Systemic blockade of P2X7 receptor protects against sepsis-induced intestinal barrier disruption.
ABSTRACT: Sepsis, during which the intestinal epithelial barrier is frequently disrupted, remains a challenging and life-threatening problem in clinical practice. The P2X7 receptor (P2X7R) is a non-selective adenosine triphosphate-gated cation channel present in macrophages that is involved in inflammatory responses. However, little is known about the role of P2X7R in macrophages during sepsis-induced intestinal barrier disruption. In this study, mice were treated with the P2X7R antagonist A740003 or the agonist BzATP by intra-peritoneal injection after the induction of gut-origin sepsis. The survival rates, inflammatory responses, intestinal barrier integrity, macrophage marker expression, and ERK and NF-?B activities were evaluated. Intestinal macrophages were also isolated and studied after exposure to Brilliant Blue G or BzATP. We found that a systemic P2X7R blockade downregulated sepsis-induced inflammatory responses and attenuated intestinal barrier dysfunction based on the evidence that mice in the A740003-treated group exhibited alleviated pro-inflammatory cytokine synthesis, intestinal hyperpermeability, epithelial apoptosis rates and tight junction damage compared with the septic mice. These changes were partly mediated by the inhibition of M1 macrophages activation via ERK/NF-?B pathways. Our data presented herein show that a P2X7R blockade could be a potential therapeutic target for the treatment of sepsis-induced intestinal barrier dysfunction.
Project description:ATP can activate a variety of pathways through P2 purinoreceptors, leading to neuroprotection and pathology in the CNS. Among all P2X receptors, the P2X7 receptor (P2X7R) is a well-defined therapeutic target for inflammatory and neuropathic pain. Activation of P2X7R can generate reactive oxygen species (ROS) in macrophages and microglia. However, the role of ROS in P2X7R-induced pain remains un?explored. Here, we investigated the downstream effects of neuronal P2X7R activation in the spinal cord. We found that ATP induces ROS production in spinal cord dorsal horn neurons, an effect eliminated by ROS scavenger N-tert-butyl-?-phenylnitrone (PBN) and P2X7R antagonist A438079. A similar effect was observed with a P2X7R agonist, BzATP, and was attenuated by a NADPH oxidase inhibitor apocynin. Intrathecal administration of BzATP resulted in ROS production in the spinal cord and oxidative DNA damage in dorsal horn neurons. BzATP also induced robust biphasic spontaneous nociceptive behavior. Pre-treatment with A438079 abolished all BzATP-induced nociceptive behaviors, while ROS scavengers dose-dependently attenuated the secondary response. Here, we provide evidence that neuronal P2X7R activation leads to ROS production and subsequent nociceptive pain in mice. Together, the data indicate that P2X7R-induced ROS play a critical role in the P2X7R signaling pathway of the CNS.
Project description:Gliomas are the most lethal tumors of central nervous system. ATP is an important signaling molecule in CNS and it is a selective P2X7 purinergic receptor ligand at high concentrations. Herein, we investigated whether the activation of P2X7R might be implicated in death of a radiosensitive human glioma lineage. The effects of P2X7R agonists (ATP and BzATP) and irradiation (2 Gy) on glioma cells were analyzed by MTT assay and annexin-V/PI determination, whereas mRNA and protein P2X7R expression was assessed by qRT-PCR and flow cytometry, respectively. P2X7R pore formation was functionality examined by analyzing ethidium bromide uptake. The human glioma cells U-138 MG and U-251 MG were resistant to death when treated with either ATP (5 mM) or BzATP (100 μM), but the radiosensitive M059J glioma cells displayed a significant decrease of cell viability (32.4 ± 4.1 % and 25.6 ± 3.3 %, respectively). The M059J lineage expresses significantly higher mRNA P2X7R levels when compared to the U-138 MG and U-251 cell lines (0.40 ± 0.00; 0.28 ± 0.01, and 0.31 ± 0.01, respectively), and irradiation upregulated P2X7R expression (0.55 ± 0.08) in this lineage. Noteworthy, P2X7R protein doubled after irradiation on M059J lineage, and increased in 50 % and 42.6 % when comparing M059J-irradiated to irradiated U-138 MG and U-251 MG cells, respectively. Ethidium bromide uptake was significantly increased in 104 % and 77.8 % when comparing M059J to U-138 MG and U-251MG, respectively. Finally, the selective P2X7R antagonist A740003 significantly decreased the cell death caused by irradiation. We provide novel evidence indicating that M059J human glioma cell line is ATP-P2X7R sensitive, pointing out the relevance of the purinergic P2X7R on glioma radiosensitivity.
