RESUMO
OBJECTIVE: To investigate the mechanisms behind the effects of acupuncture in Traditional Chinese Medicine, we delved into the adenosine triphosphate/peripheral purinergic P2X receptor 3 (ATP/P2X3) receptor signaling system as an indicator of the body's energy state, commonly referred to as "Qi". METHODS: The tail-flick test was utilized to explore the impact of acupuncture on pain tolerance threshold (PTT) in mice, while also assessing adenosine (ADO) levels and adenylate energy charge (EC) at Zusanli (ST36). The study further investigated the dose-dependent effects of acupuncture on PTT and ADO levels at Zusanli (ST36). To shed light on the underlying mechanisms of acupuncture's effects, the study examined the impact of ATP, a P2X3 receptor antagonist, and adenosine disodium on PTT following acupuncture administration. RESULTS: Acupuncture at Zusanli (ST36) led to significant improvements in PTT in mice, with the most effective interventions being twirling for 2 min and needle retention for 28 min. These interventions also resulted in significant increases in ATP levels. The effects of acupuncture were further augmented by administration of different doses of ATP at Zusanli (ST36), and pretreatment with a P2X3 receptor antagonist decreased PTT. Adenylate EC peaked at 30 min after intraperitoneal injection of ATP, and pretreatment with various doses of i.p. ATP 30 min prior to acupuncture increased PTT in a dose-dependent manner. Additionally, pretreatment with an i.p. or intramuscular injection of adenosine disodium enhanced the effects of acupuncture. CONCLUSION: This research provides compelling evidence that ATP is involved in the regulation of PTT through acupuncture, revealing new avenues for achieving enhanced clinical outcomes.
Assuntos
Pontos de Acupuntura , Terapia por Acupuntura , Trifosfato de Adenosina , Animais , Trifosfato de Adenosina/metabolismo , Camundongos , Masculino , Humanos , Limiar da Dor/efeitos dos fármacos , Receptores Purinérgicos P2X3/metabolismo , Receptores Purinérgicos P2X3/genéticaRESUMO
We previously reported the presence of P2X3 purinoceptors (P2X3)-expressing subserosal afferent nerve endings consisting of net- and basket-like nerve endings in the rat gastric antrum. These nerve endings may morphologically be vagal mechanoreceptors activated by antral peristalsis. The present study investigated immunoreactivities for vesicular glutamate transporter (VGLUT) 1 and VGLUT2 as well as exocytosis-related proteins, i.e., core components of the SNARE complex (SNAP25, Stx1, and VAMP2) and synaptotagmin-1 (Syt1), in whole-mount preparations of the rat gastric antrum using double immunofluorescence. VGLUT1 immunoreactivity was not detected, whereas VGLUT2 immunoreactivity was observed in P2X3-immunoreactive subserosal nerve endings composed of both net- and basket-like endings. In net-like nerve endings, intense VGLUT2 immunoreactivity was localized in polygonal bulges of reticular nerve fibers and peripheral axon terminals. Furthermore, intense immunoreactivities for SNAP25, Stx1, and VAMP2 were localized in net-like nerve endings. Intense immunoreactivities for VAMP2 and Syt1 were observed in VGLUT2-immunoreactive net-like nerve endings. In basket-like nerve endings, VGLUT2 immunoreactivity was localized in pleomorphic terminal structures and small bulges surrounding the subserosal ganglion, whereas immunoreactivities for SNAP25, Stx1, and VAMP2 were weak in these nerve endings. VGLUT2-immunoreactive basket-like nerve endings were weakly immunoreactive for VAMP2 and Syt1. These results suggest that subserosal afferent nerve endings release glutamate by exocytosis mainly from net-like nerve endings to modulate their mechanoreceptor function.
