Purinergic P2X7 Receptor Research Articles

Electroacupuncture downregulates P2X3 receptor expression in dorsal root ganglia of the spinal nerve-ligated rat.

Electroacupuncture has been shown to effectively reduce chronic pain in patients with nerve injury. The underlying mechanisms are not well understood. Accumulated evidence suggests that purinergic P2X3 receptors (P2X3Rs) in dorsal root ganglion neurons play a major role in mediating chronic pain associated with nerve injury. The aim of this study is to determine if electroacupuncture stimulation alters P2X3R activity in dorsal root ganglia to produce analgesia under neuropathic pain condition. Peripheral neuropathy was produced by ligation of the left lumbar 5 (L5) spinal nerve in rats. Low-frequency (2 Hz) electrical stimulation was applied to ipsilateral ST36 and BL60 acupoints in rats. The P2X3R agonist (α,β-meATP)-induced flinch responses were reduced after electroacupuncture treatment. Western analyses showed that P2X3R expression was upregulated in nerve-uninjured lumbar 4 (L4) dorsal root ganglion neurons ipsilateral to the spinal nerve ligation. Electroacupuncture-stimulation reversed the upregulation. In nerve-injured L5 dorsal root ganglia, P2X3R expression was substantially reduced. Electroacupuncture had no effect on the reduction. We also determined the injury state of P2X3R expressing dorsal root ganglion neurons using the neuronal injury marker, activating transcription factor 3 (ATF3). Immunohistochemical assay showed that in L4 dorsal root ganglia, almost all P2X3Rs were expressed in uninjured (ATF3−) neurons. Spinal nerve ligation increased the expression of P2X3Rs. Electroacupuncture reduced the increase in P2X3R expression without affecting the percentage of ATF + neurons. In ipsilateral L5 dorsal root ganglion neurons, spinal nerve ligation reduced the percentage of P2X3R + neurons and markedly increased the percentage of ATF3 + cells. Almost all of P2X3Rs were expressed in damaged (ATF3+) neurons. Electroacupuncture had no effect on spinal nerve ligation-induced changes in the percentage of P2X3R or percentage of ATF3 + cells in L5 dorsal root ganglia. These observations led us to conclude that electroacupuncture effectively reduces injury-induced chronic pain by selectively reducing the expression of P2X3Rs in nerve-uninjured L4 dorsal root ganglion neurons.

Investigation on 2',3'-O-Substituted ATP Derivatives and Analogs as Novel P2X3 Receptor Antagonists.

Antagonists of the purinergic P2X3 receptors represent promising drugs for the treatment of inflammation and pain. The ATP derivative 2',3'-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP) has been described as a potent competitive inhibitor of this receptor. In this work, the design and synthesis of novel TNP-ATP analogues bearing alkyl groups in the 2',3'-position are reported. These compounds were biologically evaluated as P2X3 antagonists using the patch clamp recording technique on mouse trigeminal ganglionic sensory neurons. Some of the compounds showed nanomolar inhibitory potency for the P2X3 receptor. Further modification of these derivatives was made by substitution of the triphosphate chain with different acidic groups. All compounds were additionally tested at five human P2X receptor subtypes stably expressed in 1321N1 astrocytoma cells to evaluate their potency and P2X3 selectivity. Results confirmed the P2X3 antagonist potency for some derivatives.

Microparticulate P2X7 and GSDM-D mediated regulation of functional IL-1β release.

The pro-inflammatory cytokine IL-1β is a secreted protein that is cleaved by caspase-1 during inflammasome activation upon recognition of internal and external insults to cells. Purinergic receptor P2X7 has been described to be involved in the release pathway of bioactive mature IL-1β by activated immune cells. Microparticle (MP) shedding has also been recently recognized as a manner of cytokine IL-1β release. However, the understanding of purinergic receptor roles in the MP-mediated IL-1β release process is still rudimentary. Gasdermin-D (GSDM-D), a protein involved in pyroptosis and inflammasome activation, has been recently described to be involved in the release of microparticles by virtue of its pore-forming ability. Hence, our current work is aimed to study the role of P2X7 in regulating GSDM-D-mediated microparticles and thereby bioactive IL-1β release. We provide evidence that cleaved functional IL-1β release in microparticles upon LPS stimulation is regulated by GSDM-D and P2X7 in a two-step fashion. GSDM-D activation first regulates release of IL-1β and P2X7 into microparticles. Then, microparticulate active P2X7 receptor then regulates the release of bioactive IL-1β encapsulated in microparticles to be able to target other cells inducing IL-8. Using an ATP model of stimulation, we further demonstrated that extracellular ATP stimulation to IL-1β containing LPS microparticles induces release of its content, which when subjected to epithelial cells induced IL-8. This effect was blocked by P2X7 inhibitor, KN62, as well as by IL-1RA. Taken together, our findings demonstrate for the first time the synergistic critical roles of GSDM-D and purinergic receptors in the regulation of microparticulate bioactive IL-1β release and induction of target cell responses.

