Introduction

Rheumatoid arthritis (RA) is one of the diseases resulting from prolonged chronic inflammation that predominantly affects joints. Recently, many advances have been made in the treatment of RA leading to better control of joint inflammation. However, pain resulting from RA is still a major problem reported by patients [1, 2]. It is believed that RA-related pain is not solely caused by inflammatory reactions. Lee and colleagues [3] revealed that 12–20% of patients reported prolonged pain without signs of inflammation. Moreover, Hewlett et al. [4] revealed that the severity of pain varies between patients and even RA patients with no indication of inflammation frequently experience pain. There is a weak association between the intensity of pain and the objective measures of inflammation [5]. Although the application of disease-modifying anti-rheumatic drugs (DMARDs) may resolve inflammation, 58% of early RA patients reported incomplete improvement in body pain after a year of treatment. Another 15% of patients reported no improvement of pain and 27% reported increased pain after one year compared to the baseline value [6]. These findings suggest that the pain associated with RA arises from non-inflammatory sources. Moreover, the nature of suffering from pain is multifactorial. Stress that resulted from sleep disturbance, depression, anxiety and catastrophising in RA patients may also influence the perception of pain. Meanwhile, RA is also believed to be derived from the presence of anti-citrullinated protein (ACPA), which is then developed into autoantibodies and may be present in the synovial fluid of the patients [7]. Enhanced pain hypersensitivity caused by ACPA was also reported by Wigerblad et al. [8] in mice model of collagen-induced arthritis administered with human or murine ACPA, which indicated that the ACPA-induced hypersensitivity is mediated by pathways depending on the IL-18 release. Although RA patients suffer from hyperalgesia as a result of lower pain threshold due to the pathogenesis of RA, the abnormalities may originate from the central nervous system (CNS), including the loss of conditioned pain modulation or central sensitisation, when there are the absent signs of chronic local inflammation or local tissue damage [1]. In a clinical trial, Christensen et al. [9] showed that 65% of RA patients experienced nociceptive pain, while the rest experienced non-nociceptive pain. As the consequence of the amplification of a nociceptive signal, neuropathic pain condition or central sensitisation is believed to be the culprit of the abnormal symptoms occurring in RA [1]. In this review, the aim was to discuss a comprehensive overview of P2X4 receptors (P2X4R) in the pathogenesis of RA and its possible mechanisms leading to RA-related pain.

The P2X4 receptor is ATP-gated purinergic signalling

Extracellular adenosine triphosphate (ATP) is a crucial signalling molecule regulating several biological processes involved in the nervous, endocrine, respiratory and cardiovascular systems. It is the energy substance in the body that is involved in the energy conversion of cells and tissues. The P2X4R activates channels in various neuronal, muscle, glandular, hair cells, microglia, astrocytes and B-lymphocytes. However, it is also heavily involved in the pathological progress of diseases such as inflammation, immune function, cancer, pain and ischaemia [10]. The effects of extracellular ATP activation implicate two types of P2 receptors cell surface; the G protein-coupled P2Y receptors and the P2X receptors, which have an integral cation channel that opens following the ATP binding process [11, 12]. They are known as ligand-gated cation-selective ion channels that bind to extracellular ATP [12, 13], mediating the inflow of sodium (Na+) and calcium ions (Ca2+) as well as the outflow of potassium ion (K+) [14]. These receptors belong to distinct structural types of Cys-loop and glutamate receptor families of ligand-gated cation channels. These channels are abundant in both excitable and non-excitable cells, including neurons, muscles, glands, hair, microglia, astrocytes and B lymphocyte cells [13]. Since ATP is a fast neurotransmitter at specific central synapses, it is also released in the periphery, which explains how pain transmission can be easily transmitted after ATP activation of P2X receptors [15].