Assuntos
Exocitose , Ácido Glutâmico , Terminações Nervosas , Antro Pilórico , Receptores Purinérgicos P2X3 , Proteína Vesicular 2 de Transporte de Glutamato , Animais , Masculino , Ratos , Ácido Glutâmico/metabolismo , Imuno-Histoquímica , Terminações Nervosas/metabolismo , Antro Pilórico/inervação , Antro Pilórico/metabolismo , Ratos Wistar , Receptores Purinérgicos P2X3/metabolismo , Proteína 25 Associada a Sinaptossoma/metabolismo , Sinaptotagmina I/metabolismo , Sintaxina 1/metabolismo , Proteína 2 Associada à Membrana da Vesícula/metabolismo , Proteína Vesicular 1 de Transporte de Glutamato/metabolismo , Proteína Vesicular 2 de Transporte de Glutamato/metabolismoRESUMO
AIMS: We report on investigations exploring the P2X3-receptor antagonist filapixant's effect on taste perception and cough-reflex sensitivity and describe its pharmacokinetics, including its CYP3A4-interaction potential. METHODS: In a randomized, placebo-controlled, double-blind study, 3 × 12 healthy men (18-45 years) were assigned (3:1) to filapixant (20, 80 or 250 mg by mouth) or placebo twice daily over 2 weeks. A single dose of midazolam (1 mg), a CYP3A4 substrate, was administered with and without filapixant. Assessments included a taste-strips test, a taste questionnaire, cough challenge with adenosine triphosphate, adverse event reports and standard safety assessments. RESULTS: Taste disturbances were observed mainly in the 250-mg group: six of nine participants (67%) in this group reported hypo- or dysgeusia in the questionnaire; eight participants (89%) reported taste-related adverse events. Five participants (56%) had a decrease in overall taste-strips-test scores ≥2 points (point estimate -1.1 points, 90% confidence interval [-3.3; 1.1]). Cough counts increased with adenosine triphosphate concentration but without major differences between treatments. Filapixant exposure increased proportionally to dose. Co-administration of filapixant had no clinically relevant effect on midazolam pharmacokinetics. Area under the concentration-time curve ratios and 90% confidence intervals were within 80-125%. No serious or severe adverse events were reported. CONCLUSIONS: Overall, filapixant was safe and well tolerated, apart from mild, transient taste disturbances. Such disturbances occurred more frequently than expected based on (in vitro) receptor-selectivity data, suggesting that other factors than P2X3:P2X2/3 selectivity might also play an important role in this context. The cough-challenge test showed no clear treatment effect. Filapixant has no clinically relevant CYP3A4 interaction potential.
Assuntos
Citocromo P-450 CYP3A , Relação Dose-Resposta a Droga , Interações Medicamentosas , Midazolam , Antagonistas do Receptor Purinérgico P2X , Humanos , Masculino , Adulto , Citocromo P-450 CYP3A/metabolismo , Antagonistas do Receptor Purinérgico P2X/administração & dosagem , Antagonistas do Receptor Purinérgico P2X/farmacocinética , Antagonistas do Receptor Purinérgico P2X/efeitos adversos , Antagonistas do Receptor Purinérgico P2X/farmacologia , Método Duplo-Cego , Adulto Jovem , Midazolam/farmacocinética , Midazolam/administração & dosagem , Midazolam/efeitos adversos , Adolescente , Voluntários Saudáveis , Pessoa de Meia-Idade , Tosse/induzido quimicamente , Paladar/efeitos dos fármacos , Receptores Purinérgicos P2X3/efeitos dos fármacos , Receptores Purinérgicos P2X3/metabolismoRESUMO
BACKGROUND AND PURPOSE: Many medications taste intensely bitter. The innate aversion to bitterness affects medical compliance, especially in children. There is a clear need to develop bitter blockers to suppress the bitterness of vital medications. Bitter taste is mediated by TAS2R receptors. Because different pharmaceutical compounds activate distinct sets of TAS2Rs, targeting specific receptors may only suppress bitterness for certain, but not all, bitter-tasting compounds. Alternative strategies are needed to identify universal bitter blockers that will improve the acceptance of every medication. Taste cells in the mouth transmit signals to afferent gustatory nerve fibres through the release of ATP, which activates the gustatory nerve-expressed purine receptors P2X2/P2X3. We hypothesized that blocking gustatory nerve transmission with P2X2/P2X3 inhibitors (e.g. 5-(5-iodo-4-methoxy-2-propan-2-ylphenoxy)pyrimidine-2,4-diamine [AF-353]) would reduce bitterness for all medications and bitter compounds. EXPERIMENTAL APPROACH: Human sensory taste testing and mouse behavioural analyses were performed to determine if oral application of AF-353 blocks perception of bitter taste and other taste qualities but not non-gustatory oral sensations (e.g. tingle). KEY RESULTS: Rinsing the mouth with AF-353 in humans or oral swabbing it in mice suppressed the bitter taste and avoidance behaviours of all compounds tested. We further showed that AF-353 suppressed other taste qualities (i.e. salt, sweet, sour and savoury) but had no effects on other oral or nasal sensations (e.g, astringency and oral tingle). CONCLUSION AND IMPLICATIONS: This is the first time a universal, reversible taste blocker in humans has been reported. Topical application of P2X2/P2X3 inhibitor to suppress bitterness may improve medical compliance.
