Study of PT1 Peptide Analgesic and Anti-Inflammatory Activity on a Local Inflammation Model in Mice CD-1.

PT1 peptide isolated from the venom of spider Geolycosa sp. is a modulator of P2X3 receptors that play a role in the development of inflammation and the transmission of pain impulses. The anti-inflammatory and analgesic efficacy of the PT1 peptide was studied in a model of complete Freund's adjuvant-induced paw inflammation in CD-1 mice. The analgesic activity of PT1 peptide was maximum after intramuscular injection at a dose of 0.01 mg/kg, which surpassed the analgesic effect of diclofenac at a dose of 1 mg/kg. The anti-inflammatory activity was maximum after intramuscular injection at a dose of 0.0001 mg/kg; a decrease in paw thickness was observed as soon as 2 h after the administration of the PT1 peptide against the background of inflammation development. All tested doses of PT1 peptide showed high anti-inflammatory activity 4 and 24 h after administration. PT1 peptide at a dose of 0.01 mg/kg when injected intramuscularly simultaneously produced high anti-inflammatory and analgesic effects compared to other doses of the peptide. Increasing the dose of PT1 peptide led to a gradual decrease in its analgesic and anti-inflammatory activity; increasing the dose of intramuscular injection to 0.1 and 1 mg/kg is inappropriate.

P2X7 and P2Y1 receptors in DRG mediate electroacupuncture to inhibit peripheral sensitization in rats with IBS visceral pain.

Although multiple purinergic receptors mediate the analgesic effects of acupuncture, it remains unclear whether there is mutual interaction between purinergic receptors to jointly mediate the electroacupuncture inhibition of peripheral sensitization in visceral pain. Visceral hypersensitivity was induced by intracolonic 2,4,6-trinitrobenzene sulfonic acid (TNBS) in rat. The antinociception effect of electroacupuncture on visceral pain was evaluated by morphology, behaviors, neuroelectrophysiology and molecular biology techniques. After labeling the colon-related primary sensory neurons with neural retrograde tracer and employing neuropharmacology, neuroelectrophysiology, and molecular biotechnology, the mechanisms of P2X7R, P2Y1R, and P2X3R in colon-related dorsal root ganglion (DRG) neurons alleviating visceral hypersensitivity of irritable bowel syndrome (IBS) by electroacupuncture at Zusanli and Sanyinjiao acupoints.were elucidated from the perspective of peripheral sensitization. Electroacupuncture significantly inhibited TNBS-induced colonic hypersensitivity in rats with IBS, and Satellite Glial Cells (SGCs) in DRG were found to be involved in electroacupuncture-mediated regulation of the electrophysiological properties of neurons. P2X7R was found to play a pain-inducing role in IBS visceral hypersensitivity by affecting P2X3R, and electroacupuncture exerted an analgesic effect by inhibiting P2X7R activation. P2Y1R was found to play an analgesic role in the process of visceral pain, mediating electroacupuncture to relieve visceral hypersensitivity. P2Y1R relieved visceral pain by inhibiting P2X3R in neurons associated with nociception, with P2X7R identified as upstream of P2Y1R up-regulation by electroacupuncture. Our study suggests that the P2X7R → P2Y1R → P2X3R inhibitory pathway in DRG mediates the inhibition of peripheral sensitization by electroacupuncture in rats with IBS visceral hypersensitivity.

Construction and evaluation of a column chart model and a random forest model for predicting the prognosis of hydrodistention surgery in BPS/IC patients based on preoperative CD117, P2X3R, NGF, and TrkA levels.