Seven subtypes of P2X receptors designated as P2X1 through P2X7 that co-assemble as homotrimeric or heterotrimeric have been identified [16] ranging from 388 (P2X4) to 595 (P2X7) subunits [10, 11]. Each of the subunits contains two hydrophobic, putative membrane-spanning sections (TM1 and TM2) which are separated by an ectodomain containing 10 cysteine residues forming disulphide bonds [15]. These structures made them resemble the form of epithelial Na+ channels (ENaC) and acid-sensing channels (ASIC) [11]. It is believed that P2X receptors are closely involved in the development and transmission of pain and inflammatory-nociceptive signals [12]. P2X receptors are widely expressed in both neurons and glia in the nervous system, and P2X2, P2X4 and P2X6 receptors are most abundantly expressed [11, 15].

Similar to other P2X receptors, P2X4R is crucial in many physiological processes. It consists of three subunits consisting of the head, body and tail. The interactions between these subunits form the triple symmetry axis trimer channel protein [17]. It contains 388 amino acids comprising of an N-terminus (30 amino acids), a C-terminus (38 amino acids), two transmembrane domains (TM1 and TM2; 20 amino acids each) and a large extracellular domain (280 amino acids) [10] (Fig. 1). P2X4R is in fact, essential for the physiological importance of the body [10, 14] as it is involved in the normal regulation of inflammation, fibrogenesis, apoptosis and neuromodulation associated with pain sensitivity [18].

Fig. 1
figure 1

Structure of the P2X4 receptor. This receptor consists of N- and C-terminals, two transmembrane domains (TM1 and TM2) and a large extracellular domain (adapted from Zhang et al. [8])

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During the physiological state, activation of P2X4R by binding of the extracellular ATP induces the opening of transmembrane pores and permits the influx of Ca2+, Na+ and K+ to modify their membrane voltage. This effect leads to the reduction of electrochemical gradients and activation of intracellular signalling components [19]. However, during tissue injury with the presence of noxious stimulation, especially in the case of chronic or neuropathic pain, the abundant ATP released from presynaptic nerves can bind to P2X4 receptors on microglia to produce distinctive, fast excitatory postsynaptic potentials (EPSPs) through the BDNF-TrkB receptor pathway [20, 21]. The inflammatory cascades lead to increased ATP-activated currents that change the voltage dependence of P2X4R and thus increasing the activation of this receptor [19]. This was demonstrated by the upregulation of P2X4R in the forelimb of the rat following complete Freund’s adjuvant (CFA) injection [19]. Since P2x4R has the highest permeability to Ca2+, the increased Ca2+ influx may activate related cells such as neurons, microglia and astrocytes. Consequently, these effects lead to the development of allodynia following nerve injury [20, 22]. These abnormal CNS changes result in the development of several neurological abnormalities including neuropathic pain [23], epilepsy [24, 25], anxiety [26], stroke [27] and Parkinson’s disease [28]. Furthermore, abnormal upregulation or deletion of P2X4R is also associated with the development of inflammation [29], auto-immune diseases [30], cardiovascular diseases [31] and endocrine disorders [32].

P2X4R alleviates inflammatory-induced pain responses

Arthritis-related pain is characterised by the occurrence of hyperalgesia and flare pain at the affected joints. Reduced pain thresholds and noxious hypersensitivity have been reported in RA patients [33, 34]. Patients with RA normally demonstrated reduced pressure pain thresholds and increased sensitivity to painful stimuli across several anatomical sites, including both inflamed joints and non-inflamed tissues [35, 36], suggesting central augmentation of pain signals. Patients may experience joint hyperalgesia when the affected joints are moved or when light pressure is exerted on the joints [37]. The essential mechanisms leading to RA-associated pain may be derived from modified central pain regulatory mechanisms such as abnormal pain modulation (central sensitisation), rather than prolonged stimulation of peripheral nociceptors [16]. It has also been reported that enhanced pain sensitivity was amplified in individuals that experienced a longer duration of RA [38].

Pain responses in animal models of arthritis are well-established. Several researchers investigating the involvement of P2X4R in the pathogenesis of chronic arthritis and inflammation have identified positive contributions of this receptor in pain behaviour responses. For instance, a rat model of collagen-induced arthritis and CFA-induced arthritis mimicked RA via several main characteristics such as systemic effects on joints, preferential injury to peripheral extremity joints, the participation of rheumatoid factors, greater severity in females and histological discoveries including synovial hyperplasia and infiltration of inflammatory cells [39, 40].