Assuntos
Antagonistas do Receptor Purinérgico P2X , Receptores Purinérgicos P2X3 , Paladar , Humanos , Paladar/efeitos dos fármacos , Animais , Receptores Purinérgicos P2X3/metabolismo , Masculino , Feminino , Antagonistas do Receptor Purinérgico P2X/farmacologia , Antagonistas do Receptor Purinérgico P2X/administração & dosagem , Adulto , Camundongos , Receptores Purinérgicos P2X2/metabolismo , Administração Tópica , Adulto Jovem , Pirimidinas/farmacologia , Pirimidinas/administração & dosagem , Camundongos Endogâmicos C57BLRESUMO
Migraine, a debilitating neurological condition, significantly affects patients' quality of life. Fenofibrate, a peroxisome proliferator-activated receptor alpha (PPAR-α) agonist approved for managing dyslipidemia, has shown promise in treating neurological disorders. Therefore, this study aims to investigate the protective effects of fenofibrate against nitroglycerin (NTG)-induced chronic migraine in rats. Migraine was induced in rats by administering five intermittent doses of NTG (10 mg/kg, i. p.) on days 1, 3, 5, 7, and 9. Rats were treated with either topiramate (80 mg/kg/day, p. o.), a standard drug, or fenofibrate (100 mg/kg/day, p. o.) from day 1-10. Fenofibrate significantly improved mechanical and thermal hypersensitivity, photophobia, and head grooming compared to topiramate. These effects were associated with reduced serum levels of nitric oxide (NO), calcitonin gene-related peptide (CGRP), and pituitary adenylate cyclase-activating polypeptide (PACAP). Furthermore, fenofibrate down-regulated c-Fos expression in the medulla and medullary pro-inflammatory cytokine contents. Additionally, fenofibrate attenuated NTG-induced histopathological changes in the trigeminal ganglia and trigeminal nucleus caudalis. These effects were associated with the inhibition of CGRP/p-CREB/purinergic 2X receptor 3 (P2X3) and nerve growth factor (NGF)/protein kinase C (PKC)/acid-sensing ion channel 3 (ASIC3) signaling pathways. This study demonstrates that fenofibrate attenuated NTG-induced migraine-like signs in rats. These effects were partially mediated through the inhibition of CGRP/p-CREB/P2X3 and NGF/PKC/ASIC3 signaling pathways. The present study supports the idea that fenofibrate could be an effective candidate for treating migraine headache without significant adverse effects. Future studies should explore its clinical applicability.
Assuntos
Peptídeo Relacionado com Gene de Calcitonina , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico , Fenofibrato , Transtornos de Enxaqueca , Fator de Crescimento Neural , Nitroglicerina , Proteína Quinase C , Receptores Purinérgicos P2X3 , Transdução de Sinais , Animais , Nitroglicerina/farmacologia , Nitroglicerina/toxicidade , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transtornos de Enxaqueca/tratamento farmacológico , Transtornos de Enxaqueca/induzido quimicamente , Transtornos de Enxaqueca/metabolismo , Masculino , Fenofibrato/farmacologia , Fenofibrato/uso terapêutico , Ratos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteína Quinase C/metabolismo , Receptores Purinérgicos P2X3/metabolismo , Fator de Crescimento Neural/metabolismo , Óxido Nítrico/metabolismo , Ratos Sprague-Dawley , Comportamento Animal/efeitos dos fármacosRESUMO
Toothache is one of the most common types of pain, but the mechanisms underlying pulpitis-induced pain remain unknown. The ionotropic purinergic receptor family (P2X) is reported to mediate nociception in the nervous system. This study aims to investigate the involvement of P2X3 in the sensitisation of the trigeminal ganglion (TG) and the inflammation caused by acute pulpitis. An acute tooth inflammation model was established by applying LPS to the pulp of SD rats. We found that the increased expression of P2X3 was induced by acute pulpitis. A selective P2X3 inhibitor (A-317491) reduced pain-like behavior in the maxillofacial region of rats and depressed the activation of neurons in the trigeminal ganglion induced by pulpitis. The upregulated MAPK signaling (p-p38, p-ERK1/2) expression in the ipsilateral TG induced by pulpitis could also be depressed by the application of the P2X3 inhibitor. Furthermore, the expression of markers of inflammatory processes, such as NF-κB, TNF-α and IL-1ß, could be induced by acute pulpitis and deduced by the intraperitoneal injection of P2X3 antagonists. Our findings demonstrate that purinergic P2X3 receptor signaling in TG neurons contributes to pulpitis-induced pain in rats and that P2X3 signaling may be a potential therapeutic target for tooth pain.