Ethanol upregulates the P2X7 purinergic receptor in human macrophages.

Alcohol consumption is considered to be the third leading cause of death in the United States. In addition to its direct toxicity, ethanol has two contrasting effects on the immune system: the nucleotide oligomerization domain-like receptor pyrin domain-containing-3 (NLRP3) inflammasome is inhibited by acute ethanol exposure but activated by chronic ethanol exposure. Purinergic receptors (especially the P2X7 receptor) are able to activate the NLRP3 inflammasome and are involved in many ethanol-related diseases (such as gout, pulmonary fibrosis, alcoholic steatohepatitis, and certain cancers). We hypothesized that ethanol regulates purinergic receptors and thus modulates the NLRP3 inflammasome's activity. In experiments with monocyte-derived macrophages, we found that interleukin (IL)-1β secretion was inhibited after 7h of exposure (but not 48h of exposure) to ethanol. The disappearance of ethanol's inhibitory effect on IL-1β secretion after 48h was not mediated by the upregulated production of IL-1β, IL-1α, IL-6 or the inflammasome components NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain, and caspase 1. P2X7R expression was upregulated by ethanol, whereas expression of the P2X4 and P2X1 receptors was not. Taken as a whole, our results suggest that ethanol induces NLRP3 inflammasome activation by upregulating the P2X7 receptor. This observation might have revealed a new mechanism for inflammation in ethanol-related diseases.

Inhibition of P2X7R-NLRP3 Inflammasome Activation by Pleurotus citrinopileatus: A Possible Protective Role in Alcoholic Hepatosteatosis.

Pleurotus citrinopileatus (golden oyster mushroom) is a widely used edible mushroom. We investigated the inhibitory effect of P. citrinopileatus aqueous extract against alcoholic steatohepatitis and its underlying mechanism. Acute and chronic ethanol-feeding murine models were established by intragastrically administering ethanol or feeding an ethanol-containing Lieber-DeCarli liquid diet to male C57BL/6 mice. In both models, P. citrinopileatus decreased serum alanine aminotransferase (ALT), aspartate transaminase (AST), triglyceride (TG), and hepatic TG levels. Hematoxylin and eosin (HE) and Oil Red O staining confirmed that P. citrinopileatus ameliorated both acute and chronic alcoholic hepatosteatosis, characterized by regulation of lipid-metabolism-related proteins, including sirtuin 1 (SIRT1), AMP-activated kinase (AMPK), and sterol regulatory element-binding protein (SREBP1). P. citrinopileatus reversed inflammatory response via modulating purinergic receptor P2X ligand-gated ion channel 7 (P2X7R)-NOD-like receptor pyrin domain 3 (NLRP3) inflammasome. P. citrinopileatus restored the expressions of those proteins to a normal level. In addition, HepG2 cells were incubated with P. citrinopileatus prior to ethanol stimulation. P. citrinopileatus reduced ethanol exposure-induced lipid deposition. Concomitantly, P. citrinopileatus increased AMPK and SIRT1 expressions, which were reduced by ethanol treatment. P. citrinopileatus ameliorated alcoholic hepatic steatosis and accompanied inflammatory response via regulating SIRT1-AMPK and P2X7R-NLRP3 inflammasome activation, highlighting a promising strategy and utility of P. citrinopileatus for alcoholic steatohepatitis as dietary health supplements.

