Disruption Of Tight Junction Proteins Research Articles

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-1β 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-1β 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-1β 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-1β and antioxidant enzyme activity levels were detected by ELISA. Oxidative stress marker ROS was examined by fluorescence analysis. The NLRP3/IL-1β 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-1β, 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-1β signaling pathway through the activation of the Nrf2 pathway.

Ginsenoside Re protects rhinovirus-induced disruption of tight junction through inhibition of ROS-mediated phosphatases inactivation in human nasal epithelial cells

Maintaining tight junction integrity significantly contributes to epithelial barrier function. If the barrier function is destroyed, the permeability of the cells increases, and the movement of the pathogens is promoted, thereby further increasing the susceptibility to secondary infection. Ginsenoside components have multiple biological activities, including antiviral effects. In this study, we examined the protective effects of ginsenoside Re against rhinovirus-induced tight junction disruption in primary human nasal epithelial cells (HNE). Incubation with human rhinovirus resulted in marked disruption of tight junction proteins (ZO-1, E-cadherin, claudin-1, and occludin) in human nasal epithelial cells. Rhinovirus-induced disruption of tight junction proteins was strongly inhibited by the treatment of cells with ginsenoside Re. Indeed, significant amounts of reactive oxygen species (ROS) have been detected in human nasal epithelial cells co-incubated with rhinovirus. Moreover, rhinovirus-induced ROS generation was markedly reduced by the ginsenoside Re. However, ginsenosides Rb1 and Rc did not inhibit tight junction disruption or ROS generation in nasal epithelial cells following incubation with rhinovirus. Furthermore, incubation with rhinovirus resulted in a marked decrease in protein phosphatase activity and an increase in protein tyrosine phosphorylation levels in nasal epithelial cells. Treatment of cells with ginsenoside Re inhibited rhinovirus-induced inactivation of phosphatases and phosphorylation of tyrosine. Our results identified ginsenoside Re as an effective compound that prevented rhinovirus-induced tight junction disruption in human nasal epithelial cells.

Mitochondrial transplantation attenuates oligomeric amyloid-beta-induced mitochondrial dysfunction and tight junction protein destruction in retinal pigment epithelium

Transplantation of mitochondria derived from mesenchymal stem cells (MSCs) has emerged as a new treatment method to improve mitochondrial dysfunction and alleviate cell impairment. Interest in using extrinsic mitochondrial transplantation as a therapeutic approach has been increasing because it has been confirmed to be effective in treating various diseases related to mitochondrial dysfunction, including ischemia, cardiovascular disease, and toxic damage. To support this application, we conducted an experiment to deliver external mitochondria to retinal pigment epithelial cells treated with oligomeric amyloid-beta (oAβ). Externally delivered amyloid-beta internalizes into cells and interacts with mitochondria, resulting in mitochondrial dysfunction and intracellular damage, including increased reactive oxygen species and destruction of tight junction proteins. Externally delivered mitochondria were confirmed to alleviate mitochondrial dysfunction and tight junction protein disruption as well as improve internalized oAβ clearance. These results were also confirmed in a mouse model in vivo. Overall, these findings indicate that the transfer of external mitochondria isolated from MSCs has potential as a new treatment method for age-related macular degeneration, which involves oAβ-induced changes to the retinal pigment epithelium.

Heat-killed Lactobacillus rhamnosus GG with different cultivation time restores intestinal epithelial barrier dysfunction via MLCK/MLC pathway activation

Heat-killed Lactobacillus rhamnosus GG (HK-LGG) has been proven to improve intestinal health, boost immunity, and exert anti-tumor effects. However, the effect of LGG with different incubation times after heat-killing on the regulatory effect on the intestinal barrier remains unclear. The analysis of HK-LGG composition obtained after 18 h and 48 h culture (HK-LGG-18 h & HK-LGG-48 h) indicated that HK-LGG-48 h exhibited higher levels of S-layer protein, peptidoglycan, and lipoteichoic acid. In contrast, the content of capsular polysaccharides in HK-LGG-48 h was found to be lower than that in HK-LGG-18 h. HK-LGG effectively attenuated the increase in electrical resistance and cell permeability and prevented the disruption of tight junction proteins in Caco-2 cells induced by lipopolysaccharide (LPS). HK-LGG suppressed the LPS-induced upregulation of protein expression related to myeloid differentiation factor 88, toll-like receptor 4, and nuclear factor-kappa B (NF-κB). The regulatory effect of HK-LGG on the intestinal barrier is through the regulation of the NF-κB-mediated Myosin Light Chain kinase -p- Myosin Light Chain signaling pathway. In particular, HK-LGG-48 h regulated the expression of inflammation cytokines, with a more pronounced effect than HK-LGG-18 h.