Assuntos
Pulpite , Ratos , Animais , Pulpite/metabolismo , NF-kappa B/metabolismo , Ratos Sprague-Dawley , Dor/metabolismo , Transdução de Sinais , Inflamação/complicações , Inflamação/metabolismo , Receptores Purinérgicos P2X3/metabolismo , Gânglio Trigeminal/metabolismoRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: PolyphyllinVI (PPâ ¥) is the main bioactive component of Chonglou which is a traditional Chinese herbal with various effects, including antitumor, anti-inflammatory, and analgesia. AIM OF THE STUDY: This study aimed to investigate the properties and mechanisms of the analgesia of PPâ ¥ by using neuropathic pain (NPP) mice. MATERIALS AND METHODS: The potential targets and mechanisms of PPâ ¥ in alleviating NPP were excavated based on the network pharmacology. Subsequently, the construction of a spared nerve injury (SNI) mice model was used to evaluate the effect of PPâ ¥ on NPP and the expression of the P2X3 receptor. We identified the signaling pathways of PPâ ¥ analgesia by RNA sequencing. RESULTS: The results of network pharmacology showed that BCL2, CASP3, JUN, STAT3, and TNF were the key targets of the analgesic effect of PPâ ¥. PPâ ¥ increased the MWT and TWL of SNI mice and decreased the level of P2X3 receptors in the dorsal root ganglion (DRG) and spinal cord (SC). Additionally, PPâ ¥ reduced the release of pro-inflammatory mediators (TNF-α, IL-1ß, and IL-6) in the DRG, SC, and serum. Based on the KEGG enrichment of differentially expressed genes (DEGs) identified by RNA-Seq, PPVI may relieve NPP by regulating the AMPK/NF-κB signaling pathway. Western blotting results showed that the AMPK signaling pathway was activated, followed by inhibition of the NF-κB signaling pathway. CONCLUSION: PPâ ¥ increased the MWT and TWL of SNI mice maybe by inhibiting the expression of the P2X3 receptor and the release of inflammatory mediators. The properties of the analgesia of PPâ ¥ may be based on the AMPK/NF-κB pathway.
Assuntos
Neuralgia , Receptores Purinérgicos P2X3 , Ratos , Camundongos , Animais , Ratos Sprague-Dawley , Receptores Purinérgicos P2X3/metabolismo , NF-kappa B/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Neuralgia/metabolismo , Gânglios EspinaisRESUMO
Hindlimb ischaemia-reperfusion (IR) is among the most prominent pathophysiological conditions observed in peripheral artery disease (PAD). An exaggerated arterial blood pressure (BP) response during exercise is associated with an elevated risk of cardiovascular events in individuals with PAD. However, the precise mechanisms leading to this exaggerated BP response are poorly elucidated. The P2X3 signalling pathway, which plays a key role in modifying the exercise pressor reflex (EPR), is the focus of the present study. We determined the regulatory role of P2X3 on the EPR in a rat model of hindlimb IR. In vivo and in vitro approaches were used to determine the expression and functions of P2X3 in muscle afferent nerves and EPR in IR rats. We found that in IR rats there was (1) upregulation of P2X3 protein expression in the L4-6 dorsal root ganglia (DRG); (2) amplified P2X currents in isolated isolectin B4 (IB4)-positive muscle DRG neurons; and (3) amplification of the P2X-mediated BP response. We further verified that both A-317491 and siRNA knockdown of P2X3 significantly decreased the activity of P2X currents in isolated muscle DRG neurons. Moreover, inhibition of muscle afferents' P2X3 receptor using A-317491 was observed to alleviate the exaggerated BP response induced by static muscle contraction and P2X-induced BP response by α,ß-methylene ATP injection. P2X3 signalling pathway activity is amplified in muscle afferent DRG neurons in regulating the EPR following hindlimb IR.
Assuntos
Gânglios Espinais , Neurônios Aferentes , Fenóis , Compostos Policíclicos , Ratos , Animais , Gânglios Espinais/metabolismo , Ratos Sprague-Dawley , Neurônios Aferentes/fisiologia , Reflexo , Neurônios/metabolismo , Músculo Esquelético/metabolismo , Isquemia/metabolismo , Membro Posterior/metabolismo , Receptores Purinérgicos P2X3/metabolismoRESUMO
In the carotid body of laboratory rodents, adenosine 5'-triphosphate (ATP)-mediated transmission is regarded as critical for transmission from chemoreceptor type I cells to P2X3 purinoceptor-expressing sensory nerve endings. The present study investigated the distribution of P2X3-immunoreactive sensory nerve endings in the carotid body of the adult male Japanese monkey (Macaca fuscata) using multilabeling immunofluorescence. Immunoreactivity for P2X3 was detected in nerve endings associated with chemoreceptor type I cells immunoreactive for synaptophysin. Spherical or flattened terminal parts of P2X3-immunoreactive nerve endings were in close apposition to the perinuclear cytoplasm of synaptophysin-immunoreactive type I cells. Immunoreactivity for ectonucleoside triphosphate diphosphohydrolase 2 (NTPDase2), which hydrolyzes extracellular ATP, was localized in the cell body and cytoplasmic processes of S100B-immunoreactive cells. NTPDase2-immunoreactive cells surrounded P2X3-immunoreactive terminal parts and synaptophysin-immunoreactive type I cells, but did not intrude into attachment surfaces between terminal parts and type I cells. These results suggest ATP-mediated transmission between type I cells and sensory nerve endings in the carotid body of the Japanese monkey, as well as those of rodents.
