Purinergic signaling systems across comparative models of spinal cord injury

Within the last several decades,the scientific community has made substantial progress in elucidating the complex pathophysiology underlying spinal cord injury.However,despite the many advances using conventional mammalian models,both cellular and axonal regeneration following spinal cord injury have remained out of reach.In this sense,turning to non-mammalian,regenerative species presents a unique opportunity to identify pro-regenerative cues and characterize a spinal cord microenvironment permissive to re-growth.Among the signaling pathways hypothesized to be dysregulated during spinal cord injury is the purinergic signaling system.In addition to its well-known role as energy currency in cells,ATP and its metabolites are small molecule neurotransmitters that mediate many diverse cellular processes within the central nervous system.While our understanding of the roles of the purinergic system following spinal cord injury is limited,this signaling pathway has been implicated in all injury-induced secondary processes,including cellular death,inflammation,reactive gliosis,and neural regeneration.Given that the purinergic system is also evolutionarily conserved between mammalian and non-mammalian species,comparisons of these roles may provide important insights into conditions responsible for recovery success.Here,we compare the secondary processes between key model species and the influence of purinergic signaling in each context.As our understanding of this signaling system and pro-regenerative conditions continues to evolve,so does the potential for the development of novel therapeutic interventions for spinal cord injury.

The role of purinergic receptors in neural repair and regeneration after spinal cord injury

Spinal cord injury is a serious injury of the central nervous system that results in neurological deficits.The pathophysiological mechanisms underlying spinal cord injury,as well as the mechanisms involved in neural repair and regeneration,are highly complex.Although there have been many studies on these mechanisms,there is no effective intervention for such injury.In spinal cord injury,neural repair and regeneration is an important part of improving neurological function after injury,although the low regenerative ability of nerve cells and the difficulty in axonal and myelin regeneration after spinal cord injury hamper functional recovery.Large amounts of ATP and its metabolites are released after spinal cord injury and participate in various aspects of functional regulation by acting on purinergic receptors which are widely expressed in the spinal cord.These processes mediate intracellular and extracellular signalling pathways to improve neural repair and regeneration after spinal cord injury.This article reviews research on the mechanistic roles of purinergic receptors in spinal cord injury,highlighting the potential role of purinergic receptors as interventional targets for neural repair and regeneration after spinal cord injury.

Electroacupuncture diminishes P2X_2 and P2X_3 purinergic receptor expression in dorsal root ganglia of rats with visceral hypersensitivity

Electroacupuncture at Shangjuxu （ST37） and Tianshu （ST25） can improve visceral hypersensitivity in rats. Colorectal distension was used to establish a rat model of chronic visceral hypersensitivity. Immunohistochemistry was used to detect P2X2 and P2X3 receptor expression in dorsal root ganglia from rats with chronic visceral hypersensitivity. Results demonstrated that abdominal withdrawal reflex scores obviously increased following establishment of the model, indicating visceral hypersensitivity. Simultaneously, P2X2 and P2X3 receptor expression increased in dorsal root ganglia. After bilateral electroacupuncture at Shangjuxu and Tianshu, abdominal withdrawal reflex scores and P2X2 and P2X3 receptor expression decreased in rats with visceral hypersensitivity. These results indicated that electroacupuncture treatment improved visceral hypersensitivity in rats with irritable bowel syndrome by reducing P2X2 and P2X3 receptor expression in dorsal root ganglia.

Purinergic signaling systems across comparative models of spinal cord injury

Within the last several decades,the scientific community has made substantial progress in elucidating the complex pathophysiology underlying spinal cord injury.However,despite the many advances using conventional mammalian models,both cellular and axonal regeneration following spinal cord injury have remained out of reach.In this sense,turning to non-mammalian,regenerative species presents a unique opportunity to identify pro-regenerative cues and chara cterize a spinal cord microenvironment permissive to re-growth.Among the signaling pathways hypothesized to be dysregulated during spinal cord injury is the purinergic signaling system.In addition to its well-known role as energy currency in cells,ATP and its metabolites are small molecule neurotransmitte rs that mediate many diverse cellular processes within the central nervous system.While our unde rstanding of the roles of the purinergic system following spinal cord injury is limited,this signaling pathway has been implicated in all injury-induced secondary processes,including cellular death,inflammation,reactive gliosis,and neural regeneration.Given that the purinergic system is also evolutionarily conserved between mammalian and non-mammalian species,comparisons of these roles may provide important insights into conditions responsible for recovery success.Here,we compare the secondary processes between key model species and the influence of purinergic signaling in each context.As our understanding of this signaling system and pro-regenerative conditions continues to evolve,so does the potential for the development of novel therapeutic interventions for spinal cord injury.