SKQ1 Improves Trimethylamine-N-Oxide-Induced Endothelial Dysfunction by Attenuating Oxidative Stress and Restoring Junctional Proteins

In recent years, the role of microbiota and microbial metabolites in the progress of aging and age-related diseases has been attracting increased attention. In this context, trimethylamine N-oxide (TMAO) is a pro-atherogenic and pro-inflammatory metabolite derived from the gut microbial metabolism of trimethylamine and is found to be elevated in patients with age-related cardiovascular disorders. We identified that TMAO treatment in mice has enhanced mitochondrial ROS production, endoplasmic reticulum stress and caused significant endothelial dysfunction. The present study investigated whether mitochondria-targeted antioxidant SKQ1 protected against TMAO-induced endothelial dysfunction. Endothelial cells were treated with or without TMAO (30μM) in the cell culture media. TMAO treated cells showed significant increase in both total ROS as well as mito-ROS production compared to untreated control group (n=8/group; p<0.05). In addition, western blot analysis demonstrated that TMAO treatment significantly decreased tight junction proteins such as occludin, ZO-1 and VE-Cadherin which were restored upon SKQ1 treatment (150nM, 12h). TMAO (30μM) treatment for 24 h induced significant mitochondrial dysfunction as evidenced by decreased mitochondrial membrane potential, increased mitochondrial ROS levels, and enhanced endothelial cell permeability compared to the control group (n=8/group; p<0.05). Treatment with a SKQ1 (150nM, 12h) significantly inhibited TMAO-induced ROS production and protected against increased endothelial cell permeability and mitochondrial ROS production (n=8/group; p<0.05). In conclusion, mitochondria-targeted antioxidant SKQ1 protects against TMAO induced endothelial dysfunction by attenuating ROS production and disruption of tight junction proteins. This work is supported by NIH R01 HL148711 award to Saisudha Koka This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

2-Methoxyestradiol Alleviates Neuroinflammation and Brain Edema in Early Brain Injury After Subarachnoid Hemorrhage in Rats.

ObjectiveNumerous studies have shown that neuroinflammation and brain edema play an important role in early brain injury (EBI) after subarachnoid hemorrhage (SAH). 2-Methoxyestradiol (2-ME) has been shown to have anti-inflammatory and anti-angiogenic effects. This study aimed to investigate the effects of 2-ME on neuroinflammation and brain edema after SAH and its underlying mechanism of action.MethodsRats were used to produce an endovascular puncture model of SAH. 2-ME or the control agent was injected intraperitoneally 1 h after SAH induction. At 24 h after surgery, the neurological score, SAH grading, brain water content, and blood–brain barrier (BBB) permeability were examined. The microglial activation level in the rat brain tissue was determined using immunofluorescence staining, whereas the cell apoptosis in the rat brain tissue was assessed using terminal deoxynucleotidyl transferase dUTP nick-end labeling assay, the levels of Interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α were measured by enzyme linked immunosorbent assay, and the expression levels of ZO-1, occludin, hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF), and matrix metallopeptidase (MMP)-9 in the rat brain tissue were determined using western blotting.ResultsTwenty-four hours after SAH, brain water content, BBB permeability, microglial activation, and cell apoptosis were significantly increased, whereas neurological function deteriorated significantly in rats. Treatment with 2-ME significantly decreased brain water content, BBB permeability, microglial cell activation, and cell apoptosis and improved neurological dysfunction in rats. Treatment with 2-ME reduced the expression levels of inflammatory factors (IL-1β, IL-6, and TNF-α), which were significantly elevated 24 h after SAH. Treatment with 2-ME alleviated the disruption of tight junction proteins (ZO-1 and occludin), which significantly decreased 24 h after SAH. To further determine the mechanism of this protective effect, we found that 2-ME inhibited the expression of HIF-1α, MMP-9, and VEGF, which was associated with the inflammatory response to EBI and BBB disruption after SAH.Conclusion2-ME alleviated neuroinflammation and brain edema as well as improved neurological deficits after SAH in rats. The neuroprotective effect of 2-ME on EBI after SAH in rats may be related to the inhibition of neuroinflammation and brain edema.

