Degradation Of Tight Junction Proteins Research Articles

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

Background and purposeThe 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. MethodsBEnd3 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. ResultsOur 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. ConclusionsOur 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.

Role of MMP-9 and NF-κB in Diabetic Retinopathy: Progression and Potential Role of Bioflavonoids in the Mitigation of Diabetic Retinopathy.

"Diabetes mellitus" is a chronic metabolic disorder manifested by elevated blood glucose levels, primarily due to insufficient insulin production or resistance to insulin. Long-term diabetes results in persistent complications like retinopathy, cardiomyopathy, nephropathy, and neuropathy, causing significant health risks. The most alarming microvascular consequence allied with diabetes is "diabetic retinopathy," distinguished by the proliferation of anomalous blood vessels in the eye, mainly in the retina, resulting in visual impairment, diabetic macular edema, and retinal detachment if left untreated. According to estimates, 27.0% of people with diabetes worldwide have retinopathy, which leads to 0.4 million blindness cases. It is believed that mitochondrial damage and the production of inflammatory mediators are the early indicators of diabetic retinopathy before any histological changes occur in the retina. Moreover, it is evident that augmented oxidative stress in the retina further initiates the NF-κB/MMP-9 downstream signaling pathway. Interestingly, these downstream regulators, Nuclear Factor Kappa B [NF- kB] and matrix metalloproteinases 9 [MMP-9], have been recognized as important regulators of the inception and advancement of diabetic retinopathy. This diabetes and oxidative stress-induced MMP-9 are believed to regulate various cellular functions, including angiogenesis and apoptosis, causing blood-retinal barrier breakdown and tight junction protein degradation that further leads to diabetic retinopathy. Thus, there is an emergency need for the treatment of diabetic retinopathy. Emerging treatment options include anti-VEGF, laser treatment, and eye surgery, but these have certain limitations. This comprehensive review explores the mechanisms of MMP-9 and NF-kB involvement in diabetic retinopathy and bioflavonoids' therapeutic potential and mechanisms of action in inhibiting MMP-9 activity and suppressing NF-kB-mediated inflammation. Clinical evidence supporting the use of bioflavonoids in mitigating diabetic complications and future perspectives are also examined.

Japanese encephalitis virus NS1 and NS1' protein disrupts the blood-brain barrier through macrophage migration inhibitory factor-mediated autophagy.

Flaviviruses in the Japanese encephalitis virus (JEV) serogroup, such as JEV, West Nile virus, and St. Louis encephalitis virus, can cause severe neurological diseases. The nonstructural protein 1 (NS1) is a multifunctional protein of flavivirus that can be secreted by infected cells and circulate in the host bloodstream. NS1' is an additional form of NS1 protein with 52 amino acids extension at its carboxy-terminal and is produced exclusively by flaviviruses in the JEV serogroup. In this study, we demonstrated that the secreted form of both NS1 and NS1' can disrupt the blood-brain barrier (BBB) of mice, with NS1' exhibiting a stronger effect. Using the in vitro BBB model, we found that treatment of soluble recombinant JEV NS1 or NS1' protein increases the permeability of human brain microvascular endothelial cells (hBMECs) and leads to the degradation of tight junction proteins through the autophagy-lysosomal pathway. Consistently, NS1' protein exhibited a more pronounced effect compared to NS1 in these cellular processes. Further research revealed that the increased expression of macrophage migration inhibitory factor (MIF) is responsible for triggering autophagy after NS1 or NS1' treatment in hBMECs. In addition, TLR4 and NF-κB signaling was found to be involved in the activation of MIF transcription. Moreover, administering the MIF inhibitor has been shown to decrease viral loads and mitigate inflammation in the brains of mice infected with JEV. This research offers a novel perspective on the pathogenesis of JEV. In addition, the stronger effect of NS1' on disrupting the BBB compared to NS1 enhances our understanding of the mechanism by which flaviviruses in the JEV serogroup exhibit neurotropism.IMPORTANCEJapanese encephalitis (JE) is a significant viral encephalitis worldwide, caused by the JE virus (JEV). In some patients, the virus cannot be cleared in time, leading to the breach of the blood-brain barrier (BBB) and invasion of the central nervous system. This invasion may result in cognitive impairment, behavioral disturbances, and even death in both humans and animals. However, the mechanism by which JEV crosses the BBB remains unclear. Previous studies have shown that the flavivirus NS1 protein plays an important role in causing endothelial dysfunction. The NS1' protein is an elongated form of NS1 protein that is particularly produced by flaviviruses in the JEV serogroup. This study revealed that both the secreted NS1 and NS1' of JEV can disrupt the BBB by breaking down tight junction proteins through the autophagy-lysosomal pathway, and NS1' is found to have a stronger effect compared to NS1 in this process. In addition, JEV NS1 and NS1' can stimulate the expression of MIF, which triggers autophagy via the ERK signaling pathway, leading to damage to BBB. Our findings reveal a new function of JEV NS1 and NS1' in the disruption of BBB, thereby providing the potential therapeutic target for JE.

