A Novel Approach to Reducing Lung Metastasis in Osteosarcoma: Increasing Cell Stiffness with Carbenoxolone.

AIM: Primary malignant bone tumor osteosarcoma can metastasize to the lung. Diminishing lung metastasis would positively affect the prognosis of patients. Our previous studies demonstrated that highly metastatic osteosarcoma cell lines are significantly softer than low-metastasis cell lines. We therefore hypothesized that increasing cell stiffness would suppress metastasis by reducing cell motility. In this study, we tested whether carbenoxolone (CBX) increases the stiffness of LM8 osteosarcoma cells and prevents lung metastasis in vivo. METHODS: We evaluated the actin cytoskeletal structure and polymerization of CBX-treated LM8 cells using actin staining. Cell stiffness was measured using atomic force microscopy. Metastasis-related cell functions were analyzed using cell proliferation, wound healing, invasion, and cell adhesion assays. Furthermore, lung metastasis was examined in LM8-bearing mice administered with CBX. RESULTS: Treatment with CBX significantly increased actin staining intensity and stiffness of LM8 cells compared with vehicle-treated LM8 cells (p < 0.01). In Young's modulus images, compared with the control group, rigid fibrillate structures were observed in the CBX treatment group. CBX suppressed cell migration, invasion, and adhesion but not cell proliferation. The number of LM8 lung metastases were significantly reduced in the CBX administration group compared with the control group (p < 0.01). CONCLUSION: In this study, we demonstrated that CBX increases tumor cell stiffness and significantly reduces lung metastasis. Our study is the first to provide evidence that reducing cell motility by increasing cell stiffness might be effective as a novel anti-metastasis approach in vivo.

Therapeutic implications of hypothalamic-pituitaryadrenal-axis modulation in Alzheimer's disease: A narrative review of pharmacological and lifestyle interventions.

With disease-modifying treatments for Alzheimer's disease (AD) still elusive, the search for alternative intervention strategies has intensified. Growing evidence suggests that dysfunction in hypothalamic-pituitaryadrenal-axis (HPAA) activity may contribute to the development of AD pathology. The HPAA, may therefore offer a novel target for therapeutic action. This review summarises and critically evaluates animal and human studies investigating the effects of pharmacological and non-pharmacological intervention on HPAA modulation alongside cognitive performance. The interventions discussed include glucocorticoid receptor antagonists and 11ß-hydroxysteroid dehydrogenase inhibitors as well as lifestyle treatments such as physical activity, diet, sleep and contemplative practices. Pharmacological HPAA modulators improve pathology and cognitive deficit in animal AD models, but human pharmacological trials are yet to provide definitive support for such benefits. Lifestyle interventions may offer promising strategies for HPAA modification and cognitive health, but several methodological caveats across these studies were identified. Directions for future research in AD studies are proposed.

Caspase-11 promotes NLRP3 inflammasome activation via the cleavage of pannexin1 in acute kidney disease.

Ischemia/reperfusion (I/R) injury is a major cause of acute kidney injury (AKI) in clinic. The activation of NLRP3 inflammasome is associated with inflammation and renal injury in I/R-induced AKI. In the current study we explored the molecular and cellular mechanisms for NLRP3 inflammasome activation following renal I/R. Mice were subjected to I/R renal injury by clamping bilateral renal pedicles. We showed that I/R injury markedly increased caspase-11 expression and the cleavage of pannexin 1 (panx1) in the kidneys accompanied by NLRP3 inflammasome activation evidenced by the activation of caspase-1 and interlukin-1ß (IL-1ß) maturation. In Casp-11-/- mice, I/R-induced panx1 cleavage, NLRP3 inflammasome activation as well as renal functional deterioration and tubular morphological changes were significantly attenuated. In cultured primary tubular cells (PTCs) and NRK-52E cells, hypoxia/reoxygenation (H/R) markedly increased caspase-11 expression, NLRP3 inflammasome activation, IL-1ß maturation and panx1 cleavage. Knockdown of caspase-11 attenuated all those changes; similar effects were observed in PTCs isolated from Casp-11-/- mice. In NRK-52E cells, overexpression of caspase-11 promoted panx1 cleavage; pretreatment with panx1 inhibitor carbenoxolone or knockdown of panx1 significantly attenuated H/R-induced intracellular ATP reduction, extracellular ATP elevation and NLRP3 inflammasome activation without apparent influence on H/R-induced caspase-11 increase; pretreatment with P2X7 receptor inhibitor AZD9056 also attenuated NLRP3 inflammasome activation. The above results demonstrate that the cleavage of panx1 by upregulated caspase-11 is involved in facilitating ATP release and then NLRP3 inflammasome activation in I/R-induced AKI. This study provides new insight into the molecular mechanism of NLRP3 inflammasome activation in AKI.