WJSC 6^(th) Anniversary Special Issues(2):Mesenchymal stem cells Purinergic receptors and nucleotide processing ectoenzymes:Their roles in regulating mesenchymal stem cell functions

Human mesenchymal stem cells(MSCs)are a rare population of non-hematopoietic stem cells with multilineage potential,originally identified in the bone marrow.Due to the lack of a single specific marker,MSCs can be recognized and isolated by a series of features such as plastic adherence,a panel of surface markers,the clonogenic and the differentiation abilities.The recognized role of MSCs in the regulation of hemopoiesis,in cell-degeneration protection and in the homeostasis of mesodermal tissues through their differentiation properties,justifies the current interest in identifying the biochemical signals produced by MSCs and their active crosstalk in tissue environments.Only recently have extracellular nucleotides(eNTPs)and their metabolites been included among the molecular signals produced by MSCs.These molecules are active on both ionotropic and metabotropic receptors present in most cell types.MSCs possess a significant display of these receptors and of nucleotide processing ectoenzymes on their plasma membrane.Thus,from their niche,MSCs give a significant contribution to the complex signaling network of eNTPs and its derivatives.Recent studies have demonstrated the multifaceted aspects of eNTP metabolism and their signal transduction in MSCs and revealed important roles in specifying differentiation lineages and modulating MSC physiology and communication with other cells.This review discusses the roles of eNTPs,their receptors and ectoenzymes,and the relevance of the signaling network and MSC functions,and also focuses on the importance of this emerging area of interest for future MSC-based cell therapies.

Purinergic signalling in neuroregeneration

Purinergic signalling,adenosine 5′-triphosphate（ATP）as an extracellular signalling molecule,was proposed in 1972（Burnstock,1972）.However,it was not generally accepted until the early 1990s when receptors for ATP and its breakdown product adenosine were cloned and characterised（Ralevic and Burnstock,1998）.Four P1（adenosine）receptors are recognised（A1,A2A,A2B and A3）.

Uridine adenosine tetraphosphate acts as a pro-angiogenic factor in vitrothrough purinergic P2Y receptors

OBJECTIVE Uridine adenosine tetraphosphate(Up4A),a dinucleotide,contains both purine and pyrimidine moieties,and exerts its vascular influence via activation of purinergic receptors.Here,we aimed to investigate the effects of Up4 A on angiogenesis and the putative purinergic receptors(PR)involved in this process.METHODS Tubule formation assay was performed in 3D matrix system.In this assay,human umbilical vein endothelial cells(HUVECs)were co-cultured with pericytes with various Up4 A doses(0,1,2.5,5,10 and 20μmol·L-1)in the absence and presence of P2Y6 R antagonist MRS2578(10μmol·L-1)for 5d.Expression profile of PR subtypes and angiogenic factors was assessed in HUVECs by q-PCR with and without P2Y6 R antagonist.RESULTS No difference in initial tubule formation was detected between Up4 A stimulation and control conditions at day 2.In contrast,a significant increase in vascular density in response to Up4 A was observed at day 5.Up4 A at a dose of 2.5and 5μmol·L-1 promoted total tubule length(by-1.89 fold and-2.23fold),number of tubules(by-1.71 fold and-1.89fold)as well as number of junctions(by-2.24 fold and-2.80fold),all of which were inhibited by MRS2578.Further increase in Up4 A dose to10 and 20μmol·L-1 did not induce an increase in these vascular parameters as compared to non-treated controls.Moreover,Up4 A increased mRNA level of P2YRs(P2Y2R,P2Y4 R and P2Y6R)but not P2XR(P2X4R and P2X7R)or P1R(A2AR and A2BR),while Up4 A upregulated VEGFA and ANGPT1 but not VEGFR2,ANGPT2,Tie1 and Tie2at mRNA level.Transcriptional upregulation of P2 YRs and angiogenic factors by Up4 A was inhibited by MRS2578.CONCLUSION Up4 A is functionally capable of promoting tubule formation in vitro co-culture system.This process is likely mediated by activation of pyrimidine-favored P2 YRs but not P2 XR or P1 Rs,and involves stimulation of well known angiogenic factors.

