This article describes the role of the P2X7 receptor in the participation of astrocytes, neurons, and microglia in neuropathic pain (NPP). Targeting the P2X7 receptor could be a molecular target for NPP therapy.
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Review Article
* These authors contributed equally
This article describes the role of the P2X7 receptor in the participation of astrocytes, neurons, and microglia in neuropathic pain (NPP). Targeting the P2X7 receptor could be a molecular target for NPP therapy.
Primary and secondary injuries of the nervous system can induce neuropathic pain (NPP), which severely impacts patients' physical and mental health and imposes a significant economic burden on individuals, families, and society. Currently, effective curative treatments remain elusive. Consequently, investigating the molecular basis of NPP's pathological mechanisms is crucial for developing targeted therapies. The ion channel P2X7 purinergic receptor (P2X7R) is widely expressed in the nervous system and plays a vital role in maintaining its functional stability. Accumulating evidence indicates that P2X7R is a pain-related molecule involved in the initiation and progression of NPP. The activation or upregulation of P2X7R contributes to NPP by mediating the activities of immune cells, glial cells, and neurons, regulating intercellular interactions among them, enhancing sensory signal transmission and synaptic plasticity, and ultimately leading to central sensitization. Notably, targeted inhibition of P2X7R activation using specific antagonists (e.g., A438079, A740003, and BBG) exhibits pharmacological analgesic properties. Furthermore, downregulating P2X7R expression in tissues/cells via alternative approaches (such as pharmacological agents, cell therapy, and physical therapy) also demonstrates therapeutic potential against NPP. This review comprehensively discusses the mechanisms by which P2X7R regulates both neuronal and non-neuronal cells in NPP. Given its characteristics, targeting P2X7R expression or antagonizing its activity represents a promising, novel pharmacological strategy for NPP treatment.
Neuropathic pain (NPP), a chronic condition resulting from lesions or diseases of the somatosensory nervous system, severely compromises patients' quality of life. It manifests as spontaneous pain (e.g., burning, tingling), hyperalgesia, and allodynia, in which innocuous stimuli such as light touch provoke pain. In addition, it is worth noting that even after the nociceptive stimulation is eliminated, the pain can still exist, which may be related to the unrepaired nerve injury. Pain induced by nerve injury can spread to adjacent areas, and its potential pathophysiology involves peripheral and central sensitization, which involves many factors, including changes in ion channels, activation of immune cells, glial-derived mediators, and epigenetic regulation1,2. Although a variety of clinical methods are used to relieve pain, including drugs, physiotherapy, and surgical intervention, in order to achieve the maximum analgesic effect, the therapeutic effect is still not very satisfactory. Therefore, it is necessary to explore the molecular basis of the pathological mechanisms of neuropathic pain and reverse these pathological changes, including immune cell activation and infiltration, glial cell activation, reduced neuroinflammation, protection of neurons, and improved neuroplasticity. Targeted treatment of NPP.
Changes in the local microenvironment around nerve injury, especially the activation of inflammatory cells and glial cells, can regulate cell activity and contribute to disease progression by releasing adenosine triphosphate (ATP) into the extracellular matrix3,4. The extracellular ATP-mediated purinergic signaling pathway plays a key role in NPP after central or peripheral nerve injury. Studies have shown that volume-regulated anion channels (VRACs) that release ATP from microglia are a key factor in NPP, reducing the increase in extracellular ATP in the spinal cord caused by peripheral nerve injury and relieving NPP5. P2X7 receptor (P2X7R) is widely expressed in the central and peripheral nervous systems and plays a fundamental role in maintaining the function and stability of the nervous system6. P2X7R can serve as an important pain mediator, participating in and regulating the progression of NPP6,7. P2X7R is expressed in immune cells, glial cells, and neurons. By regulating cell activity and interactions, these cells participate in neuroinflammation, immunomodulation, and cytokine release, which mediate the occurrence and development of NPP8,9,10,11. The expression of P2X7R on peripheral blood lymphocytes and monocytes, and the level of serum IL-1β, were significantly increased in patients with chronic nociceptive and NPP12.