Novel Evidence That Extracellular Nucleotides and Nucleosides Regulate the Expression of Heme Oxygenase 1 (HO-1) in Opposite Ways in Hematopoietic Stem/Progenitor Cells (HSPCs), Which Explains Why ATP Enhances Mobilization of HSPCs, While Its Metabolite Adenosine Inhibits This Process

Background. One of the problems with the mobilization of hematopoietic stem/progenitor cells (HSPCs) in the clinic is that a significant number of patients are poor mobilizers. In order to develop more efficient mobilization strategies, we have to better understand the mobilization process at the molecular and cellular levels. We have reported that HSPCs express heme oxygenase 1 (HO-1), which is a negative regulator of the complement cascade (ComC), which in turn is required for stem cell mobilization (Stem Cell Rev. 2015, 11:110-8, Leukemia. 2017, 31:446-458). Moreover, very recently we became interested in the novel role of extracellular nucleotides (EXNs) and nucleosides in this process (Leukemia 2018, in press, doi: 10.1038/s41375-018-0122-0). The most important EXN is adenosine triphosphate (ATP), which is involved in intracellular energy transfer but if released from activated cells into the extracellular space becomes an important signaling molecule involved in purinergic signaling. Secretion of ATP from activated cells occurs in a pannexin channel-dependent manner and is additionally augmented by a positive regulatory loop after ATP binding to the P2X7 purinergic receptor. We recently found that the ATP concentration during the mobilization process increases to micromolar concentrations in the BM microenvironment. Moreover, we found that in the extracellular BM space ATP is processed by the CD39 and CD73 ectonucleotidases to ADP, AMP, and finally to adenosine, which is also an important mediator of purinergic signaling. Hypothesis. We hypothesized that EXNs and purinergic signaling via extracellular ATP and its metabolite adenosine are involved in triggering the mobilization of HSPCs and that this effect is mediated by the expression of HO-1 in HSPCs. Materials and Methods. To address this question, 2-month-old P2X7-/-, CD39-/-, and CD73-/-mice as well as their normal wild type (WT) littermates were mobilized with G-CSF or AMD3100. Following mobilization, we measured i) the total number of white blood cells (WBCs) and ii) the number of circulating clonogenic colony-forming unit granulocyte/macrophage (CFU-GM) progenitors and Sca-1+c-kit+lineage- (SKL) cells circulating in PB. The secretion of ATP from BM cells was inhibited by employing the pannexin 1 blocking drug probenecid or a synthetic pannexin 1 blocking peptide. In parallel, we evaluated i) the expression of HO-1 in HSPCs by employing RQ-PCR and western blot analysis and ii) the activation of the ComC by C5a ELISA. Results. Here we provide evidence that ATP, as an EXN secreted in a pannexin-1-dependent manner from BM cells, triggers activation of the ComC and initiates mobilization of HSPCs. Inhibition of the pannexin 1 channel by probenecid or a pannexin 1 blocking peptide inhibited this process. Furthermore, mobilization of HSPCs was augmented in a P2X7 receptor-dependent manner, evidenced by the finding that P2X7-/- mice were poor mobilizers. Furthermore, ATP is processed to adenosine in the extracellular space by CD39 and CD73 ectonucleotidases, and to our surprise we observed that CD73-deficient mice mobilize more HSPCs than their control wild type (WT) littermates, which indicates a novel negative role for adenosine in the mobilization process. This finding was confirmed by injecting mice with adenosine during AMD3100 administration. Finally, we found that ATP and adenosine modulate the mobilization process by regulating in opposite ways HSPC expression of HO-1, which is a negative regulator of mobilization. Therefore, while ATP downregulates expression of HO-1 in HSPCs and enhances mobilization, adenosine upregulates its expression and negatively affects the egress of HSPCs from BM into PB. Conclusions. We demonstrate for the first time that purinergic signaling involving ATP and its metabolite adenosine regulate the mobilization of HSPCs in an HO-1-dependent manner. While ATP triggers and promotes this process, adenosine has an inhibitory effect. The opposite effects of ATP and adenosine on the mobilization process can be explained by their different effects on the regulation of HO-1 expression in HSPCs. Finally, administration of ATP as a signaling molecule (together with G-CSF or AMD3100), inhibition of CD73 or HO-1 by small-molecule antagonists may provide the basis for more efficient mobilization strategies. DisclosuresNo relevant conflicts of interest to declare.

