AST-004 is an adenosine A1R/A3R agonist and promising cerebroprotectant currently in Phase II clinical development. Clinical evaluations have been initiated in concussion, with trials in preparation for mild complicated traumatic brain injury (TBI) and acute ischemic stroke (AIS). AST-004 was discovered as the result of basic research on the P2Y1R-activated homeostatic roles of astrocytes in cerebroprotection, including control of edema, excitotoxicity and oxidative stress. AST-004 was identified as the active nucleoside metabolite of the P2Y1R agonist MRS2365. Subsequent research demonstrated that astrocyte mitochondrial ATP production could be activated by AST-004, resulting in significant efficacy in rodent models of permanent and transient occlusion AIS, porcine models of TBI and a non-human primate model of AIS as well as models of neurodegeneration and addiction. The purpose of this review is to highlight the research that led to the discovery of AST-004, summarize its widespread preclinical efficacy in a variety of disease models, and describe how this compound’s excellent safety and pharmaceutical profile is differentiated from prototypical A1R and A3R agonists formerly or currently in the clinic.

Glioblastoma multiforme (GBM) is among the most aggressive and lethal primary brain tumors. P2X7 is a purinergic receptor overexpressed in GBM, whose activation can promote tumor growth. Two major splice variants of the P2X7 receptor, isoforms A and B, are expressed in GBM cells. In our study, both isoforms A and B were present in GBM cells, with a slight prevalence of isoform B expression in the U87MG cell line compared to the T98G cell line. In GBM cell cultures, we evaluated the effects of P2X7 receptor activation/inhibition on cell growth. Exposure of GBM cells to various concentrations of ATP did not alter cellular activity, whereas inhibition of the P2X7 receptor with the antagonist AZ10606120, but not with A740003, significantly reduced GBM cell viability and proliferation. The U87MG cell line, which expresses the B isoform more highly, was more sensitive to the antiproliferative effects of AZ10606120. To clarify the mechanisms of AZ10606120-induced tumor inhibition, we measured the expression of proteins involved in cell repair and survival processes. AZ10606120 induced an increase in caspase-3 and p21 expression, demonstrating that P2X7R blockade can induce tumor cell death, likely by apoptosis. AZ10606120 also stimulated interleukin-6 (IL-6) release in GBM cells and induced phosphorylation of CREB and STAT3. Blockade of P2X7R by AZ10606120 appears to activate a pro-tumor IL-6/STAT3 axis. Increased IL-6 secretion and activation of the CREB and STAT3 pathways are negative prognostic signs that highlight the need to combine P2X7R inhibition with AZ10606120 with other therapies.

This study investigates the impact of a 16-week mixed circuit training (MCT) program on purinergic signaling and oxidative stress markers in women with type 2 diabetes mellitus (T2DM), focusing on its potential to modulate platelet-related purinergic signaling and oxidative stress markers that are mechanistically linked to platelet activation. A total of 21 women with T2DM and 23 non-diabetic controls, all sedentary and middle-aged, underwent MCT twice weekly. Biochemical, hemodynamic, and oxidative stress parameters, along with platelet ectonucleotidase activity and extracellular ATP levels, were assessed pre- and post-intervention. MCT significantly decreased ectonucleotidase diphosphohydrolase (E-NTPDase) activity for ADP hydrolysis in platelets, along with a reduction in extracellular ATP levels, indicating a modulation of purinergic signaling. Additionally, exercise enhanced antioxidant defenses, increasing glutathione-S-transferase (GST) activity and vitamin C levels, while reducing myeloperoxidase (MPO) activity, a key pro-oxidant enzyme. These changes suggest a shift toward a biochemical profile that may be relevant to thromboinflammatory pathways; however, no direct measures of platelet aggregation were performed. Mixed circuit training emerges as a valuable non-pharmacological strategy for improving oxidative balance and modulating purinergic markers in women with T2DM, warranting future studies including functional platelet assays and clinical outcomes.

The pathogenesis of COVID-19 involves complex interactions between viral replication and host immune dysregulation, mediated by toll-like receptors (TLRs) and P2X receptors (P2XRs). These receptors detect pathogen- and damage-associated molecular patterns (PAMPs and DAMPs), triggering inflammatory cascades. Opioids, beyond their analgesic role, modulate innate and adaptive immune responses via opioid receptors and indirectly through TLR and P2X signaling. This narrative review integrates experimental, clinical, and bioinformatic evidence to explore the mechanistic crosstalk between opioid signaling, TLRs (notably TLR2, TLR4, TLR9), and P2XRs (notably P2X4 and P2X7) in COVID-19 immunopathology. Chronic opioid exposure may either enhance or suppress inflammation depending on dose, duration, and immune context. In COVID-19, the hyperactivation of TLR4, TLR7, and TLR9 drives cytokine storms, while the release of ATP from damaged cells activates P2X7, thereby amplifying the inflammatory response. ATP breakdown into adenosine further modulates immunity via A2A and A2B receptors. Targeting TLR4 and P2X7 offers a promising therapeutic strategy to mitigate hyperinflammation and improve outcomes in COVID-19 and related inflammatory diseases. In addition, we outline how the Contact System, the Kallikrein-Kinin System (KKS), the Renin-Angiotensin System (RAS), and the NLRP3 inflammasome provide the innate inflammatory backdrop through which opioids and P2 receptor signaling may shape immune dysregulation in COVID-19. Notably, direct clinical evidence for opioid-P2 receptor interactions in COVID-19 remains limited, highlighting the need for targeted translational studies.