Project description:Objectives: Systemic sclerosis (SSc) is a connective tissue disorder presenting fibrosis of the skin and internal organs, for which no effective treatments are currently available. Increasing evidence indicates that the P2X7 receptor (P2X7R), a nucleotide-gated ionotropic channel primarily involved in the inflammatory response, may also have a key role in the development of tissue fibrosis in different body districts. This study was aimed at investigating P2X7R expression and function in promoting a fibrogenic phenotype in dermal fibroblasts from SSc patients, also analyzing putative underlying mechanistic pathways. Methods: Fibroblasts were isolated by skin biopsy from 9 SSc patients and 8 healthy controls. P2X7R expression, and function (cytosolic free Ca2+ fluxes, ?-smooth muscle actin [?-SMA] expression, cell migration, and collagen release) were studied. Moreover, the role of cytokine (interleukin-1?, interleukin-6) and connective tissue growth factor (CTGF) production, and extracellular signal-regulated kinases (ERK) activation in mediating P2X7R-dependent pro-fibrotic effects in SSc fibroblasts was evaluated. Results: P2X7R expression and Ca2+ permeability induced by the selective P2X7R agonist 2'-3'-O-(4-benzoylbenzoyl)ATP (BzATP) were markedly higher in SSc than control fibroblasts. Moreover, increased ?SMA expression, cell migration, CTGF, and collagen release were observed in lipopolysaccharides-primed SSc fibroblasts after BzATP stimulation. While P2X7-induced cytokine changes did not affect collagen production, it was completely abrogated by inhibition of the ERK pathway. Conclusion: In SSc fibroblasts, P2X7R is overexpressed and its stimulation induces Ca2+-signaling activation and a fibrogenic phenotype characterized by increased migration and collagen production. These data point to the P2X7R as a potential, novel therapeutic target for controlling exaggerated collagen deposition and tissue fibrosis in patients with SSc.
Project description:Introduction: P2X7R is an extracellular ATP-gated receptor involved in inflammatory and autoimmune processes mainly acting through NLPR3-inflammasome activation and IL-1? release, also implicated in lymphocyte proliferation and cellular apoptosis. Several observations from animal models and patients' studies highlight a possible link between P2X7R-NLRP3 axis and Systemic Lupus Erythematosus (SLE) pathogenesis. The P2X7R-inflammasome axis in addition to the direct production of IL-1? and IL-18, indirectly mediates the release of other cytokines implicated in the pathogenesis of SLE, such as IL-6. The aim of this study was to investigate the role of P2X7R and NLRP3-inflammasome in SLE. Methods: Forty-eight SLE patients, 16 with (SLE-S) and 32 without (SLE-NS) history of serositis, and 20 healthy control (HC) subjects were enrolled. Demographic, clinical, and therapeutic data were collected. IL-1? and IL-6 plasma levels were evaluated by ELISA. Peripheral blood mononuclear cells (PBMCs) were isolated from venous blood by Ficoll gradient sedimentation and employed as follows: (1) evaluation of P2X7R and NLRP3 expression by RT-PCR; (2) determination of P2X7R activity as Benzoyl ATP (BzATP)-induced [Ca2+]i increments using Fura-2-AM fluorescent probe; (3) isolation of monocytes/macrophages and assessment of in vitro IL-1? and IL-6 release following stimulation with lipopolysaccharide (LPS) and BzATP, either separately or in combination. Results: Plasma IL-1? levels were unmodified in SLE respect to HC whereas IL-6 levels were higher in SLE than in HC, resulting significantly increased in SLE-S. Macrophages isolated from SLE patients released lower quantities of IL-1? after stimulation with BzATP, whereas IL-6 release was significantly augmented in SLE-NS respect to both HC and SLE-S after all types of stimulation. The [Ca2+]i increase following BzATP stimulation was significantly lower in PBMCs from SLE patients than in PBMCs from HC. RT-PCR showed significantly reduced P2X7R and significantly augmented NLRP3 expression in PBMCs from SLE patients. Conclusion: Our data indicate reduced P2X7R expression and function in SLE patients compared with HC and, conversely, increased IL-6 signaling. The possible consequences of reduced P2X7R, mainly on cytokines network deregulation and lymphocyte proliferation, will be further investigated as well as the role of IL-6 as a possible therapeutic target especially in lupus serositis.