In a P2X4R-knockout mice model, Tsuda and colleagues [41] reported significant suppression of pain hypersensitivity to innocuous mechanical stimuli (tactile allodynia) and hind paw swelling following the intraplantar injection of CFA. Similar attenuation of mechanical allodynia was shown in a rat model of chronic constriction injury following the inhibition of P2X4R activation [42]. Furthermore, the inhibition of P2X4R via the intrathecal route significantly attenuated its upregulation in the spinal cord, thereby suppressing the development of mechanical allodynia in a mice model of herpetic pain [22]. Meanwhile, a reduced tail pressure pain threshold was reported in the adjuvant-induced arthritic rat [43]. A similar result was also reported by previous studies [44,45,46], with the polyarthritic rat showing a reduction in tail-flick latency, indicating reduced spinal reflex starting from day 7 to 22 following arthritic induction [44]. Other than that, the arthritic rats demonstrated the development of hyperalgesia as determined by the reduction in latency to cold [45, 47, 48] and hot stimuli [49]. In the paw pressure test, the CFA-injected arthritic rat demonstrated a significantly reduced threshold compared to the non-arthritic rat [50]. It was reported that the absence of P2X4R produced no effects in the mice with acute physiological pain or pain induced by tissue injury. The study highlighted P2X4R’s critical role in the development of chronic pain rather than acute pain [41, 51]. Therefore, it is essential to understand the underlying pathophysiology of modified central pain regulation which may be the leading cause of RA-associated pain.

Involvement of the P2X4 receptor in the peripheral nervous system

Since the symptoms of RA include peripheral neuropathy due to neurogenic lesions [52], similar pathomechanisms of neuropathic pain may be partially shared in RA. Previous studies revealed an extensive distribution of P2X4R in the dorsal root ganglia (DRG), autonomic and sensory ganglia [13, 14, 53]. The expression of peripheral P2X4R has been reported in the DRG or macrophages during inflammatory pain [54,55,56], but possibly not during neuropathic pain [56]. In the DRG, immunofluorescence staining demonstrated that P2X4R expression was mainly in satellite glial cells [57].

Following peripheral nerve lesion or inflammation, the enhanced peripheral nerve signals drive ɣ-aminobutyric acidergic (GABAergic) neurons to increase presynaptic ɣ-aminobutyric acid A (GABAA) receptors, leading to depolarisation of central terminals of the sensory neuron (i.e. primary afferent depolarisation), which in turn develops an impulse train that travels peripherally. These effects, therefore, trigger the release of inflammatory peptides from peripheral terminals of C-fibres that lead to the activation of inflammatory cascades in the affected tissues [58]. ATP released from injured nerves may bind and activate P2X4R on the related cells [53]. However, the inflammatory mediators can also stimulate P2X4R activation expressed on macrophages, which in turn leads to the production of prostaglandins (PGs) via the p38 mitogen-activated protein kinase (p38 MAPK) activation pathway [54].

This mechanism may also lead to the increased release of brain-derived neurotrophic factor (BDNF) from monocytes, B- and T-cells as shown in the culture of human peripheral blood mononuclear cells [59]. In the synovial fluid culture of RA patients, Klein et al. [60] reported that increased BDNF release in the synovial fluid was not mediated by the indirect action of PGs, but possibly via the actions of pro-inflammatory cytokines such as tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-17 (IL-17) as demonstrated in the culture of monocytes [61] and microglia in the central nervous system [62] (Fig. 2). In microglial cell cultures, the activation of P2X4R on their cell surfaces leads to an increased Ca2+ influx into cells that further activates p38 MAPK, resulting in increased synthesis and release of BDNF via soluble N-ethylmaleimide-sensitive factor attachment protein (SNARE)-dependent exocytosis [63]. The production of BDNF is secreted by both fibroblast cells and non-fibroblast cells, including endothelial cells and macrophages, at the later stage of osteoarthritis [9].