Wogonin inhibits tight junction disruption via suppression of inflammatory response and phosphorylation of AKT/NF-κB and ERK1/2 in rhinovirus-infected human nasal epithelial cells.

The maintenance of tight junction integrity contributes significantly to epithelial barrier function. If barrier function is destroyed, cell permeability increases and the movement of pathogens is promoted, further increasing the susceptibility to secondary infection. Here, we examined the protective effects of wogonin on rhinovirus (RV)-induced tight junction disruption. Additionally, we examined the signaling molecules responsible for anti-inflammatory activities in human nasal epithelial (HNE) cells. Primary HNE cells grown at an air-liquid interface and RPMI 2650 cells were infected apically with RV. Incubation with RV resulted in disruption of tight junction proteins (ZO-1, E-cadherin, claudin-1, and occludin) in the HNE cells. Cell viability of wogonin-treated HNE cells was measured using the MTT assay. Pretreatment with wogonin decreased RV-induced disruption of tight junctions in HNE cells. Furthermore, wogonin significantly decreased RV-induced phosphorylation of Akt/NF-κB and ERK1/2. Additionally, RV-induced generation of reactive oxygen species and RV-induced up-regulation of the production of inflammatory cytokines IL-8 and IL-6 were diminished by wogonin in HNE cells. Wogonin inhibits HRV-induced tight junction disruption via the suppression of inflammatory responses and phosphorylation of Akt/NF-κB and ERK1/2 in HNE cells. These finds will facilitate the development of novel therapeutic strategies.

Alterations to microbial secretome by estrogen may contribute to sex bias in irritable bowel syndrome.

Irritable bowel syndrome (IBS) is a female predominant functional gastrointestinal disorder, underpinned by microbial dysbiosis and microinflammation. We suggest that changes in trace amine (TA) load and metabolism may link together diet, inflammation and sex in this context. The effect of E2 treatment on microbial growth and TA generation was assessed using liquid chromatography and tandem mass spectrometry methodology. To investigate the effects of TAs on the gut, WST-1, prostaglandin E2 and tight junction protein dynamics were investigated in TA treated (HT-29) colon epithelial monolayer cultures. Differential E2-dependent alterations of the TA production capabilities of microbes were observed. Significantly, E2 treatment resulted in a 50% increase in tryptamine secretion from a probiotic microbe (p < 0.0001). Moreover, in vitro experiments indicated that TA treatment exerted type-specific effects in the gut, e.g., reducing mitochondrial functionality, even at low doses of tryptamine (p < 0.0001) and ρ-tyramine (p < 0.001). Additionally, prostaglandin E2 levels were significantly increased following ρ-tyramine and agmatine treatment (p < 0.05). In terms of functionality, all investigated TAs resulted in occludin redistribution and loss of zona occludens-1 and occludin co-localization. Increases in the gastrointestinal TA load may contribute to a relatively pro-inflammatory outcome in the intestine, along with tight junction protein disruption. Additionally, fluctuating levels of endogenous E2 may modulate microbially-derived TA levels, potentially explaining exaggerating gastrointestinal symptomology in females during low E2 phases. Thus, current data warrants subsequent investigations in appropriate in vivo models to fully elucidate the role of the trace aminergic system in the sex bias observed in IBS.

Protective Effect of an Exopolysaccharide Produced by Lactiplantibacillus plantarum BGAN8 Against Cadmium-Induced Toxicity in Caco-2 Cells.