Project description:Accumulating evidence indicates that the pluripotency of periodontal ligament stem cells (PDLSCs) is compromised under inflammatory conditions; however, the underlying mechanisms remain largely unexplored. In this study, we hypothesize that the P2X7 receptor (P2X7R) is a key molecule linked to inflammation-associated impairment of PDLSCs. We first investigated P2X7R expression in PDLSCs under normal and inflammatory conditions and then determined the effect of a P2X7R agonist (BzATP) or antagonist (BBG) on PDLSC osteogenesis under various conditions. Gene-modified PDLSCs were used to further examine the role of P2X7R and the signaling pathway underlying P2X7R-enhanced osteogenesis. We found that inflammatory conditions decreased P2X7R expression in PDLSCs and reduced osteogenesis in these cells. In addition, activation of P2X7R by BzATP or overexpression of P2X7R via gene transduction reversed the inflammation-mediated decrease in PDLSC osteogenic differentiation. When selected osteogenesis-related signaling molecules were screened, the PI3K-AKT-mTOR pathway was identified as potentially involved in P2X7R-enhanced PDLSC osteogenesis. Our data reveal a crucial role for P2X7R in PDLSC osteogenesis under inflammatory conditions, suggesting a new therapeutic target to reverse or rescue inflammation-mediated changes in PDLSCs for future mainstream therapeutic uses.
Project description:Acute lung injury (ALI) is characterized by pulmonary endothelial and epithelial cell damage, and loss of the alveolar-capillary barrier. We have previously shown that P2X7 receptor (P2X7R), a cell death receptor, is specifically expressed in alveolar epithelial type I cells (AEC I). In this study, we hypothesized that P2X7R-mediated purinergic signaling and its interaction with Wnt/?-catenin signaling contributes to AEC I death. We examined the effect of P2X7R agonist 2'-3'-O-(4-benzoylbenzoyl)-ATP (BzATP) and Wnt agonist Wnt3a on AEC I death in vitro and in vivo. We also assessed the therapeutic potential of Wnt3a in a clinically relevant ALI model of intratracheal lipopolysaccharide (LPS) exposure in ventilated mice. We found that the activation of P2X7R by BzATP caused the death of AEC I by suppressing Wnt/?-catenin signaling through stimulating glycogen synthase kinase-3? (GSK-3?) and proteasome. On the other hand, the activation of Wnt/?-catenin signaling by Wnt3a, GSK-3? inhibitor, or proteasome inhibitor blocked the P2X7R-mediated cell death. More importantly, Wnt3a attenuated the AEC I damage caused by intratracheal instillation of BzATP in rats or LPS in ventilated mice. Our results suggest that Wnt3a overrides the effect of P2X7R on the Wnt/?-catenin signaling to prevent the AEC I death and restrict the severity of ALI.
Project description:Recent evidence indicates an involvement of P2X7 purinergic receptor (P2X7R) in the fine tuning of immune functions, as well as in driving enteric neuron apoptosis under intestinal inflammation. However, the participation of this receptor in the regulation of enteric neuromuscular functions remains undetermined. This study was aimed at investigating the role of P2X7Rs in the control of colonic motility in experimental colitis.Colitis was induced in rats by 2,4-dinitrobenzenesulfonic acid. P2X7R distribution was examined by immunofluorescence analysis. The effects of A804598 (selective P2X7R antagonist) and BzATP (P2X7R agonist) were tested on contractions of longitudinal smooth muscle evoked by electrical stimulation or by carbachol in the presence of tetrodotoxin.P2X7Rs were predominantly located in myenteric neurons, but, in the presence of colitis, their expression increased in the neuromuscular layer. In normal preparations, A804598 elicited a negligible increase in electrically induced contractions, while a significant enhancement was recorded in inflamed tissues. In the presence of N?-propyl-L-arginine (NPA, neuronal nitric oxide synthase inhibitor) the A804598 effects were lost. P2X7R stimulation with BzATP did not significantly affect electrical-induced contractions in normal colon, while a marked reduction was recorded under inflammation. The inhibitory effect of BzATP was antagonized by A804598, and it was also markedly blunted by NPA. Both P2X7R ligands did not affect carbachol-induced contractions.The purinergic system contributes to functional neuromuscular changes associated with bowel inflammation via P2X7Rs, which modulate the activity of excitatory cholinergic nerves through a facilitatory control on inhibitory nitrergic pathways.
