Nociceptive signaling of P2X receptors in chronic pain states

Purinergic Signalling - P2X3 monomeric receptors (P2X3Rs) and P2X2/3 heteromeric receptors (P2X2/3Rs) in primary sensory neurons and microglial P2X4 monomeric receptors (P2X4Rs) in the spinal...     

P2X4 purinoceptor signaling in chronic pain

ATP, acting via P2 purinergic receptors, is a known mediator of inflammatory and neuropathic pain. There is increasing evidence that the ATP-gated P2X4 receptor (P2X4R) subtype is a locus through which activity of spinal microglia and peripheral macrophages instigate pain hypersensitivity caused by inflammation or by injury to a peripheral nerve. The present article highlights the recent advances in our understanding of microglia-neuron interactions in neuropathic pain by focusing on the signaling and regulation of the P2X4R. We will also develop a framework for understanding converging lines of evidence for involvement of P2X4Rs expressed on macrophages in peripheral inflammatory pain.     

Analgesic effect of electroacupuncture on chronic neuropathic pain mediated by P2X3 receptors in rat dorsal root ganglion neurons

Adenosine 5'-triphosphate disodium (ATP) gated P2X receptors, especially the subtype P2X(3), play a key role in transmission of pain signals in neuropathic pain, ATP has been documented to play a significant role in the progression of pain signals, suggesting that control of these pathways through electroacupuncture (EA) is potentially an effective treatment for chronic neuropathic pain. EA has been accepted to effectively manage chronic pain by applying the stimulating current to acupoints through acupuncture needles. To determine the significance of EA on neuropathic pain mediated by P2X(3) receptors in the dorsal root ganglion (DRG) neurons, mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were recorded, and the expression of P2X(3) receptors in the DRG neurons was assessed by immunohistochemistry (IHC) and in situ hybridization (ISH). In addition, the currents which were evoked in DRG neurons isolated from rats following chronic constriction injury (CCI) by the P2X(3) receptors agonists i.e. ATP and α,β-methylen-ATP (α,β-meATP) were examined through the experimental use of whole cell patch clamp recording. The present study demonstrates that EA treatment can increase the MWT and TWL values and decrease the expression of P2X(3) receptors in DRG neurons in CCI rats. Simultaneously, EA treatment attenuates the ATP and α,β-meATP evoked currents. EA may be expected to induce an apparent induce analgesic effect by decreasing expression and inhibiting P2X(3) receptors in DRG neurons of CCI rats. There is a similar effect on analgesic effect between rats with contralateral EA and those with ipsilateral EA.     

Electroacupuncture alleviates diabetic neuropathic pain in rats by suppressing P2X3 receptor expression in dorsal root ganglia

Diabetic neuropathic pain (DNP) is a troublesome diabetes complication all over the world. P2X3 receptor (P2X3R), a purinergic receptor from dorsal root ganglion (DRG), has important roles in neuropathic pain pathology and nociceptive sensations. Here, we investigated the involvement of DRG P2X3R and the effect of 2 Hz electroacupuncture (EA) on DNP. We monitored the rats’ body weight, fasting blood glucose level, paw withdrawal thresholds, and paw withdrawal latency, and evaluated P2X3R expression in DRG. We found that P2X3R expression is upregulated on DNP, while 2 Hz EA is analgesic against DNP and suppresses P2X3R expression in DRG. To evaluate P2X3R involvement in pain modulation, we then treated the animals with A317491, a P2X3R specific antagonist, or α β-me ATP, a P2X3R agonist. We found that A317491 alleviates hyperalgesia, while α β-me ATP blocks EA’s analgesic effects. Our findings indicated that 2 Hz EA alleviates DNP, possibly by suppressing P2X3R upregulation in DRG.