Purinergic signaling in the gastrointestinal tract

Geoffrey Burnstock completed a BSc at King's College London and a PhD at University College London. He held postdoctoral fellowships with Wilhelm Feldberg(National Institute for Medical Research),Edith Bülbring(University of Oxford) and C. Ladd Prosser(University of Illinois) . He was appointed to a Senior Lectureship in Melbourne University in 1959 and became Professor and Chairman of Zoology in 1964. In 1975 he became Head of Department of Anatomy and Developmental Biology at UCL and Convenor of the Center of Neuroscience. He has been Director of the Autonomic Neuroscience Institute at the Royal Free Hospital School of Medicine since 1997. He was elected to the Australian Academy of Sciences in 1971,the Royal Society in 1986,the Academy of Medical Sciences in 1998 and an Honorary Fellow of the Royal College of Surgeons and the Royal College of Physicians in 1999 and 2000. He was awarded the Royal Society Gold Medal in 2000. He is editor-in-chief of the journals Autonomic Neuroscience and Purinergic Signalling and on the editorial boards of many other journals. Geoffrey Burnstock's major research interest has been autonomic neurotransmission and he is best known for his seminal discovery of purinergic transmission and receptors,their signaling pathways and functional relevance. He has supervised over 100 PhD and MD studentsand published over 1400 original papers,re-views and books. He was first in the Institute of Scientific Information list of most cited scientists in Pharmacology and Toxicology from 1994-2004 [59.083 citations(March 2011) and an h-index of 109].

Purinergic signaling and human immunodeficiency virus/acquired immune deficiency syndrome: From viral entry to therapy

Human immunodeficiency virus(HIV) infection is a serious condition associated to severe immune dysfunction and immunodeficiency. Mechanisms involved in HIV-associated immune activation, inflammation and loss of CD4+ T cells have been extensively studied, including those concerning purinergic signaling pathways. Purinergic signaling components are involved in viral entry and replication and disease progression. Research involving the participation of purinergic signaling in HIV infection has been not only important to elucidate disease mechanisms but also to introduce new approaches to therapy. The involvement of purinergic signaling in the pathogenesis of HIV infection and its implications in the control of the HIV infection are reviewed in this paper.

Genomic Organization of Purinergic P2X Receptors

Purinergic P2X receptors are a family of ligand-gated cationic channels activated by extracellular ATP. P2X subunit protein sequences are highly conserved between vertebrate species. However, they can generate a great diversity of coding splicing variants to fulfill several roles in mammalian physiology. Despite intensive research in P2X expression in both central and peripheral nervous system, there is little information about their homology, genomic structure and other key features that can help to develop selective drugs or regulatory strategies of pharmacological value which are lacking today. In order to obtain clues on mammalian P2X diversity, we have performed a bioinformatics analysis of the coding regions and introns of the seven P2X subunits present in human, simian, dog, mouse, rat and zebrafish. Here we report the arrangements of exon and intron sequences, considering its number, size, phase and placement;proposing some ideas about the gain and loss of exons and retention of introns. Taken together, these evidences show traits that can be used to gain insight into the evolutionary history of vertebrate P2X receptors and better understand the diversity of subunits coding the purinergic signaling in mammals.

Targeting the guanine-based purinergic system in Alzheimer's disease

Alzheimer’s disease（AD）is the main cause of dementia worldwide and affects approximately 5%of people with 65 years or older.The estimated increase in the elderly population suggests that cases of AD will rise in the next years.

Central Glutamatergic-Purinergic System Importance in Brain/Neural Plasticity

The proteolysis of the extracellular matrix plays a key role in the synaptic neuroplasticity of the central nervous system (CNS), which results in learning and memory. Proteases from the serine family and metalloproteinases of the extracellular matrix are localized within the synapses and are released into the extracellular space in proportion to the degree of neuronal excitation. These enzymes cause changes in the morphology, shape and size, and the overall number of synapses and synthesize new synaptic connections. The proteinase also changes the function of receptors, and consequently, the secretion of neurotransmitter/neuromodulator from the presynaptic glutamatergic and/or purinergic elements are either strengthened or weakened. Neuroglia involved in homeostasis, melanin synthesis and defense of the brain contain different combinations of purinergic receptors, which contributes to many neurotransmitters. This review summarizes a concept of brain plasticity, the role of ATP and P2 receptors interaction with glutamatergic system during plasticity of the brain in the one hand and after physical exercise in the other, which may be triggering phenomena facilitative synaptic plasticity as well as potentiates an personal efficiency to react to biobehavioral adaptation and disorders.

