Upregulation Of Tight Junction Proteins Research Articles

Biofabrication of a novel biomolecule-assisted reduced graphene oxide: an excellent biocompatible nanomaterial.

Graphene has been shown much interest, both in academics and industry due to its extraordinary physical, chemical, and biological proprieties. It shows great promises in biotechnological and biomedical applications as an antibacterial and anticancer agent, nanocarrier, sensor, etc. However, many studies demonstrated the toxicity of graphene in several cell lines, which is an obstacle to its use in biomedical applications. In this study, to improve the biocompatibility of graphene, we used nicotinamide (NAM) as a reducing and stabilizing agent to catalyze the reduction of graphene oxide (GO) to reduced graphene oxide (rGO). The resulted smaller-sized GO (NAM-rGO) showed excellent biocompatibility with mouse embryonic fibroblast cells, evidenced by various cellular assays. Furthermore, NAM-rGO had no effect on mitochondrial membrane permeability and caspase-3 activity compared to GO. Reverse transcription polymerase chain reaction analysis allowed us to identify the molecular mechanisms responsible for NAM-rGO-induced biocompatibility. NAM-rGO significantly induced the expression of genes encoding tight junction proteins (TJPs) such as zona occludens-1 (Tjp1) and claudins (Cldn3) without any effect on the expression of cytoskeleton proteins. Furthermore, NAM-rGO enhances the expression of alkaline phosphatase (ALP) gene, and it does this in a time-dependent manner. Overall, our study depicted the molecular mechanisms underlying NAM-rGO biocompatibility depending on upregulation of TJPs and ALP. This potential quality of graphene could be used in diverse applications including tissue regeneration and tissue engineering.

Up-regulation of Tight-Junction Proteins by p38 Mitogen-Activated Protein Kinase/p53 Inhibition Leads to a Reduction of Injury to the Intestinal Mucosal Barrier in Severe Acute Pancreatitis.

The aim of this study was to explore the role of the p38 mitogen-activated protein kinase (p38MAPK)/p53 signaling pathway in injury to the intestinal mucosal barrier during severe acute pancreatitis (SAP). Both sham operation and SAP groups had 3 subgroups analyzed 3, 6, or 12 hours after the SAP induction. The concentrations of amylase, endotoxin, diamine oxidase, tumor necrosis factor α, and phospho-p38MAPK, p53, and caspase-3 and the messenger RNA levels of zonula occludens protein-1 and occludin in the intestine were measured. Immunohistochemical staining was used to determine the expression of zonula occludens protein-1 and occludin. Pathological changes of the pancreas and intestine were also assessed. Then, rats were randomly assigned to 5 groups-sham operation group, SAP group, 3 groups treated with different concentrations of p38MAPK-inhibitor SB203580-and the abovementioned experiment was repeated and analyzed 6 hours after the SAP induction. The phospho-p38MAPK reached a peak value at 6 hours after the SAP induction with obvious pathological injury to the pancreas and intestine. Treatment with SB203580 led to a less damage to the pancreatic and intestinal tissues. These results suggest that SAP activates the p38MAPK/p53 signaling pathway and induces injury to the intestinal mucosal barrier, which can be alleviated by inhibiting the p38MAPK/p53 pathway.

Serum-Derived Bovine Immunoglobulin/Protein Isolate for the Management of Diarrhea Associated with Chronic Pancreatitis and Pancreatic Insufficiency: A Novel Approach

Chronic pancreatitis (CP) is a debilitating disorder that ultimately leads to pancreatic insufficiency (PI) and its sequelae. Patients with CP often experience abdominal pain, bloating, nausea, vomiting, weight loss, and steatorrhea. The dearth of pancreatic enzyme secretion in these patients leads to maldigestion, excess fat in the stool, and compromised nutritional status. A typical treatment for management of CP is pancreatic digestive enzyme therapy with pancrelipase, but it is usually ineffective in controlling pain and clinical symptoms, with potential risk of intestinal strictures. Serum-derived bovine immunoglobulin/protein isolate (SBI) is a prescription medical food that has been shown to manage chronic loose and frequent stools in patients with IBS-D, IBD, and HIV enteropathy. SBI has a multifaceted mode of action that through binding of microbial components, results in a downstream reduction in inflammatory markers and up-regulation of tight junction proteins, thus increasing barrier function. This results in improved nutrient utilization, as reported in non-clinical and clinical studies. For this reason, SBI was added to the therapy regimen of a CP/PI patient. The patient is a 73 year old female diagnosed with idiopathic CP and PI in 2012. She has a history of papillary stenosis diagnosed by ERCP with sphincterotomy. She also had chronic pancreatic steatorrhea that was managed with 5-6 doses of pancrelipase per day (at meals and snacks). This patient developed persistent bloating, distension, abdominal pain, and constipation, secondary to pancrelipase therapy. SBI 5g BID was added to her therapy regimen. Upon follow-up 7 weeks later, the patient reported improved discomfort, bloating and regularity of bowel movements to 1 every 2-3 days. The dosing of pacrealipase therapy was reduced due to this favorable response to SBI. She continues on SBI and is doing remarkably well in managing her condition, including her steatorrhea. Three other patients with CP/PI have also responded well to SBI therapy. For this patient, SBI proved to be a novel, safe option for the management of gastrointestinal issues associated with CP/PI. The ability of SBI to return the gut to homeostasis, restore barrier function, and thus allow for the proper absorption of nutrients from the diet may be the reason for its efficacy in this patient. A larger, well-controlled study in CP/PI patients is recommended.