Cadmium (Cd) ranks seventh on the list of most significant potential threats to human health based on its suspected toxicity and the possibility of exposure to it. It has been reported that some bacterial exopolysaccharides (EPSs) have the ability to bind heavy metal ions. We therefore investigated the capacity of eight EPS-producing lactobacilli to adsorb Cd in the present study, and Lactiplantibacillus plantarum BGAN8 was chosen as the best candidate. In addition, we demonstrate that an EPS derived from BGAN8 (EPS-AN8) exhibits a high Cd-binding capacity and prevents Cd-mediated toxicity in intestinal epithelial Caco-2 cells. Simultaneous use of EPS-AN8 with Cd treatment prevents inflammation, disruption of tight-junction proteins, and oxidative stress. Our results indicate that the EPS in question has a strong potential to be used as a postbiotic in combatting the adverse effects of Cd. Moreover, we show that higher concentrations of EPS-AN8 can alleviate Cd-induced cell damage.

Prolonged Antibiotic Use Worsens Neuroinflammation and Increases the Risk of Neurodegeneration via Elevated Expression of Systemic IL‐6 in Gulf War Illness Symptom Persistence Murine Model

Prolonged use of fluoroquinolone class of antibiotics has been studied widely. This class of antibiotic has been reported to have side effects linked to cardiovascular, gastrointestinal and neuronal ramifications following long-term usage. Though the Veterans Affairs has reported a recent drop in antibiotic prescriptions, it is still used in higher number of veterans and use of fluoroquinolones is no exception. Gulf War Illness (GWI) pathology is widely believed to be the result of environmental co-exposures and use of the pyridostigmine bromide pills. Advancing our recent studies on GWI pathology in a mouse model we hypothesized that prolonged use (5 months) of fluoroquinolones coupled with Neomycin might augment poor outcomes in systemic inflammation and neuroinflammation. In this study using a symptom persistence mouse model of GWI, we studied the combinatorial effect of prolonged broad spectrum antibiotic Neomycin and Enrofloxacin treatment for 5 months. Expression of serum proinflammatory cytokine IL-6 saw a sharp increase in mice co-exposed with antibiotics and GW chemicals group (GW-Ab) over GW chemical treated group only (GW). Results also showed decreased expression of blood brain barrier (BBB) tight junction protein Claudin 5 in GW-Ab group when compared to GW group. There was an increased microglial activation, expression of cytokine IL-1β, IL-6 and 3-nitrotyrosine and decreased expression of brain derived neurotrophic factor (BDNF) was also observed in GW-Ab group when compared to GW group. To study whether observed disruption of BBB membrane integrity, increased neuroinflammation had any effect on neurodegeneration and neuroplasticity, we probed the mouse brain for Tau and BDNF receptor expression. Results showed that there was an increased expression of phospho Tau (neurodegenerative marker) and decreased expression of phospho-tropomycin receptor kinase B (TrkB)-receptor of BDNF in GW-Ab group when compared to GW group. Interestingly, elevated expression of glycoprotein 130, a receptor for IL-6 trans signaling was also observed in GW-Ab over GW group. This led us to infer that circulatory IL-6 might be an active mediator in worsening brain pathology observed in GW-Ab, especially since IL-6 has been reported to cross BBB through disruption of tight junction proteins. Mechanistically, studies in mouse astrocytes primed with IL-6 also showed a decreased expression of TrkB with increased expression of Tau protein and further confirmed our in vivo results. Thus we conclude that prolonged antibiotic usage exacerbated GWI-associated neuroinflammation and neurodegeneration in GWI symptom persistence.

Incense smoke-induced oxidative stress disrupts tight junctions and bronchial epithelial barrier integrity and induces airway hyperresponsiveness in mouse lungs