Clemastine Alleviates Depressive-Like Behavior Through Reversing the Imbalance of Microglia-Related Pro-inflammatory State in Mouse Hippocampus.

Backgrounds: Abundant reports indicate that neuroinflammatory signaling contributes to behavioral complications associated with depression and may be related to treatment response. The glial cells, especially microglia and astrocytes in brain regions of hippocampus and medial prefrontal cortex (mPFC), are major components of CNS innate immunity. Moreover, purinergic receptor P2X, ligand-gated ion channel 7 (P2X7R) was recently reckoned as a pivotal regulator in central immune system. Besides, it was pointed out that clemastine, a first-generation histamine receptor H1 (HRH1) antagonist with considerable safety profile and pharmacological effect, may suppress immune activation through modulating P2X7R. Herein, we investigated the potential anti-neuroinflammatory effects of clemastine on chronic unpredictable mild stress (CUMS)-induced depressive-like behavior in a mouse model.Methods: Male BALB/c mice were subjected to CUMS for 4 weeks, some of them were injected with clemastine fumarate solution. After the stress procedure, behavioral tests including Sucrose Preference Tests (SPTs), Tail Suspension Tests (TSTs) and locomotor activities were performed to evaluate depressive-like phenotype. Subsequently, expression of cytokines and microglia-related inflammatory biomarkers were assessed.Results: In the present research, we found that clemastine significantly reversed both the declination of SPT percentage and the extension of TST immobility durations in depression mouse model without affecting locomotor activity. Also, we observed that clemastine regulated the imbalance of pro-inflammatory cytokines including interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α) in the hippocampus and serum of depressive-like mice. Additionally, clemastine significantly suppressed microglial M1-like activation specifically in the hippocampus, and also improved hippocampal astrocytic loss. Furthermore, clemastine downregulated hippocampal P2X7R without interfering with the expression of HRH1.Conclusion: As a safe and efficient anti-allergic agent, clemastine could impressively alleviate stress-related depressive-like phenotype in mice. Further evidence supported that it was because of the potential function of clemastine in modulating the expression of P2X7 receptor possibly independent of HRH1, therefore suppressing the microglial M1-like activation and pro-inflammatory cytokines release in brain regions of hippocampus rather than mPFC.

Purinergic P2X7 receptor blockade mitigates alcohol-induced steatohepatitis and intestinal injury by regulating MEK1/2-ERK1/2 signaling and egr-1 activity

The P2X7 receptor is an ATP-binding cation channel involved in a broad range of inflammatory diseases. However, little is known about the potential role of P2X7R in alcohol-induced steatohepatitis and intestinal injury. In our study, C57BL/6 mice were intraperitoneally injected with P2X7R antagonists Brilliant Blue G and A438079 from the 4th day to the 10th day during the induction of chronic plus binge alcohol feeding model. Our results showed that alcohol feeding induced significant steatohepatitis and liver injury, which were mitigated by P2X7R blockade as evidenced by decreased serum levels of ALT, AST, T-CHO and TG, reduced lipid accumulation, and less inflammation. The increased intestinal inflammatory cytokines production and the prominent intestinal barrier disruption caused by alcohol were also modulated by P2X7R antagonism. Interestingly, alcohol feeding increased the relative abundance of phylum Bacteroidetes while decreased the number of phylum Verrucomicrobia and genus Akkermansia in the cecal content, which were reversed by P2X7R antagonist. Importantly, the improvement of intestinal barrier function and the restoration of partial taxonomic alterations in the gut microbiota might contribute to protect the liver from gut microbiota dysbiosis-induced second hit. Furthermore, P2X7R blockade inhibited MEK1/2-ERK1/2 phosphorylation and egr-1 expression in both liver and intestine from alcohol-fed mice. Collectively, P2X7R blockade mitigates alcohol-induced steatohepatitis and intestinal injury by inhibiting MEK1/2-ERK1/2 signaling and egr-1 expression. These studies strongly suggest that P2X7R blockade may be a promising therapeutic approach for treating alcoholic liver disease.

Noncoding transcribed ultraconserved region (T-UCR) UC.48+ is a novel regulator of high-fat diet induced myocardial ischemia/reperfusion injury.