Carbenoxolone Ameliorates Allergic Airway Inflammation through NF-κB/NLRP3 Pathway in Mice.

Asthma is a respiratory disease characterized by heterogeneous chronic airway inflammation. Activation of nucleotide-binding oligomerization domain-like receptor pyrin domain containing 3 (NLRP3) inflammasome is involved in the development of many pulmonary inflammatory diseases. The role and regulatory mechanism of carbenoxolone (CBX) in ovalbumin (OVA)-induced asthma models are not fully clear. Therefore, the study investigated whether CBX ameliorates airway inflammation and remodeling, as well as its mechanism in OVA induced-inflammation in mice. Wright-Giemsa staining was used to count inflammatory cells in bronchoalveolar lavage fluid (BALF). The level of inflammatory cells infiltration, mucus cell proliferation, and collagen deposition in lung tissue were separately assessed by hematoxylin and eosin, periodic acid-Schiff, and Masson trichrome staining, respectively. Airway resistance (AR) was measured by non-invasive airway system. Immunohistochemical assay was used to observe NLRP3 expression area. The expression of nuclear factor-kappaB (NF-κB), p-NF-κB, inhibitor of kappaB (IκB)-α, p-IκB-α, NLRP3, pro-caspase-1, caspase-1, and interleukin (IL)-1ß in lung tissue were measured using quantitative real-time PCR or Western blotting. Our results showed that CBX can significantly attenuate the leukocyte count and the percentage of eosinophils and neutrophils in the BALF, peribronchial inflammation, airway mucus secretion, collagen deposition area, and AR in OVA-induced airway inflammation. In addition, the expression of p-NF-κB, p-IκB-α, NLRP3 and related factors were dramatically alleviated after CBX treatment. These data suggest that CBX has a significant protective effect on allergic airway inflammation by suppressing the activation of NLRP3 inflammasome through NF-κB pathway in asthmatic mice.

Comparative effect of vitamin D3 and carbenoxolone treatments in metabolic syndrome rats.

Metabolic syndrome (MetS) is a cluster of cardiovascular risk factors including central obesity, hypertension, insulin resistance, dyslipidemia, and hyperglyemia. MetS is found to be a positive predictor of cardiovascular morbidity and mortality. The present study was planned to test the efficacy of vitamin D3 supplementation as compared with cortisol inhibition on MetS parameters. Wistar rats were allocated into four groups: control, untreated MetS, and MetS treated with either vitamin D3 (10 µg/kg) or carbenoxolone (50 mg/kg). MetS was induced by combination of high-fat diet and oral fructose. After the induction period (8 weeks), MetS was confirmed, and treatment modalities started for a further 4 weeks. Compared with untreated MetS, vitamin D3- and carbenoxolone-treated rats showed significant reduction in blood pressure, body mass index, Lee index, waist circumference, retroperitoneal fat, and improvement of dyslipidemia. Meanwhile, treatment with carbenoxolone significantly lowered the elevated liver enzymes, and vitamin D3 resulted in improved insulin sensitivity, enhanced glucose uptake by muscles, and replenished glycogen content in the liver and muscles near control levels. In conclusion, although treatment with vitamin D3 or carbenoxolone reduced the risk factors associated with MetS, vitamin D3 was effective in ameliorating insulin resistance which is the hallmark of MetS.

Astrocytes in the rostral ventromedial medulla contribute to the maintenance of oro-facial hyperalgesia induced by late removal of dental occlusal interference.