Recent clinical studies have suggested that inhalation of incense smoke (IS) may result in impaired lung function and asthma. However, there is little experimental evidence to link IS with airway hyperresponsiveness (AHR) and bronchial epithelial barrier function. Using mouse and cell culture models, we evaluated the effects of IS exposure on AHR, expression of multiple epithelial tight junction (TJ)- and adherens junction-associated mRNAs and proteins in the lungs, and the barrier function of bronchial epithelial cells assessed by transepithelial electronic resistance (TEER). Exposure of BALB/c mice to IS increased AHR and inflammatory macrophage recruitment to BALF; reduced claudin-1, -2, -3, -7, -10b, -12, -15, and -18, occludin, zonula occludens-1 [ZO-1], and E-cadherin mRNA expression; and caused discontinuity of claudin-2 and ZO-1 protein immunostaining in lung tissue. IS extract dose-dependently decreased TEER and increased reactive oxygen species production in bronchial epithelial cell cultures. Treatment with N-acetyl-l-cysteine, but not glucocorticosteroids or long-acting β2-agonists, prevented the detrimental effects of IS. IS exposure can be problematic for respiratory health, as evidenced by AHR, increased recruitment of inflammatory macrophages and disruption of TJ proteins in the lung, and damage to epithelial barrier function. However, antioxidants may be useful for the treatment of IS-induced airway dysfunction.

2-Methoxyestradiol Protects Against Lung Ischemia/Reperfusion Injury by Upregulating Annexin A1 Protein Expression.

Background: 2-Methoxyestradiol (2ME), a natural 17-β estradiol metabolite, is a potent anti-inflammatory agent, but its effect on ischemia/reperfusion (IR)-induced acute lung inflammation remains unknown. Annexin A1 (AnxA1), a glucocorticoid-regulated protein, is effective at inhibiting neutrophil transendothelial migration by binding the formyl peptide receptors (FPRs). We aimed to investigate whether 2ME upregulates the expression of AnxA1 and protects against IR-induced lung damage.Methods: IR-mediated acute lung inflammation was induced by ischemia for 40 min followed by reperfusion for 60 min in an isolated, perfused rat lung model. The rat lungs were randomly treated with vehicle or 2ME, and the functional relevance of AnxA1 was determined using an anti-AnxA1 antibody or BOC2 (a pan-receptor antagonist of the FPR). In vitro, human primary alveolar epithelial cells (HPAECs) and rat neutrophils were pretreated with 2ME and an AnxA1 siRNA or anti-AnxA1 antibody and subjected to hypoxia-reoxygenation (HR).Results: 2ME significantly decreased all lung edema parameters, neutrophil infiltration, oxidative stress, proinflammatory cytokine production, lung cell apoptosis, tight junction protein disruption, and lung tissue injury in the IR-induced acute lung inflammation model. 2ME also increased the expression of the AnxA1 mRNA and protein and suppressed the activation of nuclear factor-κB (NF-κB). In vitro, 2ME attenuated HR-triggered NF-κB activation and interleukin-8 production in HPAECs, decreased transendothelial migration, tumor necrosis factor-α production, and increased apoptosis in neutrophils exposed to HR. These protective effects of 2ME were significantly abrogated by BOC2, the anti-AnxA1 antibody, or AnxA1 siRNA.Conclusions: 2ME ameliorates IR-induced acute lung inflammation by increasing AnxA1 expression. Based on these results, 2ME may be a promising agent for attenuating IR-induced lung injury.

Avenanthramide C Prevents Neuronal Apoptosis via PI3K/Akt/GSK3β Signaling Pathway Following Middle Cerebral Artery Occlusion.

Avenanthramides are a group of phenolic alkaloids that have been shown to have anti-inflammatory, anti-oxidant, anti-atherogenic, and vasodilation effects. The aim of the present study was to investigate the neuroprotective effect of avenanthramide-c (Avn-c) in focal brain ischemia and reperfusion injury using middle cerebral artery occlusion (MCAo) model with mice. Male C57BL/6 mice were divided into 4 groups: sham, control (MCAo), Avn-c, and Avn-c + LY294002 (phosphoinositide 3-kinase inhibitor) group. They were subjected to 60 min MCAo followed by reperfusion. Brain infarct volume and neurological deficit scores were measured after 24 h of reperfusion. We evaluated the blood brain barrier (BBB) integrity (ZO-1, VE-cadherin and occludin) and apoptosis (Bax, Bcl2, caspase3, Cytochrome C, and poly ADP ribose polymerase(PARP)-1). We also measured GSK3β for evaluation of the downstream mechanism of Akt. We examined the effect of the Avn-c in the phosphoinositide 3-kinase pathway. Avn-c reduced neurological score and infarction size. Avn-c inhibited the MCAo-induced disruption of tight junction proteins. Avn-c decreased apoptotic protein expression (Bax, Cytochrome C, and cleaved PARP-1) and increased anti-apoptotic protein expression (Bcl2) after MCAo. Akt and GSK3β were decreased in MCAo group and were restored in Avn-c group. This effect of Avn-c was abolished by PI3K inhibitor. In summary, Avn-c showed neuroprotective effects through PI3K-Akt-GSK3β signaling pathway.