359. Gene Transfer of the Na, K-ATPase beta1 Subunit and Occludin Using Electroporation Rescues Lipopolysaccaride (LPS)-Induced Acute Lung Injury

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are characterized by acute respiratory failure and associated with diverse disorders. Gene therapy is a potentially powerful approach to treat ALI and ARDS through repair of alveolar epithelial function. We previously showed that electroporation-mediated gene transfer of the b1 subunit of the Na,K-ATPase to the lung not only increased alveolar fluid clearance, but also improved epithelium/endothelium barrier functions via up-regulation of tight junction proteins to provide partial treatment of lipopolysaccaride- (LPS-) injured lungs. To further investigate if we could improve treatment, we co-delivered plasmids expressing occludin, which is the one of major constituents of tight junctions located at the plasma membrane. Lung injury was induced 1 day before gene delivery by intratracheal administration of LPS (5 mg/kg) and assessed by wet-to-dry ratios, histological analysis, myeloperoxidase (MPO) measurement, and bronchoalveolar lavage (BAL) protein levels and cellularity. Delivery of b1-Na,K-ATPase and occludin alone or in combination using electroporation treated previously injured lungs in a mouse model, compared to the control plasmid pCDNA3. However, combined gene transfer of b1-Na,K-ATPase and occludin only provided similar treatment benefit to either gene delivery of b1-Na,K-ATPase or occludin alone although the expression of tight junction proteins had been increased. These results demonstrate that both b1-Na,K-ATPase and occludin regulate formation of tight junctions in severely injured lungs and thus may play a critical role in the treatment of ALI/ARDS. It indicates that epithelial or endothelial barrier functions may be involved in the treatment of ALI/ARDS.

Molecular mechanisms for NG-nitro-L-arginine methyl ester action against cerebral ischemia–reperfusion injury-induced blood–brain barrier dysfunction

Abstract Background: Ischemic stroke, an acute neurological injury lacking an effective therapy, is a leading cause of death worldwide. The unmet need in stroke research is to identify viable therapeutic targets and to understand their interplay during cerebral ischemia-reperfusion (I/R) injury. Objective: To explore the protective effects and molecular mechanism of NG-nitro-L-arginine methyl ester (L-NAME) in cerebral ischemia-reperfusion injury-induced blood-brain barrier (BBB) dysfunction. Methods: Two hundred fifty-six rats were randomly assigned to a sham operation group, I/R group, and I/R with L-NAME treatment group. Brain water content was determined by calculating dry/wet weight. The permeability of the BBB was observed using an electron microscope and by determining the Evans Blue leakage from brain tissue on the ischemic side. The expression of brain MMP-9 and GFAP was determined using an immunohistochemical method. The expression of ZO-1 protein was determined by western blotting. Results: We found that L-NAME remarkably attenuated the permeability of the BBB after I/R as assessed by Evans Blue leakage and brain water content (p < 0.05). This was further confirmed by examination of the ultrastructural morphology of the BBB using a transmission electron microscope. Furthermore, we found that expression of the zonae occludens-1 (ZO-1) was decreased in endothelial cells, and expression of MMP-9 and GFAP was increased in the basement membrane and astrocyte end-feet in vehicle control groups, respectively, but these changes could be prevented by L-NAME pretreatment. Conclusion: These results suggested that the neuroprotective effects of L-NAME against BBB damage induced by I/R might be related to the upregulation of tight junction proteins and inhibition of MMP-9 and GFAP expression. L-NAME can be used as a potential MMP-9-based multiple targeting therapeutic strategy in cerebral I/R injury.

