Modulation Of Tight Junction Proteins Research Articles

Quercetin Abrogates Oxidative Neurotoxicity Induced by Silver Nanoparticles in Wistar Rats

This study aimed to investigate the oxidative neurotoxicity induced by silver nanoparticles (AgNPs) and assess the neuroprotective effects of quercetin against this toxicity. Forty adult male rats were divided into four equal groups: control, AgNPs (50 mg/kg intraperitoneally), quercetin (50 mg/kg orally), and quercetin + AgNPs. After 30 days, blood and brain tissue samples were collected for further studies. AgNP exposure increased lipid peroxidation and decreased glutathione peroxidase, catalase, and superoxide dismutase activities in brain tissue. AgNPs decreased serum acetylcholine esterase activity and γ-aminobutyric acid concentrations. AgNPs upregulated tumor necrosis factor-α, interleukin-1β, and Bax transcript levels. AgNPs reduced the transcripts of claudin-5, brain-derived neurotrophic factor, paraoxonase, nuclear factor-erythroid factor 2 (Nrf2), and Bcl-2. Histopathologically, AgNPs caused various degenerative changes and neuronal necrosis associated with glial cell reactions. AgNPs increased the immunohistochemical staining of glial fibrillary acidic protein (GFAP) in the cerebrum and cerebellum. Oral treatment with quercetin efficiently counteracted the opposing effects of AgNPs on brain tissue via modulation of tight junction proteins, Nrf2, and paraoxonase, and its positive mechanism in modulating pro-inflammatory cytokines and the downregulation of GFAP expression, and the apoptotic pathway. AgNPs also altered the severity of histopathological lesions and modulated GFAP immunostaining in the examined tissue.

Small ncRNA binding protein, PIWI: A potential molecular bridge between blood brain barrier and neuropathological conditions

The blood brain barrier (BBB) is a neuroprotective layer that maintains the homeostasis of central nervous system and provides an appropriate environment for neurons to execute their functions. The fundamental role of the dynamic semi-permeable BBB is selective and stringent transport of molecules from circulating blood and surrounding extracellular matrix across brain. Disruption of BBB has critical implications that can lead to various neuropathological disorders (NPDs) namely multiple sclerosis, Alzheimer’s disease, epilepsy, traumatic brain injuries and neuropsychiatric disorders, etc. Therapeutic management of NPDs is still a daunting challenge in the field of neuromedicine and there is a great need for identifying novel drug targets and biomarkers. Recently, noncoding RNAs (ncRNA) have emerged as promising prognostic markers in NPDs. Piwi interacting RNAs (piRNA), a family of short noncoding RNAs which in association with PIWI-like proteins have shown to regulate neuronal function and memory formation. In addition, piRNAs are differentially expressed in Alzheimer’s brain tissues and studies also revealed the association of denovo mutations in PIWI genes with autism. Moreover, the role of PIWI-like proteins in neuronal long-term potentiation and neurite outgrowth is now evident, confirming their importance in normal physiology of the brain. Notably, we have reported the significance of PIWI-like proteins in the maintenance of Blood Retinal Barrier (BRB) and its potential role in diseases like diabetic retinopathy through modulation of tight junction proteins. Further studies in hydra and cancer cells confirmed the important function of PIWI-like proteins in the organization of tight junctions. Interestingly, we also observed that loss of PIWI-like proteins affected the activity of Ephrin receptors which have an established functional link to tight junction assembly. Collectively, the evidences profoundly support the novel concept that piRNAs/PIWI-like proteins may have a potential role on the governance of BBB by altering the tight junctions through Ephrin effectors, commotion of which could be the preceding event to various NPDs. Here, we propose PIWI-like proteins and associated piRNAs as potential restorative drug targets for combating neuropathological conditions.

