Dissection of P2X4 and P2X7 Receptor Current Components in BV-2 Microglia

Microglia cells represent the immune system of the central nervous system. They become activated by ATP released from damaged and inflamed tissue via purinergic receptors. Ionotropic purinergic P2X4 and P2X7 receptors have been shown to be involved in neurological inflammation and pain sensation. Whether the two receptors assemble exclusively as homotrimers or also as heterotrimers is still a matter of debate. We investigated the expression of P2X receptors in BV-2 microglia cells applying the whole-cell voltage-clamp technique. We dissected P2X4 and P2X7 receptor-mediated current components by using specific P2X4 and P2X7 receptor blockers and by their characteristic current kinetics. We found that P2X4 and P2X7 receptors are activated independently from each other, indicating that P2X4/P2X7 heteromers are not of functional significance in these cells. The pro-inflammatory mediators lipopolysaccharide and interferon γ, if applied in combination, upregulated P2X4, but not P2X7 receptor-dependent current components also arguing against phenotypically relevant heteromerization of P2X4 and P2X7 receptor subunits.     

Structural and Functional Basis for Understanding the Biological Significance of P2X7 Receptor

The P2X7 receptor (P2X7R) possesses a unique structure associated to an as yet not fully understood mechanism of action that facilitates cell permeability to large ionic molecules through the receptor itself and/or nearby membrane proteins. High extracellular adenosine triphosphate (ATP) levels—inexistent in physiological conditions—are required for the receptor to be triggered and contribute to its role in cell damage signaling. The inconsistent data on its activation pathways and the few studies performed in natively expressed human P2X7R have led us to review the structure, activation pathways, and specific cellular location of P2X7R in order to analyze its biological relevance. The ATP-gated P2X7R is a homo-trimeric receptor channel that is occasionally hetero-trimeric and highly polymorphic, with at least nine human splice variants. It is localized predominantly in the cellular membrane and has a characteristic plasticity due to an extended C-termini, which confers it the capacity of interacting with membrane structural compounds and/or intracellular signaling messengers to mediate flexible transduction pathways. Diverse drugs and a few endogenous molecules have been highlighted as extracellular allosteric modulators of P2X7R. Therefore, studies in human cells that constitutively express P2X7R need to investigate the precise endogenous mediator located nearby the activation/modulation domains of the receptor. Such research could help us understand the possible physiological ATP-mediated P2X7R homeostasis signaling.     

The P2X7 Receptor: A Promising Pharmacological Target in Diabetic Retinopathy

Diabetes is a worldwide emergency. Its chronic complications impose a heavy burden on patients, health systems, and on society as a whole. Diabetic retinopathy is one of the most common and serious complications of diabetes, and an established risk factor for blindness in adults. Over 15 years of investigation led to the identification of vascular endothelial growth factor (VEGF) as a main pathogenic factor in diabetic retinopathy and to the introduction of highly effective anti-VEGF-based therapies, such as the monoclonal antibody bevacizumab or its fragment ranibizumab, which helped to prevent diabetes-related blindness in millions of patients. Recently, a pathogenic role for uncontrolled increases in the extracellular ATP concentration (eATP) and for overactivation of the purinergic receptor P2X7 (P2X7R) has been suggested. The P2X7R is an eATP-gated plasma membrane channel expressed in multiple tissues and organs, with a pleiotropic function in inflammation, immunity, cancer, and hormone and growth factor release. P2X7R stimulation or overexpression positively regulate the secretion and buildup of VEGF, thus promoting neo-angiogenesis in a wide variety of disease processes. In this review, we explore current evidence that supports the role of P2X7R receptor signaling in the pathogenesis of diabetic retinopathy, as well as the most appealing current therapeutical options for P2X7R targeting.     

The P2X7R-NLRP3 and AIM2 Inflammasome Platforms Mark the Complexity/Severity of Viral or Metabolic Liver Damage