Type I Interferon Protects against Pneumococcal Invasive Disease by Inhibiting Bacterial Transmigration across the Lung

Streptococcus pneumoniae infection is a leading cause of bacterial pneumonia, sepsis and meningitis and is associated with high morbidity and mortality. Type I interferon (IFN-I), whose contribution to antiviral and intracellular bacterial immunity is well established, is also elicited during pneumococcal infection, yet its functional significance is not well defined. Here, we show that IFN-I plays an important role in the host defense against pneumococci by counteracting the transmigration of bacteria from the lung to the blood. Mice that lack the type I interferon receptor (Ifnar1 −/−) or mice that were treated with a neutralizing antibody against the type I interferon receptor, exhibited enhanced development of bacteremia following intranasal pneumococcal infection, while maintaining comparable bacterial numbers in the lung. In turn, treatment of mice with IFNβ or IFN-I-inducing synthetic double stranded RNA (poly(I:C)), dramatically reduced the development of bacteremia following intranasal infection with S. pneumoniae. IFNβ treatment led to upregulation of tight junction proteins and downregulation of the pneumococcal uptake receptor, platelet activating factor receptor (PAF receptor). In accordance with these findings, IFN-I reduced pneumococcal cell invasion and transmigration across epithelial and endothelial layers, and Ifnar1 −/− mice showed overall enhanced lung permeability. As such, our data identify IFN-I as an important component of the host immune defense that regulates two possible mechanisms involved in pneumococcal invasion, i.e. PAF receptor-mediated transcytosis and tight junction-dependent pericellular migration, ultimately limiting progression from a site-restricted lung infection to invasive, lethal disease.

Effects of selective hypothermia on blood-brain barrier integrity and tight junction protein expression levels after intracerebral hemorrhage in rats

Hypothermia has neuroprotective effects on global cerebral ischemic injuries. However, its efficacy after intracerebral hemorrhage (ICH) is inconclusive. In this study, bacterial collagenase was used to induce ICH stroke in male Wistar rats. We assessed the effects of normothermia and 4 h of local hypothermia (~33.2°C) initiated 1, 6, or 12 h after collagenase infusion on hemorrhage volume and neurological outcomes. Following early cooling initiated after 1 h, blood-brain barrier (BBB) disruption and brain water content were tested. Furthermore, the expression levels of tight junction (TJ) proteins (claudin 5 and occludin) and the proinflammatory cytokines interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α) were determined using Western blotting, real-time quantitative PCR, and immunohistochemical staining at 1 and 3 d after ICH. Early local hypothermia tends to reduce hemorrhagic volume and neurological deficits, but the difference is not statistically significant compared with other groups. However, early hypothermia significantly reduces BBB disruption, edema formation, the expression levels of IL-1β and TNF-α, and the loss of TJ proteins. Together, these data suggest that local hypothermia is an effective treatment for edema formation and BBB disruption via the upregulation of TJ proteins and the suppression of TNF-α and IL-1β.

Plasma Aldosterone Levels Increase After Cerebral Ischemia and Mineralocorticoid Receptor Antagonism at the Time of Reperfusion Reduces the Size of the Infarct

Ischemic stroke causes blood brain barrier (BBB) breakdown by an unknown mechanism. Upregulation of tight junction proteins Claudin‐5 (CLN‐5) and Occludin (OCC) is linked to increased BBB permeability with hypoxia. Chronic MR antagonism reduces stroke damage but the acute effects of antagonism are unknown. We hypothesized that aldo increases post‐stroke, and acute MR antagonism reduces damage by regulating CLN‐5 and OCC. Wistar Kyoto (WKY) rats had 2 hours (h) of middle cerebral artery occlusion (MCAO) and reperfusion (R) (n=8), control rats had sham surgery (CON; n=4). Plasma was collected before and, 6 and 48 h post‐surgery. Aldo, measured by ELISA was increased 6 h after MCAO (848±60 vs 449±87 pg, MCAO vs CON, p<0.05) and remained elevated at 48 h (1104±48 vs 625±100, MCAO vs CON, p<0.05). To study acute MR inhibition, WKY rats had 3 h of MCAO and 45 h of R. Spironolactone (SPIR; 25mg/kg I.P.) was given at R (n=11), CON rats received vehicle (n=11). SPIR reduced the infarct size. CLN‐5 mRNA expression, assessed by qRT‐PCR was reduced by SPIR; OCC mRNA unchanged. Interestingly, OCC and CLN‐5 protein expression were decreased by SPIR. These data suggest that aldo increases after stroke and MR inhibition may prevent the damage caused by ischemia by maintaining BBB integrity Protein mRNA %HI CLN‐5 OCC CLN‐5 OCC SPIR 25.7±3.5* 0.2±0.1* 2.2±0.3* 0.2±0.04* 0.2±0.1 CON 35.1± 5.0 0.6±0.1 5.8±1.3 0.4±0.1 0.4±0.04 Mean±SE, p<0.05

Doxycycline-Mediated Protective Effect Against Focal Cerebral Ischemia–Reperfusion Injury Through the Modulation of Tight Junctions and PKCδ Signaling in Rats