OBJECTIVE: This study seeks to investigate independent risk factors affecting the prognoses of patients with bladder pain syndrome/interstitial cystitis (BPS/IC) following hydrodistention surgery and to develop a column chart model and a random forest model to help predict clinical outcomes. METHOD: A retrospective analysis was conducted on the clinical data of 1006 BPS/IC patients who visited the urology department of the Fifth Hospital of Shanxi Medical University (Shanxi Provincial People's Hospital) between June 2012 and June 2022. The patients were randomly divided into a model group (n = 704) and a validation group (n = 302). In the model group, logistic regression analysis was used to identify independent risk factors, which were used to construct a prognostic nomogram. The nomogram was evaluated by analyzing the area under the curve (AUC), calibration curve, and decision curve. These results were subsequently validated via consistency analysis (n = 302). And based on the random forest algorithm, we calculate the same data and construct a random forest model. RESULT: Multivariate logistic regression analysis revealed that age and the expression of the biomarkers CD117, P2X3R, NGF, and TrkA were independent prognostic factors for patients with BPS/IC (P < 0.05). Using these five indicators, a nomogram was developed to predict the risk factors for BPS/IC (scores ranged from 0 to 400). Based on the indicators, the nomogram demonstrated good prognostic performance (AUC = 0.982 and 95% confidence interva is 0.960-0.100). The correction curve indicated a high level of differentiation in the model, and the decision curve suggested positive clinical benefits. The random forest model has high accuracy and good calibration in predicting the prognosis of patients with interstitial cystitis after hydrodistention surgery. CONCLUSION: Age, CD117, P2X3R, NGF, and TrkA are independent prognostic factors for bladder pain syndrome/interstitial cystitis. The column chart model and random forest model constructed based on these indicators have good predictive performance for patient prognosis.

Electroacupuncture Alleviates Dry Eye Ocular Pain Through TNF-ɑ Mediated ERK1/2/P2X3R Signaling Pathway in SD Rats.

Purpose: This study aimed to examine electroacupuncture's influence on ocular pain and its potential modulation of the TNF-ɑ mediated ERK1/2/P2X3R signaling pathway in dry eye-induced rat models. Methods: Male Sprague-Dawley rats with induced dry eye, achieved through extraorbital lacrimal gland removal, were treated with electroacupuncture. Comprehensive metrics such as the corneal mechanical perception threshold, palpebral fissure height, eyeblink frequency, eye wiping duration, behavioral changes in the open field test, and the forced swimming test were employed. Additionally, morphological changes in microglia and neurons were observed. Expression patterns of key markers, TNF-ɑ, TNFR1, p-ERK1/2, and P2X3R, in the trigeminal ganglion (TG) and spinal trigeminal nucleus caudalis (SpVc) regions, were studied with etanercept serving as a control to decipher the biochemistry of electroacupuncture's therapeutic effects. Results: Electroacupuncture treatment demonstrated a notable decrease in the corneal mechanical perception threshold, improvement in palpebral fissure height, and significant reductions in both eyeblink frequency and eye wiping duration. Moreover, it exhibited a promising role in anxiety alleviation. Notably, the technique effectively diminished ocular pain by curbing microglial and neuronal activation in the TG and SpVc regions. Furthermore, it potently downregulated TNF-ɑ, TNFR1, p-ERK1/2, and P2X3R expression within these regions. Conclusion: Electroacupuncture attenuated damage to sensory nerve pathways, reduced pain, and eased anxiety in dry eye-afflicted rats. The findings suggest a crucial role of TNF-ɑ mediated ERK1/2/P2X3R signaling pathway inhibition by electroacupuncture in these benefits.

Electroacupuncture Reduces Ocular Surface Neuralgia in Dry-Eyed Guinea Pigs by Inhibiting the Trigeminal Ganglion and Spinal Trigeminal Nucleus Caudalis P2X3R-PKC Signaling Pathway.