In contrast to other classic therapeutic targets and strategies for NPP, P2X7R targeting exhibits unique advantages in efficacy and mechanism specificity. For instance, conventional first-line pharmacotherapies for NPP, such as certain analgesics and anti-inflammatory drugs, often only relieve symptoms temporarily without addressing the core pathological processes of neuroinflammation and glial activation. Glial cell modulation strategies, which focus on inhibiting spinal microglial activation4, only target a single cell type in the complex NPP pathological network, while P2X7R antagonists or targeted downregulation can simultaneously regulate the activity of glial cells, immune cells, and neurons, key cell populations involved in NPP progression6,7,12. Unlike strategies targeting general neuroinflammatory pathways3, P2X7R targeting specifically blocks the purinergic signaling cascade that is closely linked to NPP initiation and maintenance, avoiding non-specific immunosuppression and potential side effects. Additionally, approaches targeting other ion channels or neuronal signaling pathways10 often fail to reverse the persistent neuroinflammation mediated by glial-neuronal crosstalk4, whereas P2X7R targeting directly inhibits this crosstalk by regulating ATP-mediated signal transmission5,6 achieving more comprehensive therapeutic effects.
This review aims to provide a comprehensive and critical synthesis of current knowledge on the cell-type-specific mechanisms by which P2X7R modulates neuronal and non-neuronal cells in NPP. However, it is interesting to note that inhibition of P2X7R with an antagonist or targeted down-regulation of P2X7R expression has pharmacological effects in relieving/treating NPP13. For example, Lu AF27139 is a highly selective and potent small-molecule antagonist of P2X7R in rats, mice, and humans, with good pharmacokinetics and central nervous system permeability14. Lu AF27139 can functionally block P2X7R levels in the brain and spinal cord and their downstream pain-related pathways, and reduce neuropathological hypersensitivity in rats with chronic compression injury14. Trimethoxyflavanone relieves neuropathic pain by inhibiting P2X7R function, CGRP production, DRG neuron sensitization, and abnormal glial cell activation in the spinal cord15. These studies reveal the key role of P2X7R in NPP. Therefore, understanding the mechanism and pharmacological properties of P2X7R in NPP is of great significance for targeted treatment of NPP.
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1. NPP
NPP is caused by primary or secondary direct or indirect damage to the central or peripheral nervous system, resulting in dysfunction or transient disorders of the nervous system and sensory information transmission, mainly manifested as spontaneous pain, hyperalgesia, or abnormal pain1. NPP arises from peripheral parts of the nervous system, such as trigeminal neuralgia, post-herpetic neuralgia, peripheral nerve injury, painful polyneuropathy, or radiculopathy. Peripheral sensory information can be transmitted to the center (a key factor in the perception of chronic pain). The increased sensitivity of the central nervous system to pain is associated with changes in the functional connectivity pattern of the neural network. While central chronic NPP can be caused by spinal cord or brain injury, stroke, or multiple sclerosis16,17,18,19,20. After peripheral tissue injury, peripheral nociceptors are sensitized, enhancing the input of pain fibers. Nerve injury can lead to a decrease in nerve fibers and demyelination of axons in the damaged area. The ion channel opening in sensory neurons in the injury area was increased (calcium influx was enhanced), and synaptic plasticity between afferent C fibers and dorsal horn neurons was enhanced, thereby increasing information input19. Although the specific pathological mechanism of neuropathic pain is still unclear, the process of neuropathic pain has been recognized to involve immunomodulation, abnormal neuronal excitation and plasticity, glial cell activation, neuroinflammatory responses, and changes in ion channel receptors (such as P2X7R)10,20,21. These cell activation and interactions, including information transmission and cytokine release, play a key role in the occurrence and development of NPP. Therefore, we will have a comprehensive, in-depth discussion of the relationship between P2X7R and these cells.
2. Regulation of microglia activity by P2X7R
Microglia are resident immune cells in the nervous system, widely distributed throughout the central nervous system, and play a key role in stabilizing nervous system function and maintaining the stability of the microenvironment. Under normal physiological conditions, microglia are in a resting state, lack immunity and phagocytosis, but retain some migratory ability. However, after the central nervous system is injured, such as by ischemia, inflammation, or trauma, microglia can be activated, proliferate, and migrate to the injured area to remove cellular metabolites and apoptotic cell fragments. It is very important for the repair and maintenance of the central nervous system following injury. Timely microglial clearance enables effective remyelination, providing a necessary basis for nerve regeneration22,23,24. The polarization and proliferation of microglia contribute to neuropathic pain. The failure of myelin regeneration in the central nervous system leads to neuroinflammatory and neurodegenerative diseases, which can induce NPP25. Studies have reported that effective remyelination requires pro-inflammatory microglia to die and reproduce to promote regeneration. The death and/or regeneration of damaged microglia may be the basis of dysactivation of microglia in nervous system diseases26. The dominant view in the field of pain is that NPP is driven by microglia in the somatosensory processing area of the spinal dorsal horn. The response of spinal microglia to extracellular stimulation alters their activation and polarization and induces the release of inflammatory cytokines through intracellular signaling pathways, such as mitogen-activated protein kinases, p38, and extracellular signal-regulated protein kinases. induce neuroinflammatory response and lead to pain. However, inhibition or alteration of microglial activation/proliferation can reduce abnormal excitability and NPP in dorsal horn neurons27,28,29. Microglia GI DREADD can reduce neuroinflammation, synaptic function, and NPP after spinal nerve transection30.