Fig. 2
figure 2

Mechanism of P2X4R at peripheral region involving peripheral nerve lesion and inflammatory cells. Following the injury of a peripheral nerve due to inflammatory reactions in synovial fluid, the released ATP and PGs from the damaged nerve activate P2X4R expressed on the macrophage. Subsequently, the highly Ca2+ influx into the macrophage cell may activate p38 MAPK leading to the release of BDNF and PGE2 into the synovial fluid and surrounding nerves. The high influx of Ca2+ also indirectly stimulates P2X7R to allow the production of pro-inflammatory factors from macrophage, which further enhances the BDNF release by monocytes, B- and T-cells. Moreover, the released pro-inflammatory cytokines can further stimulate NLRP1 inflammasome on the macrophage. The synergistic effects between injured peripheral nerves and inflammatory cells lead to the leukocyte infiltration in joints and synovial hyperplasia. The prolonged synovial inflammation may further lead to bone erosion and cartilage destruction (osteoarthritis) and therefore transmit the nociceptive signals to a central level that develop thermal hyperalgesia and mechanical allodynia; the common symptoms of RA-associated chronic pain

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Li and colleagues [64] reported that the inhibition of P2X4R resulted in decreased serum interleukin-1β (IL-1β), TNF-α, IL-6 and IL-17 levels in a mouse model of collagen-induced arthritis. Synovial inflammation and joint damage indicated by prominent leukocyte infiltration, synovial hyperplasia, bone erosion and evident cartilage damage were attenuated following the administration of a P2X4R antisense nucleotide; a P2X4R gene suppressor. In addition, the activated NLR family, pyrin domain containing 1 (NLRP1), PYD- and CARD-domain containing (ASC) and caspase-1 (NLRP inflammasome) were inhibited following the suppression of P2X4R in this mouse model and synovial cells of RA patients. This study highlighted the importance of NRLP1 involvement in the inflammasome signalling pathway in RA pathogenesis following P2X4R activation.

Furthermore, Lalisse and colleagues [65] demonstrated that P2X4R regulates Ca2+ influx in DRG neurons in a mouse model for chronic inflammation. The activation of P2X4R may also implicate other peripheral signalling pathways. In an in vitro study of mouse macrophage, PX4R was found to inhibit P2X7R-mediated autophagy [66]. Kawano et al. [66] revealed that P2X4R modulates several mechanisms of P2X7R-mediated inflammation, including the production of IL-1β that targets macrophages, causing their death. These pathological events can lead to an abnormal discharge of primary sensory neurons, enhanced nociceptive input transmission and enhanced synaptic plasticity of the spinal cord, as revealed in an in-vitro study using superficial spinal dorsal horn slices [67]. Through the above studies, it was demonstrated that P2X4R activation may damage nervous systems. It is important to attenuate its activation following chronic pain and inflammatory conditions. Minimising P2X4R overexpression may become a potential target for the treatment of RA and other nervous system-related diseases.

Involvement of the P2X4 receptor during central pain transmission

In the CNS, P2X4R is extensively distributed in the brain and cerebral cortex, basal ganglia, diencephalon, dorsal thalamus and spinal cord, but more densely found in the pons and medulla of the brain [13, 14, 53]. This receptor is specifically found in excitatory synapses in the brain, together with P2X2 receptors [11]. In pathological conditions such as neuropathic pain or epilepsy, the upregulation of P2X4R expression was detected in the neurons and microglia of the hippocampus and spinal cord [25, 64]. The activation of P2X4R in these cells is triggered by episodic tetanus stimuli contributing to synaptic plasticity in the hippocampal pyramidal neurons [67]. Moreover, the activation of P2X4R increases Ca2+ signalling to allow gene expression, increase the release of neurotransmitters to augment the transmission of sensory signals and to enhance sensory neuronal hyperexcitability and central sensitisation that subsequently develop prolonged inflammatory responses and pain [10, 65].