P2X7R-NLRP3 and AIM2 inflammasomes activate caspase-1 and the release of cytokines involved in viral-related liver disease. Little is known about their role in non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steato-hepatitis (NASH). We characterized the role of inflammasomes in NAFLD, NASH, and HCV. Gene expression and subcellular localization of P2X7R/P2X4R-NLRP3 and AIM2 inflammasome components were examined in histopathological preparations of 46 patients with biopsy-proven viral and metabolic liver disease using real-time PCR and immunofluorescence. P2X7R, P2X4R, and Caspase-1 are two- to five-fold more expressed in patients with NAFLD/NASH associated with chronic HCV infection than those with metabolic damage only (p ≤ 0.01 for all comparisons). The AIM2 inflammasome is 4.4 times more expressed in patients with chronic HCV infection, regardless of coexistent metabolic abnormalities (p = 0.0006). IL-2, a cytokine playing a pivotal role during chronic HCV infection, showed a similar expression in HCV and NASH patients (p = 0.77) but was virtually absent in NAFLD. The P2X7R-NLRP3 complex prevailed in infiltrating macrophages, while AIM2 was localized in Kupffer cells. Caspase-1 expression correlated with elastography-based liver fibrosis (r = 0.35, p = 0.02), whereas P2X7R, P2X4R, NRLP3, Caspase-1, and IL-2 expression correlated with circulating markers of disease severity. P2X7R and P2X4R play a major role in liver inflammation accompanying chronic HCV infection, especially when combined with metabolic damage, while AIM2 is specifically expressed in chronic viral hepatitis. We describe for the first time the hepatic expression of IL-2 in NASH, so far considered a peculiarity of HCV-related liver damage.     

P2X7 Receptors and TMEM16 Channels Are Functionally Coupled with Implications for Macropore Formation and Current Facilitation

P2X7 receptors (P2X7) are cationic channels involved in many diseases. Following their activation by extracellular ATP, distinct signaling pathways are triggered, which lead to various physiological responses such as the secretion of pro-inflammatory cytokines or the modulation of cell death. P2X7 also exhibit unique behaviors, such as “macropore” formation, which corresponds to enhanced large molecule cell membrane permeability and current facilitation, which is caused by prolonged activation. These two phenomena have often been confounded but, thus far, no clear mechanisms have been resolved. Here, by combining different approaches including whole-cell and single-channel recordings, pharmacological and biochemical assays, CRISPR/Cas9 technology and cell imaging, we provide evidence that current facilitation and macropore formation involve functional complexes comprised of P2X7 and TMEM16, a family of Ca²⁺-activated ion channel/scramblases. We found that current facilitation results in an increase of functional complex-embedded P2X7 open probability, a result that is recapitulated by plasma membrane cholesterol depletion. We further show that macropore formation entails two distinct large molecule permeation components, one of which requires functional complexes featuring TMEM16F subtype, the other likely being direct permeation through the P2X7 pore itself. Such functional complexes can be considered to represent a regulatory hub that may orchestrate distinct P2X7 functionalities.     

Inhibition of P2X7 Purinergic Receptor Ameliorates Fibromyalgia Syndrome by Suppressing NLRP3 Pathway

Fibromyalgia is a chronic condition characterized by persistent widespread pain that significantly reduces quality of life in patients. The purinergic P2X7 receptor (P2X7R) seems to be involved in different pain states and neuroinflammation. The purpose of this study is to investigate the positive effects of P2X7R inhibition by the antagonist Brilliant Blue G (BBG) in a rat model of reserpine-induced fibromyalgia. Sprague–Dawley male rats were injected with 1 mg/kg of reserpine for three consecutive days. Later, animals were administered BBG (50 mg/kg) intraperitoneally for seven days. Reserpine injections induced a significant increase in pain pro-inflammatory mediators as well as a significant increase in neuroinflammation. Chronic pain, in turn, led to depressive-like symptoms and reduced neurogenesis. Blockage of P2X7R by BBG administrations is able to attenuate the behavioral deficits, pain mediators and microglial activation induced by reserpine injection. Additionally, BBG prevents NLRP3 inflammasome activation and consequently the release of active interleukin (IL)-1 and IL-18, involved in the activation of nociceptors. In conclusion, these results suggest that inhibition of P2X7R should be further investigated to develop a potential approach for the management of fibromyalgia.     

Beyond Seizure Control: Treating Comorbidities in Epilepsy via Targeting of the P2X7 Receptor

Epilepsy is one of the most common chronic diseases of the central nervous system (CNS). Treatment of epilepsy remains, however, a clinical challenge with over 30% of patients not responding to current pharmacological interventions. Complicating management of treatment, epilepsy comes with multiple comorbidities, thereby further reducing the quality of life of patients. Increasing evidence suggests purinergic signalling via extracellularly released ATP as shared pathological mechanisms across numerous brain diseases. Once released, ATP activates specific purinergic receptors, including the ionotropic P2X7 receptor (P2X7R). Among brain diseases, the P2X7R has attracted particular attention as a therapeutic target. The P2X7R is an important driver of inflammation, and its activation requires high levels of extracellular ATP to be reached under pathological conditions. Suggesting the therapeutic potential of drugs targeting the P2X7R for epilepsy, P2X7R expression increases following status epilepticus and during epilepsy, and P2X7R antagonism modulates seizure severity and epilepsy development. P2X7R antagonism has, however, also been shown to be effective in treating conditions most commonly associated with epilepsy such as psychiatric disorders and cognitive deficits, which suggests that P2X7R antagonisms may provide benefits beyond seizure control. This review summarizes the evidence suggesting drugs targeting the P2X7R as a novel treatment strategy for epilepsy with a particular focus of its potential impact on epilepsy-associated comorbidities.     