P2X7R is widely expressed in the nervous system, especially in microglia. P2X7R regulates the activity of microglia by binding to their ligands (ATP or its analogue BzATP) and plays a key role in NPP31,32. The expression level of P2X7R is closely related to the degree of pain. NPP is induced by increased activation or expression of P2X7R in nerve injury.
Seven days after peripheral nerve injury, the expression of P2X7R mRNA and protein was significantly increased in the spinal cord, which was mainly localized in the microglia of the dorsal horn. Intrathecal injection of the P2X7R antagonist (A438079) could effectively inhibit microglial activation and alleviate mechanical hypersensitivity33. It is well established that P2X7R activation contributes to neuropathic pain (NPP) by triggering microglial activation, promoting their polarization toward a pro-inflammatory (M1) phenotype, mediating the release of pro-inflammatory cytokines, initiating a neuroinflammatory cascade, exacerbating nerve tissue damage, and ultimately inducing or aggravating pain34,35. Notably, overexpression of P2X7R alone is sufficient to promote the activation and proliferation of microglia. Specifically, P2X7R activation in microglia leads to the release of pro-inflammatory cytokines, such as interleukin-1β (IL-1β), and this process is closely associated with the development and maintenance of NPP7. IL-1β acts as a key mediator in microglial activation and proliferation, and its release or production is dependent on the macropore function of P2X7R34. Treatment with the P2X7R agonist BzATP can induce microglia to release IL-1α, IL-1β, and IL-18, while the release of other cytokines/chemokines is independent of P2X7R activation. These findings highlight the specific role of P2X7R in regulating the release of IL-1 family cytokines36. Collectively, these results demonstrate that P2X7R is a critical mediator of microglia-induced neuroinflammatory responses in NPP. In primary cultured rat microglia, cilnidipine can inhibit P2X7R-mediated calcium responses and IL-1β release; in vivo studies further confirm that cilnidipine can reverse mechanical allodynia induced by microglial P2X7R-mediated inflammation7.
In addition, P2X7R can regulate the interaction between microglia and neurons, thereby participating in the pathogenesis of NPP. In NPP models, the P2X7R-p38 signaling pathway is closely associated with microglial shedding of microvesicles and plays an important role in NPP progression by mediating microglial-neuronal interactions37. Electroacupuncture has been shown to reduce the density of dendritic spines, inhibit abnormal synaptic remodeling, alleviate neuroinflammation, and improve neuropathic pain symptoms; these effects are associated with decreased P2X7R expression and improved neurobehavioral function. In contrast, activation of P2X7R by BzATP can enhance abnormal dendritic spine/synaptic remodeling and promote neuroinflammation, thereby exacerbating pain38.
Therefore, modulating P2X7R activity to shift microglia from a pro-inflammatory M1 phenotype toward an anti-inflammatory M2 phenotype represents a promising therapeutic strategy for NPP. The M1 phenotype of microglia is characterized by the release of pro-inflammatory cytokines (such as IL-1β and TNF-α) and by promoting neuroinflammation, while the M2 phenotype is associated with the release of anti-inflammatory factors and the promotion of tissue repair. Inhibition of P2X7R activity can reverse neuropathic pain by reducing the expression of tumor necrosis factor-α (TNF-α), interleukin-1α, and IL-6 through inhibiting p38MAPK phosphorylation in microglia, which is accompanied by the transition of microglia from M1 to M2 phenotype39.