Stress‐induced Effects on Endometriosis and Tight Junction Protein Expression are Counteracted by VSL#3 Administration in an Animal Model

IntroductionEndometriosis is a gynecological disorder in which tissue that normally lines the endometrium grows outside the uterus and commonly results in peritoneal inflammation and infertility. Our previous studies show that stress exacerbates the development of endometriosis symptoms with worsening of colonic damage, and probiotic administration can decrease lesion size. Tight junction proteins are important to maintain the integrity of the intestinal barrier, and their disturbance can contribute to a variety of pathological conditions. It is possible that in our model stress contributes to the intestinal effects via modulation of tight junction proteins, but it is unclear whether probiotic use could counteract any of these effects. In this project we focused on tight junction protein expression intensity using the specific integral membrane proteins occludin and claudin‐2 to investigate how probiotics might alleviate endometriotic damage in the colon. Downregulation of occludin can enhance paracellular permeability, while claudin‐2 overexpression has been linked to intestinal pathophysiology by decreasing barrier function of other claudins.HypothesisProbiotic administration will reverse the effects of stress on colonic tight junction protein expression in an animal model of endometriosis.MethodsEndometriosis was induced by suturing uterine horn tissue next to the intestinal mesenteries in female Sprague‐Dawley rats (11–12/group) on day 0. One week later, rats were given the probiotic mixture VSL#3 or placebo in their drinking water before subjecting to 7 days of water avoidance stress (60 mins/day; days 14–20). Non‐stressed controls also received VSL#3 or placebo. On day 60 the rats were sacrificed, the vesicles were retrieved and measured, and the tissues collected before paraffin embedding. Colonic damage was measured and immunofluorescence for occludin and claudin‐2 was performed.ResultsThe vesicle area from rats exposed to stress were significantly larger in size than non‐stressed controls (p<0.05), while after probiotic administration the size was reduced (p<0.01). Colonic macroscopic damage had a trend to increase in endometriosis animals and was reduced after probiotic administration (p<0.05). Colonic occludin expression showed a trend to increase when probiotic was administered after stress, while claudin‐2 expression was downregulated after stress (p<0.01) and probiotic administration (p<0.01).ConclusionStress aggravates endometriosis symptoms causing larger vesicles and increased colonic damage. After probiotic administration, these symptoms are reversed and integrity of the tight junctions in colonic tissue is increased. This tendency provides further support for the potential use of probiotics as a complementary treatment to reduce the effects of inflammation in this condition.Support or Funding InformationSupported in part by P20 GM103475‐15, R25GM096955 & R25GM082406This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Tanycytes: A rich morphological history to underpin future molecular and physiological investigations.

Tanycytes are located at the base of the brain and retain characteristics from their developmental origins, such as radial glial cells, throughout their life span. With transport mechanisms and modulation of tight junction proteins, tanycytes form a bridge connecting the cerebrospinal fluid with the external limiting basement membrane. They also retain the powers of self-renewal and can differentiate to generate neurones and glia. Similar to radial glia, they are a heterogeneous family with distinct phenotypes. Although the four subtypes so far distinguished display distinct characteristics, further research is likely to reveal new subtypes. In this review, we have re-visited the work of the pioneers in the field, revealing forgotten work that is waiting to inspire new research with today's cutting-edge technologies. We have conducted a systematic ultrastructural study of α-tanycytes that resulted in a wealth of new information, generating numerous questions for future study. We also consider median eminence pituicytes, a closely-related cell type to tanycytes, and attempt to relate pituicyte fine morphology to molecular and functional mechanism. Our rationale was that future research should be guided by a better understanding of the early pioneering work in the field, which may currently be overlooked when interpreting newer data or designing new investigations.

Lactobacillus brevis 23017 Relieves Mercury Toxicity in the Colon by Modulation of Oxidative Stress and Inflammation Through the Interplay of MAPK and NF-κB Signaling Cascades.

Aims: Lactobacillus strains have protective effects against heavy metals while relieving oxidative stress and modulating the immune response. Mechanisms that ameliorate heavy metal toxicity and the relationship between probiotics and gut barrier protection in the process of heavy metal pathogenesis was poorly understood.Methods and Results: In this study, Lactobacillus brevis 23017 (LAB, L. brevis 23017), a selected probiotics strain with strong mercury binding capacities, was applied to evaluate the efficiency against mercury toxicity in a mouse model. Histopathological results along with HE stains show that L. brevis 23017 protects the integrity of the small intestinal villus, which slows weight loss in response to Hg exposure. The qRT-PCR results demonstrate that L. brevis 23017 maintains a normal mucosal barrier via modulation of tight junction proteins. Importantly, the present study demonstrates that L. brevis 23017 effectively ameliorates injury of the small intestine by reducing intestinal inflammation and alleviating oxidative stress in animal models. Moreover, L. brevis 23017 blocks oxidative stress and inflammation through MAPK and NF-κB pathways, as shown by western blot.Conclusions: Together, these results reveal that L. brevis 23017 may have applications in the prevention and treatment of oral Hg exposure with fermented functional foods by protecting gut health in daily life.