P2Y1 receptor in Alzheimer’s disease

Alzheimer’s disease is the most frequent form of dementia characterized by the deposition of amyloid-beta plaques and neurofibrillary tangles consisting of hyperphosphorylated tau.Targeting amyloid-beta plaques has been a primary direction for developing Alzheimer’s disease treatments in the last decades.However,existing drugs targeting amyloid-beta plaques have not fully yielded the expected results in the clinic,necessitating the exploration of alternative therapeutic strategies.Increasing evidence unravels that astrocyte morphology and function alter in the brain of Alzheimer’s disease patients,with dysregulated astrocytic purinergic receptors,particularly the P2Y1 receptor,all of which constitute the pathophysiology of Alzheimer’s disease.These receptors are not only crucial for maintaining normal astrocyte function but are also highly implicated in neuroinflammation in Alzheimer’s disease.This review delves into recent insights into the association between P2Y1 receptor and Alzheimer’s disease to underscore the potential neuroprotective role of P2Y1 receptor in Alzheimer’s disease by mitigating neuroinflammation,thus offering promising avenues for developing drugs for Alzheimer’s disease and potentially contributing to the development of more effective treatments.

Purinergic 2X7Receptor is Involved in the Podocyte Damage of Obesity-Related Glomerulopathy via Activating Nucleotide-Binding and Oligomerization Domain-Like Receptor Protein 3 Inflammasome

Background:The nucleotide-binding and oligomerization domain-like receptor protein 3 (NLRP3) inflammasome composed of NLRP3,apoptosis-associated speck-like protein containing CARD (ASC),and caspase-1 is engaged in the inflammatory response of many kidney diseases and can be activated by purinergic 2X7 receptor (P2X7R).This study was conducted to explore whether P2X7R plays a pathogenic role in the podocyte damage of obesity-related glomerulopathy (ORG) and whether this role is mediated by the activation ofNLRP3 inflammasome.Methods:A mouse model of ORG was established by high-fat diet feeding.The conditionally immortalized mouse podocytes were cultured with leptin or with leptin and P2X7R antagonist (KN-62 or A438079).The mRNA and protein expression of the P2X7R and NLRP3 inflammasome components including NLRP3,ASC,and caspase-1,as well as the podocyte-associated molecules including nephrin,podocin,and desmin in mouse renal cortex or cultured mouse podocytes were tested by real-time-polymerase chain reaction and Westem blot analysis,respectively.Results:The significantly upregulated expression of P2X7R and NLRP3 inflammasome components and the NLRP3 inflammasome activation were observed in the renal cortex (in fact their location in podocytes was proved by confocal microscopy) of ORG mice in vivo,which were accompanied with the morphological changes of podocyte damage and the expression changes of podocyte-associated molecules.Similar changes in the expression of P2X7R and NLRP3 inflammasome components as well as in the expression ofpodocyte-associated molecules were also observed in the cultured podocyte studies treated by leptin in vitro,and all of the above changes were significantly attenuated by the P2X7R antagonist KN-62 or A438079.Conclusions:P2X7R could trigger the activation ofNLRP3 inflammasome,and the activated P2X7R/NLRP3 inflammasome in podocytes might be involved in the podocyte damage of ORG.

Autoimmune Hepatitis: Clinical Review with Insights into the Purinergic Mechanism of Disease

Autoimmune hepatitis (AIH) is an important disorder that predominantly results in inflammatory liver disease in genetically predisposed women.The clinicopathological picture is characterized by symptoms associated with both systemic inflammation and hepatic dysfunction,and with increased serum aminotransferases,elevated IgG,autoantibodies,and interface hepatitis on liver biopsy.AIH usually results in liver injury as a consequence of chronic hepatitis and cirrhosis.However,rarely,patients may present with fulminant liver failure.Early diagnosis is important in all instances because the disease can be highly responsive to immunosuppressive therapeutic options.Left untreated,the disease is associated with high morbidity and mortality.Here we provide an overview of the current state of knowledge on AIH and summarize the treatment options for this serious condition in adults.We also discuss the pathogenesis of the disease as a possible consequence of autoimmunity and the breakdown of hepatic tolerance.We focus on regulatory T cell impairments as a consequence of changes in CD39 ectonucleotidase expression and altered purinergic signaling.Further understanding of hepatic tolerance may aid in the development of specific and well-tolerated therapies for AIH.