Dihydrotanshinone,a Natural Diterpenoid,Preserves Blood-Retinal Barrier Integrity via P2X7 Receptor

Activation of P2X7 signaling, due to high glucose levels, leads to blood retinal barrier (BRB) breakdown, which is a hallmark of diabetic retinopathy (DR). Furthermore, several studies report that high glucose (HG) conditions and the related activation of the P2X7 receptor (P2X7R) lead to the over-expression of pro-inflammatory markers. In order to identify novel P2X7R antagonists, we carried out virtual screening on a focused compound dataset, including indole derivatives and natural compounds such as caffeic acid phenethyl ester derivatives, flavonoids, and diterpenoids. Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) rescoring and structural fingerprint clustering of docking poses from virtual screening highlighted that the diterpenoid dihydrotanshinone (DHTS) clustered with the well-known P2X7R antagonist JNJ47965567. A human-based in vitro BRB model made of retinal pericytes, astrocytes, and endothelial cells was used to assess the potential protective effect of DHTS against HG and 2′(3′)-O-(4-Benzoylbenzoyl)adenosine-5′-triphosphate (BzATP), a P2X7R agonist, insult. We found that HG/BzATP exposure generated BRB breakdown by enhancing barrier permeability (trans-endothelial electrical resistance (TEER)) and reducing the levels of ZO-1 and VE-cadherin junction proteins as well as of the Cx-43 mRNA expression levels. Furthermore, HG levels and P2X7R agonist treatment led to increased expression of pro-inflammatory mediators (TLR-4, IL-1β, IL-6, TNF-α, and IL-8) and other molecular markers (P2X7R, VEGF-A, and ICAM-1), along with enhanced production of reactive oxygen species. Treatment with DHTS preserved the BRB integrity from HG/BzATP damage. The protective effects of DHTS were also compared to the validated P2X7R antagonist, JNJ47965567. In conclusion, we provided new findings pointing out the therapeutic potential of DHTS, which is an inhibitor of P2X7R, in terms of preventing and/or counteracting the BRB dysfunctions elicited by HG conditions.     

NMDA and P2X7 Receptors Require Pannexin 1 Activation to Initiate and Maintain Nociceptive Signaling in the Spinal Cord of Neuropathic Rats

Pannexin 1 (Panx1) is involved in the spinal central sensitization process in rats with neuropathic pain, but its interaction with well-known, pain-related, ligand-dependent receptors, such as NMDA receptors (NMDAR) and P2X7 purinoceptors (P2X7R), remains largely unexplored. Here, we studied whether NMDAR- and P2X7R-dependent nociceptive signaling in neuropathic rats require the activation of Panx1 channels to generate spinal central sensitization, as assessed by behavioral (mechanical hyperalgesia) and electrophysiological (C-reflex wind-up potentiation) indexes. Administration of either a selective NMDAR agonist i.t. (NMDA, 2 mM) or a P2X7R agonist (BzATP, 150 μM) significantly increased both the mechanical hyperalgesia and the C-reflex wind-up potentiation, effects that were rapidly reversed (minutes) by i.t. administration of a selective pannexin 1 antagonist (10panx peptide, 300 μM), with the scores even reaching values of rats without neuropathy. Accordingly, 300 μM 10panx completely prevented the effects of NMDA and BzATP administered 1 h later, on mechanical hyperalgesia and C-reflex wind-up potentiation. Confocal immunofluorescence imaging revealed coexpression of Panx1 with NeuN protein in intrinsic dorsal horn neurons of neuropathic rats. The results indicate that both NMDAR- and P2X7R-mediated increases in mechanical hyperalgesia and C-reflex wind-up potentiation require neuronal Panx1 channel activation to initiate and maintain nociceptive signaling in neuropathic rats.     