Irisin links Claudin-5 preservation and Mfn2-mediated mitochondrial dynamics to resist doxorubicin-induced cardiac endothelial damage

Irisin is protective in the cardiac microenvironment and can resist doxorubicin-induced cardiotoxicity. The purpose of this study was to investigate the connection between Irisin, endothelial cell integrity, and mitochondrial dynamics. Primary cardiac microvascular endothelial cells (CMECs) were used to explore the regulatory effects of Irisin on tight junction proteins, mitochondrial dynamics, β-catenin expression, and transcriptional activity. Results showed that Irisin can suppress doxorubicin-induced upregulation of MMP2 and MMP9, thereby reducing the degradation of tight junction proteins (ZO-1 and Claudin-5) and VE-cadherin. The preservation of Claudin-5 contributes to maintaining Mfn2 expression, which in turn supports mitochondrial fusion. Although Irisin restores doxorubicin-induced downregulation of β-catenin, it concurrently limits β-catenin transcriptional activity via Mfn2-mediated sulfenylation. Therefore, this study revealed a novel mechanism linking the protective effects of Irisin on the tight junction proteins and mitochondrial dynamics upon doxorubicin exposure.

ASK1-K716R reduces neuroinflammation and white matter injury via preserving blood–brain barrier integrity after traumatic brain injury

BackgroundTraumatic brain injury (TBI) is a significant worldwide public health concern that necessitates attention. Apoptosis signal-regulating kinase 1 (ASK1), a key player in various central nervous system (CNS) diseases, has garnered interest for its potential neuroprotective effects against ischemic stroke and epilepsy when deleted. Nonetheless, the specific impact of ASK1 on TBI and its underlying mechanisms remain elusive. Notably, mutation of ATP-binding sites, such as lysine residues, can lead to catalytic inactivation of ASK1. To address these knowledge gaps, we generated transgenic mice harboring a site-specific mutant ASK1 Map3k5-e (K716R), enabling us to assess its effects and elucidate potential underlying mechanisms following TBI.MethodsWe employed the CRIPR/Cas9 system to generate a transgenic mouse model carrying the ASK1-K716R mutation, aming to investigate the functional implications of this specific mutant. The controlled cortical impact method was utilized to induce TBI. Expression and distribution of ASK1 were detected through Western blotting and immunofluorescence staining, respectively. The ASK1 kinase activity after TBI was detected by a specific ASK1 kinase activity kit. Cerebral microvessels were isolated by gradient centrifugation using dextran. Immunofluorescence staining was performed to evaluate blood–brain barrier (BBB) damage. BBB ultrastructure was visualized using transmission electron microscopy, while the expression levels of endothelial tight junction proteins and ASK1 signaling pathway proteins was detected by Western blotting. To investigate TBI-induced neuroinflammation, we conducted immunofluorescence staining, quantitative real-time polymerase chain reaction (qRT-PCR) and flow cytometry analyses. Additionally, immunofluorescence staining and electrophysiological compound action potentials were conducted to evaluate gray and white matter injury. Finally, sensorimotor function and cognitive function were assessed by a battery of behavioral tests.ResultsThe activity of ASK1-K716R was significantly decreased following TBI. Western blotting confirmed that ASK1-K716R effectively inhibited the phosphorylation of ASK1, JNKs, and p38 in response to TBI. Additionally, ASK1-K716R demonstrated a protective function in maintaining BBB integrity by suppressing ASK1/JNKs activity in endothelial cells, thereby reducing the degradation of tight junction proteins following TBI. Besides, ASK1-K716R effectively suppressed the infiltration of peripheral immune cells into the brain parenchyma, decreased the number of proinflammatory-like microglia/macrophages, increased the number of anti-inflammatory-like microglia/macrophages, and downregulated expression of several proinflammatory factors. Furthermore, ASK1-K716R attenuated white matter injury and improved the nerve conduction function of both myelinated and unmyelinated fibers after TBI. Finally, our findings demonstrated that ASK1-K716R exhibited favorable long-term functional and histological outcomes in the aftermath of TBI.ConclusionASK1-K716R preserves BBB integrity by inhibiting ASK1/JNKs pathway in endothelial cells, consequently reducing the degradation of tight junction proteins. Additionally, it alleviates early neuroinflammation by inhibiting the infiltration of peripheral immune cells into the brain parenchyma and modulating the polarization of microglia/macrophages. These beneficial effects of ASK1-K716R subsequently result in a reduction in white matter injury and promote the long-term recovery of neurological function following TBI.