Assuntos
Corpo Carotídeo , Ratos , Animais , Masculino , Corpo Carotídeo/metabolismo , Macaca fuscata/metabolismo , Receptores Purinérgicos P2X3/metabolismo , Sinaptofisina/metabolismo , Ratos Wistar , Células Receptoras Sensoriais/metabolismo , Trifosfato de Adenosina/metabolismoRESUMO
The role of heat shock protein 27 (HSP27), a chaperone, in neuropathic pain after nerve injury has not been systematically surveyed despite its neuroprotective and regeneration-promoting effects. In this study, we found that HSP27 expression in sensory neurons of the dorsal root ganglia (DRG) mediated nerve injury-induced neuropathic pain. Neuropathic pain behaviors were alleviated by silencing HSP27 in the DRG of a rat spinal nerve ligation (SNL) model. Local injection of an HSP27-overexpression construct into the DRG of naïve rats elicited neuropathic pain behaviors. HSP27 interacted with a purinergic receptor, P2X3, and their expression patterns corroborated the induction and reversal of neuropathic pain according to two lines of evidence: colocalization immunohistochemically and immunoprecipitation biochemically. In a cell model cotransfected with HSP27 and P2X3, the degradation rate of P2X3 was reduced in the presence of HSP27. Such an alteration was mediated by reducing P2X3 ubiquitination in SNL rats and was reversed after silencing HSP27 in the DRGs of SNL rats. In summary, the interaction of HSP27 with P2X3 provides a new mechanism of injury-induced neuropathic pain that could serve as an alternative therapeutic target.
Assuntos
Proteínas de Choque Térmico HSP27 , Neuralgia , Animais , Ratos , Gânglios Espinais/metabolismo , Proteínas de Choque Térmico HSP27/metabolismo , Hiperalgesia/metabolismo , Neuralgia/metabolismo , Ratos Sprague-Dawley , Nervos Espinhais/metabolismo , Receptores Purinérgicos P2X3/metabolismoRESUMO
Heart failure is associated with multiple mechanisms, including sympatho-excitation, and is one of the leading causes of death worldwide. Enhanced carotid body chemoreflex function is strongly related to excessive sympathetic nerve activity and sleep-disordered breathing in heart failure. How to reduce the excitability of the carotid body is still scientifically challenging. Both clinical and experimental evidence have suggested that targeting purinergic receptors is of great potential to combat heart failure. In a recent study, Lataro et al. (Lataro et al. in Nat Commun 14:1725, 5) demonstrated that targeting purinergic P2X3 receptors in the carotid body attenuates the progression of heart failure. Using a series of molecular, biochemical, and functional assays, the authors observed that the carotid body generates spontaneous, episodic burst discharges coincident with the onset of disordered breathing in male rats with heart failure, which was generated by ligating the left anterior descending coronary artery. Moreover, P2X3 receptor expression was found to be upregulated in the petrosal ganglion chemoreceptive neurons of rats with heart failure. Of particular note, treatment with a P2X3 antagonist rescued pathological breathing disturbances, abolished episodic discharges, reinstated autonomic balance, attenuated cardiac dysfunction, and reduced the immune cell response and plasma cytokine levels in those rats.
Assuntos
Corpo Carotídeo , Insuficiência Cardíaca , Ratos , Masculino , Animais , Corpo Carotídeo/metabolismo , Receptores Purinérgicos P2X/metabolismo , Insuficiência Cardíaca/metabolismo , Neurônios/metabolismo , Sistema Nervoso Simpático , Receptores Purinérgicos P2X3/metabolismo , Receptores Purinérgicos P2X2/metabolismoRESUMO
P2X3 receptors and group II metabotropic glutamate receptors (mGluRs) have been found to be expressed in primary sensory neurons. P2X3 receptors participate in a variety of pain processes, while the activation of mGluRs has an analgesic effect. However, it's still unclear whether there is a link between them in pain. Herein, we reported that the group II mGluR activation inhibited the electrophysiological activity of P2X3 receptors in rat dorsal root ganglia (DRG) neurons. Group II mGluR agonist LY354740 concentration-dependently decreased P2X3 receptor-mediated and α,ß-methylene-ATP (α,ß-meATP)-evoked inward currents in DRG neurons. LY354740 significantly suppressed the maximum response of P2X3 receptor to α,ß-meATP, but did not change their affinity. Inhibition of ATP currents by LY354740 was blocked by the group II mGluR antagonist LY341495, also prevented by the intracellular dialysis of either the Gi/o protein inhibitor pertussis toxin, the cAMP analog 8-Br-cAMP, or the protein kinase A (PKA) inhibitor H-89. Moreover, LY354740 decreased α,ß-meATP-induced membrane potential depolarization and action potential bursts in DRG neurons. Finally, intraplantar injection of LY354740 also relieved α,ß-meATP-induced spontaneous nociceptive behaviors and mechanical allodynia in rats by activating peripheral group â ¡ mGluRs. These results indicated that peripheral group II mGluR activation inhibited the functional activity of P2X3 receptors via a Gi/o protein and cAMP/PKA signaling pathway in rat DRG neurons, which revealed a novel mechanism underlying analgesic effects of peripheral group II mGluRs. This article is part of the Special Issue on "Purinergic Signaling: 50 years".