Hypercholesterolemia Negatively Regulates P2X7-Induced Cellular Function in CD4+ and CD8+ T-Cell Subsets from B6 Mice Fed a High-Fat Diet

We have previously showed that plasma membrane cholesterol and GM1 ganglioside content are responsible for the opposite sensitivity of mouse leukemic T cells to ATP. We also reported that the sensitivity of CD4⁺ and CD8⁺ T cells to ATP depends on their stage of differentiation. Here, we show that CD4⁺ and CD8⁺ T cells from B6 mice express different levels of membrane GM1 and P2X7 but similar levels of cholesterol. Thus, in CD4⁺ T cells, membrane cholesterol content negatively correlated with ATP/P2X7-induced CD62L shedding but positively correlated with pore formation, phosphatidylserine externalization, and cell death. By contrast, in CD8⁺ T cells, cholesterol, GM1, and P2X7 levels negatively correlated with all these ATP/P2X7-induced cellular responses. The relationship between cholesterol and P2X7-induced cellular responses was confirmed by modulating cholesterol levels either ex vivo or through a high-fat diet. Membrane cholesterol enrichment ex vivo led to a significant reduction in all P2X7-induced cellular responses in T cells. Importantly, diet-induced hypercholesterolemia in B6 mice was also associated with decreased sensitivity to ATP in CD4⁺ and CD8⁺ T cells, highlighting the relationship between cholesterol intake and the amplitudes of P2X7-induced cellular responses in T cells.     

Purinergic Signalling in Allogeneic Haematopoietic Stem Cell Transplantation and Graft-versus-Host Disease

Allogeneic haematopoietic stem cell transplantation (allo-HSCT) is a curative therapy for blood cancers and other haematological disorders. However, allo-HSCT leads to graft-versus-host disease (GVHD), a severe and often lethal immunological response, in the majority of transplant recipients. Current therapies for GVHD are limited and often reduce the effectiveness of allo-HSCT. Therefore, pro- and anti-inflammatory factors contributing to disease need to be explored in order to identify new treatment targets. Purinergic signalling plays important roles in haematopoiesis, inflammation and immunity, and recent evidence suggests that it can also affect haematopoietic stem cell transplantation and GVHD development. This review provides a detailed assessment of the emerging roles of purinergic receptors, most notably P2X7, P2Y₂ and A_2A receptors, and ectoenzymes, CD39 and CD73, in GVHD.     

High,in Contrast to Low Levels of Acute Stress Induce Depressive-like Behavior by Involving Astrocytic,in Addition to Microglial P2X7 Receptors in the Rodent Hippocampus

Extracellular adenosine 5′-triphosphate (ATP) in the brain is suggested to be an etiological factor of major depressive disorder (MDD). It has been assumed that stress-released ATP stimulates P2X7 receptors (Rs) at the microglia, thereby causing neuroinflammation; however, other central nervous system (CNS) cell types such as astrocytes also possess P2X7Rs. In order to elucidate the possible involvement of the MDD-relevant hippocampal astrocytes in the development of a depressive-like state, we used various behavioral tests (tail suspension test [TST], forced swim test [FST], restraint stress, inescapable foot shock, unpredictable chronic mild stress [UCMS]), as well as fluorescence immunohistochemistry, and patch-clamp electrophysiology in wild-type (WT) and genetically manipulated rodents. The TST and FST resulted in learned helplessness manifested as a prolongation of the immobility time, while inescapable foot shock caused lower sucrose consumption as a sign of anhedonia. We confirmed the participation of P2X7Rs in the development of the depressive-like behaviors in all forms of acute (TST, FST, foot shock) and chronic stress (UCMS) in the rodent models used. Further, pharmacological agonists and antagonists acted in a different manner in rats and mice due to their diverse potencies at the respective receptor orthologs. In hippocampal slices of mice and rats, only foot shock increased the current responses to locally applied dibenzoyl-ATP (Bz-ATP) in CA1 astrocytes; in contrast, TST and restraint depressed these responses. Following stressful stimuli, immunohistochemistry demonstrated an increased co-localization of P2X7Rs with a microglial marker, but no change in co-localization with an astroglial marker. Pharmacological damage to the microglia and astroglia has proven the significance of the microglia for mediating all types of depression-like behavioral reactions, while the astroglia participated only in reactions induced by strong stressors, such as foot shock. Because, in addition to acute stressors, their chronic counterparts induce a depressive-like state in rodents via P2X7R activation, we suggest that our data may have relevance for the etiology of MDD in humans.     