MiR-671-5p Upregulation Attenuates Blood–Brain Barrier Disruption in the Ischemia Stroke Model Via the NF-кB/MMP-9 Signaling Pathway

Blood-brain barrier (BBB) disruption can induce further hemorrhagic transformation in ischemic stroke (IS). miR-671-5p, a micro-RNA, is abundant in the cortex of mammalian brains. Herein, we investigated the roles and potential mechanisms for the effects of miR-671-5p on BBB permeability in IS. Results showed that miR-671-5p levels were significantly downregulated in the cerebral cortex of middle cerebral artery occlusion/reperfusion (MCAO/R) C57/BL6 mice in vivo. miR-671-5p agomir administration via right intracerebroventricular injection significantly reduced infarct volume, improved neurological deficits, the axon of neurons and nerve fiber, attenuated cell injury and apoptosis, as well as reduced BBB permeability in MCAO/R mice. Treatment with miR-671-5p agomir alleviated tight junction proteins degradation, including claudin, occludin, and ZO-1 in MCAO/R mice, and these effects were reversed following NF-κB overexpression. Bend.3 brain endothelial cells were subjected to oxygen and glucose deprivation/reoxygenation (OGD/R) treatment in vivo, and then miR-671-5p agomir was transfected into the cells. This resulted in reduction of cytotoxicity, improved cell viability, trans-endothelial electrical resistance, reduced fluorescein sodium permeability, and inhibited tight junction degradation in Bend.3 OGD/R cells. However, these effects were reversed following NF-κB overexpression. These results demonstrated that upregulation of miR-671-5p in IS models in vivo and in vitro alleviated BBB permeability by targeting NF-κB/MMP-9. In summary, miR-671-5p is a potential therapeutic target for protecting BBB permeability in IS to minimize cerebral hemorrhage transformation.

Local immune dysregulation and subsequent inflammatory response contribute to pulmonary edema caused by Enterovirus infection in mice.

Pulmonary edema that comes on suddenly is the leading cause of mortality in hand-foot-and-mouth disease (HFMD) patients; however, its pathogenesis is still largely unclear. A range of research suggest immunopathogenesis during the occurrence of pulmonary edema in severe HFMD patients. Herein, to investigate the potential mechanism of immune dysregulation in the development of pulmonary edema upon Enterovirus (EV) infection, we established mouse infection models for Enteroviruses (EVs) including Coxsackievirus (CV) A6, Enterovirus A71 (EVA71), and CVA2 exhibiting a high incidence of pulmonary edema. We found that EVs infection induced an immune system disorder by reducing the numbers of pulmonary and circulatory T cells, B cells, macrophages, and monocytes and increasing the numbers of lung neutrophils, myeloid-derived suppressor cells (MDSCs), and activated T cells. In addition, the concentrations of C-X-C motif chemokine ligand 1 (CXCL-1), tumor necrosis factor-alpha, monocyte chemoattractant protein-1, and interleukin 6 were increased in EV-infected lungs. Moreover, we found that EVs replication in mice lungs lead to apoptosis of lung cells and degradation of tight junction proteins. In conclusion, EVs infection likely triggered a complexed immune defense mechanism and caused dysregulation of innate immune cells (MDSCs, neutrophils, monocytes, and macrophages) and adaptive cellular immunity (B cells, T cells). This dysregulation increased the release of cytokines and other inflammatory factors from activated immune-related cells and caused lung barrier damage and pulmonary edema.