Assuntos
Receptores de Glutamato Metabotrópico , Ratos , Animais , Receptores de Glutamato Metabotrópico/metabolismo , Gânglios Espinais/metabolismo , Receptores Purinérgicos P2X3/metabolismo , Dor/metabolismo , Neurônios , Trifosfato de Adenosina/metabolismo , Analgésicos/farmacologiaRESUMO
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,ß-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,ß-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,ß-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.
Assuntos
Neoplasias Ósseas , Dor do Câncer , Eletroacupuntura , Ratos , Animais , Hiperalgesia/metabolismo , Dor do Câncer/metabolismo , Receptores Purinérgicos P2X3/metabolismo , Ratos Sprague-Dawley , Eletroacupuntura/métodos , Dor/metabolismo , Neoplasias Ósseas/metabolismo , Analgésicos , Gânglios Espinais/metabolismoRESUMO
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.
Assuntos
Diabetes Mellitus , Neuropatias Diabéticas , Eletroacupuntura , Ratos , Animais , Hiperalgesia/metabolismo , Regulação para Baixo , Gânglios Espinais/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Neuropatias Diabéticas/metabolismo , Receptores Purinérgicos P2X3/metabolismo , Diabetes Mellitus/metabolismoRESUMO
Diabetic neuropathic pain (DNP) is frequent among patients with diabetes. We previously showed that P2X3 upregulation in dorsal root ganglia (DRG) plays a role in streptozotocin (STZ)-induced DNP but the underlying mechanism is unclear. Here, a rat model of DNP was established by a single injection of STZ (65 mg/kg). Fasting blood glucose was significantly elevated from the 1st to 3rd week. Paw withdrawal thresholds (PWTs) and paw withdrawal latencies (PWLs) in diabetic rats significantly reduced from the 2nd to 3rd week. Western blot analysis revealed that elevated p-CaMKIIα levels in the DRG of DNP rats were accompanied by pain-associated behaviors while CaMKIIα levels were unchanged. Immunofluorescence revealed significant increase in the proportion of p-CaMKIIα immune positive DRG neurons (stained with NeuN) in the 2nd and 3rd week and p-CaMKIIα was co-expressed with P2X3 in DNP rats. KN93, a CaMKII antagonist, significantly reduce mechanical hyperalgesia and thermal hyperalgesia and these effects varied dose-dependently, and suppressed p-CaMKIIα and P2X3 upregulation in the DRGs of DNP rats. These results revealed that the p-CaMKIIα upregulation in DRG is involved in DNP, which possibly mediated P2X3 upregulation, indicating CaMKIIα may be an effective pharmacological target for DNP management.