BACKGROUND: Astrocytes in the rostral ventromedial medulla (RVM) contribute to descending pain modulation, but their role in oro-facial pain induced by persistent experimental dental occlusal interference (PEOI) or following EOI removal (REOI) is unknown. OBJECTIVE: To explore the involvement of RVM astrocytes in PEOI-induced oro-facial hyperalgesia or its maintenance following REOI. METHODS: Male rats were randomly assigned into five groups: sham-EOI, postoperative day 6 and 14 of PEOI (PEOI 6 d and PEOI 14 d), postoperative day 6 following REOI on day 3 (REOI 3 d) and postoperative day 14 following REOI on day 8 (REOI 8 d). The nociceptive head withdrawal threshold (HWT) and activities of RVM ON- or OFF-cells were recorded before and after intra-RVM astrocyte gap junction blocker carbenoxolone (CBX) microinjection. RVM astrocytes were labelled immunohistochemically with glial fibrillary acidic protein (GFAP) and analysed semi-quantitatively. RESULTS: Persistent experimental dental occlusal interference-induced oro-facial hyperalgesia, as reflected in decreased HWTs, was partially inhibited by REOI at day 3 but not at day 8 after EOI placement. Increased GFAP-staining area occurred only in REOI 8 d group in which CBX could inhibit the maintained hyperalgesia; CBX was ineffective in inhibiting hyperalgesia in PEOI 14 d group. OFF-cell activities showed no change, but the spontaneous activity and responses of ON-cells were significantly enhanced that could be suppressed by CBX in REOI 8 d group. CONCLUSION: Rostral ventromedial medulla astrocytes may not participate in PEOI-induced oro-facial hyperalgesia or hyperalgesia inhibition by early REOI but are involved in the maintenance of oro-facial hyperalgesia by late REOI.

The Anti-Epileptic Effects of Carbenoxolone In Vitro and In Vivo.

Gap junctions (GJs) are intercellular junctions that allow the direct transfer of ions and small molecules between neighboring cells, and GJs between astrocytes play an important role in the development of various pathologies of the brain, including regulation of the pathological neuronal synchronization underlying epileptic seizures. Recently, we found that a pathological change is observed in astrocytes during the ictal and interictal phases of 4-aminopyridin (4-AP)-elicited epileptic activity in vitro, which was correlated with neuronal synchronization and extracellular epileptic electrical activity. This finding raises the question: Does this signal depend on GJs between astrocytes? In this study we investigated the effect of the GJ blocker, carbenoxolone (CBX), on epileptic activity in vitro and in vivo. Based on the results obtained, we came to the conclusion that the astrocytic syncytium formed by GJ-associated astrocytes, which is responsible for the regulation of potassium, affects the formation of epileptic activity in astrocytes in vitro and epileptic seizure onset. This effect is probably an important, but not the only, mechanism by which CBX suppresses epileptic activity. It is likely that the mechanisms of selective inhibition of GJs between astrocytes will show important translational benefits in anti-epileptic therapies.

High extracellular ATP levels released through pannexin-1 channels mediate inflammation and insulin resistance in skeletal muscle fibres of diet-induced obese mice.