Polystyrene nanoplastics promote CHIP-mediated degradation of tight junction proteins by activating IRE1α/XBP1s pathway in mouse Sertoli cells

Microplastics (MPs) and nanoplastics (NPs) widely exist in human living environment and enter the body through water, food chain and breathing. Several studies have shown that MPs or NPs disrupt the blood-testis barrier in rodents. However, the molecular mechanism by which MPs and NPs damage the blood-testis barrier remains unclear. In the present study, our aim was to investigate the molecular mechanism of the destruction of blood-testis barrier induced by polystyrene (PS)-NPs. Mice were treated with 50 μg/kg·day PS-NPs by tail vein injection once daily for two consecutive days. The results showed that PS-NPs exposure significantly decreased the levels of tight junction (TJ) proteins ZO-2, occludin and claudin-11 in testis of mice. In vitro, we used TM4 Sertoli cells to explore the underlying mechanism of the decrease in TJ proteins induced by PS-NPs. We found that PS-NPs activated IRE1α and induced its downstream XBP1 splicing, which in turn elevated the expression of the E3 ubiquitin ligase CHIP, then CHIP triggers proteasomal degradation of ZO-2, occludin, and claudin-11 proteins. Our findings suggest that IRE1α/XBP1s/CHIP pathway is a pivotal mechanism of TJ proteins degradation induced by PS-NPs in mouse Sertoli cells. In conclusion, our results reveal that the degradation of TJ proteins is one of the mechanisms of blood-testis barrier destruction caused by acute exposure to PS-NPs.

Lycium Barbarum polysaccharides ameliorates hyperglycemia-exacerbated cerebral ischemia/reperfusion injury via protecting blood-brain barrier

BackgroundHyperglycemia exacerbates brain damage in cerebral ischemia/reperfusion injury. Previous study found that Lycium barbarum polysaccharides (LBP) has a neuroprotective effect on hyperglycemia-aggravated ischemic brain injury, which raising the possibility for treatment of neurodegenerative diseases. However, the underlying mechanism of LBP-induced protection by ameliorating hyperglycemia-aggravated ischemia/reperfusion injury needs to be tested. This study aimed to investigate the effects of LBP on blood–brain barrier (BBB) integrity with a hyperglycemia-aggravated cerebral ischemia/reperfusion injury model. MethodsSprague-Dawley male rats were randomly divided into three groups: normoglycemic (NG), hyperglycemic (HG), and LBP-pretreated hyperglycemic (HG + LBP). Animals underwent middle cerebral artery occlusion (MCAO) for 30 min, followed by 1-, 3-, and 7-day of reperfusion. ResultsOur results showed that the neurological deficit, infarct volume, cell apoptosis, and IgG leakage in the HG group significantly increased separately, compared with that of the NG group, (p < 0.05). Pre-treatment with LBP reversed these injury indicators (p < 0.05). And much more severe degree of swelling endothelium, swollen astrocyte, and decreased tight junctions in the micro-vessel were detected in the HG group comparing to that of the NG group. In addition, increased degree of basement membrane degradation, dissociation between the astrocyte endfeet and basement membrane, and tight junction's protein degradation was found in the HG group compared with the NG group (p < 0.05). However, when exposure to LBP therapy could reverse the above alterations (p < 0.05). ConclusionsThese results demonstrated that LBP could ameliorate hyperglycemia-exacerbated cerebral ischemia/reperfusion injury via protecting the blood-brain barrier.

MMP-12 knockdown prevents secondary brain damage after ischemic stroke in mice

We previously reported that increased expression of matrix metalloproteinase-12 (MMP-12) mediates blood-brain barrier disruption via tight junction protein degradation after focal cerebral ischemia in rats. Currently, we evaluated whether MMP-12 knockdown protects the post-stroke mouse brain and promotes better functional recovery. Adult male mice were injected with negative siRNA or MMP-12 siRNA (intravenous) at 5 min of reperfusion following 1 h transient middle cerebral artery occlusion. MMP-12 knockdown significantly reduced the post-ischemic infarct volume and improved motor and cognitive functional recovery. Mechanistically, MMP-12 knockdown ameliorated degradation of tight junction proteins zonula occludens-1, claudin-5, and occludin after focal ischemia. MMP-12 knockdown also decreased the expression of inflammatory mediators, including monocyte chemoattractant protein-1, tumor necrosis factor-α, and interleukin-6, and the expression of apoptosis marker cleaved caspase-3 after ischemia. Overall, the present study indicates that MMP-12 promotes secondary brain damage after stroke and hence is a promising stroke therapeutic target.

Improvement of the bladder perfusion curative effect through tight junction protein degradation induced by magnetic temperature-sensitive hydrogels.