Angiostrongylus cantonensis infection induces MMP-9 and causes tight junction protein disruption associated with Purkinje cell degeneration.

Angiostrongylus cantonensis causes a human central nervous system (CNS) infection characterized by eosinophilic meningitis or meningoencephalitis. Individuals infected with A. cantonensis exhibit unbalanced walking. The mechanism of extensive neurological impairments of hosts caused by A. cantonensis larvae remains unclear. Tight junction proteins (e.g., claudin-5 and zonula occludens-1) are the most important regulators of paracellular permeability and cellular adhesion. In a previous study, we found that increased matrix metalloproteinase-9 (MMP-9) activity may be associated with blood-CNS barrier disruption and/or the degeneration of Purkinje cells in eosinophilic meningitis caused by A. cantonensis. In the present study, the co-localization of MMP-9 and tight junction proteins on the degeneration of Purkinje cells was measured via confocal laser scanning immunofluorescence microscopy. The statistical evidence indicated that MMP-9 correlated between tight junction protein disruption and Purkinje cell degeneration at 20 days post-infection with A. cantonensis. In conclusion, Purkinje cell degeneration is highly correlated with tight junction protein disruption via the MMP-9 activation pathway.

Rebamipide promotes lacrimal duct epithelial cell survival via protecting barrier function

Nasolacrimal duct obstruction (NLDO) is thought to be due to inflammation and fibrosis of lacrimal duct epithelial cells (LDECs). Here we investigated the effect of rebamipide, a drug that is used for the protection of the mucosa and the treatment of gastritis and gastroduodenal ulcers, on LDECs, both in vitro and in vivo. In this study, LDECs were cultured from rabbit lacrimal duct tissues, and the barrier function of LEDCs was examined in vitro via transepithelial electrical resistance (TER) measurement, with or without interleukin (IL)-6 and/or rebamipide. For the in vivo examination, benzalkonium chloride (BAC) was injected into the rabbit lacrimal ducts, followed by the application of rebamipide or a placebo vehicle alone. The results of the in vitro examination revealed a significant decrease in TER in the group treated with IL-6 alone compared with the placebo-vehicle group (p < 0.05) and the group treated with IL-6 and rebamipide (p < 0.01). The results of the in vivo examination revealed that the infiltration of neutrophils under the basement membrane and the disruption of tight junction proteins with BAC injection and rebamipide attenuates the disturbance of tissue construction. These results suggest that rebamipide protects LDECs via an anti-inflammatory effect and preserves the barrier function of those cells.

Early intravenous administration of tranexamic acid ameliorates intestinal barrier injury induced by neutrophil extracellular traps in a rat model of trauma/hemorrhagic shock