Botulinum toxin A (BTX-A) alleviates NPP by inhibiting P2X7R and promoting M2 polarization of Hapi microglia stimulated by lipopolysaccharide40. High-frequency spinal cord stimulation significantly reduces the immune response in the spinal dorsal horn and promotes lasting pain relief by inactivating the Kaiso-P2X7R axis in microglia. Targeting Kaiso-P2X7R in microglia significantly improved the efficacy of high-frequency spinal cord stimulation, thus reducing neuroinflammation and providing lasting pain relief41. Electroacupuncture can significantly increase the pain threshold induced by sciatic nerve injury or P2X7R agonist BzATP in mice, inhibit the activation of spinal cord P2X7R microglia, inhibit the overexpression of IL-1β and IL-18 in the spinal cord, and reduce tactile hypersensitivity and thermal hyperalgesia42 (Figure 1). Other studies have also shown that intrathecal injection of thermally processed Aconitum jaluense may inhibit the production and expression of P2X7R in the spinal cord and reduce the activation of microglia, which has an anti-hypersensitive effect on NPP43. In addition, the activation of microglia after peripheral nerve injury plays an important role in the abnormal convergence of nociceptive signals to spinal dorsal horn neurons and the occurrence of NPP. The crosstalk between microglia and neurons is finally involved in the expression of nociceptive hypersensory pain. Galectin-3 derived from injured sensory neurons enhances the excitatory synaptic transmission of excitatory neurons in the spinal dorsal horn by activating microglia, which leads to NPP44. These studies have revealed that P2X7R-mediated microglia activity plays a key role in NPP. Targeting microglial activation by inhibiting or downregulating P2X7R expression is a new approach to treating NPP.
3. P2X7R mediates astrocytes
Astrocytes account for about 20%–40% of the total number of glial cells in the central nervous system, which is the key to maintaining the stability of the environment in the central nervous system. Astrocytes are considered to provide structural and nutritional support for neurons and can be distinguished from other types of glial cells by expressing glial fibrillary acidic protein (GFAP). GFAP is expressed in all major branches and processes of astrocytes and changes dynamically during the transition from injury to a reactive state45,46,47. There is growing evidence that chronic pain may be caused by astrocytic gliosis. In addition, astrocytes can regulate nociceptive synaptic transmission through neuron-glial and neuroglial-glial cell interactions, and participate in the modulation of pain signals and the maintenance of NPP45,48,49,50. Astrocytes have extensive contact with neuronal synapses. Activation of astrocytes can initiate spinal plasticity, reducing synapses formed shortly after sciatic nerve ligation and inducing pain behavior50. Studies have shown that CXCL13, produced by neurons, activates astrocytes through CXCR5, thus promoting neuropathic pain, revealing the neuron/astrocyte interaction in the spinal cord51. The activation of reactive astrocytes involves a variety of signal transduction mechanisms and molecules, including intracellular kinases, channels, receptors, and transcription factors. Once pathological changes occur, they play a role in regulating pain after injury52. The expression of spinal cord aquaporin AQP4 is significantly up-regulated after spinal cord injury, mainly expressed in astrocytes, while inhibition of AQP4 can weaken the development and maintenance of NPP by inhibiting glial cell activation and the anti-neuritis mechanism. These reveal that astrocyte activation is involved in the pathogenesis of NPP. Therefore, targeting astrocytes and inhibiting their activation is helpful to alleviate NPP.
P2X7R is also expressed in astrocytes and can participate in NPP by regulating the activity of astrocytes. Different studies have confirmed that, in the model of neuropathic pain, the expression of P2X7R is significantly increased in astrocytes, and astrocyte activation is closely related to the development and maintenance of pain53,54. In such models, increased mRNA and protein levels of P2X7R, along with its co-localization with the astrocytic marker GFAP, have been observed in the hippocampus. Concomitantly, levels of inflammasome-related components (NLRP3, ASC, Caspase-1), the pore-forming protein Gasdermin D, and the mature cytokines IL-1β and IL-18 are elevated, suggesting inflammasome pathway engagement55 (Figure 1). From 11 to 21 days after the neuropathic pain model, chronic treatment with P2X7R antagonist A438079 increased the mechanical foot contraction threshold and reduced depression-like and anxiety-like behaviors56. A438079 can significantly reduce the immune response of IBA-1 and GFAP in microglia and astrocytes after pathological pain induced by nerve injury, and relieve pain behavior in rats56. The excitatory postsynaptic currents induced by single synaptic C fibers were recorded in the sections of the lumbar spinal cord of rats. BzATP activates microglia and astrocytes, or stimulates primary afferent C fibers at high frequency, resulting in long-term potentiation of C fiber afferents9. The enhanced fiber afferent response induced by BzATP could be blocked by P2X7R antagonist A438079 and astrocytotoxin fluoroacetate, respectively9. The coordinated activation of microglia and astrocytes can induce glial-mediated LTP at these synapses by releasing D-serine and tumor necrosis factor-α (TNF-α9. Diabetic neuropathic pain and depression induced by high glucose, substance P (SP), and corticosterone can increase the expression of P2X7R and the phosphorylation levels of tumor necrosis factor-α, IL-1β, free calcium, and ERK1/2 in primary cultured hippocampal astrocytes. However, this effect can be inhibited by dihydromyricetin or P2X7R shRNA treatment57.