     

Dorsal root ganglia P2X4 and P2X7 receptors contribute to diabetes-induced hyperalgesia and the downregulation of electroacupuncture on P2X4 and P2X7

Diabetic neuropathic pain (DNP) is highly common in diabetes patients. P2X receptors play critical roles in pain sensitization. We previously showed that elevated P2X3 expression in dorsal root ganglion (DRG) contributes to DNP. However, the role of other P2X receptors in DNP is unclear. Here, we established the DNP model using a single high-dose streptozotocin (STZ) injection and investigated the expression of P2X genes in the DRG. Our data revealed elevated P2X2, P2X4, and P2X7 mRNA levels in DRG of DNP rats. The protein levels of P2X4 and P2X7 in DNP rats increased, but the P2X2 did not change significantly. To study the role of P2X4 and P2X7 in diabetes-induced hyperalgesia, we treated the DNP rats with TNP-ATP (2’,3’-O-(2,4,6-trinitrophenyl)-adenosine 5’-triphosphate), a nonspecific P2X1–7 antagonist, and found that TNP-ATP alleviated thermal hyperalgesia in DNP rats. 2 Hz electroacupuncture is analgesic against DNP and could downregulate P2X4 and P2X7 expression in DRG. Our findings indicate that P2X4 and P2X7 in L4–L6 DRGs contribute to diabetes-induced hyperalgesia, and that EA reduces thermal hyperalgesia and the expression of P2X4 and P2X7.     

P2 receptors and chronic pain

There is abundant evidence that extracellular ATP and other nucleotides have an important role in pain signaling at both the periphery and in the CNS. The focus of attention now is on the possibility that endogenous ATP and its receptor system might be activated in chronic pathological pain states, particularly in neuropathic and inflammatory pain. Neuropathic pain is often a consequence of nerve injury through surgery, bone compression, diabetes or infection. This type of pain can be so severe that even light touching can be intensely painful; unfortunately, this state is generally resistant to currently available treatments. In this review, we summarize the role of ATP receptors, particularly the P2X₄, P2X₃ and P2X₇ receptors, in neuropathic and inflammatory pain. The expression of P2X₄ receptors in the spinal cord is enhanced in spinal microglia after peripheral nerve injury, and blocking pharmacologically and suppressing molecularly P2X₄ receptors produce a reduction of the neuropathic pain behaviour. Understanding the key roles of these ATP receptors may lead to new strategies for the management of intractable chronic pain.     

Analgesic effect of electroacupuncture on bone cancer pain in rat model: the role of peripheral P2X3 receptor

Upregulation of P2X3 receptor (P2X3R) has been strongly implicated in nociceptive signaling including bone cancer pain (BCP). The present study, using rat bone cancer model, aimed to explore the role of P2X3R in regulating rat pain behavior under the intervention of electroacupuncture (EA). The BCP model was successfully established by injection with MRMT-1 breast cancer cell into the medullary cavity of left tibia for 3 × 10⁴ cells/3 μL PBS in rats as revealed by obvious bone destruction, decreased paw withdrawal thresholds (PWTs), and reduced paw withdrawal latencies (PWLs). Western blot analyses showed that P2X3R expression was significantly upregulated in ipsilateral lumbar 4–6 (L4-6) dorsal root ganglia (DRG), but the difference not seen in spinal cord dorsal horn (SCDH). With the in-depth study of P2X3R activation, we observed that intrathecal injection of P2X3R agonist α,β-meATP aggravated MRMT-1 induced BCP, while injection of P2X3R inhibitor A-317491 alleviated pain. Subsequently, we demonstrated that BCP induced mechanical allodynia and thermal hyperalgesia were attenuated after EA treatment. Under EA treatment, total P2X3R protein expression in ipsilateral DRGs was decreased, and it is worth mentioning that decreased expression of P2X3R membrane protein, which indicated that both the expression and membrane trafficking of P2X3R were inhibited by EA. The immunofluorescence assay showed that EA stimulation exerted functions by reducing the expression of P2X3R-positive cells in ipsilateral DRGs of BCP rats. Ca²⁺ imaging analysis revealed that the EA stimulation decreased the percentage of α,β-meATP responsive neurons in DRGs and inhibited calcium influx. Notably, the inhibitory effect of EA on mechanical allodynia and nociceptive flinches was abolished by intrathecal injection of α,β-meATP. These findings demonstrated EA stimulation ameliorated mechanical allodynia and thermal hyperalgesia in rat model of MRMT-1-induced BCP. EA exerts analgesic effect on BCP by reducing the overexpression and functional activity of P2X3R in ipsilateral DRGs of BCP rats. Our work first demonstrates the critical and overall role of P2X3R in EA’s analgesia against peripheral sensitization of MRMT-1-induced BCP and further supports EA as a potential therapeutic option for cancer pain in clinic.     