Spinal cord

Several studies revealed that blocking P2X4R activation by ATP binding may reduce the occurrence of tactile allodynia in the spinal cord following intrathecal antisense oligonucleotides [20]. This may occur due to the inhibition of microglial activation since the binding of ATP to P2X receptors expressed on microglia may lead to its activation. Extracellular ATP stimulates P2X4R, triggering a transient increase in Ca2+ influx in microglia. In fact, microglial activation relies on high concentrations of Ca2+ influx [68]. Ulmann and colleagues [25] revealed the upregulation of P2X4R in activated spinal microglia at the level of transcription, translation and post-translation following peripheral nerve injury. Although P2X4R is capable of high Ca2+ concentration fluxes comparable to that of N-methyl-d-aspartate receptor (NMDAR) subtypes, it cannot implicate NMDAR because this receptor is blocked by Mg2+. Therefore, P2X4R potentially matches the regulatory function of the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) subtype of glutamate receptors to develop synaptic plasticity [69].

The activation of P2X4R activation also implicates BDNF-tyrosine kinase B (BDNF-TrKB) signalling pathway, resulting in the augmentation of BDNF production, c-fos in microglia, p38 level and stimulation of the trigeminal neuralgia [70]. In the spinal cord of P2X4R-deficient mouse, it was discovered that increased BDNF signalling, extracellular signal-regulated kinase 1/2 (ERK ½) phosphorylation of NMDAR GluN1 subtype and KCC2 downregulation following peripheral inflammation were impaired [65], suggesting that P2X4R played a significant role leading to chronic inflammatory pain. In another study, certain signalling mediators such as fibronectin, Lyn tyrosine kinase and chemokine ligand 2 (CCL2) can stimulate the upregulation of P2X4R on microglia or induce its trafficking to the cell surface of microglia following peripheral nerve injury [42]. The activated P2X4R may lead to the activation of the phosphatidylinositol 3-kinases-protein kinase B (P13K/Akt) pathway, which further leads to microglial activation and promoting its migration [47].

The increased activation of P2X4R is also linked to the enhanced level of catestatin (CST) and subsequently leads to pain aggravation [71]. Although the development of CST has yet to be investigated during the central pathogenesis of RA, it may have some effects on causing further aberrant stimulation of P2X4R on the surface of microglia. Deng et al. [71] revealed that following nerve injury, phosphorylation of ERK 1/2 in microglia leads to the increased expression of CST which further enhances the activated P2X4R. CST can also chemotactically accumulate monocytes and mediate neurogenic inflammation [72]. Moreover, the activation of ERK 1/2 can lead to the release of pro-inflammatory mediators in the spinal dorsal horn region [71]. Likewise, activated T-helper cell 1 (Th1 lymphocytes) from peripheral blood can infiltrate the blood-brain barrier and communicate with spinal microglia via the release of pro-inflammatory mediators, including interferon-ɣ (IFN-ɣ), to activate P2X4R [42, 73]. Consequently, these mechanisms lead to the stimulation of microglia and astrocytes productions in the spinal cord, produce multiple neuroinflammatory signalling mediators (i.e. IL-6, IL-10, IL-1β, IL-6 and IL-18), stimulate a variety of cyclooxygenase-2 ((COX-2), cysteine protease-1, nitric oxide synthase 2, carboxypeptidase, metalloproteinase and chymotrypsin) and promote neuroinflammatory responses [74,75,76] which can be deleterious, or facilitate synaptic plasticity and cell death. The highly activated microglia can further enhance the expression levels of P2X4R and, therefore, prolong the occurrence of hyperalgesia. The simplified proposed mechanism following P2X4R activation in the spinal cord is illustrated in Fig. 3.