Increasing evidence has suggested high-fat diet (HFD) is an independent risk factor for myocardial ischemia/reperfusion (MI/R) injury. Long noncoding RNAs (lncRNAs) recently attracted much attraction in the study of MI/R injury. However, the functional questions of specific lncRNAs in HFD-induced MI/R injury have not been well elucidated. Uc.48+ is a lncRNA from a transcribed ultraconserved region (T-UCR) of human, mouse, and rat genomes. Here, we explored the aggravating role of uc.48+and identified purinergic P2X7 receptor (P2X7R) as a downstream regulator of uc.48+ in HFD-induced MI/R vulnerability. We demonstrated uc.48+ expression was upregulated, accompanied by the corresponding upregulation of P2X7R in HFD I/R myocardium and HFD-induced MI/R vulnerability. Overexpression of uc.48+enhanced, whereas silencing of uc.48 + decreased the expression of P2X7R, cardiomyocyte apoptosis, and MI/R injury. The functional relevance of uc.48+ regulated P2X7R expression and the subsequent NF-κB signaling to promote cardiomyocyte apoptosis was supported by inhibition of P2X7R with its specific antagonist (A438079) as well as the inhibitor of NF-κB signaling (pyrrolidine dithiocarbamate, PDTC) in H9c2 hypoxia/reoxygenation (H/R) cells transfected with pcDNA3.0-uc.48 + plasmid, and RNA immunoprecipitation (RIP) suggested uc.48+ could interact with transcription factor Sp1. Importantly, Sp1 inhibitor (mithramycin, MIT) was found to suppress uc.48+ -induced P2X7R expression and the NF-κB signaling and cardiomyocyte apoptosis. Our findings provide a potential novel mechanism through which uc.48+ boosts cardiomyocyte apoptosis and MI/R vulnerability to HFD. Thus, uc.48+ is a novel regulator of HFD-induced MI/R injury; targeting uc.48+ may be a novel therapeutic approach of MI/R vulnerability to HFD.

P2X4R silence suppresses glioma cell growth through BDNF/TrkB/ATF4 signaling pathway.

Purinergic receptor P2X 4 (P2X4R), a member of purinergic channels family and a subtype of ionotropic adenosine triphosphate receptors, plays a critical role in tumorigenesis. Evidence suggested that P2X4R is expressed in rat C6 glioma model, however, its role and the underlying mechanism of action are still unclear in human glioblastoma multiforme (GBM). In the current study, our aim is to examine the function and the molecular basis of P2X4R in GBM. We first observed that GBM cells, U251, T98, U87, U373, and A172 were all high expressed P2X4R, when compared with the normal human astrocytes (NHA) cells. To gain the function of P2X4R, P2X4R silence cells were constructed by transfection with P2X4R small interfering RNA (siRNA). We found that P2X4R deletion impeded T98 and U87 cell viability and proliferation, and further studies indicated that cell apoptosis and caspase-3 activity was increased in T98 and U87 cell transfected with P2X4R siRNA. Subsequently, we confirmed that P2X4R silence suppressed brain-derived neurotrophic factor (BDNF), Trk receptor tyrosine kinases (TrkB), and activating transcription factor 4 (ATF4) expression in T98 and U87 cells. And P2X4R siRNA-induced ATF4-expression inhibition dependent on BDNF/TrkB signaling pathway. The impact of P2X4R silence on T98 and U87 cell growth and apoptosis was reversed by ATF4 overexpression. In summary, this study provides the first evidence that P2X4R plays important roles in GBM cell growth and apoptosis.

A non-synonymous polymorphism in purinergic P2X7 receptor gene confers reduced susceptibility to essential hypertension in Chinese postmenopausal women

Essential hypertension (EH), characterized by elevated blood pressure (systolic blood pressure, SBP ≥ 140 mmHg and/or diastolic blood pressure, DBP ≥ 90 mmHg), has become a major public health conc...