BackgroundEarly intravenous administration of tranexamic acid has been shown to protect the intestinal barrier after a model of trauma-hemorrhagic shock in the rat, but the potential mechanism remains unclear. Our previous studies have demonstrated that neutrophil extracellular traps contribute to the intestinal barrier dysfunction during sepsis and other critical conditions. Meanwhile, there are high levels of neutrophil infiltration in the intestine during trauma-hemorrhagic shock. Here, we hypothesized that neutrophil extracellular trap formation played a vital role during trauma-hemorrhagic shock-induced intestinal injury and that tranexamic acid, a serine protease inhibitor, may inhibit neutrophil extracellular trap formation and protect intestinal barrier function in trauma-hemorrhagic shock. MethodsA model of trauma-hemorrhagic shock in male rats was established. The rats were divided into 6 groups: (1) sham group; (2) trauma-hemorrhagic shock group; (3) trauma-hemorrhagic shock + DNase I group; (4) trauma-hemorrhagic shock + tranexamic acid group; (5) trauma-hemorrhagic shock + tranexamic acid (different time) group; and (6) trauma-hemorrhagic shock + tranexamic acid (different doses) group. The DNase I solution was injected intravenously to disrupt neutrophil extracellular traps immediately after the trauma-hemorrhagic shock model was completed. After 24 hours, the small intestine and blood were collected for analysis. Human neutrophils were harvested and incubated with phorbol-12-myristate-13-acetate or tranexamic acid, generation of reactive oxygen species, and key proteins expression were detected. ResultsTrauma-hemorrhagic shock induced the formation of intestinal neutrophil extracellular traps and disrupted the intestinal tight junction proteins. Clearing of neutrophil extracellular traps by DNase I resulted in increased expression of tight junction proteins and alleviated the intestinal injury. Early intravenous tranexamic acid administration (1 hour after trauma-hemorrhagic shock) decreased neutrophil extracellular trap formation and prevented tight junction protein disruption compared to the non-tranexamic acid group; however, after delayed administration of tranexamic acid (6 hours), there were no changes in neutrophil extracellular trap formation and intestinal injuries compared to the non-tranexamic acid group. Furthermore, tranexamic acid inhibited neutrophil extracellular trap formation and protected the intestinal barrier in a dose-dependent manner and high-dose (20 mg/kg) treatment of tranexamic acid showed a better effect compared with the therapeutic dose (10 mg/kg). The results of thromboelastography demonstrated that the R and K values in the high-dose group decreased (R, 1.85 ± 0.14 vs 3.87 ± 0.16 minutes, P < .001; K, 0.95 ± 0.04 vs 1.48 ± 0.07 minutes, P < .001), accompanied by a decrease in LY30, indicating that treatment with a high dose of tranexamic acid may cause hypercoagulability and shutdown of fibrinolysis. In addition, less neutrophil extracellular trap formation was detected in neutrophils incubated with neutrophils via an reactive oxygen species-dependent pathway. ConclusionWe first demonstrated a novel role of neutrophil extracellular traps in the pathophysiology of intestinal barrier dysfunction during trauma-hemorrhagic shock. Notably, early but not delayed intravenous administration of tranexamic acid effectively inhibits neutrophil extracellular trap formation and protects intestinal barrier function. Therefore, these results suggested a potential theoretic intervention for the protection of the intestinal barrier during trauma-hemorrhagic shock. In such a process, tranexamic acid appears to regulate neutrophil extracellular trap formation via the classic reactive oxygen species/mitogen-activated protein kinase pathway.

High Mobility Group Box 1 Mediates TMAO-Induced Endothelial Dysfunction.

The intestinal microbe-derived metabolite trimethylamine N-oxide (TMAO) is implicated in the pathogenesis of cardiovascular diseases (CVDs). The molecular mechanisms of how TMAO induces atherosclerosis and CVDs’ progression are still unclear. In this regard, high-mobility group box protein 1 (HMGB1), an inflammatory mediator, has been reported to disrupt cell–cell junctions, resulting in vascular endothelial hyper permeability leading to endothelial dysfunction. The present study tested whether TMAO associated endothelial dysfunction results via HMGB1 activation. Biochemical and RT-PCR analysis showed that TMAO increased the HMGB1 expression in a dose-dependent manner in endothelial cells. However, prior treatment with glycyrrhizin, an HMGB1 binder, abolished the TMAO-induced HMGB1 production in endothelial cells. Furthermore, Western blot and immunofluorescent analysis showed significant decrease in the expression of cell–cell junction proteins ZO-2, Occludin, and VE-cadherin in TMAO treated endothelial cells compared with control cells. However, prior treatment with glycyrrhizin attenuated the TMAO-induced cell–cell junction proteins’ disruption. TMAO increased toll-like receptor 4 (TLR4) expression in endothelial cells. Inhibition of TLR4 expression by TLR4 siRNA protected the endothelial cells from TMAO associated tight junction protein disruption via HMGB1. In conclusion, our results demonstrate that HMGB1 is one of the important mediators of TMAO-induced endothelial dysfunction.

Faecal Proteases from Pouchitis Patients Activate Protease Activating Receptor-2 to Disrupt the Epithelial Barrier.