AIMS/HYPOTHESIS: Skeletal muscle is a key target organ for insulin's actions and is the main regulator of blood glucose. In obese individuals and animal models, there is a chronic low-grade inflammatory state affecting highly metabolic organs, leading to insulin resistance. We have described that adult skeletal muscle fibres can release ATP to the extracellular medium through pannexin-1 (PANX1) channels. Besides, it is known that high extracellular ATP concentrations can act as an inflammatory signal. Here, we propose that skeletal muscle fibres from obese mice release high levels of ATP, through PANX1 channels, promoting inflammation and insulin resistance in muscle cells. METHODS: C57BL/6J mice were fed with normal control diet (NCD) or high-fat diet (HFD) for 8 weeks. Muscle fibres were isolated from flexor digitorum brevis (FDB) muscle. PANX1-knockdown FDB fibres were obtained by in vivo electroporation of a short hairpin RNA Panx1 plasmid. We analysed extracellular ATP levels in a luciferin/luciferase assay. Gene expression was studied with quantitative real-time PCR (qPCR). Protein levels were evaluated by immunoblots, ELISA and immunofluorescence. Insulin sensitivity was analysed in a 2-NBDG (fluorescent glucose analogue) uptake assay, immunoblots and IPGTT. RESULTS: HFD-fed mice showed significant weight gain and insulin resistance compared with NCD-fed mice. IL-6, IL-1ß and TNF-α protein levels were increased in FDB muscle from obese mice. We observed high levels of extracellular ATP in muscle fibres from obese mice (197 ± 55 pmol ATP/µg RNA) compared with controls (32 ± 10 pmol ATP/µg RNA). ATP release in obese mice fibres was reduced by application of 100 µmol/l oleamide (OLE) and 5 µmol/l carbenoxolone (CBX), both PANX1 blockers. mRNA levels of genes linked to inflammation were reduced using OLE, CBX or 2 U/ml ATPase apyrase in muscle fibres from HFD-fed mice. In fibres from mice with pannexin-1 knockdown, we observed diminished extracellular ATP levels (78 ± 10 pmol ATP/µg RNA vs 252 ± 37 pmol ATP/µg RNA in control mice) and a lower expression of inflammatory markers. Moreover, a single pulse of 300 µmol/l ATP to fibres from control mice reduced insulin-mediated 2-NBDG uptake and promoted an elevation in mRNA levels of inflammatory markers. PANX-1 protein levels were increased two- to threefold in skeletal muscle from obese mice compared with control mice. Incubation with CBX increased Akt activation and 2-NBDG uptake in HFD fibres after insulin stimulation, rescuing the insulin resistance condition. Finally, in vivo treatment of HFD-fed mice with CBX (i.p. injection of 10 mg/kg each day) for 14 days, compared with PBS, reduced extracellular ATP levels in skeletal muscle fibres (51 ± 10 pmol ATP/µg RNA vs 222 ± 28 pmol ATP/µg RNA in PBS-treated mice), diminished inflammation and improved glycaemic management. CONCLUSIONS/INTERPRETATION: In this work, we propose a novel mechanism for the development of inflammation and insulin resistance in the skeletal muscle of obese mice. We found that high extracellular ATP levels, released by overexpressed PANX1 channels, lead to an inflammatory state and insulin resistance in skeletal muscle fibres of obese mice.

11ß hydroxysteroid dehydrogenases regulate circulating glucocorticoids but not central gene expression.

Regulation of glucocorticoids (GCs), important mediators of physiology and behavior at rest and during stress, is multi-faceted and dynamic. The 11ß hydroxysteroid dehydrogenases 11ß-HSD1 and 11ß-HSD2 catalyze the regeneration and inactivation of GCs, respectively, and provide peripheral and central control over GC actions in mammals. While these enzymes have only recently been investigated in just two songbird species, central expression patterns suggest that they may function differently in birds and mammals, and little is known about how peripheral expression regulates circulating GCs. In this study, we utilized the 11ß-HSD inhibitor carbenoxolone (CBX) to probe the functional effects of 11ß-HSD activity on circulating GCs and central GC-dependent gene expression in the adult zebra finch (Taeniopygia guttata). Peripheral CBX injection produced a marked increase in baseline GCs 60 min after injection, suggestive of a dominant role for 11ß-HSD2 in regulating circulating GCs. In the adult zebra finch brain, where 11ß-HSD2 but not 11ß-HSD1 is expressed, co-incubation of micro-dissected brain regions with CBX and stress-level GCs had no impact on expression of several GC-dependent genes. These results suggest that peripheral 11ß-HSD2 attenuates circulating GCs, whereas central 11ß-HSD2 has little impact on gene expression. Instead, rapid 11ß-HSD2-based regulation of local GC levels might fine-tune membrane GC actions in brain. These results provide new insights into the dynamics of GC secretion and action in this important model organism.

Carbenoxolon Is Capable to Regulate the Mitochondrial Permeability Transition Pore Opening in Chronic Alcohol Intoxication.

BACKGROUND: carbenoxolone, which is a derivative of glyceretic acid, is actively used in pharmacology for the treatment of diseases of various etiologies. In addition, we have shown carbenoxolone as an effective inducer of mitochondrial permeability transition pore in rat brain and liver mitochondria. METHODS: in the course of this work, comparative studies were carried out on the effect of carbenoxolone on the parameters of mPTP functioning in mitochondria isolated from the liver of control and alcoholic rats. RESULTS: within the framework of this work, it was found that carbenoxolone significantly increased its effect in the liver mitochondria of rats with chronic intoxication. In particular, this was expressed in a reduction in the lag phase, a decrease in the threshold calcium concentration required to open a pore, an acceleration of high-amplitude cyclosporin-sensitive swelling of mitochondria, as well as an increase in the effect of carbenoxolone on the level of mitochondrial membrane-bound proteins. Thus, as a result of the studies carried out, it was shown that carbenoxolone is involved in the development/modulation of alcohol tolerance and dependence in rats.