P2X7 Receptor Antagonist A804598 Inhibits Inflammation in Brain and Liver in C57BL/6J Mice Exposed to Chronic Ethanol and High Fat Diet

Chronic low-grade neuroinflammation is increasingly implicated in organ damage caused by alcohol abuse. Purinergic P2X7 receptors (P2X7Rs) play an important role in the generation of inflammatory responses during a number of CNS pathologies as evidenced from studies using pharmacological inhibition approach. P2X7Rs antagonism has not been tested during chronic alcohol abuse. In the present study, we tested the potential of P2X7R antagonist A804598 to reduce/abolish alcohol-induced neuroinflammation using chronic intragastric ethanol infusion and high-fat diet (Hybrid) in C57BL/6J mice. We have previously demonstrated an increase in neuroinflammatory response in 8weeks of Hybrid paradigm. In the present study, we found neuroinflammatory response to 4weeks of Hybrid exposure. A804598 treatment reversed the changes in microglia and astrocytes, reduced/abolished increases in mRNA levels of number of inflammatory markers, including IL-1β, iNOS, CXCR2, and components of inflammatory signaling pathways, such as TLR2, CASP1, NF-kB1 and CREB1, as well in the protein levels of pro-IL-1β and Nf-kB1. The P2X7R antagonist did not affect the increase in mRNA levels of fraktalkine (CX3CL1) and its receptor CX3CR1, an interaction that plays a neuroprotective role in neuron-glia communication. P2X7R antagonism also resulted in reduction of the inflammatory markers but did not alter steatosis in the liver. Taken together, these findings demonstrate how P2X7R antagonism suppresses inflammatory response in brain and liver but does not alter the neuroprotective response caused by Hybrid exposure. Overall, these findings support an important role of P2X7Rs in inflammation in brain and liver caused by combined chronic alcohol and high-fat diet. Graphical Abstract ᅟ.

The purinergic receptor P2X5 contributes to bone loss in experimental periodontitis

Purinergic receptor signaling is increasingly recognized as an important regulator of inflammation. The P2X family purinergic receptors P2X5 and P2X7 have both been implicated in bone biology, and it has been suggested recently that P2X5 may be a significant regulator of inflammatory bone loss. However, a role for P2X5 in periodontitis is unknown. The present study aimed to evaluate the functional role of P2X5 in ligature-induced periodontitis in mice. Five days after placement of ligature, analysis of alveolar bone revealed decreased bone loss in P2rx5−/− mice compared to P2rx7−/− and WT control mice. Gene expression analysis of the gingival tissue of ligated mice showed that IL1b, IL6, IL17a and Tnfsf11 expression levels were significantly reduced in P2rx5−/− compared to WT mice. These results suggest the P2X5 receptor may regulate bone loss related to periodontitis and it may thus be a novel therapeutic target in this oral disease.

Early P2X7R-dependent activation of microglia during the asymptomatic phase of autoimmune encephalomyelitis

Microglia-mediated neuroinflammation accompanies many central nervous system (CNS) diseases, including multiple sclerosis (MS), and is strongly dependent on the purinergic P2X7 receptor. The nature of the inflammatory response in MS is studied for decades indicating, that proinflammatory microgliosis is involved in advanced stages of MS and is associated with active tissue damage and neurological dysfunctions. Evidence on the role of microgliosis in initial stages of the disease is scarce. Thus, in the present study, we investigated the time course of microglial activation in rat brain subjected to experimental autoimmune encephalomyelitis (EAE) which is the animal model of MS. We show that activation of microglia occurs in brains of immunized rats at a very early stage of EAE, well before the development of neurological symptoms of the disease. Enhanced immunoreactivity of microglia/macrophage-specific protein Iba-1, together with morphological features of microgliosis, was identified beginning at day 4 post immunization. Concomitantly, microglial expression of P2X7R was also examined. Moreover, our results reveal that administration of Brilliant Blue G, an antagonist of P2X7R, delays the onset of the disease and partially inhibits development of neurological symptoms in EAE rats. Blockage of P2X7R significantly reduces activation of microglia as confirmed by decreased Iba-1 immunoreactivity and suppresses neuroinflammation in EAE rat brains, as indicated by decreased protein levels of investigated proinflammatory cytokines: IL-1β, IL-6 and TNF-α. Our results indicate that microglia are involved in inducing neuroinflammation at a very early stage of MS/EAE via a P2X7R-dependent mechanism.