Design,Synthesis, and in vitro Evaluation of P2X7 Antagonists

The P2X7 receptor is a promising target for the treatment of various diseases due to its significant role in inflammation and immune cell signaling. This work describes the design, synthesis, and in vitro evaluation of a series of novel derivatives bearing diverse scaffolds as potent P2X7 antagonists. Our approach was based on structural modifications of reported (adamantan-1-yl)methylbenzamides able to inhibit the receptor activation. The adamantane moieties and the amide bond were replaced, and the replacements were evaluated by a ligand-based pharmacophore model. The antagonistic potency of the synthesized analogues was assessed by two-electrode voltage clamp experiments, using Xenopus laevis oocytes that express the human P2X7 receptor. SAR studies suggested that the replacement of the adamantane ring by an aryl-cyclohexyl moiety afforded the most potent antagonists against the activation of the P2X7 cation channel, with analogue 2-chloro-N-[1-(3-(nitrooxymethyl)phenyl)cyclohexyl)methyl]benzamide ( 56 ) exhibiting the best potency with an IC₅₀ value of 0.39 μM.     

Relaxin Inhibits the Cardiac Myofibroblast NLRP3 Inflammasome as Part of Its Anti-Fibrotic Actions via the Angiotensin Type 2 and ATP (P2X7) Receptors

Chronic NLRP3 inflammasome activation can promote fibrosis through its production of interleukin (IL)-1β and IL-18. Conversely, recombinant human relaxin (RLX) can inhibit the pro-fibrotic interactions between IL-1β, IL-18 and transforming growth factor (TGF)-β1. Here, the broader extent by which RLX targeted the myofibroblast NLRP3 inflammasome to mediate its anti-fibrotic effects was elucidated. Primary human cardiac fibroblasts (HCFs), stimulated with TGF-β1 (to promote myofibroblast (HCMF) differentiation), LPS (to prime the NLRP3 inflammasome) and ATP (to activate the NLRP3 inflammasome) (T+L+A) or benzoylbenzoyl-ATP (to activate the ATP receptor; P2X7R) (T+L+Bz), co-expressed relaxin family peptide receptor-1 (RXFP1), the angiotensin II type 2 receptor (AT₂R) and P2X7R, and underwent increased protein expression of toll-like receptor (TLR)-4, NLRP3, caspase-1, IL-1β and IL-18. Whilst RLX co-administration to HCMFs significantly prevented the T+L+A- or T+L+Bz-stimulated increase in these end points, the inhibitory effects of RLX were annulled by the pharmacological antagonism of either RXFP1, AT₂R, P2X7R, TLR-4, reactive oxygen species (ROS) or caspase-1. The RLX-induced amelioration of left ventricular inflammation, cardiomyocyte hypertrophy and fibrosis in isoproterenol (ISO)-injured mice, was also attenuated by P2X7R antagonism. Thus, the ability of RLX to ameliorate the myofibroblast NLRP3 inflammasome as part of its anti-fibrotic effects, appeared to involve RXFP1, AT₂R, P2X7R and the inhibition of TLR-4, ROS and caspase-1.     

Functional Coupling between the P2X7 Receptor and Pannexin-1 Channel in Rat Trigeminal Ganglion Neurons

The ionotropic P2X receptor, P2X₇, is believed to regulate and/or generate nociceptive pain, and pain in several neuropathological diseases. Although there is a known relationship between P2X₇ receptor activity and pain sensing, its detailed functional properties in trigeminal ganglion (TG) neurons remains unclear. We examined the electrophysiological and pharmacological characteristics of the P2X₇ receptor and its functional coupling with other P2X receptors and pannexin-1 (PANX1) channels in primary cultured rat TG neurons, using whole-cell patch-clamp recordings. Application of ATP and Bz-ATP induced long-lasting biphasic inward currents that were more sensitive to extracellular Bz-ATP than ATP, indicating that the current was carried by P2X₇ receptors. While the biphasic current densities of the first and second components were increased by Bz-ATP in a concentration dependent manner; current duration was only affected in the second component. These currents were significantly inhibited by P2X₇ receptor antagonists, while only the second component was inhibited by P2X_{1, 3,} and ₄ receptor antagonists, PANX1 channel inhibitors, and extracellular ATPase. Taken together, our data suggests that autocrine or paracrine signaling via the P2X₇-PANX1-P2X receptor/channel complex may play important roles in several pain sensing pathways via long-lasting neuronal activity driven by extracellular high-concentration ATP following tissue damage in the orofacial area.     

