Adenosine triphosphate release inhibitors targeting pannexin1 improve recovery after spinal cord injury.

Traumatic spinal cord injury is characterized by immediate and irreversible tissue loss at the lesion site and secondary tissue damage. Secondary injuries should, in principle, be preventable, although no effective treatment options currently exist for patients with acute spinal cord injury. Traumatized tissues release excessive amounts of adenosine triphosphate and activate the P2X purinoceptor 7/pannexin1 complex, which is associated with secondary injury. We investigated the neuroprotective effects of the blue dye Brilliant Blue FCF, a selective inhibitor of P2X purinoceptor 7/pannexin1 that is approved for use as a food coloring, by comparing it with Brilliant Blue G, a P2X7 purinoceptor antagonist, and carbenoxolone, which attenuates P2X purinoceptor 7/pannexin1 function, in a rat spinal cord injury model. Brilliant Blue FCF administered early after spinal cord injury reduced spinal cord anatomical damage and improved motor recovery without apparent toxicity. Brilliant Blue G had the highest effect on this neurological recovery, with Brilliant Blue FCF and carbenoxolone having comparable improvement. Furthermore, Brilliant Blue FCF administration reduced local astrocytic and microglial activation and neutrophil infiltration, and no differences in these histological effects were observed between compounds. Thus, Brilliant Blue FCF protects spinal cord neurons after spinal cord injury and suppresses local inflammatory responses as well as Brilliant Blue G and carbenoxolone.

Inhibition of Schwann cell pannexin 1 attenuates neuropathic pain through the suppression of inflammatory responses.

BACKGROUND: Neuropathic pain is still a challenge for clinical treatment as a result of the comprehensive pathogenesis. Although emerging evidence demonstrates the pivotal role of glial cells in regulating neuropathic pain, the role of Schwann cells and their underlying mechanisms still need to be uncovered. Pannexin 1 (Panx 1), an important membrane channel for the release of ATP and inflammatory cytokines, as well as its activation in central glial cells, contributes to pain development. Here, we hypothesized that Schwann cell Panx 1 participates in the regulation of neuroinflammation and contributes to neuropathic pain. METHODS: A mouse model of chronic constriction injury (CCI) in CD1 adult mice or P0-Cre transgenic mice, and in vitro cultured Schwann cells were used. Intrasciatic injection with Panx 1 blockers or the desired virus was used to knock down the expression of Panx 1. Mechanical and thermal sensitivity was assessed using Von Frey and a hot plate assay. The expression of Panx 1 was measured using qPCR, western blotting, and immunofluorescence. The production of cytokines was monitored through qPCR and enzyme-linked immunosorbent assay (ELISA). Panx1 channel activity was detected by ethidium bromide (EB) uptake. RESULTS: CCI induced persistent neuroinflammatory responses and upregulation of Panx 1 in Schwann cells. Intrasciatic injection of Panx 1 blockers, carbenoxolone (CBX), probenecid, and Panx 1 mimetic peptide (10Panx) effectively reduced mechanical and heat hyperalgesia. Probenecid treatment of CCI-induced mice significantly reduced Panx 1 expression in Schwann cells, but not in dorsal root ganglion (DRG). In addition, Panx 1 knockdown in Schwann cells with Panx 1 shRNA-AAV in P0-Cre mice significantly reduced CCI-induced neuropathic pain. To determine whether Schwann cell Panx 1 participates in the regulation of neuroinflammation and contributes to neuropathic pain, we evaluated its effect in LPS-treated Schwann cells. We found that inhibition of Panx 1 via CBX and Panx 1-siRNA effectively attenuated the production of selective cytokines, as well as its mechanism of action being dependent on both Panx 1 channel activity and its expression. CONCLUSION: In this study, we found that CCI-related neuroinflammation correlates with Panx 1 activation in Schwann cells, indicating that inhibition of Panx 1 channels in Schwann cells reduces neuropathic pain through the suppression of neuroinflammatory responses.

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.

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.

The role of connexin43 in neuropathic pain induced by spinal cord injury.