Assuntos
Diabetes Mellitus Experimental , Neuropatias Diabéticas , Neuralgia , Ratos , Animais , Ratos Sprague-Dawley , Diabetes Mellitus Experimental/metabolismo , Cálcio/metabolismo , Estreptozocina/metabolismo , Estreptozocina/farmacologia , Receptores Purinérgicos P2X3/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/farmacologia , Gânglios Espinais/metabolismo , Neuralgia/metabolismo , Hiperalgesia/metabolismo , Neuropatias Diabéticas/metabolismoRESUMO
OBJECTIVE: To observe the effect of electroacupuncture (EA) at "Ciliao" (BL 32) and "Huiyang" (BL 35) on the pain, urodynamic and the expressions of transient receptor poteintial vanilloid 1 (TRPV1) and P2X3 receptors in bladder of rats with interstitial bladder (IC), and to explore the possible mechanism on EA for IC. METHODS: A total of 24 Wistar female rats were randomly divided into a blank group, a model group and an EA group, 8 rats in each group. In the model group and the EA group, IC model was established by intraperitoneal injection of cyclophosphamide by 150 mg/kg at once. EA was applied at "Ciliao" (BL 32) and "Huiyang" (BL 35) in the EA group for 20 min, with continuous wave, 30 Hz in frequency, once a day for 3 consecutive days. Mechanical pain threshold of bladder and urodynamic indexes (first urination time, bladder effective volume and urination pressure) were observed after model establishment and after intervention, the expressions of TRPV1 and P2X3 receptors in the bladder were detected by Western blot. RESULTS: After model establishment, the mechanical pain threshold of bladder was decreased in the model group and the EA group compared with that in the blank group (P<0.01). After intervention, the mechanical pain threshold of bladder in the model group was lower than the blank group (P<0.01), and that in the EA group was higher than the model group (P<0.01). The urodynamic of the rats in the blank group was normal, obvious abnormal contraction during the filling period of bladder was found in the rats of the model group, while no abnormal contraction during the filling period was found in the rats of the EA group. After model establishment, in the model group and the EA group, the first urination time was earlier than the blank group (P<0.01), while bladder effective volume and urination pressure were lower than the blank group (P<0.01). After intervention, in the model group, the first urination time was earlier than the blank group (P<0.01), while bladder effective volume and urination pressure were lower than the blank group (P<0.05); in the EA group, the first urination time was later than the model group (P<0.05), while bladder effective volume and urination pressure were higher than the model group (P<0.05). Compared with the blank group, the protein expressions of TRPV1 and P2X3 receptors in bladder were up-regulated in the model group (P<0.01); compared with the model group, the protein expressions of TRPV1 and P2X3 receptors in bladder were down-regulated in the EA group (P<0.05). CONCLUSION: EA can relieve bladder pain and improve urodynamic in IC rats. The mechanism may be related to the down-regulation on the expressions of TRPV1 and P2X3 receptors and the further inhibition on the abnormal input of bladder signal.
Assuntos
Antineoplásicos , Cistite Intersticial , Eletroacupuntura , Ratos , Feminino , Animais , Cistite Intersticial/genética , Cistite Intersticial/terapia , Bexiga Urinária , Receptores Purinérgicos P2X3/genética , Receptores Purinérgicos P2X3/metabolismo , Ratos Sprague-Dawley , Ratos Wistar , Dor , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismoRESUMO
Purinergic signaling is involved in multiple pain processes. P2X3 receptor is a key target in pain therapeutics, while A1 adenosine receptor signaling plays a role in analgesia. However, it remains unclear whether there is a link between them in pain. The present results showed that the A1 adenosine receptor agonist N6-cyclopentyladenosine (CPA) concentration dependently suppressed P2X3 receptor-mediated and α,ß-methylene-ATP (α,ß-meATP)-evoked inward currents in rat dorsal root ganglion (DRG) neurons. CPA significantly decreased the maximal current response to α,ß-meATP, as shown a downward shift of the concentration-response curve for α,ß-meATP. CPA suppressed ATP currents in a voltage-independent manner. Inhibition of ATP currents by CPA was completely prevented by the A1 adenosine receptor antagonist KW-3902, and disappeared after the intracellular dialysis of either the Gi/o protein inhibitor pertussis toxin, the adenylate cyclase activator forskolin, or the cAMP analog 8-Br-cAMP. Moreover, CPA suppressed the membrane potential depolarization and action potential bursts, which were induced by α,ß-meATP in DRG neurons. Finally, CPA relieved α,ß-meATP-induced nociceptive behaviors in rats by activating peripheral A1 adenosine receptors. These results indicated that CPA inhibited the activity of P2X3 receptors in rat primary sensory neurons by activating A1 adenosine receptors and its downstream cAMP signaling pathway, revealing a novel peripheral mechanism underlying its analgesic effect.
Assuntos
Gânglios Espinais , Receptores Purinérgicos P2X3 , Adenosina/metabolismo , Adenosina/farmacologia , Trifosfato de Adenosina/metabolismo , Adenilil Ciclases/metabolismo , Analgésicos/farmacologia , Animais , Colforsina/farmacologia , Gânglios Espinais/metabolismo , Neurônios/metabolismo , Dor/metabolismo , Toxina Pertussis/metabolismo , Toxina Pertussis/farmacologia , Agonistas do Receptor Purinérgico P1/metabolismo , Agonistas do Receptor Purinérgico P1/farmacologia , Antagonistas de Receptores Purinérgicos P1/farmacologia , Ratos , Receptores Purinérgicos P1/metabolismo , Receptores Purinérgicos P2X3/metabolismoRESUMO
Drug discovery programs targeting P2X3 receptors (P2X3R), an extracellular adenosine 5'-triphosphate (ATP) gated cation channel family, have been actively investigated for several CNS-related diseases. The current unmet need in the field of P2X3R targeted drugs is to avoid a side effect, the loss of taste, that could be reduced by increase of the P2X3R selectivity vs P2X2/3R. In this study, 5-methyl-1H-benzo[d]imidazole derivatives were designed and synthesized from the analysis of key pharmacophores of current antagonists. In the structure-activity relationship study, the most potent compounds 17a-b was discovered as potent P2X3R antagonists with IC50 values of 145 and 206 nM, and selectivity index of 60 and 41, respectively. In addition, 17a-b showed the not-competitive antagonism, but poor binding score in the docking study at the known allosteric binding site of Gefapixant binding site, indicating that another allosteric binding site might be existing for the novel P2X3R antagonists.