Purinergic Receptors in Basal Ganglia Diseases:Shared Molecular Mechanisms between Huntington’s and Parkinson’s Disease

Huntington’s(HD)and Parkinson’s diseases(PD)are neurodegenerative disorders caused by the death of GABAergic and dopaminergic neurons in the basal ganglia leading to hyperkinetic and hypokinetic symptoms,respectively.We review here the participation of purinergic receptors through intracellular Ca^2+signaling in these neurodegenerative diseases.The adenosine A2A receptor stimulates striatopallidal GABAergic neurons,resulting in inhibitory actions on GABAergic neurons of the globus pallidus.A2A and dopamine D2 receptors form functional heteromeric complexes inducing allosteric inhibition,and A2A receptor activation results in motor inhibition.Furthermore,the A2A receptor physically and functionally interacts with glutamate receptors,mainly with the mGlu5 receptor subtype.This interaction facilitates glutamate release,resulting in NMDA glutamate receptor activation and an increase of Ca2+influx.P2X7 receptor activation also promotes glutamate release and neuronal damage.Thus,modulation of purinergic receptor activity,such as A2A and P2X7 receptors,and subsequent aberrant Ca^2+signaling,might present interesting therapeutic potential for HD and PD.

Purinergic Modulation of Activity in the Developing Auditory Pathway

Purinergic P2 receptors,activated by endogenous ATP,are prominently expressed on neuronal and nonneuronal cells during development of the auditory periphery and central auditory neurons.In the mature cochlea,extracellular ATP contributes to ion homeostasis,and has a protective function against noise exposure.Here,we focus on the modulation of activity by extracellular ATP during early postnatal development of the lower auditory pathway.In mammals,spontaneous patterned activity is conveyed along afferent auditory pathways before the onset of acoustically evoked signal processing.During this critical developmental period,inner hair cells fire bursts of action potentials that are believed to provide a developmental code for synaptic maturation and refinement of auditory circuits,thereby establishing a precise tonotopic organization.Endogenous ATP-release triggers such patterned activity by raising the extracellular K+concentration and contributes to firing by increasing the excitability of auditory nerve fibers,spiral ganglion neurons,and specific neuron types within the auditory brainstem,through the activation of diverse P2 receptors.We review recent studies that provide new models on the contribution of purinergic signaling to early development of the afferent auditory pathway.Further,we discuss potential future directions of purinergic research in the auditory system.

Effect of electroacupuncture on inflammatory signal expression in local tissues of rats with chronic pelvic pain syndrome based on purinergic 2X7 receptor/NOD-like receptor pyrin domaincontaining 3 signal pathway

OBJECTIVES: To study the expression of inflammatory signal in local prostate tissue of chronic pelvic pain syndrome(CPPS) rats by electroacupuncture(EA) of Guanyuan(CV4), Zhongji(CV3), Huiyang(BL35) and Sanyinjiao(SP6), and to explore the possible mechanism of anti-inflammatory and analgesic effects of EA. METHODS : A total of 36 Sprague-Dawley male rats were randomly divided into three groups: control, model and EA(n=12 rats/group). The CPPS model was made by injection of CFA into ventral lobes of the prostate(0.1 m L). Electric acupuncture apparatus was applied to stimulate Guanyuan(CV4), Zhongji(CV3), bilateral Huiyang(BL35) and Sanyinjiao(SP6) acupoints in EA group. The general condition of rats was observed and the prostate index(PI) was calculated. The thermal pain threshold was collected after each therapeutic course. Histopathological changes of the prostate tissue were examined by hematoxylin-eosin staining method. The expression levels of tumor necrosis factor α(TNF-α), interleukin-1β(IL-1β) and prostaglandin E2(PGE2) in prostatic homogenates were measured by enzyme linked immunosorbent assay(ELISA). Moreover, the expression levels of purinergic 2X7 receptor(P2X7R), NOD-like receptor pyrin domain-containing 3(NLRP3), caspase-1 and interleukin-18(IL-18) m RNA were quantified by quantitative real-time polymerase chain reaction. RESULTS: Compared with control group, the PI of rats increased, and the thermal pain threshold decreased significantly in model group. The morphological structure of prostate tissues of rats in model group was severely damaged with a large number of inflammatory cells infiltration. Additionally, the levels of TNF-α, IL-1β and PGE2 were higher, and the expressions of P2X7R, NLRP3, caspase-1 and IL-18 m RNA were higher than those in control group. After EA treatment, the PI was significantly decreased, the thermal pain threshold was significantly increased, and the tissue damage was significantly improved. The expressions of inflammatory cytokines were lower in EA group, and expression of P2X7R/NLRP3 pathway was down-regulated. CONCLUSION: The effect of EA at Guanyuan(CV4), Zhongji(CV3), Huiyang(BL35) and Sanyinjiao(SP6) can improve inflammation and pain symptoms of CPPS rats induced by Complete Freund’s adjuvant(CFA). This suggests that EA at Guanyuan(CV4), Zhongji(CV3), Huiyang(BL35) and Sanyinjiao(SP6) can produce antiinflammatory analgesia effect by preventing the activation of P2X7R/NLRP3 signal pathway, inhibit the release of inflammatory cytokines in CPPS rats, which may provide a putative novel target for the treatment of CPPS.