Development of a pro-arrhythmic ex vivo intact human and porcine model: cardiac electrophysiological changes associated with cellular uncoupling.

We describe a human and large animal Langendorff experimental apparatus for live electrophysiological studies and measure the electrophysiological changes due to gap junction uncoupling in human and porcine hearts. The resultant ex vivo intact human and porcine model can bridge the translational gap between smaller simple laboratory models and clinical research. In particular, electrophysiological models would benefit from the greater myocardial mass of a large heart due to its effects on far-field signal, electrode contact issues and motion artefacts, consequently more closely mimicking the clinical setting. Porcine (n = 9) and human (n = 4) donor hearts were perfused on a custom-designed Langendorff apparatus. Epicardial electrograms were collected at 16 sites across the left atrium and left ventricle. A total of 1 mM of carbenoxolone was administered at 5 ml/min to induce cellular uncoupling, and then recordings were repeated at the same sites. Changes in electrogram characteristics were analysed. We demonstrate the viability of a controlled ex vivo model of intact porcine and human hearts for electrophysiology with pharmacological modulation. Carbenoxolone reduces cellular coupling and changes contact electrogram features. The time from stimulus artefact to (-dV/dt)max increased between baseline and carbenoxolone (47.9 ± 4.1-67.2 ± 2.7 ms) indicating conduction slowing. The features with the largest percentage change between baseline and carbenoxolone were fractionation + 185.3%, endpoint amplitude - 106.9%, S-endpoint gradient + 54.9%, S point - 39.4%, RS ratio + 38.6% and (-dV/dt)max - 20.9%. The physiological relevance of this methodological tool is that it provides a model to further investigate pharmacologically induced pro-arrhythmic substrates.

Gap junctions regulate vessel diameter in chick chorioallantoic membrane vasculature by both tone-dependent and structural mechanisms.

OBJECTIVE: In this study, we examined the impact of gap junction blockade on chick chorioallantoic membrane microvessels. METHODS: Expression of Cx37, Cx40/42, and Cx43 in chick chorioallantoic membrane tissue was studied by PCR, Western blot, and confocal immunofluorescence microscopy. Vessel diameter changes occurring under gap junction blockade with carbenoxolone (175 µmol/L), palmitoleic acid (100 µmol/L), 43 GAP27 (1 mmol/L) were analyzed by intravital microscopy. To analyze vascular tone, chick chorioallantoic membrane vessels were exposed to a vasodilator cocktail consisting of acetylcholine (10 µmol/L), adenosine (100 µmol/L), papaverine (200 µmol/L), and sodium nitroprusside (10 µmol/L). RESULTS: In chick chorioallantoic membrane lysates, Western blot analysis revealed the expression of Cx40 and Cx43. Immunofluorescence in intact chick chorioallantoic membrane vasculature showed only Cx43, limited to arterial vessel walls. Upon gap junction blockade (3 hours) arterial and venous diameters decreased to 0.50 ± 0.03 and 0.36 ± 0.06 (carbenoxolone), 0.72 ± 0.08 and 0.63 ± 0.15 (palmitoleic acid) and 0.77 ± 0.004 and 0.58 ± 0.05 (GAP27), relative to initial values. Initially, diameter decrease was dominated by increasing vascular tone. After 6 hours, however, vessel tone was reduced, suggesting structural network remodeling. CONCLUSIONS: Our findings suggest a major role for connexins in mediating acute and chronic diameter changes in developing vascular networks.

Drug-induced endocrine blood pressure elevation.

Patients with uncontrolled hypertension are at risk for cardiovascular complications. The majority of them suffers from unidentified forms of hypertension and a fraction has so-called secondary hypertension with an identifiable cause. The patient's medications, its use of certain herbal supplements and over-the-counter agents represent potential causal factors for secondary hypertension that are often overlooked. The current review focuses on drugs that are likely to elevate blood pressure by affecting the human endocrine system at the level of steroid synthesis or metabolism, mineralocorticoid receptor activity, or by affecting the catecholaminergic system. Drugs with known adverse effects but where benefits outweigh their risks, drug candidates and market withdrawals are reviewed. Finally, potential therapeutic strategies are discussed.