Project description:Neuroinflammation contributes to the pathogenesis of early brain injury (EBI) after subarachnoid hemorrhage (SAH). Cytotoxic events following SAH, such as extracellular accumulation of adenosine triphosphate (ATP), may activate the P2X purinoceptor 7 (P2X7R)/cryopyrin inflammasome axis, thus inducing the proinflammatory cytokine IL-1?/IL-18 secretion. We therefore hypothesized that inhibition of P2X7R/cryopyrin inflammasome axis would ameliorate neuroinflammation after SAH. In the present study, SAH was induced by the endovascular perforation in rats. Small interfering RNAs (siRNAs) of P2X7R or cryopyrin were administered intracerebroventricularly 24h before SAH. Brilliant blue G (BBG), a non-competitive antagonist of P2X7R, was administered intraperitoneally 30min following SAH. Post-assessments including SAH severity score, neurobehavioral test, brain water content, Western blot and immunofluorescence, were performed. Administration of P2X7R and cryopyrin siRNA as well as pharmacologic blockade of P2X7R by BBG ameliorated neurological deficits and brain edema at 24h following SAH. Inhibition of P2X7R/cryopyrin inflammasome axis suppressed caspase-1 activation, which subsequently decreased maturation of IL-1?/IL-18. To investigate the link between P2X7R and cryopyrin inflammasome in vivo, Benzoylbenzoyl-ATP (BzATP), a P2X7R agonist, was given to lipopolysaccharide (LPS) primed naive rats with scramble or cryopyrin siRNAs. In LPS-primed naive rats, BzATP induced caspase-1 activation and mature IL-1? release were neutralized by cryopyrin siRNA. Thus, the P2X7R/cryopyrin inflammasome axis may contribute to neuroinflammation via activation of caspase-1 and thereafter mature IL-1?/IL-18 production following SAH. Therapeutic interventions targeting P2X7R/cryopyrin pathway may be a novel approach to ameliorate EBI following SAH.
Project description:Blockading P2X7 receptor(P2X7R) provides neuroprotection toward various neurological disorders, including stroke, traumatic brain injury, and subarachnoid hemorrhage. However, whether and how P2X7 receptor suppression protects blood-brain barrier(BBB) after intracerebral hemorrhage(ICH) remains unexplored. In present study, intrastriatal autologous-blood injection was used to mimic ICH in rats. Selective P2X7R inhibitor A438079, P2X7R agonist BzATP, and P2X7R siRNA were administrated to evaluate the effects of P2X7R suppression. Selective RhoA inhibitor C3 transferase was administered to clarify the involvement of RhoA. Post-assessments, including neurological deficits, Fluoro-Jade C staining, brain edema, Evans blue extravasation and fluorescence, western blot, RhoA activity assay and immunohistochemistry were performed. Then the key results were verified in collagenase induced ICH model. We found that endogenous P2X7R increased at 3 hrs after ICH with peak at 24 hrs, then returned to normal at 72 hrs after ICH. Enhanced immunoreactivity was observed on the neurovascular structure around hematoma at 24 hrs after ICH, along with perivascular astrocytes and endothelial cells. Both A438079 and P2X7R siRNA alleviated neurological deficits, brain edema, and BBB disruption after ICH, in association with RhoA activation and down-regulated endothelial junction proteins. However, BzATP abolished those effects. In addition, C3 transferase reduced brain injury and increased endothelial junction proteins' expression after ICH. These data indicated P2X7R suppression could preserve BBB integrity after ICH through inhibiting RhoA activation.
Project description:Ischemia/reperfusion (I/R) elicits renin release from cardiac mast cells (MC), thus activating a local renin-angiotensin system (RAS), culminating in ventricular fibrillation. We hypothesized that in I/R, neurogenic ATP could degranulate juxtaposed MC and that ecto-nucleoside triphosphate diphosphohydrolase 1/CD39 (CD39) on MC membrane could modulate ATP-induced renin release. We report that pharmacological inhibition of CD39 in a cultured human mastocytoma cell line (HMC-1) and murine bone marrow-derived MC with ARL67156 (100 µM) increased ATP-induced renin release (?2-fold), whereas purinergic P2X7 receptors (P2X7R) blockade with A740003 (3 µM) prevented it. Likewise, CD39 RNA silencing in HMC-1 increased ATP-induced renin release (?2-fold), whereas CD39 overexpression prevented it. Acetaldehyde, an I/R product (300 µM), elicited an 80% increase in ATP release from HMC-1, in turn, causing an autocrine 20% increase in renin release. This effect was inhibited or potentiated when CD39 was overexpressed or silenced, respectively. Moreover, P2X7R silencing prevented ATP- and acetaldehyde-induced renin release. I/R-induced RAS activation in ex vivo murine hearts, characterized by renin and norepinephrine overflow and ventricular fibrillation, was potentiated (?2-fold) by CD39 inhibition, an effect prevented by P2X7R blockade. Our data indicate that by regulating ATP availability at the MC surface, CD39 modulates local renin release and thus, RAS activation, ultimately exerting a cardioprotective effect.