Astrocytes are active participants in the signaling of ATP and glutamate in the brain. ATP and glutamate coordinate astrocyte activation by mobilizing calcium ions, which, in turn, trigger astrocytes to release several neuroactive molecules, including ATP and glutamate itself, to signal neurons and regulate synaptic transmission and neuronal excitability58,59. The gelatinous substance (SG) of the spinal cord is responsible for transmitting the afferent pain information to the ascending projection neurons. Neurons and some astrocytes have inward current responses to NMDA, AMPA, and mycophenol58,59. Astrocytes excite neurons through calcium-dependent glutamate release on NMDA receptors of extrasynaptic neurons, while delayed transformation of astrocytes leads to neuronal inhibition58. Critically, the P2X7R agonist BzATP enhances inward currents in both neurons and SG astrocytes. P2X7R upregulation and activation in these astrocytes promote glutamate release, which then stimulates cationic currents in adjacent neurons via NMDA and AMPA receptors, facilitating pain transmission60. In addition, dorsal root ganglion neurons can induce strong ATP release from their somatic cells. The released ATP activates P2X7R in astroglial satellite cells that enclose each dorsal root ganglion, and triggers communication between neurons and glial cells61. Activation of P2X7R results in the release of tumor necrosis factor-α from astrocyte-like satellite cells. Tumor necrosis factor-α, in turn, enhanced the P2X3R-mediated response and increased the excitability of DRG neurons61. These studies have shown that P2X7R can participate in NPP by regulating the interaction between astrocytes and neurons.
4. P2X7R mediates macrophage activation
The interaction between the immune system and nervous system is very important in neuropathic pain. As the chief culprit of neuropathic pain, the immune response in the nervous system has been established, which not only supports the initiation and development of pain but also supports its resolution62. In fact, immune cells are recognized promoters of neuroinflammation, leading to pain and allergy at every level of the neuralgia pathway. Immune cell activation and infiltration in the progression of pain contribute to the progression of NPP62,63,64. Parallel to the activation of microglia in the dorsal horn after peripheral nerve injury is the significant expansion and proliferation of macrophages around the injured sensory neurons in the dorsal root ganglion (DRG). Removal of dorsal root ganglion macrophages can reduce mechanical hypersensitivity and DRG macrophage expansion caused by nerve injury65. Systemic or local removal of macrophages can prevent pain allergy66.
The increased expression of P2X7R in macrophages/monocytes may alter the innate immune system. Macrophages appear as M1 (pro-inflammatory) or M2 (anti-inflammatory) phenotypes and express P2X7 receptors. Under pathological conditions, P2X7R responds to high concentrations of extracellular ATP by allowing the non-selective flow of cations (Na+, Ca2+, K+)67,68. Activation of P2X7R with higher concentrations of ATP, especially after repeated administration of agonists, will cause membrane pores to open, allow the passage of ~900Da molecules, and mediate changes in macrophage function67. Another important factor in P2X7R-mediated neuroinflammatory response is the regulation of macrophage activity, which induces or maintains neuropathic pain by promoting macrophage activation, producing pro-inflammatory cytokines, and driving an inflammatory cascade. Macrophage P2X7R must be co-activated with endotoxin-sensitive Toll-like receptor 4 (TLR4) to induce the formation of inflammatory body 3 (NLRP3). Then, the inflammatory body 3 (NLRP3) activates pre-interleukin-1β (Pro-IL-1β) to degrade cysteine aspartase-1 (Caspase-1), resulting in the release of IL-1β67,69 (Figure 1). Naringenin derivative, Trimethoxyflavanone (Y3) inhibited the function of P2X7R after administration of PTX, decreased the infiltration of IBA1 positive macrophage-like cells in dorsal root ganglion, and the overactivation of astrocytes and microglia in spinal cord, alleviated neuropathic pain and neurotoxicity15 RAW264.7 macrophages were treated with high glucose and free fatty acids to induce P2X7R-mediated immune, neuropathic pain and inflammation by secreting cytokines, ROS and activating ERK signaling pathway70.