Parkinson's disease (PD) is marked by the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). Adenosine receptors (A2ARs) modulate the striatopallidal non-dopaminergic pathway to alleviate PD symptoms. In the present study, the neuroprotective mechanism of the selective A2AR antagonist exhibiting a non-purine scaffold, IDPU (Ki = 0.0038nM), was explored using the primary mid-brain neuronal (PMDN) cells isolated from P0/P1 rat pups that differentiated to form dopaminergic neurons, as validated using tyrosine hydroxylase. PMDN cells, when treated with IDPU (0-10μM) alone, showed insignificant toxicity. However, they exhibited < 60% cell viability when treated with 6-OHDA (150μM) after 24h. Cell viability improved to > 80%, and dopamine levels were restored in 6-OHDA- (150μM) induced PMDN cells when 3h post-treated with IDPU (0.7μM, 1μM), including the depletion in ROS generation and [Ca2+]i levels observed after 24h. IDPU treatment further impacts the mitochondrial control by attenuating both mitochondrial SOD production and its membrane potential loss in PD-like conditions. To investigate the mechanism of A2AR intervention on governing the mitochondrial-associated signalling cascades, the proteins were isolated from 6-OHDA (150μM) induced PMDN cells 3h post-treated with IDPU (0.7μM, 1μM) and ZM241385 (1μM) for western blot analysis. Our results exhibited that the phosphorylation of both DRP1 (Ser616) (78kDa) and p38MAPK (Tyr182) (41kDa) proteins was enhanced when exposed to 6-OHDA; however, the protein levels reduced post-treatment with both A2AR antagonists. In contrast, 6-OHDA toxicity alleviated the levels of both Parkin (58kDa) and DJ-1(23kDa), while exposure to A2AR antagonists (IDPU and ZM241385) improved their protein levels. This suggests the possible involvement of A2AR blockade in regulating mitochondrial dynamics, thus promoting survival. These findings present the first evidence that IDPU demonstrates neuroprotection in PDlike conditions via the p38MAPK/DRP1/Parkin signalling, offering a potential therapeutic mechanism for targeting mitochondrial dynamics through A2AR antagonism.

Neutrophils are essential effector cells of the innate immune system, acting as the first line of defense against infection and tissue injury. Among the purinergic receptors expressed in these cells, P2Y14 has gained increasing attention in recent years for its role in modulating neutrophil recruitment and activation in inflammatory contexts. This receptor is activated mainly by uridine diphosphoglucose (UDP-glucose) and other UDP-sugars released during cellular stress or damage. Through the activation of G protein–coupled pathways, particularly via Gi/o and RhoA signaling, P2Y14 influences key neutrophil functions, including chemotaxis, cytoskeletal rearrangements, and oxidative responses. Despite its pro-inflammatory potential, and the increasing amount of literature data in recent years, P2Y14’s complete physiological and pathological roles remain underexplored. Literature data also highlight its involvement in diseases like glioblastoma and COVID-19, where, due to increased neutrophil infiltration, it exacerbates inflammation, tissue damage, and stress. Therefore, targeting P2Y14 may be a promising strategy to modulate neutrophil chemotaxis and mitigate unwanted harmful inflammatory responses. This review discusses the characteristics and signaling mechanisms of P2Y14 in neutrophils, as well as the relevant implications of this pathway for neutrophil function.

Retinal diseases affect the health of millions of people worldwide and activated P2X7 receptors (P2X7Rs) are associated with the pathophysiology of a variety of retina-related diseases, including diabetic retinopathy, age-related macular degeneration, and glaucoma. Increasing evidence indicated that P2X7R is over-activated in retinopathy and is involved in the occurrence and development of diabetic retinopathy. Purine vasotoxicity caused by over-activation of P2X7R can lead to decreased retinal blood flow and vascular dysfunction and activation of P2X7R can lead to the production of a large number of inflammatory factors, causing local inflammatory cells to infiltrate and form a vascular microenvironment, thus constituting the pathophysiological basis for the occurrence and development of retinopathy. A variety of P2X7R antagonists have been studied in clinical trials as potential treatments for retinal diseases. However, currently no P2X7R antagonists has been approved for retina diseases. In this review, we mainly focus on recent progress on the involvement of P2X7R in retinal diseases and its therapeutic potential in the future.