Postoperative intravesical instillation of chemotherapy is a routine procedure for non-muscular invasive bladder cancer (NMIBC). However, traditional bladder perfusion methods have insufficient exposure time, resulting in unsatisfactory therapeutic effects. In the present study, a chitosan (CS)-based in situ forming depot (ISFD) delivery system, including Fe3O4 magnetic nanoparticles (Fe3O4-MNP), CS, and β-glycerophosphate (GP) as main components, was synthesized. Pirarubicin (THP), as a chemotherapeutic drug, was loaded into the new system. Results showed that our carrier system (Fe3O4-THP-CS/GP) was converted into gel and attached to the bladder wall, possessing loose network structures with magnetic targeting and sustained release properties. Moreover, its retention time in bladder was more than 72 h accompanied by a suitable expansion rate and good degradation characteristics. The antitumor activities of Fe3O4-THP-CS/GP were more effective both in vitro and in vivo than the free THP solution. In the study of its mechanism, results showed that Fe3O4-THP-CS/GP suppressed the expression of occludin (OCLN) and affected tight junctions (TJ) between urothelial cells to promote THP absorption.

Discoidin domain receptor 1(DDR1) promote intestinal barrier disruption in Ulcerative Colitis through tight junction proteins degradation and epithelium apoptosis

Background and aimsDiscoidin domain receptor 1 (DDR1) encodes a receptor tyrosine kinase involved in multiple physiological and pathological processes. DDR1 is expressed in the intestinal epithelium, but its role in Ulcerative Colitis (UC) is poorly understand. This study aimed to identify the function of DDR1 in maintaining the homeostasis of UC. MethodsThe DDR1 expression level in non-inflamed and inflamed colon samples from IBD patients were assessed. DDR1 knock-out (DDR1-/-) and wild-type (WT) mice were administered dextran sulfate sodium (DSS) to induce colitis and assessed based on colitis symptoms. In addition, intestinal epithelial barrier injury was induced by TNF-α and IFN-γ incubation to cell monolayers transfected with PCDH-DDR1 or pLKO.1-sh-DDR1–1 plasmids. The effect of DDR1 in regulating barrier integrity, tight junctions (TJ) protein status, and cell apoptosis was investigated in vivo and in vitro. Furthermore, the activation of the NF-κB p65-MLCK-p-MLC2 pathway was also investigated. ResultsDecreased DDR1 expression levels were observed at the inflamed sites compared with the non-inflamed. DDR1-/- mice had alleviated intestinal mucosal barrier injuries, upregulated TJ proteins, decreased epithelium apoptosis from DSS-induced colitis, and reduced proinflammatory cytokines production in the colon. These findings were further confirmed in vitro. DDR1 over-expression aggravated the TNF-α/IFN-γ-induced TJ disruption, while DDR1 shRNA prevented TJ damage even in the presence of JSH-23. DDR1 dependently destroyed the intestinal barrier via the NF-κB p65-MLCK-p-MLC2 pathway. ConclusionOur findings revealed that DDR1 regulated the intestinal barrier in colitis by modulating TJ proteins expression and epithelium apoptosis, making it a potential target of UC.

TSG-6 released from adipose stem cells-derived small extracellular vesicle protects against spinal cord ischemia reperfusion injury by inhibiting endoplasmic reticulum stress

BackgroundSpinal cord ischemia reperfusion injury (SCIRI) is a complication of aortic aneurysm repair or spinal cord surgery that is associated with permanent neurological deficits. Mesenchymal stem cell (MSC)-derived small extracellular vesicles (sEVs) have been shown to be potential therapeutic options for improving motor functions after SCIRI. Due to their easy access and multi-directional differentiation potential, adipose‐derived stem cells (ADSCs) are preferable for this application. However, the effects of ADSC-derived sEVs (ADSC-sEVs) on SCIRI have not been reported.ResultsWe found that ADSC-sEVs inhibited SCIRI-induced neuronal apoptosis, degradation of tight junction proteins and suppressed endoplasmic reticulum (ER) stress. However, in the presence of the ER stress inducer, tunicamycin, its anti-apoptotic and blood–spinal cord barrier (BSCB) protective effects were significantly reversed. We found that ADSC-sEVs contain tumor necrosis factor (TNF)-stimulated gene-6 (TSG-6) whose overexpression inhibited ER stress in vivo by modulating the PI3K/AKT pathway.ConclusionsADSC-sEVs inhibit neuronal apoptosis and BSCB disruption in SCIRI by transmitting TSG-6, which suppresses ER stress by modulating the PI3K/AKT pathway.