Alpha linolenic acid intake alleviates myocardial ischemia/reperfusion injury via the P2X7R/NF-κB signalling pathway

This study investigated the protective role of α-linolenic acid (ALA) in myocardial ischemia/reperfusion (MI/R) injury. ALA was indicated to dose-dependently protect myocardium against I/R injury, attenuate purinergic P2X7 receptor (P2X7R) activation, prevent NF-κB activation, and inhibit tumor necrosis factor (TNF)-α and interleukin-1β (IL-1β) production in I/R myocardium. Overexpression of P2X7R enhanced NF-κB activation, inflammation and MI/R injury, which could be alleviated by ALA. In vitro hypoxia/reoxygenation (H/R) H9c2 cells, ALA could also inhibit P2X7R activation, NF-κB activation and TNF-α and IL-1β production. Molecular docking analysis indicated ALA may hamper the activation of P2X7R. The functional relevance of ALA directly regulated P2X7R and the subsequent NF-κB signalling was supported by inhibition of P2X7R with A438079 (P2X7R antagonist) as well as suppression of NF-κB by PDTC (NF-κB inhibitor) in H9c2 H/R cells transfected with pcDNA3.0-P2X7R plasmid. These findings demonstrated that ALA attenuates inflammation and MI/R injury through down-regulating P2X7R/NF-κB signalling pathway.

From the Editor’s desk....: September 2018

From the Editor’s desk....: September 2018

Administration of an antagonist of P2X7 receptor to EAE rats prevents a decrease of expression of claudin-5 in cerebral capillaries

Purinergic P2X receptors, when activated under pathological conditions, participate in induction of the inflammatory response and/or cell death. Both neuroinflammation and neurodegeneration represent hallmarks of multiple sclerosis (MS), an autoimmune disease of the central nervous system. In the current study, we examined whether P2X7R is expressed in brain microvasculature of rats subjected to experimental autoimmune encephalomyelitis (EAE) and explore possible relationships with blood-brain barrier (BBB) protein—claudin-5 after administration of P2X7R antagonist—Brilliant Blue G (BBG). Capillary fraction isolated from control and EAE rat brains was subjected to immunohistochemical and Western blot analyses. We document the presence of P2X7R in brain capillaries isolated from brain tissue of EAE rats. P2X7R is found to be localized on the abluminal surface of the microvessels and is co-expressed with PDGFβR, a marker of pericytes. We also show over-expression of this receptor in isolated capillaries during the course of EAE, which is temporally correlated with a lower protein level of PDGFβR, as well as claudin-5, a tight junction-building protein. Administration of a P2X7R antagonist to the immunized rats significantly reduced clinical signs of EAE and enhances protein expression of both claudin-5 and PDGFβR. These results indicate that P2X7 receptor located on pericytes may contribute to pathological mechanisms operated during EAE in cerebral microvessels influencing the BBB integrity.

Blockade of Pannexin-1 Channels and Purinergic P2X7 Receptors Shows Protective Effects Against Cytokines-Induced Colitis of Human Colonic Mucosa.

Introduction: The pannexin-1 (Panx1) channels are found in many cell types, and ATP released from these channels can act on nearby cells activating purinergic P2X7 receptors (P2X7R) which lead to inflammation. Although Panx1 and P2X7R are implicated in the process of inflammation and cell death, few studies have looked at the role they play in inflammatory bowel disease in human. Hence, the aim of the present study was to investigate the function of Panx1 and P2X7R in an ex vivo colitis model developed from human colonic mucosal explants.Materials and Methods: Healthy human colonic mucosal strips (4 × 10 mm) were incubated in carbogenated culture medium at 37°C for 16 h. Proinflammatory cytokines TNFα and IL-1β (each 10 ng/mL) were used to induce colitis in mucosal strips, and the effects of Panx1 and P2X7R on cytokines-induced tissue damage were determined in the presence of the Panx1 channel blocker 10Panx1 (100 μM) and P2X7R antagonist A438079 (100 μM). The effects of 10Panx1 and A438079 on cytokines-enhanced epithelial permeability were also studied using Caco-2 cells.Results: Histological staining showed that the mucosal strips had severe structural damage in the cytokines-only group but not in the incubation-control group (P < 0.01). Compared to the cytokines-only group, crypt damage was significantly decreased in groups receiving cytokines with inhibitors (10Panx1, A438079, or 10Panx1 + A438079, P < 0.05). The immunoreactive signals of tight junction protein zonula occludens-1 (ZO-1) were abundant in all control tissues but were significantly disrupted and lost in the cytokines-only group (P < 0.01). The diminished ZO-1 immunoreactivity induced by cytokines was prevented in the presence of 10Panx1 (P = 0.04). Likewise, 10Panx1 significantly attenuated the cytokines-evoked increase in paracellular permeability of Caco-2 cells. Although the inhibition of P2X7R activity by A438079 diminished cytokines-induced crypt damage, its effect on the maintenance of ZO-1 immunoreactivity and Caco-2 epithelial cell integrity was less evident.Conclusion: The blockade of Panx1 and P2X7R reduced the inflammatory cytokines-induced crypt damage, loss of tight junctions and increase in cell permeability. Thus, Panx1 and P2X7R may have roles in causing mucosal damage, a common clinical feature of inflammatory bowel disease.