Neuropathic pain is caused by the damage or dysfunction of the nervous system. In many neuropathic pain models, there is an increase in the number of gap junction (GJ) channels, especially the upregulation of the expression of connexin43 (Cx43), leading to the secretion of various types of cytokines and involvement in the formation of neuropathic pain. GJs are widely distributed in mammalian organs and tissues, and Cx43 is the most abundant connexin (Cx) in mammals. Astrocytes are the most abundant glial cell type in the central nervous system (CNS), which mainly express Cx43. More importantly, GJs play an important role in regulating cell metabolism, signaling, and function. Many existing literatures showed that Cx43 plays an important role in the nervous system, especially in the CNS under normal and pathological conditions. However, many internal mechanisms have not yet been thoroughly explored. In this review, we summarized the current understanding of the role and association of Cx and pannexin channels in neuropathic pain, especially after spinal cord injury, as well as some of our own insights and thoughts which suggest that Cx43 may become an emerging therapeutic target for future neuropathic pain, bringing new hope to patients.

Carbenoxolone as a novel therapy for attenuation of cancer-induced bone pain.

Pain is a major complication for patients with cancer significantly compromising their quality of life. Current treatment is far from optimal and particularly bone-related cancer pain poses an increasing clinical and socioeconomical problem. Connexins, key proteins in cell-cell communication, have the potential to affect cancer-induced bone pain at multiple levels, including nociceptive signaling and bone degradation. This study tested the analgesic potential of carbenoxolone, a broad-acting connexin blocker, in a mouse model of cancer-induced bone pain. In addition, a pharmacological approach was used to elucidate the underlying mechanisms using the 2 specific blockers Gap27 and Gap26. Compared with vehicle treatment, chronic systemic administration of 20 or 40 mg/kg carbenoxolone caused a significantly later onset and attenuation of movement-evoked and on-going pain, assessed with limb use and weight bearing, respectively. In addition, the carbenoxolone-treated groups demonstrated a significant delay in time to reach the humane endpoint. Acute intrathecal administration of Gap27 significantly attenuated both limb use and weight bearing, whereas Gap26 had a less pronounced effect. Carbenoxolone treatment had a minor effect on the bone degradation in the early phase of disease progression, whereas no effect was observed in the late phase. Surprisingly, connexin43 was downregulated in the cancer-bearing animals compared with shams. The results suggest that connexins are involved in cancer-induced bone pain, and that carbenoxolone could be a novel analgesic treatment for the pain state.

TAT-Gap19 and Carbenoxolone Alleviate Liver Fibrosis in Mice.

Although a plethora of signaling pathways are known to drive the activation of hepatic stellate cells in liver fibrosis, the involvement of connexin-based communication in this process remains elusive. Connexin43 expression is enhanced in activated hepatic stellate cells and constitutes the molecular building stone of hemichannels and gap junctions. While gap junctions support intercellular communication, and hence the maintenance of liver homeostasis, hemichannels provide a circuit for extracellular communication and are typically opened by pathological stimuli, such as oxidative stress and inflammation. The present study was set up to investigate the effects of inhibition of connexin43-based hemichannels and gap junctions on liver fibrosis in mice. Liver fibrosis was induced by administration of thioacetamide to Balb/c mice for eight weeks. Thereafter, mice were treated for two weeks with TAT-Gap19, a specific connexin43 hemichannel inhibitor, or carbenoxolone, a general hemichannel and gap junction inhibitor. Subsequently, histopathological analysis was performed and markers of hepatic damage and functionality, oxidative stress, hepatic stellate cell activation and inflammation were evaluated. Connexin43 hemichannel specificity of TAT-Gap19 was confirmed in vitro by fluorescence recovery after photobleaching analysis and the measurement of extracellular release of adenosine-5'-triphosphate. Upon administration to animals, both TAT-Gap19 and carbenoxolone lowered the degree of liver fibrosis accompanied by superoxide dismutase overactivation and reduced production of inflammatory proteins, respectively. These results support a role of connexin-based signaling in the resolution of liver fibrosis, and simultaneously demonstrate the therapeutic potential of TAT-Gap19 and carbenoxolone in the treatment of this type of chronic liver disease.

Acute anti-obesity effects of intracerebroventricular 11ß-HSD1 inhibitor administration in diet-induced obese mice.