Activation of P2X7R is the mechanism that stimulates macrophages to mature rapidly and release IL-1β, which participates in the inflammatory process and nociceptive nerve transmission71,72. P2X7R antagonist A839977 blocked BzATP-induced calcium influx of recombinant human, rat, and mouse P2X7R, effectively blocked Yo-Pro uptake and release of IL-1β by human THP-1 cells induced by agonists, and produced a strong anti-hyperalgesia effect73. Interestingly, in IL-1αβ knockout mice, the analgesic effect of A839977 completely disappeared73. It is suggested that the anti-hyperalgesic effect of P2X7R blockade in a mouse pain model is mediated by blocking IL-1β release. Studies have shown that the P2X7R antagonist A438079 can reduce the amount of IL-1β released by peripheral macrophages in a dose-dependent manner, inhibit the spontaneous activity of spinal cord neurons in neuropathic rats, and regulate pain behavior by modulating the immune-neural interaction released by endogenous cytokines74.
5. P2X7R-regulated neuron
Peripheral nerve injury causes excessive nociceptive response of spinal dorsal horn neurons and activates convergent nociceptive afferents of spinal dorsal horn neurons. This change is considered an indicator of the development of NPP75. In the rat spinal cord, short-interference RNA-mediated collapsin response mediator protein 2 (CRMP2) gene knockout decreased the frequency and amplitude of spontaneous excitatory postsynaptic currents in acute superficial spinal dorsal horn neurons, but did not reduce the frequency and amplitude of spontaneous inhibitory postsynaptic currents, and had no effect on small excitatory postsynaptic currents and inhibitory postsynaptic currents76. The expression of P2X7R on neurons has been historically debated. In the past, it has been controversial whether P2X7R is expressed in neurons. With the progress of research, some studies have revealed that P2X7R is expressed in neurons, which can regulate neuronal activity and participate in sensory information transmission and pain generation77,78,79. In the rat NPP model, DRG neurons were treated with the HIV envelope glycoprotein 120 (Gp120). Activation of P2X7R agonist (BzATP) increased neuronal current, while P2X7R antagonist BBG significantly inhibited BzATP-activated current and relieved hyperalgesia in gp120-treated DRG neurons80. ATP can induce ROS production in spinal dorsal horn neurons, which can be eliminated by the ROS scavenger N-tert-butyl-α-benzonitrile and the P2X7R antagonist A43807981. Intrathecal injection of P2X7R agonist BzATP can induce spinal cord ROS production and DNA oxidative damage of dorsal horn neurons, and enhance bipolar spontaneous nociceptive behavior in rats81. This provides evidence that neuronal P2X7R activation leads to ROS production and subsequent nociceptive pain in rats.
The activation of P2X7R has an extensive effect on the activity of spinal cord neurons after chronic injury. In the animal model of pathological pain, the P2X7R antagonist A438079 can reduce the evoked activity of different types of spinal cord neurons (low threshold, nociceptive-specific, wide dynamic range), regulate central sensitization, and produce an antinociceptive effect82. Other studies have shown that the up-regulation of DRG BDNF protein induced by sciatic nerve injury can enhance P2X7R activity and contribute to the up-regulation of DRG P2X7R protein expression induced by sciatic nerve injury83. Further, BDNF overexpression increases P2X7R expression in DRG neurons and mediates NPP development. It was further shown that BDNF overexpression increased P2X7R expression in DRG neurons and mediated NPP83. These studies have revealed that P2X7R is expressed in neurons, and neuronal currents and activities mediate neuropathic pain. However, some studies have found that in neuropathic pain, P2X7R is not expressed in neurons but in glial cells (such as satellite glial cells in the dorsal root ganglion) that regulate NPP. Therefore, further studies are needed to confirm and elucidate the mechanisms underlying P2X7R expression and regulation of neuronal function.