Erythrocyte P2X1 receptor expression is correlated with change in haematocrit in patients admitted to the ICU with blood pathogen-positive sepsis

BackgroundPore-forming proteins released from bacteria or formed as result of complement activation are known to produce severe cell damage. Inhibition of purinergic P2X receptors markedly reduces damage inflicted by cytolytic bacterial toxin and after complement activation in both erythrocytes and monocytes. P2X expression generally shows variation throughout the population. Here, we investigate correlation between P2X receptor abundance in blood cell plasma membranes and haematocrit during sepsis, in patients admitted to the emergency department (ED) or intensive care unit (ICU).MethodPatients admitted to the ED and successively transferred to ICU with the diagnosis sepsis (< 2 systemic inflammatory response syndrome (SIRS) criteria and suspected infection), were grouped as either blood pathogen-positive (14 patients) or blood pathogen-negative (20 patients). Blood samples drawn at ICU admission were analysed for P2X1 and P2X7 receptor abundance using indirect flow cytometry.ResultsHere, we find inverse correlation between P2X1 receptor expression and change in haematocrit (rs − 0.80) and haemoglobin (rs − 0.78) levels from admission to ED to arrival at ICU in patients with pathogen-positive sepsis. This correlation was not found in patients without confirmed bacteraemia. Patients with high P2X1 expression had a significantly greater change in both haematocrit (− 0.59 ± 0.36) and haemoglobin levels (− 0.182 ± 0.038 mg/dl) per hour, during the first hours after hospital admission compared to patients with low P2X1 expression (0.007 ± 0.182 and − 0.020 ± 0.058 mg/dl, respectively).ConclusionHigh levels of P2X1 are correlated with more pronounced reduction in haematocrit and haemoglobin in patients with confirmed bacteraemia. This supports previous in vitro findings of P2X activation as a significant component in cell damage caused by pore-forming bacterial toxins and complement-dependent major attack complex. These data suggest a new potential target for future therapeutics in initial stages of sepsis.

Green Pharma: A New Strategy for Drug Discovery in Academia by Targeting Glial Cells and ATP Receptors

Neuropathic pain associated with cancer, diabetic neuropathy, and postherpetic neuralgia is a type of intractable chronic pain characterized by mechanical allodynia and abnormal pain hypersensitivity evoked by innocuous stimuli. However, this disorder has no specific treatment. We previously showed that the purinergic receptor P2X4 (P2X4R), a subtype of ATP-gated nonselective cation channels, is highly upregulated in spinal microglia after peripheral nerve injury, and blocking the function of P2X4R reverses mechanical allodynia. In the present study, we screened a chemical library of 1979 clinically approved compounds (a gift from the Drug Discovery Initiative at the University of Tokyo) aimed at achieving "Eco-Pharma," which refers to seeking new effects of existing drugs. We demonstrated that duloxetine, a serotonin and noradrenaline reuptake inhibitor, has an inhibitory effect on rat and human P2X4R. In rat primary cultured microglial cells, duloxetine also inhibited P2X4R-mediated responses. Moreover, intrathecal administration of duloxetine in a model of neuropathic pain reversed nerve injury-induced mechanical allodynia. Based on those results, we suggest that the inhibition of P2X4R expressed in microglial cells may be involved in the antiallodynic effect of duloxetine in neuropathic pain. Furthermore, in this review, we discuss a new strategy for drug discovery called "Green Pharma" (a merger of "Eco-Pharma" and "Green chemistry" and referring to the development of eco-friendly pharmaceuticals).