Proteomic analysis of differential protein expression in neuropathic pain and electroacupuncture treatment models

Acupuncture has long been used for pain relief. Although recent studies have shown that acupuncture can reduce neuropathic pain, the mechanism of this effect is not clear and little information is available regarding proteins that are involved in the development of neuropathic pain and the effects of acupuncture. We have developed an animal model for neuropathic pain using young adult male Sprague-Dawley rats. The model was confirmed by behavioral tests. Electroacupuncture (EA) treatment was applied to Zusanli (ST36) of neuropathic pain model to examine the analgesic effect of EA. The protein expression profile of the hypothalamus in both neuropathic pain and EA treatment models was analyzed using two-dimensional electrophoresis-based proteomics. We detected thirty-six proteins that were differentially expressed in the neuropathic pain model compared with normal rats and that restored to normal expression levels after EA treatment. Twenty-one of these proteins were identified in the MS-FiT database and are involved in a number of biological processes, including inflammation, enzyme metabolism and signal transduction. Potential applications of our results include the identification and characterization of signaling pathways involved in EA treatment and further exploration of the role of selected identified proteins in the animal model.     

Analysis of the analgesic effects of tricyclic antidepressants on neuropathic pain,diabetic neuropathic pain,and fibromyalgia in rat models

ObjectiveTo investigate the analgesic effect of amitriptyline on neuropathic pain model rats, diabetic neuropathic pain model rats and fibromyalgia model rats.MethodsThe healthy male Sprague wrote – Dawley (SD) rats were taken as the research object, and they were randomly divided into model group (group A), beside the sciatic nerve and injection of 5 mm amitriptyline group (group B), beside the sciatic nerve and injection of 10 mm amitriptyline group (group C), beside the sciatic nerve and injection of 15 mm amitriptyline group (group D), intraperitoneal injection of amitriptyline group (group E). Pain induced by selective injury of sciatic nerve branches in rats, pain induced by chronic compression of sciatic nerve, diabetic neuropathic pain and fibromyalgia were conducted to determine the pain threshold of mechanical stimulation in rats after drug administration.ResultsThe pain threshold of mechanical stimulation in the local amitriptyline group (group B, C, D) was significantly higher than that in the group A and group E at each time point after drug treatment, and the pain threshold of mechanical stimulation gradually increased with the increase of concentration. There was no statistically significant difference in mechanical stimulation pain threshold between group A and group E at each time point after drug treatment.ConclusionPara-sciatic injection of amitriptyline at different concentrations has analgesic effects on neuropathic pain, diabetic neuropathic pain and fibromyalgia in rat models, and amitriptyline directly ACTS on the local sciatic nerve.     