The strategy for the development of effective and safe neuroprotective agents has great potential to reduce cerebral ischemia-reperfusion injury and improve the functional outcome in stroke patients. Recently, doxycycline, a tetracycline antibiotic, has been shown to have neuroprotective efficiency in reduction of a variety of ischemia-reperfusion injuries as well as ischemic brain damage. We used the rat models of middle cerebral artery occlusion (MCAO) and reperfusion to investigate the effects of treatments with doxycycline against the blood-brain barrier (BBB) leakage at 3, 12, 72, and 120h of reperfusion. Male Sprague-Dawley rats were subjected to MCAO for 2h followed by reperfusion for 3, 12, 72, and 120h and received either doxycycline (45mg/kg) or saline. The results showed that the treatment of doxycycline significantly reduced the BBB leakage and cerebral infarct volume, which were proved by Evans blue assay and TTC staining. Real-time PCR, immunohistochemistry, and western blot assay verified that the administration of doxycycline significantly up-regulated the expression of tight junction claudin-5, occludin, and ZO-1 from 3 to 120h after reperfusion. The results of real-time PCR, western blot, and gelatin zymography analyses revealed that the gene and protein expression and activities of matrix metalloproteinases (MMPs) MMP-2 and MMP-9 were significantly elevated in a different time-dependent manner after ischemia-reperfusion but significantly inhibited by doxycycline treatment. Moreover, doxycycline could also significantly down-regulate the expression of PKCδ mRNA and protein after ischemia-reperfusion. These results suggested that the protective effects of doxycycline against BBB damage induced by reperfusion might be related to the up-regulation of tight junction proteins and inhibition of MMP-2, MMP-9, and PKCδ.

Thymic stromal lymphopoietin enhances tight-junction barrier function of human nasal epithelial cells

Epithelial-derived thymic stromal lymphopoietin (TSLP) triggers dendritic cell (DC)-mediated Th2-type inflammatory responses and is a master switch for allergic inflammatory diseases. In the present study, the expression and induction of TSLP and the effects of TSLP on the tight-junctional barrier of human nasal epithelial cells (HNECs) have been investigated in order to elucidate the role of TSLP in allergic rhinitis. We have found high expression of TSLP in the epithelium from patients with allergic rhinitis with recruitment and infiltration of DCs. In vitro, TSLP is significantly produced in HNECs after treatment with a toll-like receptor 2 (TLR2) ligand, Pam(3)Cys-Ser-(Lys)(4), and a mixture of interleukin-1beta and tumor necrosis factor-alpha. Treatment with TSLP rapidly enhances the barrier function of cultured HNECs, together with an increase of tight-junction proteins claudin-1, -4, -7, and occludin. The nasal-epithelial-derived TSLP thus not only activates DCs but also preserves the epithelial barrier via the upregulation of tight-junction proteins, thereby regulating antigen sensitization during the early stage of allergic rhinitis.

Protein Kinase C Enhances Tight Junction Barrier Function of Human Nasal Epithelial Cells in Primary Culture by Transcriptional Regulation

The epithelium of upper respiratory tissues such as human nasal mucosa forms a continuous barrier via tight junctions, which is thought to be regulated in part through a protein kinase C (PKC) signaling pathway. To investigate the mechanisms of the regulation of PKC-mediated tight junction barrier function of human nasal epithelium in detail, primary human nasal epithelial cells were treated with the PKC activator 12-O-tetradecanoylophorbol-13-acetate (TPA). In primary human nasal epithelial cells, treatment with TPA led not only to activation of phosphorylation of PKC, myristoylated alanine-rich C kinase substrate, and mitogen-activated protein kinase but also expression of novel PKC-delta, PKC-theta, and PKC-epsilon. Treatment with TPA increased transepithelial electrical resistance, with tight junction barrier function more than 4-fold that of the control, together with up-regulation of tight junction proteins, occludin, zona occludens (ZO)-1, ZO-2 and claudin-1 at the transcriptional level. Furthermore, it affected the subcellular localization of the tight junction proteins and the numbers of tight junction strands. The up-regulation of barrier function and tight junction proteins was prevented by a pan-PKC inhibitor, and the inhibitors of PKC-delta and PKC-theta but not PKC-epsilon. In primary human nasal epithelial cells, transcriptional factors GATA-3 and -6 were detected by reverse transcription-polymerase chain reaction. The knockdown of GATA-3 using RNA interference resulted in inhibition of up-regulation of ZO-1 and ZO-2 by treatment with TPA. These results suggest that TPA-induced PKC signaling enhances the barrier function of human nasal epithelial cells via transcriptional up-regulation of tight junction proteins, and the mechanisms may contribute to a drug delivery system.