The Effects of Carbenoxolone against Experimental Autoimmune Encephalomyelitis in a Mouse Model.

INTRODUCTION: Multiple sclerosis (MS) is a complex demyelinating disease involving central nervous system (CNS). It is still a challenge to secure an effective therapeutic strategy against this disease. Carbenoxolone (CBX) is a derivative of glycyrrhetinic acid, which is widely used in brain research for its gap-junction inhibition effects. Many researchers have observed CBX-mediated suppression of CNS inflammation in their studies. OBJECTIVE: We want to further examine its anti-inflammation effects in CNS demyelinating disease like MS. METHODS: Thus, our study applied an experimental autoimmune encephalomyelitis (EAE) mouse model and examined the effects of CBX on it. RESULTS AND CONCLUSIONS: We found that CBX significantly reversed the EAE severity and pathology in EAE. IL-17-secreting and IFN-γ-secreting CD4+ T lymphocytes were remarkably lower in the spleen of CBX-treated mice. Production of IL-23 and IL-17 from cortex in EAE animals was markedly reduced by CBX. Furthermore, CBX treatment increased the expression of brain-derived neurotrophic factor. This study provides evidence for the protective role of CBX against EAE.

Calcium-Dependent Arrhythmogenic Foci Created by Weakly Coupled Myocytes in the Failing Heart.

RATIONALE: Intercellular uncoupling and Ca2+ (Ca) mishandling can initiate triggered ventricular arrhythmias. Spontaneous Ca release activates inward current which depolarizes membrane potential (Vm) and can trigger action potentials in isolated myocytes. However, cell-cell coupling in intact hearts limits local depolarization and may protect hearts from this arrhythmogenic mechanism. Traditional optical mapping lacks the spatial resolution to assess coupling of individual myocytes. OBJECTIVE: We investigate local intercellular coupling in Ca-induced depolarization in intact hearts, using confocal microscopy to measure local Vm and intracellular [Ca] simultaneously. METHODS AND RESULTS: We used isolated Langendorff-perfused hearts from control (CTL) and heart failure (HF) mice (HF induced by transaortic constriction). In CTL hearts, 1.4% of myocytes were poorly synchronized with neighboring cells and exhibited asynchronous (AS) Ca transients. These AS myocytes were much more frequent in HF (10.8% of myocytes, P<0.05 versus CTL). Local Ca waves depolarized Vm in HF but not CTL hearts, suggesting weaker gap junction coupling in HF-AS versus CTL-AS myocytes. Cell-cell coupling was assessed by calcein fluorescence recovery after photobleach during intracellular [Ca] recording. All regions in CTL hearts exhibited faster calcein diffusion than in HF, with HF-AS myocyte being slowest. In HF-AS, enhancing gap junction conductance (with rotigaptide) increased coupling and suppressed Vm depolarization during Ca waves. Conversely, in CTL hearts, gap junction inhibition (carbenoxolone) decreased coupling and allowed Ca wave-induced depolarizations. Synchronization of Ca wave initiation and triggered action potentials were observed in HF hearts and computational models. CONCLUSIONS: Well-coupled CTL myocytes are effectively voltage-clamped during Ca waves, protecting the heart from triggered arrhythmias. Spontaneous Ca waves are much more common in HF myocytes and these AS myocytes are also poorly coupled, enabling local Ca-induced inward current of sufficient source strength to overcome a weakened current sink to depolarize Vm and trigger action potentials.

Disruption of Gap Junction-Mediated Intercellular Communication in the Spiral Ligament Causes Hearing and Outer Hair Cell Loss in the Cochlea of Mice.

It is well-known that outer hair cell (OHC) loss occurs in the cochlea of animal models of permanent hearing loss induced by intense noise exposure. Our earlier studies demonstrated the production of hydroxynonenal and peroxynitrite, as well as the disruption of gap junction-mediated intercellular communication (GJIC), in the cochlear spiral ligament prior to noise-induced sudden hearing loss. The goal of the present study was to evaluate the mechanism underlying cochlear OHC loss after sudden hearing loss induced by intense noise exposure. In organ of Corti explant cultures from mice, no significant OHC loss was observed after in vitro exposure to 4-hydroxynonenal (a product of lipid peroxidation), H2O2, SIN-1 (peroxynitrite generator), and carbenoxolone (a gap junction inhibitor). Interestingly, in vivo intracochlear carbenoxolone injection through the posterior semicircular canal caused marked OHC and hearing loss, as well as the disruption of gap junction-mediated intercellular communication in the cochlear spiral ligament. However, no significant OHC loss was observed in vivo in animals treated with 4-hydroxynonenal and SIN-1. Taken together, our data suggest that disruption of GJIC in the cochlear lateral wall structures is an important cause of cochlear OHC loss in models of hearing loss, including those induced by noise.