Protective effect of S-nitrosoglutathione administration against hyperglycemia induced disruption of blood brain barrier is mediated by modulation of tight junction proteins and cell adhesion molecules

Diabetes is associated with increased blood brain barrier (BBB) permeability resulting in neurological deficits. The present study investigated the role of S-nitrosoglutathione (GSNO) on tight junction proteins and cell adhesion molecules in streptozotocin-induced diabetic mice. Diabetes was induced by intraperitoneal injection of streptozotocin (40 mg/kg body weight) for 5 days in mice. GSNO was administered daily (100 μg/kg body weight, orally) for 8 weeks after the induction of diabetes. A significant decline was observed in the cognitive ability of diabetic animals assessed using radial arm maze test. A significant increase was observed in nitrotyrosine levels in cortex and hippocampus of diabetic mice. Relative mRNA and protein expression of tight junction proteins viz; zona occludens-1 (ZO-1) and occludin were significantly lower in the microvessels isolated from cortex and hippocampus of diabetic animals, whereas expression of claudin-5 was unaltered. Immunofluorescence of tight junction proteins confirmed loss of ZO-1 and occludin in the diabetic brain. Furthermore, significant increase in interstitial cell adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 mRNA and protein levels was observed in diabetic animals. Ultrastructure of microvessels from diabetic brain was also altered thereby confirming BBB disruption. GSNO administration to diabetic animals, on the other hand, was able to ameliorate loss of ZO-1 and occludin as well as normalize ICAM-1 and VCAM-1 expression, restore BBB integrity, and improve cognitive deficits. The findings clearly suggest that GSNO is a therapeutic molecule with potential to protect BBB and prevent diabetes induced neurological deficits.

Alcohol Consumption Increases Claudin‐18 Turnover in Alveolar Epithelial Cells

Alveolar epithelial cell (AEC) tight junctions are crucial for maintaining barrier function between fluid filled tissues and the airspace in the lung. Failure of alveolar tight junctions leads to acute respiratory distress syndrome (ARDS), a frequently lethal condition characterized by airspace flooding in the lung. Chronic alcohol abuse is a great risk factor for ARDS. Alcohol consumption impairs alveolar barrier function by modulation of tight junction proteins, called claudins. Claudins are a family of tetraspan transmembrane proteins that confer specific barrier properties to epithelial cell monolayers by regulating tight junction permeability. We used a rodent model of chronic alcohol ingestion to investigate the effects of alcohol on claudins and barrier function. Primary AECs from alcohol and control fed rats were cultured on Transwell permeable supports and allowed to differentiate into model type I cells. Transepithelial resistance measurements (TER) and dye flux assays were used to determine barrier function in cultured AECs. Tight junction morphology was assessed by immunofluorescence. In response to alcohol, alveolar tight junctions showed altered claudin composition leading to an increase in paracellular leak and formation of projections perpendicular to tight junctions (“spikes”) when immunostained for claudin‐18. Quantification of cells containing tight junction spikes revealed that alcohol increases the portion of cells containing tight junction spikes, which correlated with decreased barrier function. Live cell imaging of AECs expressing YFP‐tagged claudin‐18 confirmed that vesicles containing tagged claudin‐18 fused with and pinched off of tight junction spikes. Therefore, we hypothesized that these tight junction spikes are sites of enhanced protein turnover which might then result in decreased barrier function. As endocytosis is an important regulator of claudin turnover, we examined the effect of treating cells with the inhibitor Dynasore that targets dynamin, a protein which has a role in the scission of endocytic vesicles. Treatment of AECs with Dynasore resulted in a significant decrease in the number of cells containing claudin‐18 spikes and also caused a concomitant decrease in paracellular flux of a 10 kDa marker, Texas Red dextran. Our data support a role for tight junction spikes in regulating paracellular permeability and suggest that dynamin or dynamin‐related proteins may be useful therapeutic targets to stabilize alveolar barrier function and impair the development of ARDS.Support or Funding InformationThis work was supported by Emory Alcohol and Lung Biology Center/National Institutes of Health (NIH) grant P50‐AA013757 (MK), R01‐HL116958 (MK), T32‐AA‐013528 (SAM), the German Academic Exchange Service (DAAD) (BS), R25‐GM099644 (SDW), and Emory+Children's Center of Excellence for Cystic Fibrosis Research (MK).