6. Antagonistic P2X7R activation and NPP therapy
As mentioned earlier, P2X7R activation mediates the occurrence and development of neuropathic pain. Therefore, inhibition of P2X7R activation has pharmacological effects on relieving/treating NPP. At present, a variety of P2X7R antagonists (such as BBG, A438079, and A740003) have been developed. These antagonists can target P2X7R activity and have been well applied in different diseases (such as spinal cord injury, tumors, and cardiovascular disease). It is confirmed that the targeted P2X7R has the function of treating related diseases84,85,86. Accordingly, these antagonists have also been used in pain treatment, and the pharmacological properties of analgesia can be exerted by inhibiting the activation of P2X7R87,88.
A740003 (N-(1-{[cyanoimino) (5-quinolinylamino) methyl] amino}-2 cyanoimino-dimethylpropyl)-2-(3-methoxyphenyl) acetamide and A438079 (3-(5-(2mae 3muricophenyl)-1H-tetrazol-1-yl) methyl pyridine hydrochloride were identified almost at the same time. A740003 is a derivative of cyanoguanidine, while A438079 is a derivative of tetrazole. They were initially reported as potent inhibitors of P2X7 receptors and were later characterized as allosteric antagonists, binding to a site distinct from the ATP-binding pocket89,90. They act on P2X7R in the same way and show the same effect under various mutations in the allosteric binding pocket, indicating that they bind in a very similar configuration. A740003 is a novel competitive antagonist of P2X7R. Its IC50 against human and rat is 40 nM and 18 nM, respectively89. The antagonistic IC50 of A438079 to human P2X7R was 130 nM91,92. In the animal model of diabetic neuropathic pain, intrathecal injection of P2X7R antagonist A438079 significantly prolonged the mechanical withdrawal threshold and thermal withdrawal latency, decreased the levels of mRNA and protein of TRPV1 in dorsal root ganglion, decreased the phosphorylation level of p38 and extracellular signal regulatory protein 1sum2 (ERK1/2), and alleviated NPP93. A438079 significantly reduced the activity of microglia and astrocytes after sciatic nerve ligation, increased Mechanical paw withdrawal threshold (MWT), decreased thermal withdrawal duration, and reduced depression-like and anxiety-like behaviors94. The PWTs of mice increased 3 h after intrathecal injection of the selective P2X7R antagonist A740003 (50 nmol/L × 5 µL), and reached the peak at 5 h. Furthermore, it can also relieve pain by knocking out the expression of P2X7R in mice95.
Another antagonist in the application of NPP is Brilliant Blue G, a commonly used derivative of the synthetic food dye compound FD&C Blue 1, which is a highly selective P2X7R antagonist96. BBG acts as a highly selective P2X7R antagonist in rat models, where it binds to an allosteric pocket in a non-competitive manner, inhibiting ATP-induced currents with an IC50 around 300 nM97,98. Its potential utility is supported by studies demonstrating its safety and ability to penetrate the blood-brain barrier in rodent models99. P2X7R inhibitor BBG could reduce the expression of NLRP3, IL-1β, IL-18, Caspase-1, and P2X7R in microglia stimulated by lipopolysaccharide, and increase the pain threshold of rats100. Intraperitoneal injection of TRPV4 agonist GSK-1016790A could significantly increase mechanical hyperalgesia and thermal hyperalgesia in rats, while P2X7R antagonist BBG could partially inhibit hyperalgesia in rats100. P2X7R antagonist BBG can reduce the expression of IL-1β and tumor necrosis factor-α receptor and the phosphorylation level of ERK1/2, increase the expression of IL-10, and relieve hyperalgesia in rats with NPP101.
Other commonly used P2X7R antagonists, such as KN-62, JNJ-54175446, and AZ10606120, have pharmacological effects on pain relief in other pain types, including inflammatory pain, visceral pain, and bone cancer pain102,103,104, but there are few reports on the application of NPP, which may be different from P2X7R in different animal model types and design schemes. Nevertheless, P2X7R antagonists have made some progress in NPP therapy and have room for further development. Therefore, there is a great prospect to explore more antagonists targeting P2X7R in NPP.