Ginsenoside Rd protects cerebral endothelial cells from oxygen-glucose deprivation/reoxygenation induced pyroptosis via inhibiting SLC5A1 mediated sodium influx

BackgroundGinsenoside Rd is a natural compound with promising neuroprotective effects. However, the underlying mechanisms are still not well-understood. In this study, we explored whether ginsenoside Rd exerts protective effects on cerebral endothelial cells after oxygen-glucose deprivation/reoxygenation (OGD/R) treatment and its potential docking proteins related to the underlying regulations. MethodCommercially available primary human brain microvessel endothelial cells (HBMECs) were used for in vitro OGD/R studies. Cell viability, pyroptosis-associated protein expression and tight junction protein degradation were evaluated. Molecular docking proteins were predicted. Subsequent surface plasmon resonance (SPR) technology was utilized for validation. Flow cytometry was performed to quantify caspase-1 positive and PI positive (caspase-1+/PI+) pyroptotic cells. ResultsGinsenoside Rd treatment attenuated OGD/R-induced damage of blood-brain barrier (BBB) integrity in vitro. It suppressed NLRP3 inflammasome activation (increased expression of NLRP3, cleaved caspase-1, IL-1β and GSDMD-N terminal (NT)) and subsequent cellular pyroptosis (caspase-1+/PI + cells). Ginsenoside Rd interacted with SLC5A1 with a high affinity and reduced OGD/R-induced sodium influx and potassium efflux in HBMECs. Inhibiting SLC5A1 using phlorizin suppressed OGD/R-activated NLRP3 inflammasome and pyroptosis in HBMECs. ConclusionGinsenoside Rd protects HBMECs from OGD/R-induced injury partially via binding to SLC5A1, reducing OGD/R-induced sodium influx and potassium efflux, thereby alleviating NLRP3 inflammasome activation and pyroptosis.

A biomimetic zeolite-based nanoenzyme contributes to neuroprotection in the neurovascular unit after ischaemic stroke via efficient removal of zinc and ROS

Zeolite-based nanomaterials have a large number of applications in the field of medicine due to their high porosity, biocompatibility and biological stability. In this study, we designed cerium (Ce)-doped Linde Type A (LTA) zeolite-based nanomaterials (Ce/Zeo-NMs) as a multifunctional mesoporous nanoenzyme to reduce dysfunction of the neurovascular unit (NVU) and attenuate cerebral ischaemia-reperfusion (I/R) injury. Owing to its unique adsorption capacity and mimetic catalytic activities, Ce@Zeo-NMs adsorbed excess zinc ions and exhibited scavenging activity against reactive oxygen species (ROS) induced by acute I/R, thus reshaping the oxidative and zinc microenvironment in the ischaemic brain. In vivo results demonstrated that Ce@Zeo-NMs significantly reduced ischaemic damage to the NVU by decreasing the infarct area, protecting against breakdown of the blood–brain barrier (BBB) via inhibiting the degradation of tight junction proteins (TJPs) and inhibiting activation of microglia and astrocytes in a rat model of middle cerebral artery occlusion-reperfusion (MCAO/R). Taken together, these findings indicated that Ce@Zeo-NMs may serve as a promising dual-targeting therapeutic agent for alleviating cerebral I/R injury. Statement of SignificanceCerium (Ce)-doped Linde Type A zeolite-based nanomaterials (Ce/Zeo-NMs) as a multifunctional mesoporous nanoenzyme were designed for inducing neuroprotection after ischaemic stroke by reducing dysfunction of the neurovascular unit (NVU). Ce@Zeo-NMs had the ability to adsorb excessive Zn2+ and showed mimetic enzymatic activities. As a result, Ce@Zeo-NMs protected against cerebral ischaemia and reduced the damage of NVU by improving the integrity of blood brain barrier (BBB) and inhibiting activation of microglia and astrocytes in a rat model of middle cerebral artery occlusion-reperfusion (MCAO/R). These findings indicated that Ce@Zeo-NMs may serve as a therapeutic strategy for neuroprotection and functional recovery upon ischaemic stroke onset.

Meningitic Escherichia coli-Induced Interleukin-17A Facilitates Blood-Brain Barrier Disruption via Inhibiting Proteinase 3/Protease-Activated Receptor 2 Axis.