P2X receptors: New players in cancer pain

Pain is unfortunately a quite common symptom for cancer patients. Normally pain starts as an episodic experience at early cancer phases to become chronic in later stages. In order to improve the quality of life of oncological patients, anti-cancer treatments are often accompanied by analgesic therapies. The P2X receptor are adenosine triphosphate (ATP) gated ion channels expressed by several cells including neurons, cancer and immune cells. Purinergic signaling through P2X receptors recently emerged as possible common pathway for cancer onset/growth and pain sensitivity. Indeed, tumor microenvironment is rich in extracellular ATP, which has a role in both tumor development and pain sensation. The study of the different mechanisms by which P2X receptors favor cancer progression and relative pain, represents an interesting challenge to design integrated therapeutic strategies for oncological patients. This review summarizes recent findings linking P2X receptors and ATP to cancer growth, progression and related pain. Special attention has been paid to the role of P2X2, P2X3, P2X4 and P2X7 in the genesis of cancer pain and to the function of P2X7 in tumor growth and metastasis. Therapeutic implications of the administration of different P2X receptor blockers to alleviate cancer-associated pain sensations contemporarily reducing tumor progression are also discussed.     

Abnormal expression of P2X family receptors in Chinese pediatric acute leukemias

Nucleotides are new players in intercellular communication network. P2X family receptors are ATP-gated plasma membrane ion channels with diverse biological functions. Their distribution patterns and significance in pediatric leukemias have not been established. Here we investigated the expression of P2X receptors in BMMC samples from Chinese pediatric acute leukemias. Real-time PCR and Western blot results showed that P2X1, P2X4, P2X5 and P2X7 receptors were simultaneously over expressed in leukemias compared with controls, whereas P2X2, P2X3 and P2X6 were absent or marginally expressed in both groups. It was worth noting that the co-expression feature of them, especially between P2X4 and P2X7, could be observed and the highest expression of P2X7 was detected in relapsed patients. Moreover, concomitant decrease of P2X4, P2X5 and P2X7 expressions was observed at CR stage in a follow-up study. Functional P2X7 was also verified. These results suggested that P2X1, P2X4, P2X5 and P2X7 were hematopoiesis-related P2X receptors, and their signaling, especially for P2X7, might play important roles in pediatric leukemias. P2X receptors might co-operatively contribute to the malignant phenotype in human pediatric leukemias.     

Modification of neuropathic pain sensation through microglial ATP receptors

Neuropathic pain that typically develops when peripheral nerves are damaged through surgery, bone compression in cancer, diabetes, or infection is a major factor causing impaired quality of life in millions of people worldwide. Recently, there has been a rapidly growing body of evidence indicating that spinal glia play a critical role in the pathogenesis of neuropathic pain. Accumulating findings also indicate that nucleotides play an important role in neuron-glia communication through P2 purinoceptors. Damaged neurons release or leak nucleotides including ATP and UTP to stimulate microglia through P2 purinoceptors expressing on microglia. It was shown in an animal model of neuropathic pain that microglial P2X₄ and P2X₇ receptors are crucial in pain signaling after peripheral nerve lesion. In this review, we describe the modification of neuropathic pain sensation through microglial P2X₄ and P2X₇, with the possibility of P2Y₆ and P2Y₁₂ involvement.     

Role of sodium ferulate in the nociceptive sensory facilitation of neuropathic pain injury mediated by P2X(3) receptor

Neuropathic pain usually is persistent and no effective treatment. ATP plays an important role in the initiation of pain. P2X(3) receptors are localized in the dorsal root ganglion (DRG) neurons and activated by extracellular ATP. Sodium ferulate (SF) is an active principle from Chinese herbal medicine and has anti-inflammatory activities. This study observed the effects of SF on the nociceptive facilitation of the primary sensory afferent after chronic constriction injury (CCI) mediated by P2X(3) receptor. In this study, the content of ATP in DRG neurons was measured by high-performance liquid chromatography (HPLC). P2X(3) agonist-activated currents in DRG neurons was recorded by the whole-cell patch-clamp skill. The expression of P2X(3) mRNA in DRG neurons was analyzed by in situ hybridization. The ATP content of DRG was increased after CCI. In CCI rats treated with SF, the content of ATP in DRG neurons was reduced. SF decreased the increment of P2X(3) agonist-activated currents and P2X(3) mRNA expression in DRG neurons during CCI. SF may inhibit the initiation of pain and primary afferent sensitization mediated by P2X(3) receptor during CCI.     