Electroacupuncture (EA), as a combination of traditional acupuncture and modern electrotherapy, and has analgesic effects on various acute and chronic pain. It has been proved to ameliorate chronic visceral pain, but its specific role in rectal visceral pain remains underexplored. A capsaicin (CAP)-induced visceral pain rat model was established, and behavioral pain responses were observed and recorded. Measurement of mechanical pain threshold was performed using electronic Von Frey system. Rectal tissues, and the activity of microglia cells were detected by Hematoxylin/eosin staining and immunofluorescence, respectively. Contents of interleukin (IL)-6, IL-1β and tumor necrosis factor-α (TNF-α) were determined by enzyme-linked immunosorbent assay. Western blot was performed to determine the expressions of P2X4 receptor (P2X4R), p38 mitogen-activated protein kinase (p38), phosphorylated p38 (p-p38), transient receptor potential vanilloid-1 (TRPV1) and ionized calcium binding adapter molecule 1 (Iba-1). Transfection efficiency of TRPV1 overexpression plasmids was examined by quantitative real-time polymerase chain reaction. EA abrogated CAP-induced upregulation of behavioral pain responses and mechanical threshold, damage on rectal tissue, activation of microglia, stimulation of inflammatory response and promotion of P2X4R-p38-TRPV1 pathway expressions in rats, while P2X4R activation reversed the effect of EA. P2X4R antagonist weakened CAP-induced upregulation of P2X4R-p38-TRPV1 pathway expressions, microglia activation as well as increase of IL-1β in HMO6 cells, which was reversed by TRPV1 overexpression. Collectively, EA improves CAP-induced rectal visceral pain via inhibiting TRPV1 expression by blocking the P2X4R-activated p38 pathway in microglia. However, the specificity role of P2X4R needs to be confirmed by more experiment.

The signaling mechanisms of nucleotides and nucleosides have been extensively studied over the past decades in various conditions affecting distinct organs and tissues. It is well-established that purinergic receptors are expressed in healthy tissues, with expression levels often increasing under pathological conditions. These receptors play crucial roles in numerous physiological and pathological processes, including inflammation, tissue repair, and cellular signaling. However, the purinergic context in the esophagus and its associated pathologies remains poorly understood, representing a significant gap in current knowledge. In this review, we compiled and analyzed the available data on the involvement of P2 purinergic receptors in esophageal diseases, such as gastroesophageal reflux disease and esophageal carcinoma. Specifically, we discuss the pharmacological modulation, functional characterization, and expression patterns of these receptors in various esophageal cell lines and immune tissue samples, under both healthy and pathological conditions. Understanding the mechanisms of action and signaling pathways involving P2 purinergic receptors in the esophagus can offer valuable insights into their biological roles and emphasize their potential as therapeutic targets for future clinical applications.

Our previous work had identified that at the acupuncture point (acupoint), acupuncture-induced ATP release was a pivotal event in the initiation of analgesia. We aimed to further elucidate the degradation of ATP by CD39. Acupuncture was administered at Zusanli acupoint on arthritis rats, and pain thresholds of the hindpaws were determined. Pharmacological tools or adeno-associated viruses were administered at the acupoints to interfere with targeting signals. Protein expression was determined with qRT-PCR, WB, or immunofluorescent labeling. Cultured keratinocytes, HaCaT line, were subjected to hypotonic shock to simulate needling stimulation. Extracellular ATP and adenosine levels were quantified using luciferase-luciferin assay and ELISA, respectively. Acupuncture-induced prompt analgesia was impaired by inhibiting CD39 activities to prevent the degradation of ATP to AMP but was mimicked by using CD39 agonists. Acupuncture-induced ATP accumulation exhibited synchronous changes. Similarly, acupuncture analgesia was hindered by suppressing CD73 to prevent the conversion of AMP to adenosine. Furthermore, the acupuncture effect was replicated by agonism at P2Y2Rs but inhibited by antagonism at them. Acupuncture upregulated CD73 and P2Y2Rs but not CD39. Immunofluorescent labeling demonstrated that keratinocytes were a primary site for these proteins. Shallow acupuncture also demonstrated antinociception. In vitro tests showed that hypotonic shock induced HaCaT cells to release ATP and adenosine, which was impaired by suppressing CD39 and CD73, respectively. Finally, agonism at P2Y2Rs promoted ATP release and [Ca2+]i rise. CD39 at the acupoints contributes to the analgesic mechanism of acupuncture. It may facilitate adenosine signaling in conjunction with CD73 or provide an appropriate ATP milieu for P2Y2Rs. Skin tissue may be one of the scenes for these signalings.