Assuntos
Nitroimidazóis , Antagonistas do Receptor Purinérgico P2X , Inibidores de 14-alfa Desmetilase , Trifosfato de Adenosina/metabolismo , Sítio Alostérico , Antifúngicos , Sítios de Ligação , Inibidores do Citocromo P-450 CYP2C9 , Inibidores do Citocromo P-450 CYP3A , Imidazóis/farmacologia , Antagonistas do Receptor Purinérgico P2X/farmacologia , Receptores Purinérgicos P2X3/metabolismoRESUMO
PURPOSE: Activation of muscarinic receptors located in bladder sensory pathways is generally considered to be the primary contributor for driving the pathogenesis of neurogenic detrusor overactivity following spinal cord injury. The present study is undertaken to examine whether moxibustion improves neurogenic detrusor overactivity via modulating the abnormal muscarinic receptor pathway. MATERIALS AND METHODS: Female Sprague-Dawley rats were subjected to spinal cord injury with T9-10 spinal cord transection. Fourteen days later, animals were received moxibustion treatment for one week. Urodynamic parameters and pelvic afferents discharge were measured. Adenosine triphosphate (ATP) content in the voided cystometry fluid was determined. Expressions of M2, M3, and P2X3 receptors in the bladder mucosa were evaluated. RESULTS: Moxibustion treatment prevented the development of detrusor overactivity in spinal cord injury rats, with an increase in the intercontraction interval and micturition pressure threshold and a decrease in afferent activity during filling. The expression of M2 was markedly suppressed by moxibustion, accompanied by a reduction in the levels of ATP and P2X3. M2 receptor antagonist methoctramine hemihydrate had similar effects to moxibustion on bladder function and afferent activity, while the M2-preferential agonist oxotremorine methiodide abolished the beneficial effects of moxibustion. CONCLUSION: Moxibustion is a potential candidate for treating neurogenic bladder overactivity in a rat model of spinal cord injury, possibly through inhibiting the M2/ATP/P2X3 pathway.
Assuntos
Trifosfato de Adenosina , Moxibustão , Receptor Muscarínico M2 , Traumatismos da Medula Espinal , Bexiga Urinária Hiperativa , Trifosfato de Adenosina/antagonistas & inibidores , Trifosfato de Adenosina/metabolismo , Animais , Diaminas/farmacologia , Feminino , Antagonistas do Receptor Purinérgico P2X/farmacologia , Ratos , Ratos Sprague-Dawley , Receptor Muscarínico M2/antagonistas & inibidores , Receptor Muscarínico M2/metabolismo , Receptores Muscarínicos , Receptores Purinérgicos P2X3/metabolismo , Traumatismos da Medula Espinal/metabolismo , Bexiga Urinaria Neurogênica/tratamento farmacológico , Bexiga Urinaria Neurogênica/metabolismo , Bexiga Urinaria Neurogênica/terapia , Bexiga Urinária Hiperativa/tratamento farmacológico , Bexiga Urinária Hiperativa/metabolismo , Bexiga Urinária Hiperativa/terapiaRESUMO
Neuropathic pain is a common clinical symptom of various diseases, and it seriously affects the physical and mental health of patients. Owing to the complex pathological mechanism of neuropathic pain, clinical treatment of pain is challenging. Therefore, there is growing interest among researchers to explore potential therapeutic strategies for neuropathic pain. A large number of studies have shown that development of neuropathic pain is related to nerve conduction and related signaling molecules. P2X3 receptors (P2X3R) are ATP-dependent ion channels that participate in the transmission of neural information and related signaling pathways, sensitize the central nervous system, and play a key role in the development of neuropathic pain. In this paper, we summarized the structure and biological characteristics of the P2X3R gene and discussed the role of P2X3R in the nervous system. Moreover, we outlined the related pathological mechanisms of pain and described the relationship between P2X3R and chronic pain to provide valuable information for development of novel treatment strategies for pain.