The pathogenesis of pouch inflammation may involve epithelial barrier disruption. We investigated whether faecal proteolytic activity is increased during pouchitis and results in epithelial barrier dysfunction through protease activating receptor [PAR] activation, and assessed whether the intestinal microbiome may be the source of the proteases. Faecal samples were measured for protease activity using a fluorescein isothiocyanate [FITC]-casein florescence assay. Caco-2 cell monolayers were exposed to faecal supernatants to assess permeability to FITC-dextran. Tight junction protein integrity and PAR activation were assessed by immunoblot and immunofluorescence. A truncated PAR2 protein in Caco-2 cells was achieved by stable transfection using CRISPR/Cas9 plasmid. PAR2 activation in pouch biopsies was examined using antibodies directed to the N-terminus of the protein. Microbial composition was analysed based on 16S rRNA gene sequence analysis. Ten pouchitis patients, six normal pouch [NP] patients and nine healthy controls [HC] were recruited. The pouchitis patients exhibited a 5.19- and 5.35-fold higher faecal protease [FP] activity [p ≤ 0.05] compared to the NP and HC participants, respectively. The genus Haemophilus was positively associated with FP activity [R = 0.718, false discovery rate < 0.1]. Faecal supernatants from pouchitis patients activated PAR2 on Caco-2 monolayers, disrupted tight junction proteins and increased epithelial permeability. PAR2 truncation in Caco-2 abrogated faecal protease-mediated permeability. Pouch biopsies obtained from pouchitis patients, but not from NP patients, displayed PAR2 activation. Protease-producing bacteria may increase faecal proteolytic activity that results in pouch inflammation through disruption of tight junction proteins and increased epithelial permeability in a PAR2-dependent manner. This mechanism may initiate or propagate pouch inflammation.

Intracerebral matrix metalloproteinase 9 in fatal diabetic ketoacidosis

There is increasing awareness that in addition to the metabolic crisis of diabetic ketoacidosis (DKA) caused by severe insulin deficiency, the immune inflammatory response is likely an active multicomponent participant in both the acute and chronic insults of this medical crisis, with strong evidence of activation for both the cytokine and complement system. Recent studies report that the matrix metalloproteinase enzymes and their inhibitors are systemically activated in young Type 1 diabetes mellitus (T1D) patients during DKA and speculate on their involvement in blood-brain barrier (BBB) disruption. Based on our previous studies, we address the question if matrix metalloproteinase 9 (MMP9) is expressed in the brain in the fatal brain edema (BE) of DKA. Our data show significant expression of MMP9 on the cells present in brain intravascular areas. The presence of MMP9 in intravascular cells and that of MMP+ cells seen passing the BBB indicates a possible role in tight junction protein disruption of the BBB, possibly leading to neurological complications including BE. We have also shown that MMP9 is expressed on neurons in the hippocampal areas of both BE/DKA cases investigated, while expression of tissue inhibitor of metalloproteinases 1 (TIMP1) was reduced in the same areas. We can speculate that intraneuronal MMP9 can be a sign of neurodegeneration. Further studies are necessary to determine the role of MMP9 in the pathogenesis of the neurologic catastrophe of the brain edema of DKA. Inhibition of MMP9 expression might be helpful in preserving neuronal function and BBB integrity during DKA.

Gastrointestinal Barrier Breakdown and Adipose Tissue Inflammation.

Obesity is a state of chronic inflammation. This review aims to summarize recent data supporting the role of the intestinal mucosal barrier and the microbiome in causing adipose tissue inflammation as well as metabolic factors that can affect the intestinal barrier. Obesity and its metabolic consequences, such as diabetes mellitus, are associated with disruption of the intestinal barrier function. Intestinal microbiota and diet play a key role in the maintenance of a healthy intestinal epithelium. Intestinal barrier dysfunction can lead to heightened inflammation, which in turn can further damage the intestinal barrier through the disruption of tight junction proteins. Intestinal barrier breakdown is associated with adipose tissue inflammation in different disease states, such as obesity, diabetes mellitus, HIV, and inflammatory bowel disease. Future therapeutic strategies to ameliorate intestinal barrier function may help reduce inflammation in obesity and other chronic conditions of increased intestinal permeability.