Fig. 3
figure 3

Central mechanisms after P2X4R activation in the spinal cord. The affected neuron secretes adenosine triphosphate (ATP), chemokine ligand 2 (CCL2) and fibronectin that upregulate microglia P2X4 receptor (P2X4R) and promote its trafficking to cell surface prior to peripheral nerve injury due to rheumatoid arthritis (RA). Helper T-cells from peripheral blood vessel may pass through the blood-brain barrier (BBB) and interact with microglia via the released pro-inflammatory cytokines including interferon-ɣ (IFN-ɣ). As a consequence of ATP binding to P2X4R, several pathways are activated including phosphorylation of extracellular signal-regulated kinase 1/2 (ERK 1/2) and p38 mitogen-activated protein kinase (p38 MAPK) and phosphatidylinositol 3-kinases-protein kinase B (PI3K/Akt) that leads to the release of brain-derived neurotrophic factor (BDNF) and pro-inflammatory mediators (IL-1β, TNF-α, IL-6 and prostaglandins) that contributes to neurogenic inflammation. Secreted BDNF may then bind to tropomyosin receptor kinase B (TrkB) receptors on nociceptive neurons that further downregulate potassium chloride cotransporter (KCC2) resulting in the accumulation of chloride anions (Cl-) in the neurons. Consequently, the subsequent binding to ɣ-aminobutyric acid A receptor (GABAA receptor) that is normally inhibitory, shifts to the disinhibition of inhibitory neurons. Meanwhile, the production of cathestatin (CST) via the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK 1/2) in microglia may further augment the P2X4R activation on the activated microglia. The hyperexcitability resulted from GABA depolarisation together with the release of pro-inflammatory cytokines from microglia and lesioned nerve, as well as N-methyl-d-aspartate receptors (NMDARs) activation, leads to sensitisation of spinal circuits and therefore, chronic pain

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Brain

A significant number of studies have shown prominent expression of P2X4R in different neuronal populations in the hippocampus, cerebellum and cortex [65]. P2X4R expression has been identified in neurons throughout CNS, mediating synaptic transmission, and maintaining biological responses in tissues. In the pyramidal neurons of the hippocampus, activation of P2X4R is triggered by the occurrence of electrical activity contributed by tetanus stimuli, leading to the production of synaptic plasticity [68]. In the rat brain, dense distribution of P2X4R was identified in granule cells of the dentate gyrus, CA1/CA3 pyramidal cells, Purkinje cells of the cerebellar cortex and neurons pontine nucleus [77, 78]. The localisation of P2X4R was also found at postsynaptic membranes as opposed to terminals of Schaffer collaterals in CA1 pyramidal cells, densely located at the peripheral region of parallel fibre postsynaptic specialisation and Schaffer collateral synapses [77]. Although electron microscopy immunostaining analysis revealed that the P2X4R was preferentially localised to post-synaptic terminals, its localisation was also found at both presynaptic and postsynaptic terminals in various brain regions [77, 79].

The mechanisms of ATP activation involving microglia activation together with neuronal interactions in the brain are also similar in the spinal cord since microglia is ubiquitously distributed throughout CNS [80]. In neurodegenerative diseases, P2X4R expression is upregulated in GABAergic interneurons and GABAergic spiny neurons of the striatum and substantia nigra as reported in a rat model of Parkinson’s disease [81]. In addition, the abundant expression of P2X4R in the hypothalamus and anterior pituitary glands indicates that P2X4R plays a significant role in hypothalamic-pituitary mechanisms in the CNS [82]. Furthermore, the reduced synaptic facilitation and long-term potentiation (LTP) in P2X4R-knockout mice indicate that P2X4R plays a crucial role in the development of central sensitisation [26]. However, there is still limited discovery regarding the role of P2X4R in a brain region associated with RA.

Previous studies have shown that, as one of the ATP-activated P2 receptors, P2X4R can play a role in the reward circuit in a particular brain region by regulating glutamate [83, 84] or dopamine release [85]. P2X4R is also involved in the development of long-term potentiation (LTP) and long-term depression (LTD) [80]. It is highly probable that the unstable emotion and depression reported by RA patients involves the activation of P2X4R via certain specific pathways. Therefore, more research is required to explore the role of P2X4R within a particular brain region associated with pain transmission so that the specific pathway can be modulated to combat chronic pain and negative emotions associated with RA.