The hypothalamus is the regulatory centre of both appetite and energy balance and endoplasmic reticulum (ER) stress in the hypothalamus is involved in the pathogenesis of obesity. Recently, inhibition of 11 ß hydroxysteroid dehydrogenase type1 (11ß-HSD1) was reported to have an anti-obesity effect by reducing fat mass. However, the link between the role of 11ß-HSD1 in the hypothalamus and obesity has yet to be determined. In the present study, embryonal primary hypothalamic neurones and high-fat diet (HFD) fed mice were used to investigate the anorexigenic effects of 11ß-HSD1 inhibitors both in vitro and in vivo. In hypothalamic neurones, carbenoxolone (a non selecitve 11ß-HSD inhibitor) alleviated ER stress and ER stress-induced neuropeptide alterations. In HFD mice, i.c.v. administration of carbenoxolone or KR67500 (nonselective and selective 11ß-HSD1 inhibitors, respectively) was associated with less weight gain compared to control mice for 24 hours after treatment, presumably by reducing food intake. Furthermore, glucose regulated protein (Grp78), spliced X-box binding protein (Xbp-1s), c/EBP homologous protein (chop) and ER DnaJ homologue protein (Erdj4) expression was decreased in the hypothalami of mice administrated 11ß-HSD1 inhibitors compared to controls. Conversely, the phosphorylation of protein kinase B (PKB/Akt), signal transducer and activator of transcription 3 (Stat3), mitogen-activated protein kinase (MAPK/ERK) and S6 kinase1 (S6K1) in the hypothalamus was induced more in mice treated using the same regimes. In conclusion, acute 11ß-HSD1 inhibition in the hypothalamus could reduce food intake by decreasing ER stress and increasing insulin, leptin, and mammalian target of rapamycin complex 1 (mTORC1) signalling.

Carbenoxolone Could Deteriorate Streptozotocin-induced Diabetes through Induction of Heat Shock Protein 70 and IFN-γ in C57BL/6 Mice.

Type 1 diabetes (T1D), a spontaneous autoimmune disease, is associated with destruction of insulin-producing ß-cells in the pancreas. Since some heat shock proteins (HSP), such as HSP70 exert a protective effect in both tissues and cells, the present study was conducted to elucidate the effects of carbenoxolone (CBX) as an HSP70 inducer on T1D. The disease was induced in male C57BL/6 mice using streptozotocin (STZ) and subjects were allocated to therapeutic 1 and therapeutic 2 groups, as well as negative and positive control groups. The treated mice (therapeutic 1 and therapeutic 2 groups) received 50 mg/kg CBX intraperitoneally every 24 hours, in the therapeutic 1  group the drug was injected before and after disease induction whereas in the therapeutic 2 group the drug was injected only after disease induction. Serum fasting blood sugar (FBS) level, cytokines production (Interferon-gamma (IFN-γ), Interleukin 10 (IL-10), and IL-17), serum HSP70 level and CD4+CD25+Foxp3+ regulatory T cell (Treg) frequency measurements were outperformed 14 days after the last STZ injection. Our results showed that in the treated groups, serum HSP70, IFN-γ, and IL-17 levels were increased in contrast to the untreated groups. The IL-10 level was markedly decreased in comparison to untreated diabetic mice (p<0.05). Moreover, it was found that the frequency of Tregs in treated mice was lower in comparison to the untreated mice but the difference was not significant (p>0.05). Our results confirm that CBX might through HSP70 induction, followed by increasing IFN-γ level leads to suppression of IL-10 production in diabetic mice resulted in toxic effects on pancreatic islet beta cells and deteriorating of disease.

Quinine and carbenoxolone enhance the anticonvulsant activity of some classical antiepileptic drugs.

Objective Quinine (QUIN) and carbenoxolone (CNX) elicit anticonvulsant effects typically characterized by the reduction of the epileptiform activity as well as changes in behavioral parameters related to seizures. Therefore, the aim of this study was to analyze the effects of these molecules on the anticonvulsant activity of some classical antiepileptic drugs. Methods Male Wistar rats were used. Valproate (VPA), phenytoin (PHT), or carbamazepine (CBZ) was administered at sub-therapeutic doses for intraperitoneal via. Subsequently, animals were administered with a single dose of QUIN or CNX. The anticonvulsant activity was evaluated with the maximal electroshock (MES) test and pentylenetetrazole (PTZ) administration. Additionally, the plasma levels of CBZ were determined using an HPLC method. Results All the control rats presented generalized tonic-clonic seizures after the MES test or the administration of PTZ. For the MES test, all of the antiepileptic drugs increased their anticonvulsant activity when were co-administered with QUIN. For the PTZ test, only the combination CBZ plus QUIN significantly increased the percentage of protection against the generalized tonic-clonic seizures. The co-administration of CBZ plus QUIN resulted in an augmented concentration of CBZ in plasma. Discussion The present study shows that QUIN and CNX enhance the anticonvulsant activity of some classical antiepileptic drugs. However, only the combination CBZ/QUIN had significant effects on both MES and PTZ models. Such anticonvulsant activity could be attributed to increased levels of CBZ in plasma. We propose that these molecules could improve the pharmacological actions of antiepileptic drugs administered at sub-therapeutic doses.