Synthesis and SAR development of novel P2X7 receptor antagonists for the treatment of pain: part 2

Novel P2X₇ antagonists were developed using a purine scaffold. These compounds were potent and selective at the P2X₇ receptor in human and rodent as well as efficacious in rodent pain models. Compound 15a was identified to have oral potency in several pain models in rodent similar to naproxen, gabapentin and pregabalin. Structure–activity relationship (SAR) development and results of pain models are presented.     

P2X receptors-mediated cytosolic phospholipase A2 activation in primary afferent sensory neurons contributes to neuropathic pain

Activation of P2X₃ and P2X_2/3 receptors (P2X₃R/P2X_2/3R), ionotropic ATP receptor subtypes, in primary sensory neurons is involved in neuropathic pain, a debilitating chronic pain that occurs after peripheral nerve injury. However, the underlying mechanisms remain unknown. We investigated the role of cytosolic phospholipase A₂ (cPLA₂) as a downstream molecule that mediates the P2X₃R/P2X_2/3R-dependent neuropathic pain. We found that applying ATP to cultured dorsal root ganglion (DRG) neurons increased the level of Ser505-phosphorylated cPLA₂ and caused translocation of Ser505-phosphorylated cPLA₂ to the plasma membrane. The ATP-induced cPLA₂ activation was inhibited by a selective antagonist of P2X₃R/P2X_2/3R and by a selective inhibitor of cPLA₂. In the DRG in vivo , the number of cPLA₂-activated neurons was strikingly increased after peripheral nerve injury but not after peripheral inflammation produced by complete Freund's adjuvant. Pharmacological blockade of P2X₃R/P2X_2/3R reversed the nerve injury-induced cPLA₂ activation in DRG neurons. Moreover, administering the cPLA₂ inhibitor near the DRG suppressed nerve injury-induced tactile allodynia, a hallmark of neuropathic pain. Our results suggest that P2X₃R/P2X_2/3R-dependent cPLA₂ activity in primary sensory neurons is a key event in neuropathic pain and that cPLA₂ might be a potential target for treating neuropathic pain.     

Synthesis and SAR development of novel P2X7 receptor antagonists for the treatment of pain: part 1

Structure-activity relationship (SAR) efforts around our initial lead compound 1 led to the identification of potent P2X₇ receptor antagonists with improved pharmacokinetic profiles. These compounds were potent and selective at the P2X₇ receptor in both human and rodent. Compound (entry 31) exhibited oral efficacy in the rat MIA and CCI pain models.     

HCN2在大鼠外周神经病理性疼痛发生中的作用

目的：初步探讨超极化激活的环核苷酸门控通道2型（HCN2）在外周神经病理性疼痛发生中的作用。方法：将24只健康成年大鼠进行随机分组（n=12）：假手术组（Sham）大鼠仅分离左侧L4、L5脊神经,模型组（SNL）分离脊神经后进行相应的结扎处理,手术7 d后用行为学方法进行模型评价;将造模成功的大鼠进行随机分组（n=6）：①阴性对照组（Saline）,左侧足底注射生理盐水;②阳性对照组（GBPT）,腹腔注射加巴喷丁;③实验组（ZD7288）,左侧足底注射HCN非特异性阻断剂ZD7288。在给药前以及给药后1 h、4 h、24 h、48 h用疼痛行为学实验检测其对神经病理性疼痛的作用;分别取手术前对照组（Control）、假手术组（Sham）和模型组（SNL）大鼠的背根神经节（DRG）（n=6）,利用qPCR和Western blot的方法研究造模前后大鼠DRG内HCN2的表达的变化情况。结果：①成功建立大鼠神经痛模型;②与Saline组比较,GBPT组和ZD7288组在注射1 h后,均能明显的减轻大鼠神经病理性疼痛的症状（P<0.01）,而GBPT组和ZD7288组之间比较则无差异;③与Control组和Sham组相比较,SNL组大鼠DRG内的HCN2 mRNA表达量明显增加（P<0.01）;与Control组和Sham组相比较,SNL组大鼠DRG内的HCN2通道蛋白表达量显著增加（P<0.05）。结论：HCN2参与外周神经病理性疼痛的发生,并有可能成为治疗神经病理性疼痛一个潜在的新靶点。     