Sex differences in microglia-P2X4 receptor signalling

Although the occurrence of RA is more worsening in female than male patients [86] with a higher prevalence rate (3:1 ratio) [87], the specific molecular mechanisms leading to this complication is less extensively studied. It is reported that the pro-inflammatory immune responses to tissue damage in females are higher than in males, therefore, directly results in more pain. It is demonstrated in an in vitro study that the female sex hormone, oestrogen, is found to inhibit the release of pro-inflammatory cytokines such as IL-1α, IL-1β, TNF-α and apoptosis from microglia following the exposure of lipopolysaccharide (LPS) [88]. Other than that, a recent study by Vacca et al. [89] discovered that the oestradiol-treated female mice showed decreased number of astrocyte after the induction of nerve injury, explaining the possible significant role of hormones in the development of pain responses. However, there is very limited report regarding the differences in P2X4R activation across the gender in RA, in which, need to be discovered.

However, the recent researches by Sorge et al. [90] and Mapplebeck et al. [91] have revealed the major differences in gender in regards to the microglial and P2X4R signalling of chronic neuropathic pain. These novel discoveries justify that the P2X4R signalling in microglia is not the underlying key leading to chronic pain hypersensitivity in the females. In contrast, the specific upregulation of P2X4R is consistently found in male variant across the species and pain models. In the mice model of peripheral nerve injury (PNI), Sorge et al. [90] reported that the P2X4 transcript was increased in male but not in the female mice as shown in the quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) analysis, although both gender showed the similar levels of allodynia. Furthermore, the similar findings have been shown by Mapplebeck et al. [91] in the similar neuropathic pain model that the P2X4R from the fluorescent-activated cell sorting-isolated CD11b + (microglia) cells in the spinal cord was upregulated in males but not female rat. In the same study, the authors also demonstrated that the stimulation of ATP alone contributes to the increase of P2X4R level in the microglia primary cell culture of the male rat following a chronic pain-inducing PNI, but not in the female rat microglia cell culture. These novel findings showed that the P2X4R is increased merely in the males following the chronic neuropathic pain, therefore justifies the sex differences in the activation of microglia-P2X4R signalling. It is possible that these findings are associated with the higher affinity of interferon regulatory factor-5 (IRF5) binding to the P2X4 promoter region in male compared to female-derived primary microglia [91]. Moreover, it is possible that the hormones may influence the microglia-P2X4R signalling since the injection of P2X4R antagonist to the testosterone-treated female mice following PNI results in the reduced allodynia compared to the castrated male mice [90]. As the dimorphism is shown in the activation of P2X4R across the sexes, it is possible that the mechanism of P2X4R during the pathogenesis of RA also differ between the gender, which needs further investigations.

Potent P2X4 receptor inhibitors

In the treatment of RA-related pain, there have only been a limited number of drugs tested to demonstrate antagonism towards P2X4R. Several studies identified potential agents to block P2X4R in the neuropathic pain models. However, there is a possibility that tested drugs could be beneficial in the treatment of RA pain. The use of a general broad-range purinergic or general P2X receptor antagonist in several diseases produced unsatisfactory outcomes. In an in vitro study, pyridoxal phosphate-6-azo (benzene-2,4-disulfonic acid (PPADS)), a general non-selective P2 receptor antagonist [83, 92], demonstrated no mark effect on basal adenosine diphosphate (ADP)-directed and morphine-induced migration of microglia [47]. Furthermore, in an in vitro study using HEK293 cells transfected with the P2X4R, the responses of this receptor to ATP were found to be insensitive to the blockade of PPADS although being administered at a very high concentration [93]. Meanwhile, TNP-ATP has been shown to produce beneficial effects as a P2X4R antagonist [47]. Nevertheless, this compound implicates other types of P2X receptors with high potency [94]. It is suggested that the use of drugs specifically targeting P2X4R is best applied for the treatment of related diseases.

Interestingly, certain drugs that strongly antagonise P2X4R and prevent Ca2+ influx such as paroxetine, an analogue of monoamine uptake blockers, also produce potent antidepressant effects [95]. In a rat model for chronic constriction injury (CCI), paroxetine showed potent inhibitory effects against P2X4R and attenuated allodynia and thermal hyperalgesia [96]. A phenoxazine derivative, PSB-12602, inhibits P2X4R via an allosteric mechanism [97]. Other than that, 1-(2,6-dibromo-4-isopropyl-phenyl)-3-(3-pyridyl) urea or BX430 has recently been identified as possessing strong antagonist properties against human P2X4R-mediated Ca2+ uptake [98]. However, these drugs have yet to be tested on animal models.