Hyperbaric oxygen preconditioning ameliorates blood-brain barrier damage induced by hypoxia through modulation of tight junction proteins in an in vitro model.

AimTo explore the effects of hyperbaric oxygen preconditioning (HBOP) on the permeability of blood-brain barrier (BBB) and expression of tight junction proteins under hypoxic conditions in vitro.MethodsA BBB in vitro model was constructed using the hCMEC/D3 cell line and used when its trans-endothelial electrical resistance (TEER) reached 80-120 Ω · cm2 (tested by Millicell-Electrical Resistance System). The cells were randomly divided into the control group cultured under normal conditions, the group cultured under hypoxic conditions (2%O2) for 24 h (hypoxia group), and the group first subjected to HBOP for 2 h and then to hypoxia (HBOP group). Occludin and ZO-1 expression were analyzed by immunofluorescence assay.ResultsNormal hCMEC/D3 was spindle-shaped and tightly integrated. TEER was significantly reduced in the hypoxia (P = 0.001) and HBOP group (P = 0.014) compared to control group, with a greater decrease in the hypoxia group. Occludin membranous expression was significantly decreased in the hypoxia group (P = 0.001) compared to the control group, but there was no change in the HBOP group. ZO-1 membranous expression was significantly decreased (P = 0.002) and cytoplasmic expression was significantly increased (P = 0.001) in the hypoxia group compared to the control group, although overall expression levels did not change. In the HBOP group, there was no significant change in ZO-1 expression compared to the control group.ConclusionHyperbaric oxygen preconditioning protected the integrity of BBB in an in vitro model through modulation of occludin and ZO-1 expression under hypoxic conditions.

Topical application of delphinidin reduces psoriasiform lesions in the flaky skin mouse model by inducing epidermal differentiation and inhibiting inflammation.

Psoriasis is a chronic inflammatory skin disease characterized by hyperproliferation and aberrant keratinocyte differentiation. We have shown that treatment of reconstituted human skin with delphinidin, an anthocyanidin, present in pigmented fruits and vegetables, increased the expression and processing of caspase-14, which is involved in cornification. Delphinidin also increases the expression of epidermal differentiation marker proteins. To determine whether topical application of delphinidin can modulate pathological markers of psoriasiform lesions in flaky skin mice and if this is associated with increased epidermal differentiation and a reduction in proliferation and inflammation. Five-week-old female homozygous flaky skin mice (fsn/fsn) were treated topically with delphinidin (0·5mg cm(-2) and 1mg cm(-2) skin areas, respectively), five times a week, up to 14weeks of age. Treatment of flaky skin mice with delphinidin resulted in a reduction in (i) pathological markers of psoriasiform lesions; (ii) infiltration of inflammatory cells; and (iii) mRNA and protein expression of inflammatory cytokines. Delphinidin treatment also increased the expression and processing of caspase-14, and expression of filaggrin, loricrin, keratin-1 and keratin-10. Furthermore, there was a decrease in the expression of markers for cell proliferation (proliferating cell nuclear antigen and keratin-14) and modulation of tight junction proteins (occludin and claudin-1). In addition, delphinidin treatment increased the expression of activator protein-1 transcription factor proteins (JunB, JunD, Fra1 and Fra2). Delphinidin could be a promising agent for treatment of psoriasis and other hyperproliferative skin disorders.