Novel P2X7 Antagonist Ameliorates the Early Phase of ALS Disease and Decreases Inflammation and Autophagy in SOD1-G93A Mouse Model

Amyotrophic lateral sclerosis (ALS) is a disease with a resilient neuroinflammatory component caused by activated microglia and infiltrated immune cells. How to successfully balance neuroprotective versus neurotoxic actions through the use of anti-inflammatory agents is still under debate. There has been a boost of awareness regarding the role of extracellular ATP and purinergic receptors in modulating the physiological and pathological mechanisms in the nervous system. Particularly in ALS, it is known that the purinergic ionotropic P2X7 receptor plays a dual role in disease progression by acting at different cellular and molecular levels. In this context, we previously demonstrated that the P2X7 receptor antagonist, brilliant blue G, reduces neuroinflammation and ameliorates some of the pathological features of ALS in the SOD1-G93A mouse model. Here, we test the novel, noncommercially available, and centrally permeant Axxam proprietary P2X7 antagonist, AXX71, in SOD1-G93A mice, by assessing some behavioral and molecular parameters, among which are disease progression, survival, gliosis, and motor neuron wealth. We demonstrate that AXX71 affects the early symptomatic phase of the disease by reducing microglia-related proinflammatory markers and autophagy without affecting the anti-inflammatory markers or motor neuron survival. Our results suggest that P2X7 modulation can be further investigated as a therapeutic strategy in preclinical studies, and exploited in ALS clinical trials.     

Gallic Acid Alleviates Visceral Pain and Depression via Inhibition of P2X7 Receptor

Chronic visceral pain can occur in many disorders, the most common of which is irritable bowel syndrome (IBS). Moreover, depression is a frequent comorbidity of chronic visceral pain. The P2X7 receptor is crucial in inflammatory processes and is closely connected to developing pain and depression. Gallic acid, a phenolic acid that can be extracted from traditional Chinese medicine, has been demonstrated to be anti-inflammatory and anti-depressive. In this study, we investigated whether gallic acid could alleviate comorbid visceral pain and depression by reducing the expression of the P2X7 receptor. To this end, the pain thresholds of rats with comorbid visceral pain and depression were gauged using the abdominal withdraw reflex score, whereas the depression level of each rat was quantified using the sucrose preference test, the forced swimming test, and the open field test. The expressions of the P2X7 receptor in the hippocampus, spinal cord, and dorsal root ganglion (DRG) were assessed by Western blotting and quantitative real-time PCR. Furthermore, the distributions of the P2X7 receptor and glial fibrillary acidic protein (GFAP) in the hippocampus and DRG were investigated in immunofluorescent experiments. The expressions of p-ERK1/2 and ERK1/2 were determined using Western blotting. The enzyme-linked immunosorbent assay was utilized to measure the concentrations of IL-1β, TNF-α, and IL-10 in the serum. Our results demonstrate that gallic acid was able to alleviate both pain and depression in the rats under study. Gallic acid also reduced the expressions of the P2X7 receptor and p-ERK1/2 in the hippocampi, spinal cords, and DRGs of these rats. Moreover, gallic acid treatment decreased the serum concentrations of IL-1β and TNF-α, while raising IL-10 levels in these rats. Thus, gallic acid may be an effective novel candidate for the treatment of comorbid visceral pain and depression by inhibiting the expressions of the P2X7 receptor in the hippocampus, spinal cord, and DRG.     

Extracellular Histones Trigger Disseminated Intravascular Coagulation by Lytic Cell Death

Histones are cationic nuclear proteins that are essential for the structure and functions of eukaryotic chromatin. However, extracellular histones trigger inflammatory responses and contribute to death in sepsis by unknown mechanisms. We recently reported that inflammasome activation and pyroptosis trigger coagulation activation through a tissue-factor (TF)-dependent mechanism. We used a combination of various deficient mice to elucidate the molecular mechanism of histone-induced coagulation. We showed that histones trigger coagulation activation in vivo, as evidenced by coagulation parameters and fibrin deposition in tissues. However, histone-induced coagulopathy was neither dependent on intracellular inflammasome pathways involving caspase 1/11 and gasdermin D (GSDMD), nor on cell surface receptor TLR2- and TLR4-mediated host immune response, as the deficiency of these genes in mice did not protect against histone-induced coagulopathy. The incubation of histones with macrophages induced lytic cell death and phosphatidylserine (PS) exposure, which is required for TF activity, a key initiator of coagulation. The neutralization of TF diminished the histone-induced coagulation. Our findings revealed lytic cell death as a novel mechanism of histone-induced coagulation activation and thrombosis.