Can glial cells save neurons in epilepsy?

Epilepsy is a neurological disorder caused by the pathological hyper-synchronization of neuronal discharges.The fundamental research of epilepsy mechanisms and the targets of drug design options for its treatment have focused on neurons.However,approximately 30%of patients suffering from epilepsy show resistance to standard anti-epileptic chemotherapeutic agents while the symptoms of the remaining 70%of patients can be alleviated but not completely removed by the current medications.Thus,new strategies for the treatment of epilepsy are in urgent demand.Over the past decades,with the increase in knowledge on the role of glia in the genesis and development of epilepsy,glial cells are receiving renewed attention.In a normal brain,glial cells maintain neuronal health and in partnership with neurons regulate virtually every aspect of brain function.In epilepsy,however,the supportive roles of glial cells are compromised,and their interaction with neurons is altered,which disrupts brain function.In this review,we will focus on the role of glia-related processes in epileptogenesis and their contribution to abnormal neuronal activity,with the major focus on the dysfunction of astroglial potassium channels,water channels,gap junctions,glutamate transporters,purinergic signaling,synaptogenesis,on the roles of microglial inflammatory cytokines,microglia-astrocyte interactions in epilepsy,and on the oligodendroglial potassium channels and myelin abnormalities in the epileptic brain.These recent findings suggest that glia should be considered as the promising next-generation targets for designing anti-epileptic drugs that may improve epilepsy and drug-resistant epilepsy.

Integrating UHPLC-MS/MS quantitative analysis and exogenous purine supplementation to elucidate the antidepressant mechanism of Chaigui granules by regulating purine metabolism

Chaigui granules(CG)are a compound composed of six herbal medicines with significant antidepressant effects.However,the antidepressant mechanism of CG remains unclear.In the present study,we attempted to elucidate the antidepressant mechanism of CG by regulating purine metabolism and purinergic signaling.First,the regulatory effect of CG on purine metabolites in the prefrontal cortex(PFC)of chronic unpredictable mild stress(CUMS)rats was analyzed by ultra high-performance liquid chromatography tandem mass spectrometry(UHPLC-MS/MS)targeted quantitative analysis.Meanwhile,purinergic receptors(P2X7 receptor(P2X7R),A1 receptor(A1R)and A2A receptor(A2AR))and signaling pathways(nod-like receptor protein 3(NLRP3)inflammasome pathway and cyclic adenosine monophosphate(cAMP)-protein kinase A(PKA)pathway)associated with purine metabolism were analyzed by western blotting and enzyme-linked immunosorbent assay(ELISA).Besides,antidepressant mechanism of CG by modulating purine metabolites to activate purinergic receptors and related signaling pathways was dissected by exogenous supplementation of purine metabolites and antagonism of purinergic receptors in vitro.An in vivo study showed that the decrease in xanthine and the increase in four purine nucleosides were closely related to the antidepressant effects of CG.Additionally,purinergic receptors(P2X7R,A1R and A2AR)and related signaling pathways(NLRP3 inflammasome pathway and cAMP-PKA pathway)were also significantly regulated by CG.The results of exogenous supplementation of purine metabolites and antagonism of purinergic receptors showed that excessive accumulation of xanthine led to activation of the P2X7R-NLRP3 inflammasome pathway,and the reduction of adenosine and inosine inhibited the A1R-cAMP-PKA pathway,which was significantly ameliorated by CG.Overall,CG could promote neuroprotection and ultimately play an antidepressant role by inhibiting the xanthine-P2X7R-NLRP3 inflammasome pathway and activating the adenosine/inosine-A1R-cAMP-PKA pathway.