Several studies have discovered 5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e]-1,4-diazepin-2-one (5-BDBD) to have strong anti-P2X4R activity in various animal models, such as chronic migraine [99], inflamed airways [42] and central sensitisation [94]. This drug specifically discriminates against P2X4R and exerts no effects on other P2X receptors when co-expressed in the same tissue [94]. Coddou and colleagues [94] revealed that 5-BDBD inhibited LTP via dissociation of PSD-95 multi-protein complex interactions that further downregulate NMDAR activity. However, this drug dissolves poorly in water and requires the best medium to dissolve in order to produce a good P2X4R antagonistic effect.

Increasing attention has been given to (5-[3-(5-thioxo-4H-[1,2,4]oxadiazol-3-yl)phenyl]-1H-naphtho[1, 2-b][1,4]diazepine-2,4(3H,5H)-dione), also known as NP-1815-PX, which has recently discovered as a novel P2X4R antagonist. In a murine model for herpetic pain, the selective antagonistic effect of NP-1815-PX against P2X4R alleviates allodynia without implicating acute nociceptive pain and motor function. However, it was found to be effective when administered intrathecally but produced no significant effects when given orally [22]. Another drug, PSB-12054, inhibits P2X4R via allosteric mechanisms, with a 30-fold selectivity towards P2X4R compared to P2X1 receptor and a 50-fold selectivity compared to other types of P2X receptors [14].

In an in vitro study using human monocyte and macrophage cell cultures, PSB-12062 strongly attenuated thapsigargin-resistant components [100]. It positively modulated the ATP-mediated activation of transforming growth factor β2 (TGF-β2) gene expression while attenuating the activation of ATP-mediated C-X-C motif chemokine-5 (CXCL5) gene expression [100]. Another novel P2X4R antagonist, PSB-15417, also showed strong attenuation of mechanical allodynia in a diabetic neuropathy rat model and inhibited P2X4R activation that was mainly expressed on satellite glial cells of DRG neurons [57].

Tricarbonyldichlororuthenium (II) dimer (CORM-2) has been identified as a potent P2X4R antagonist in a study by Jurga et al. [101] that attenuated hyperalgesia and boosted the antihyperalgesic and antiallodynic effects of morphine and buprenorphine in a CCI rat model. It was further discovered that antagonising P2X4R by CORM-2 suppressed microglial and astrocyte activations decreased the expressions of matrix metallopeptidase 9 (MMP-9), IL-1β, IL-18 and IL-6 and reduced nociceptive responses, possibly through the inhibition of p38 MAPK, ERK 1/2 and PI3K/Akt signalling pathways [102]. However, there was a contradiction by Hervera et al. [103] asserting that P2X4R was potentially not the main target of CORM-2, as the modulation of HO-1/CO pathways by CORM-2 was implicated by the inhibition of NO produced by the NOS1 expression from activated neurons. A small number of novel P2X4R antagonists have been tested on chronic pain associated with RA, and the potential drugs available need to be tested to assess their efficiency in managing RA-related pain.

Conclusion and future research

Flare pain in RA is not solely caused joint inflammatory reactions, but also through several possible mechanisms resulting from other abnormalities. There is currently no optimum treatment method available to manage the chronic pain associated with RA. The discovery of P2X4R has provided a new avenue for understanding the molecular mechanisms underlying chronic pain associated with RA and other related diseases. The understanding of the role of P2X4R activation in the pathophysiology of chronic pain related to RA is still at an early stage. However, a collection of literature currently available provides hope that the flaring pain experienced by RA patients can be minimised when P2X4R is attenuated. Hence, identifying and exploring the relevant pathological mechanisms involving P2X4R in RA could be a novel approach to manage pain resulting from RA.