Analysis of carbenoxolone by ultra-high-performance liquid chromatography tandem mass spectrometry in mouse brain and blood after systemic administration.

Carbenoxolone is a derivative of glycyrrhetinic acid found in the root of Glycyrrhiza glabra, colloquially known as licorice. It has been used as a treatment for peptic and oral ulcers. In recent years, carbenoxolone has been utilized in basic research for its ability to block gap junctional communication. Better understanding the distribution of carbenoxolone after systemic administration can lead to a better understanding of its potential sites of action. Presented is an ultra high-performance liquid chromatography tandem mass spectrometer (UHPLC-MS/MS) method for the identification and quantification of carbenoxolone in mouse blood and brain tissue. Twenty mice were injected intraperitoneally with 25 mg/kg carbenoxolone and brain tissue and blood were collected for analysis. Blood concentrations (mean ± SD) at 15, 30, 60 and 120 min were determined to be (n = 5) 5394 ± 778, 2636 ± 836, 1564 ± 541 and 846 ± 252 ng/mL, respectively. Brain concentrations (mean ± SD) at 15, 30, 60 and 120 mins were determined to be (n = 5) 171 ± 62, 102 ± 35, 55 ± 10 and 27 ± 9 ng/g, respectively. The analysis of these specimens at the four different time points resulted in blood and brain half-lives in mice of ~43 and 41 min, respectively. The UHPLC-MS/MS method was determined to be sensitive and robust for quantification of carbenoxolone.

Modulating membrane fluidity corrects Batten disease phenotypes in vitro and in vivo.

The neuronal ceroid lipofuscinoses are a class of inherited neurodegenerative diseases characterized by the accumulation of autofluorescent storage material. The most common neuronal ceroid lipofuscinosis has juvenile onset with rapid onset blindness and progressive degeneration of cognitive processes. The juvenile form is caused by mutations in the CLN3 gene, which encodes the protein CLN3. While mouse models of Cln3 deficiency show mild disease phenotypes, it is apparent from patient tissue- and cell-based studies that its loss impacts many cellular processes. Using Cln3 deficient mice, we previously described defects in mouse brain endothelial cells and blood-brain barrier (BBB) permeability. Here we expand on this to other components of the BBB and show that Cln3 deficient mice have increased astrocyte endfeet area. Interestingly, this phenotype is corrected by treatment with a commonly used GAP junction inhibitor, carbenoxolone (CBX). In addition to its action on GAP junctions, CBX has also been proposed to alter lipid microdomains. In this work, we show that CBX modifies lipid microdomains and corrects membrane fluidity alterations in Cln3 deficient endothelial cells, which in turn improves defects in endocytosis, caveolin-1 distribution at the plasma membrane, and Cdc42 activity. In further work using the NIH Library of Integrated Network-based Cellular Signatures (LINCS), we discovered other small molecules whose impact was similar to CBX in that they improved Cln3-deficient cell phenotypes. Moreover, Cln3 deficient mice treated orally with CBX exhibited recovery of impaired BBB responses and reduced autofluorescence. CBX and the compounds identified by LINCS, many of which have been used in humans or approved for other indications, may find therapeutic benefit in children suffering from CLN3 deficiency through mechanisms independent of their original intended use.

Roles of astrocytic connexin-43, hemichannels, and gap junctions in oxygen-glucose deprivation/reperfusion injury induced neuroinflammation and the possible regulatory mechanisms of salvianolic acid B and carbenoxolone.