Inhibition of c-Jun N-terminal kinase prevents blood–brain barrier disruption and normalizes the expression of tight junction proteins clautin-5 and ZO-1 in a rat model of subarachnoid hemorrhage

The c-Jun N-terminal kinase (JNK) proteins are encoded by three genes (JNK1, JNK2, and JNK3), giving rise to multiple isoforms via alternative splicing. JNK inhibition using a chemical inhibitor SP600125 confers neuroprotection in an animal model of subarachnoid hemorrhage (SAH). The aim of this study is to investigate whether the protective effects of SP600125 were associated with modulation of tight junction proteins including claudin-5 and ZO-1 and to define which JNK isoforms were involved in the early brain injury after SAH. Seventy-five male Sprague Dawley rats (weighing 300-350g) were randomly assigned to five groups (n = 15): (1) sham, (2) SAH, (3) SAH + DMSO (dimethyl sulfoxide), (4) SAH + 10mg/kg SP600125, and (5) SAH + 30mg/kg SP600125. SP600125 or DMSO was injected intraperitoneally 1 h before and 6h after the induction of SAH. Animals from all the groups were killed 24h after SAH, and brain tissues were dissected and subjected to electron microscopic examination, Western blot analysis, and histological evaluation. SP600125 pretreatment restored tight junctions and attenuated blood-brain barrier (BBB) disruption and cerebral edema after SAH, coupled with reduced apoptosis in the cerebral cortex. SP600125 exposure restored the reduced expression of both claudin-5 and ZO-1 following SAH and normalized the levels of JNK1 and JNK3. Our data demonstrate that the JNK signaling plays an important role in the regulation of tight junction proteins and BBB integrity, and thus represents a promising target against brain injuries after SAH.

Modulation of tight junction proteins in the perineurium for regional pain control

Peripheral neurons are surrounded by the perineurium that forms the blood-nerve barrier and protects the nerve. Although the barrier serves as protection, it also hampers drug delivery of analgesic drugs to the peripheral nerve. We previously showed that opening of the barrier using hypertonic solutions facilitates drug delivery, for example, of hydrophilic opioids, which selectively target nociceptors. The perineurial barrier is formed by tight junction proteins, including claudin-1, claudin-5, and occludin. Under pathophysiological conditions such as nerve crush injury, the perineurial barrier is opened and tight junction proteins are no longer present. After several days, tight junction proteins reappear and the barrier reseals. Similarly, perineurial injection of hypertonic saline transiently opens the barrier, claudin-1 disappears, and hydrophilic analgesic drugs are effective. In the future, these findings could be used to reseal the barrier breakdown and could be applied to other barriers like the blood-brain or the intestinal mucosal barrier.

Modulation of Tight Junction Proteins in the Perineurium to Facilitate Peripheral Opioid Analgesia

Peripheral application of opioids reduces inflammatory pain but is less effective in noninflamed tissue of rats and human patients. Hypertonic solutions can facilitate the antinociceptive activity of hydrophilic opioids in noninflamed tissue in vivo. However, the underlying mechanisms are not well understood. We hypothesized that the enhanced efficacy of opioids may be because of opening of the perineurial barrier formed by tight junction-proteins like claudin-1. Male Wistar rats were treated intraplantarly with 10% NaCl. Pain behavior (n = 6) and electrophysiological recordings (n = 9 or more) from skin-nerve preparations after local application of the opioid [d-Ala2,N-Me-Phe4,Gly5-ol]enkephalin (DAMGO) were explored. Tight junction-proteins as well as permeability of the barrier were examined by immunohistochemistry and Western blot (n = 3 or more). Local administration of 10% NaCl facilitated increased mechanical nociceptive thresholds in response to DAMGO, penetration of horseradish peroxidase into the nerve, as well as a reduced response of C- but not Aδ-nociceptors to mechanical stimulation after application of DAMGO in the skin-nerve preparation. In noninflamed paw tissue, claudin-1 was expressed in the epidermis, blood vessels, and the perineurium, surrounding neurons immunoreactive for calcitonin gene-related peptide or protein gene product 9.5. Claudin-1 but not claudin-5 or occludin was significantly reduced after pretreatment with 10% NaCl. Intraplantar application of a metalloproteinase inhibitor (GM6001) completely reversed these effects. Hypertonic saline opens the perineurial barrier via metalloproteinase activation and claudin-1 regulation, thereby allowing access of hydrophilic drugs to peripheral opioid receptors. This principle may be used to specifically target hydrophilic drugs to peripheral neurons.

WITHDRAWN: ‘Genistein reduced the invasive activity of human breast carcinoma cells as a result of decreased tight junction permeability and modulation of tight junction proteins’

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