate production, and ROS-induced DNA damage in HepG2 cells. Hence, Caco-2/HepG2 co-culture system may faithfully reproduce the breach in the intestinal barrier integrity that occurs in NAFLD, thus resulting in the increased inflammatory response and ER and oxidative and stress, which promote the switch towards NASH.

Vonoprazan decreases tight junction-associated proteins at the blood-brain barrier

Vonoprazan decreases tight junction-associated proteins at the blood-brain barrier