Bacterial meningitis is a life-threatening infectious disease with high morbidity and mortality worldwide, among which meningitic Escherichia coli is a common Gram-negative pathogenic bacterium causing meningitis. It can penetrate the blood–brain barrier (BBB), invoke local inflammatory responses and consequently disrupt the integrity of the BBB. Interleukin-17A (IL-17A) is recognized as a pro-inflammatory cytokine that is released during meningitic E. coli infection. It has been reported that IL-17A is involved in several pathological tissue injuries. However, the function of IL-17A in BBB breakdown remains rarely discussed. Here, our study found that E. coli-induced IL-17A led to the degradation of tight junction proteins (TJs) and adherens junction proteins (AJs) in human brain microvascular endothelial cells (hBMECs) through inhibiting protease proteinase 3 (PRTN3)/protease-activated receptor 2 (PAR-2) axis, thus increasing the permeability of BBB. In summary, this study uncovered the involvement of IL-17A in regulating BBB integrity and proposed a novel regulatory mechanism, which could be potential therapeutic targets of E. coli meningitis.

Secreted gingipains from Porphyromonas gingivalis increase permeability in human cerebral microvascular endothelial cells through intracellular degradation of tight junction proteins

Despite a clear correlation between the infiltration of periodontal pathogens in the brain and cognitive decline in Alzheimer's disease (AD), the precise mechanism underlying bacteria crossing the blood-brain barrier (BBB) remains unclear. The periodontal pathogen Porphyromonas gingivalis produces a unique class of cysteine proteases termed gingipains. Gingipains appear to be key virulence factors that exacerbate sporadic AD. We herein report that gingipains are involved in increasing permeability of hCMEC/D3 cell monolayer, human cerebral microvascular endothelial cell lines, through degradation of tight junction proteins including Zonula occludens-1 (ZO-1) and occludin. There was a significant decrease in the mean protein levels of ZO-1 and occludin after infection of hCMEC/D3 cells with wild-type (WT) P. gingivalis. However, infection of these cells with a gingipain-deficient P. gingivalis strain showed significantly lower reduction of the mean protein levels of either ZO-1 and occludin, compared to the WT strain. Similar results were obtained after treatment with culture supernatant from WT and gingipain-deficient P. gingivalis strains. In vitro digestion of human recombinant ZO-1 and occludin by WT P. gingivalis culture supernatant in the absence or presence of gingipain inhibitors indicated that gingipains directly degraded these tight junction proteins. A close immunohistochemical examination using anti-gingipain antibody further revealed that gingipains localized in the cytosol and nuclei of hCMEC/D3 cells after infection with WT P. gingivalis and treatment with its culture supernatant. Furthermore, intracellular localization of outer membrane vesicles (OMVs) bound gingipains from WT P. gingivalis and OMV-induced degradation of ZO-1 and occludin were also observed in hCMEC/D3 cells. Thus, the delivery of gingipains into the cerebral microvascular endothelial cells, probably through OMV, may be responsible for the BBB damage through intracellular degradation of ZO-1 and occludin.

Neurovascular protection by adropin in experimental ischemic stroke through an endothelial nitric oxide synthase-dependent mechanism

Adropin is a highly-conserved peptide that has been shown to preserve endothelial barrier function. Blood-brain barrier (BBB) disruption is a key pathological event in cerebral ischemia. However, the effects of adropin on ischemic stroke outcomes remain unexplored. Hypothesizing that adropin exerts neuroprotective effects by maintaining BBB integrity, we investigated the role of adropin in stroke pathology utilizing loss- and gain-of-function genetic approaches combined with pharmacological treatment with synthetic adropin peptide. Long-term anatomical and functional outcomes were evaluated using histology, MRI, and a battery of sensorimotor and cognitive tests in mice subjected to ischemic stroke. Brain ischemia decreased endogenous adropin levels in the brain and plasma. Adropin treatment or transgenic adropin overexpression robustly reduced brain injury and improved long-term sensorimotor and cognitive function in young and aged mice subjected to ischemic stroke. In contrast, genetic deletion of adropin exacerbated ischemic brain injury, irrespective of sex. Mechanistically, adropin treatment reduced BBB damage, degradation of tight junction proteins, matrix metalloproteinase-9 activity, oxidative stress, and infiltration of neutrophils into the ischemic brain. Adropin significantly increased phosphorylation of endothelial nitric oxide synthase (eNOS), Akt, and ERK1/2. While adropin therapy was remarkably protective in wild-type mice, it failed to reduce brain injury in eNOS-deficient animals, suggesting that eNOS is required for the protective effects of adropin in stroke. These data provide the first causal evidence that adropin exerts neurovascular protection in stroke through an eNOS-dependent mechanism. We identify adropin as a novel neuroprotective peptide with the potential to improve stroke outcomes.