PURPOSE: To observe the effects of electroacupuncture on ocular surface neuralgia and the P2X3R-PKC signaling pathway in guinea pigs with dry eye. METHODS: A dry eye guinea pig model was established by subcutaneous injection of scopolamine hydrobromide. Guinea pigs were monitored for body weight, palpebral fissure height, number of blinks, corneal fluorescein staining score, phenol red thread test, and corneal mechanical perception threshold. Histopathological changes and mRNA expression of P2X3R and protein kinase C in the trigeminal ganglion and spinal trigeminal nucleus caudalis were observed. We performed a second part of the experiment, which involved the P2X3R-specific antagonist A317491 and the P2X3R agonist ATP in dry-eyed guinea pigs to further validate the involvement of the P2X3R-protein kinase C signaling pathway in the regulation of ocular surface neuralgia in dry eye. The number of blinks and corneal mechanical perception threshold were monitored before and 5 min after subconjunctival injection and the protein expression of P2X3R and protein kinase C was detected in the trigeminal ganglion and spinal trigeminal nucleus caudalis of guinea pigs. RESULTS: Dry-eyed guinea pigs showed pain-related manifestations and the expression of P2X3R and protein kinase C in the trigeminal ganglion and spinal trigeminal nucleus caudalis was upregulated. Electroacupuncture reduced pain-related manifestations and inhibited the expression of P2X3R and protein kinase C in the trigeminal ganglion and spinal trigeminal nucleus caudalis. Subconjunctival injection of A317491 attenuated corneal mechanoreceptive nociceptive sensitization in dry-eyed guinea pigs, while ATP blocked the analgesic effect of electroacupuncture. CONCLUSIONS: Electroacupuncture reduced ocular surface sensory neuralgia in dry-eyed guinea pigs, and the mechanism of action may be associated with the inhibition of the P2X3R-protein kinase C signaling pathway in the trigeminal ganglion and spinal trigeminal nucleus caudalis by electroacupuncture.

Dorsal root ganglion receptor subtype P2X3R mediates postoperative-hyperalgesic priming in mice / 中国药理学通报

Aim To investigate the differences in the role of different purinergic receptor subtypes at different sites in postoperative-hyperalgesic priming in mice. Methods A postoperative-hyperalgesic priming model was constructed by injecting PGE

Discovery of 5-methyl-1H-benzo[d]imidazole derivatives as novel P2X3 Receptor antagonists.

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.

Nociceptive signaling mediated by P2X3, P2X4 and P2X7 receptors.

Chronic pain is a debilitating condition that often occurs following peripheral tissue inflammation and nerve injury. This pain, especially neuropathic pain, is a significant clinical problem because of the ineffectiveness of clinically available drugs. Since Burnstock proposed new roles of nucleotides as neurotransmitters, the roles of extracellular ATP and P2 receptors (P2Rs) in pain signaling have been extensively studied, and ATP-P2R signaling has subsequently received much attention as it can provide clues toward elucidating the mechanisms underlying chronic pain and serve as a potential therapeutic target. This review summarizes the literature regarding the role of ATP signaling via P2X3Rs (as well as P2X2/3Rs) in primary afferent neurons and via P2X4Rs and P2X7Rs in spinal cord microglia in chronic pain, and discusses their respective therapeutic potentials.

Electroacupuncture downregulates P2X3 receptor expression in dorsal root ganglia of the spinal nerve-ligated rat.

Electroacupuncture has been shown to effectively reduce chronic pain in patients with nerve injury. The underlying mechanisms are not well understood. Accumulated evidence suggests that purinergic P2X3 receptors (P2X3Rs) in dorsal root ganglion neurons play a major role in mediating chronic pain associated with nerve injury. The aim of this study is to determine if electroacupuncture stimulation alters P2X3R activity in dorsal root ganglia to produce analgesia under neuropathic pain condition. Peripheral neuropathy was produced by ligation of the left lumbar 5 (L5) spinal nerve in rats. Low-frequency (2 Hz) electrical stimulation was applied to ipsilateral ST36 and BL60 acupoints in rats. The P2X3R agonist (α,ß-meATP)-induced flinch responses were reduced after electroacupuncture treatment. Western analyses showed that P2X3R expression was upregulated in nerve-uninjured lumbar 4 (L4) dorsal root ganglion neurons ipsilateral to the spinal nerve ligation. Electroacupuncture-stimulation reversed the upregulation. In nerve-injured L5 dorsal root ganglia, P2X3R expression was substantially reduced. Electroacupuncture had no effect on the reduction. We also determined the injury state of P2X3R expressing dorsal root ganglion neurons using the neuronal injury marker, activating transcription factor 3 (ATF3). Immunohistochemical assay showed that in L4 dorsal root ganglia, almost all P2X3Rs were expressed in uninjured (ATF3-) neurons. Spinal nerve ligation increased the expression of P2X3Rs. Electroacupuncture reduced the increase in P2X3R expression without affecting the percentage of ATF + neurons. In ipsilateral L5 dorsal root ganglion neurons, spinal nerve ligation reduced the percentage of P2X3R + neurons and markedly increased the percentage of ATF3 + cells. Almost all of P2X3Rs were expressed in damaged (ATF3+) neurons. Electroacupuncture had no effect on spinal nerve ligation-induced changes in the percentage of P2X3R or percentage of ATF3 + cells in L5 dorsal root ganglia. These observations led us to conclude that electroacupuncture effectively reduces injury-induced chronic pain by selectively reducing the expression of P2X3Rs in nerve-uninjured L4 dorsal root ganglion neurons.