Antinociceptive and neurochemical effects of a single dose of IB-MECA in chronic pain rat models

This study aimed to evaluate the effect of a single administration of IB-MECA, an A3 adenosine receptor agonist, upon the nociceptive response and central biomarkers of rats submitted to chronic pain models. A total of 136 adult male Wistar rats were divided into two protocols: (1) chronic inflammatory pain (CIP) using complete Freund’s adjuvant and (2) neuropathic pain (NP) by chronic constriction injury of the sciatic nerve. Thermal and mechanical hyperalgesia was measured using von Frey (VF), Randal-Selitto (RS), and hot plate (HP) tests. Rats were treated with a single dose of IB-MECA (0.5 μmol/kg i.p.), a vehicle (dimethyl sulfoxide—DMSO), or positive control (morphine, 5 mg/kg i.p.). Interleukin 1β (IL-1β), brain-derived neurotrophic factor (BDNF), and nerve growth factor (NGF) levels were measured in the brainstem and spinal cord using enzyme-linked immunosorbent assay (ELISA). The establishment of the chronic pain (CIP or NP) model was observed 14 days after induction by a decreased nociceptive threshold in all three tests (GEE, P < 0.05). The antinociceptive effect of a single dose of IB-MECA was observed in both chronic pain models, but this was more effective in NP model. There was an increase in IL-1β levels promoted by CIP. NP model promoted increase in the brainstem BDNF levels, which was reversed by IB-MECA     

Quercetin relieved diabetic neuropathic pain by inhibiting upregulated P2X4 receptor in dorsal root ganglia

The upregulation of nociceptive ion channels expressed in dorsal root ganglia (DRG) contributes to the development and retaining of diabetic pain symptoms. The flavonoid quercetin (3,3′,4′,5,7-pentahydroxyflavone) is a component extracted from various fruits and vegetables and exerts anti-inflammatory, analgesic, anticarcinogenic, antiulcer, and antihypertensive effects. However, the exact mechanism underlying quercetin's analgesic action remains poorly understood. The aim of this study was to investigate the effects of quercetin on diabetic neuropathic pain related to the P2X₄ receptor in the DRG of type 2 diabetic rat model. Our data showed that both mechanical withdrawal threshold and thermal withdrawal latency in diabetic rats treated with quercetin were higher compared with those in untreated diabetic rats. The expression levels of P2X₄ messenger RNA and protein in the DRG of diabetic rats were increased compared with the control rats, while quercetin treatment significantly inhibited such enhanced P2X₄ expression in diabetic rats. The satellite glial cells (SGCs) enwrap the neuronal soma in the DRG. Quercetin treatment also lowered the elevated coexpression of P2X₄ and glial fibrillary acidic protein (a marker of SGCs) and decreased the upregulation of phosphorylated p38 mitogen-activated protein kinase (p38MAPK) in the DRG of diabetic rats. Quercetin significantly reduced the P2X₄ agonist adenosine triphosphate-activated currents in HEK293 cells transfected with P2X₄ receptors. Thus, our data demonstrate that quercetin may decrease the upregulation of the P2X₄ receptor in DRG SGCs, and consequently inhibit P2X₄ receptor-mediated p38MAPK activation to relieve the mechanical and thermal hyperalgesia in diabetic rats.