BACKGROUND: Glia-mediated neuroinflammation is related to brain injury exacerbation after cerebral ischemia/reperfusion (I/R) injury. Astrocytic hemichannels or gap junctions, which were mainly formed by connexin-43, have been implicated in I/R damage. However, the exact roles of astrocytic hemichannels and gap junction in neuroinflammatory responses induced by I/R injury remain unknown. METHODS: Primary cultured astrocytes were subjected to OGD/R injury, an in vitro model of I/R injury. Salvianolic acid B (SalB) or carbenoxolone (CBX) were applied for those astrocytes. Besides, Cx43 mimetic peptides Gap19 or Gap26 were also applied during OGD/R injury; Cx43 protein levels were determined by western blot and cytoimmunofluorescene staining, hemichannel activities by Ethidium bromide uptake and ATP concentration detection, and gap junction intercellular communication (GJIC) permeability by parachute assay. Further, astrocyte-conditioned medium (ACM) was collected and incubated with microglia. Meanwhile, ATP or apyrase were applied to explore the role of ATP during OGD/R injury. Microglial activation, M1/M2 phenotypes, and M1/M2-related cytokines were detected. Also, microglia-conditioned medium (MEM) was collected and incubated with astrocytes to further investigate its influence on astrocytic hemichannel activity and GJIC permeability. Lastly, effects of ACM and MCM on neuronal viability were detected by flow cytometry. RESULTS: We found that OGD/R induced abnormally opened hemichannels with increased ATP release and EtBr uptake but reduced GJIC permeability. WB tests showed decreased astrocytic plasma membrane's Cx43, while showing an increase in cytoplasma. Treating OGD/R-injured microglia with ATP or OGD/R-ACM induced further microglial activation and secondary pro-inflammatory cytokine release, with the M1 phenotype predominating. Conversely, astrocytes incubated with OGD/R-MCM exhibited increased hemichannel opening but reduced GJIC coupling. Both SalB and CBX inhibited abnormal astrocytic hemichannel opening and ATP release and switched the activated microglial phenotype from M1 to M2, thus providing effective neuroprotection. Application of Gap19 or Gap26 showed similar results with CBX. We also found that OGD/R injury caused both plasma membrane p-Cx43(Ser265) and p-Src(Tyr416) significantly upregulated; application of SalB may be inhibiting Src kinase and attenuating Cx43 internalization. Meanwhile, CBX treatment induced obviously downregulation of p-Cx43(Ser368) and p-PKC(Ser729) protein levels in plasma membrane. CONCLUSIONS: We propose a vicious cycle exists between astrocytic hemichannel and microglial activation after OGD/R injury, which would aggravate neuroinflammatory responses and neuronal damage. Astrocytic Cx43, hemichannels, and GJIC play critical roles in OGD/R injury-induced neuroinflammatory responses; treatment differentially targeting astrocytic Cx43, hemichannels, and GJIC may provide novel avenues for therapeutics during cerebral I/R injury.

Astrocytic gap junction inhibition by carbenoxolone enhances the protective effects of ischemic preconditioning following cerebral ischemia.

BACKGROUND: Stroke is the second leading cause of death worldwide and the most common cause of adult-acquired disability in many nations. Thus, attenuating the damage after ischemic injury and improving patient prognosis are of great importance. We have indicated that ischemic preconditioning (IP) can effectively reduce the damage of ischemia reperfusion and that inhibition of gap junctions may further reduce this damage. Although we confirmed that the function of gap junctions is closely associated with glutamate, we did not investigate the mechanism. In the present study, we aimed to clarify whether the blockade of cellular communication at gap junctions leads to significant reductions in the levels of glutamate released by astrocytes following cerebral ischemia. METHODS: To explore this hypothesis, we utilized the specific blocking agent carbenoxolone (CBX) to inhibit the opening and internalization of connexin 43 channels in an in vitro model of oxygen-glucose deprivation/re-oxygenation (OGD/R), following IP. RESULTS: OGD/R resulted in extensive astrocytic glutamate release following upregulation of hemichannel activity, thus increasing reactive oxygen species (ROS) generation and subsequent cell death. However, we observed significant increases in neuronal survival in neuron-astrocyte co-cultures that were subjected to IP prior to OGD/R. Moreover, the addition of CBX enhanced the protective effects of IP during the re-oxygenation period following OGD, by means of blocking the release of glutamate, increasing the level of the excitatory amino acid transporter 1, and downregulating glutamine expression. CONCLUSIONS: Our results suggest that combined use of IP and CBX represents a novel therapeutic strategy to attenuate damage from cerebral ischemia with minimal adverse side effects.