Update on the clinical development of gefapixant, a P2X3 receptor antagonist for the treatment of refractory chronic cough.

Chronic cough, or cough lasting >8 weeks, is often associated with underlying medical conditions (ie, asthma, gastroesophageal reflux disease, nonasthmatic eosinophilic bronchitis, and upper-airway cough syndrome). In some patients with chronic cough, treatment of these underlying conditions does not resolve the cough (refractory chronic cough [RCC]), or none of these conditions are present (unexplained chronic cough [UCC]). Despite appropriate medical evaluation, patients with RCC or UCC frequently experience cough persisting for many years, as there are currently no targeted pharmacological approaches approved for the treatment of these conditions. However, the adenosine triphosphate (ATP)-gated P2X3 receptor, a key modulator of the activation of sensory neurons central to the cough reflex, has recently garnered attention as a potential therapeutic target for the treatment of chronic cough. Gefapixant, a first-in-class, non-narcotic, selective antagonist of the P2X3 receptor, recently demonstrated efficacy and was generally well tolerated in phase 2 clinical trials in patients with RCC, validating the utility of targeting this receptor in patients with chronic cough. On the basis of these data, 2 global phase 3 trials, with combined anticipated enrolment exceeding 2000 patients and with treatment durations of up to 1 year, have been initiated. Together, these trials will further evaluate efficacy and safety of gefapixant in the control of cough in patients with RCC or UCC.

Variable transcriptional responsiveness of the P2X3 receptor gene during CFA-induced inflammatory hyperalgesia.

The purinergic receptor P2X3 (P2X3-R) plays important roles in molecular pathways of pain, and reduction of its activity or expression effectively reduces chronic inflammatory and neuropathic pain sensation. Inflammation, nerve injury, and cancer-induced pain can increase P2X3-R mRNA and/or protein levels in dorsal root ganglia (DRG). However, P2X3-R expression is unaltered or even reduced in other pain studies. The reasons for these discrepancies are unknown and might depend on the applied traumatic intervention or on intrinsic factors such as age, gender, genetic background, and/or epigenetics. In this study, we sought to get insights into the molecular mechanisms responsible for inflammatory hyperalgesia by determining P2X3-R expression in DRG neurons of juvenile male rats that received a Complete Freund's Adjuvant (CFA) bilateral paw injection. We demonstrate that all CFA-treated rats showed inflammatory hyperalgesia, however, only a fraction (14-20%) displayed increased P2X3-R mRNA levels, reproducible across both sides. Immunostaining assays did not reveal significant increases in the percentage of P2X3-positive neurons, indicating that increased P2X3-R at DRG somas is not critical for inducing inflammatory hyperalgesia in CFA-treated rats. Chromatin immunoprecipitation (ChIP) assays showed a correlated (R2 = 0.671) enrichment of the transcription factor Runx1 and the epigenetic active mark histone H3 acetylation (H3Ac) at the P2X3-R gene promoter in a fraction of the CFA-treated rats. These results suggest that animal-specific increases in P2X3-R mRNA levels are likely associated with the genetic/epigenetic context of the P2X3-R locus that controls P2X3-R gene transcription by recruiting Runx1 and epigenetic co-regulators that mediate histone acetylation.