7. Targeted down-regulation of P2X7R expression to alleviate NPP
It is understood that central nervous system (spinal cord) or peripheral nerve injury (such as the sciatic or trigeminal nerves) can significantly up-regulate P2X7R expression in tissues, which is closely related to the occurrence and progression of NPP. Different studies have shown that P2X7R expression is significantly up-regulated in a neuropathic pain model. Therefore, targeting P2X7R and down-regulating its expression can alleviate or treat NPP. At present, some studies have shown that physiotherapy, cell migration, plant components (such as Gallic acid), botulinum toxin, and resveratrol can down-regulate P2X7R expression in spinal cord or nerve tissue, and considerable results have been reported in the treatment of NPP105,106,107,108,109,110. Cannabinoid receptor 2 (CB2R) is one of the important receptors in the endogenous cannabinoid system. It is widely expressed in the central nervous system, especially in glial cells, and plays an important role in the development and progression of nervous system inflammation111. After spared nerve injury, the content of P2X7R in the spinal dorsal horn increased with time, but after continuous intrathecal injection of CB2R agonist PM226, the content of P2X7R protein in brain tissue decreased significantly, reduced the number of microglia and the release of inflammatory cytokines, played a neuroprotective role, and alleviated mechanical hyperalgesia in rats111. Gallic acid (3-4-5-trihydroxybenzoic acid), which occurs naturally in many plants, has anti-inflammatory, antioxidant, and analgesic effects. Studies have shown that Gallic acid can inhibit ferroptosis in spinal cord microglia and reduce mitochondrial injury by regulating the P2X7R-ROS signaling pathway, thus alleviating behavioral changes in rats with pain and depression112. Dihydromyricetin can reduce the expression of P2X7R in DRG, spinal cord, and hippocampus, reduce the levels of phosphorylated extracellular signal-regulated protein 1, p38, TNF-α, and IL-1, and effectively relieve NPP in rats113.
In recent years, with the continuous exploration of pain treatment methods, the concept of cell transplantation therapy has been introduced. The researchers cultured active and functional cells (such as mesenchymal stem cells, neural stem cells, Schwann cells, and olfactory ensheathing cells) in vitro and transplanted them into the host through neuroprotection, anti-inflammation, and promoting axonal regeneration, down-regulated the expression of P2X7R in tissue, thus playing a role in alleviating NPP114,115,116,117,118. A study showed that 4 weeks after injection of neural stem cells into the injured spinal cord segment in a rat spinal cord injury model, the expression of neurofilament protein increased significantly, while the expression of glial fibrillary acidic protein and P2X7R (P2X4R) decreased, motor and sensory function improved significantly, and neuropathic pain alleviated119. In our previous study, after transplantation of olfactory ensheathing cells and microencapsulated olfactory ensheathing cells into the sciatic nerve injury model, the expression of P2X7R in the spinal cord was assessed using behavioral methods. The results showed that cell transplantation could significantly reduce the expression of P2X7R protein and mRNA, and the number of P2X7R-labeled positive cells in spinal cord tissue, and alleviate hyperalgesia in rats118. We further enhanced the downregulation of P2X7R expression in olfactory ensheathing cells combined with biomaterials (chitosan) at the injured sciatic nerve site and significantly alleviated NPP in rats120. All these studies reveal that targeting P2X7R and down-regulating its expression can alleviate / treat NPP, providing a more widely available treatment for NPP.
Clarifying NPP model heterogeneity is crucial for clinical transformation of P2X7R-targeted therapy. Compared with other common NPP treatments, P2X7R-targeted strategies (antagonists, targeted downregulation) have unique advantages in regulating key cell populations and blocking purinergic signaling3,4,6,7,10,12.
NPP can be divided into traumatic, diabetic, chemotherapy-induced and viral types, with distinct P2X7R-mediated mechanisms2,5,12,55,80,101: traumatic models involve glial activation and purinergic disorder4,5,33; diabetic models relate to hyperglycemia-induced neuroinflammation and NLRP3 inflammasome activation5,113; chemotherapy-induced models involve macrophage/glial activation and sensitization2,15; viral models are mediated by immune/glial activation and enhanced sensory neuron excitability6,80,101, consistent with clinical cases12.
Initiation of NPP relies on ATP-induced P2X7R activation and initial neuroinflammation3,5,2,55,80; maintenance depends on persistent P2X7R activation and abnormal glial-neuronal communication4,6,56,60. Female animals show higher P2X7R expression, more severe pain, and greater sensitivity to antagonists in traumatic and diabetic models55,66, while sex differences in other models remain understudied.
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The mechanism of NPP after nerve injury is complex, which can lead to sensory and motor dysfunction, seriously affect the quality of life of patients, and there is no effective treatment. Therefore, mining the molecular basis of NPP is helpful to the treatment of NPP. Fortunately, ion channel P2X7R has been shown to play a key role in the occurrence and development of NPP. Activation or overexpression of P2X7R can regulate glial cell and macrophage activation, mediate the release of pro-inflammatory cytokines and the inf...
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