DCA Protects against Oxidation Injury Attributed to Cerebral Ischemia-Reperfusion by Regulating Glycolysis through PDK2-PDH-Nrf2 Axis.

Cerebral ischemic stroke (IS) is still a difficult problem to be solved; energy metabolism failure is one of the main factors causing mitochondrion dysfunction and oxidation stress damage within the pathogenesis of cerebral ischemia, which produces considerable reactive oxygen species (ROS) and opens the blood-brain barrier. Dichloroacetic acid (DCA) can inhibit pyruvate dehydrogenase kinase (PDK). Moreover, DCA has been indicated with the capability of increasing mitochondrial pyruvate uptake and promoting oxidation of glucose in the course of glycolysis, thereby improving the activity of pyruvate dehydrogenase (PDH). As a result, pyruvate flow is promoted into the tricarboxylic acid cycle to expedite ATP production. DCA has a protective effect on IS and brain ischemia/reperfusion (I/R) injury, but the specific mechanism remains unclear. This study adopted a transient middle cerebral artery occlusion (MCAO) mouse model for simulating IS and I/R injury in mice. We investigated the mechanism by which DCA regulates glycolysis and protects the oxidative damage induced by I/R injury through the PDK2-PDH-Nrf2 axis. As indicated from the results of this study, DCA may improve glycolysis, reduce oxidative stress and neuronal death, damage the blood-brain barrier, and promote the recovery of oxidative metabolism through inhibiting PDK2 and activating PDH. Additionally, DCA noticeably elevated the neurological score and reduced the infarct volume, brain water content, and necrotic neurons. Moreover, as suggested from the results, DCA elevated the content of Nrf2 as well as HO-1, i.e., the downstream antioxidant proteins pertaining to Nrf2, while decreasing the damage of BBB and the degradation of tight junction proteins. To simulate the condition of hypoxia and ischemia in vitro, HBMEC cells received exposure to transient oxygen and glucose deprivation (OGD). The DCA treatment is capable of reducing the oxidative stress and blood-brain barrier of HBMEC cells after in vitro hypoxia and reperfusion (H/R). Furthermore, this study evidenced that HBMEC cells could exhibit higher susceptibility to H/R-induced oxidative stress after ML385 application, the specific inhibitor of Nrf2. Besides, the protection mediated by DCA disappeared after ML385 application. To sum up, as revealed from the mentioned results, DCA could exert the neuroprotective effect on oxidative stress and blood-brain barrier after brain I/R injury via PDK2-PDH-Nrf2 pathway activation. Accordingly, the PDK2-PDH-Nrf2 pathway may play a key role and provide a new pharmacology target in cerebral IS and I/R protection by DCA.

Bryodulcosigenin a natural cucurbitane-type triterpenoid attenuates dextran sulfate sodium (DSS)-induced colitis in mice

BackgroundBryodulcosigenin (BDG) a cucurbitane-type triterpenoid has been isolated from the roots of Bryonia dioca and possesses marked anti-inflammatory effects, although its beneficial effect against intestinal disorders remains unclear. PurposeTo explore the underlying mechanism of BDG on the dysbiosis of chronic ulcerative colitis (UC) and its associated side-effects on lung tissues. MethodsA chronic UC model was established using 2.5% dextran sulfate sodium (DSS) in mice treated for 64 days and diagnostic assessments, western blot analysis and quantitative real time-PCR were employed to determine the protective mechanism of BDG. ResultsOral administration of BDG (10 mg/kg/day) significantly improved colon length, disease activity index, and alleviated colonic histopathological damage in the DSS-induced colitis mice. BDG not only reversed the TNF-α-induced degradation of tight junction proteins (occludin and ZO-1) but also suppressed the elevated apoptosis seen in intestinal epithelial cells (NCM460). In addition, BDG significantly attenuated damage in alveolar epithelial cells (MLE-12) co-cultured with NCM460 cells under inflammatory conditions. Furthermore, BDG in vivo significantly prevented the symptoms of respiratory disorders and repressed alveolar inflammation by regulating DSS-induced chronic colitis in mice. ConclusionBDG effectively inhibited the apoptosis of intestinal epithelial cells and suppressed the activation of the NLRP3 inflammasome which resulted in the restoration of the intestinal barrier. Therefore, the enhanced integrity of intestinal epithelial cells produced by BDG intervention contributed to its anti-colitis effects, indicating its great potential as an inhibitor of UC and lung injury. Therefore, restoring intestinal integrity may represent a promising strategy in the prevention of pulmonary disease.