Regulatory effect of mild moxibustion on P2X3 receptors in spinal cord,anterior cingulate cortex and thalamic ventral posterolateral nucleus of rats with IBS visceral hyperalgesia

Objective To observe the therapeutic effect of mild moxibustion on irritable bowel syndrome(IBS)visceral hyperalgesia model rats and its regulatory effect on P2X3 receptors in the spinal cord,anterior cingutate cortex(ACC)and thalamic ventral posterolateral nucleus(VPL).Methods Thirty 8-day-old newborn rats were randomly divided into a normal group(n=6)and a modeling group(n=24)according to the completely random number table method.Rats in the normal group were bred routinely,and those in the modeling group were subjected to preparing IBS chronic visceral hyperalgesia model using colorectal distention(CRD)in stimulation method.Rats successfully modelled were re-divided into a model group,a mild moxibustion group,a P2X3 receptor antagonist group,and a normal saline group according to the completely random number table method with 6 rats in each group.Rats in each group received corresponding interventions from the 37-day old,once a day for 7 consecutive days.Immunohistochemistry and Western blot assays were used to detect P2X3 protein expressions in the spinal cord,ACC and VPL of rats.Results Under different intensities of CRD stimulation,the abdominal withdrawal reflex(AWR)scores of the model group were significantly increased versus the normal group(all P<0.05);the AWR scores of the mild moxibustion group and the P2X3 receptor antagonist group were significantly reduced versus the model group(all P<0.01).The P2X3 protein expressions in rat spinal cord,ACC and VPL tissues of the model group were significantly increased versus the normal group(all P<0.01);the P2X3 protein expressions in rat spinal cord,ACC and VPL tissues of the mild moxibustion group and the P2X3 receptor antagonist group were significantly reduced versus the model group(all P<0.01).Conclusion Mild moxibustion can inhibit the P2X3 receptor expressions in the spinal cord,ACC,and VPL tissues of IBS visceral hyperalgesia model rats,which may be the mechanism of mild moxibustion in relieving the central sensitization of rats with IBS visceral hyperalgesia.

Microencapsulated Schwann cell transplantation inhibits P2X3 receptor expression in dorsal root ganglia and neuropathic pain

Schwann cell transplantation is a promising method to promote neural repair, and can be used for peripheral nerve protection and myelination. Microcapsule technology largely mitigates immune rejection of transplanted cells. We previously showed that microencapsulated olfactory ensheathing cells can reduce neuropathic pain and we hypothesized that microencapsulated Schwann cells can also inhibit neuropathic pain. Rat Schwann cells were cultured by subculture and then microencapsulated and were tested using a rat chronic constriction injury（CCI） neuropathic pain model. CCI rats were treated with Schwann cells or microencapsulated Schwann cells and were compared with sham and CCI groups. Mechanical withdrawal threshold and thermal withdrawal latency were assessed preoperatively and at 1, 3, 5, 7, 9, 11 and 14 days postoperatively. The expression of P2X3 receptors in L4-5 dorsal root ganglia of the different groups was detected by double-label immunofluorescence on day 14 after surgery. Compared with the chronic constriction injury group, mechanical withdrawal threshold and thermal withdrawal latency were higher, but the expression of P2X3 receptors was remarkably decreased in rats treated with Schwann cells and microencapsulated Schwann cells, especially in the rats transplanted with microencapsulated Schwann cells. The above data show that microencapsulated Schwann cell transplantation inhibits P2X3 receptor expression in L4-5 dorsal root ganglia and neuropathic pain.

Microencapsulation improves inhibitory effects of transplanted olfactory ensheathing cells on pain after sciatic nerve injury

Olfactory bulb tissue transplantation inhibits P2X2/3 receptor-mediated neuropathic pain. However, the olfactory bulb has a complex cellular composition, and the mechanism underlying the action of purified transplanted olfactory ensheathing cells（OECs） remains unclear. In the present study, we microencapsulated OECs in alginic acid, and transplanted free and microencapsulated OECs into the region surrounding the injured sciatic nerve in rat models of chronic constriction injury. We assessed mechanical nociception in the rat models 7 and 14 days after surgery by measuring paw withdrawal threshold, and examined P2X2/3 receptor expression in L4–5 dorsal root ganglia using immunohistochemistry. Rats that received free and microencapsulated OEC transplants showed greater withdrawal thresholds than untreated model rats, and weaker P2X2/3 receptor immunoreactivity in dorsal root ganglia. At 14 days, paw withdrawal threshold was much higher in the microencapsulated OEC-treated animals. Our results confirm that microencapsulated OEC transplantation suppresses P2X2/3 receptor expression in L4–5 dorsal root ganglia in rat models of neuropathic pain and reduces allodynia, and also suggest that transplantation of microencapsulated OECs is more effective than transplantation of free OECs for the treatment of neuropathic pain.

Adenosine triphosphate mediates the pain tolerance effect of manual acupuncture at Zusanli(ST36) in mice

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.

Effect of Electroacupuncture on the Pathomorphology of the Sciatic Nerve and the Sensitization of P2X_3 Receptors in the Dorsal Root Ganglion in Rats with Chronic Constrictive Injury

Objective： To explore the effect of electroacupuncture （EA） on the pathomorphology of the sciatic nerve and the role of P2X3 receptors in EA analgesia. Methods： The chronic constriction injury （CCI） model was adopted in this study. A total of 32 rats were randomly divided into four groups： sham CCI, CCI, CCI plus contralateral EA （CCI ＋ conEA） and CCI plus ipsilateral EA （CCI ＋ ipsEA）. Mechanical withdrawal threshold （MWT） and thermal withdrawal latency OWL） were measured. EA began at day 7 after the CCI operation and was applied to the Zusanli （ST 36） and Yanglingquan acupoints （GB 34）. At day 14, the pathomorphologic changes of the operated sciatic nerve were demonstrated by hematoxylin and eosin staining. In addition, dorsal root ganglion （DRG） neurons isolated from rats were examined by electrophysiological recording to determine if the P2X3 receptor agonists, adenosine 5＇-tdphosphate disodium （ATP） and α, 13 -methylen-ATP （or, 13 -meATP） evoked inward currents. Results： Pain thresholds in the CCI group were obviously decreased post CCI surgery （P〈0.01）. In the EA groups, thermal and mechanical threshold values were increased after the last EA treatment （P〈0.05, P〈0.01）. There was no significant difference in light microscopic examination among the four groups （P〉0.05）. Current amplitude after application of ATP and or, 13 -meATP in DRG neurons were much larger in the CCI group compared to those obtained in sham CCI （P〈0.05）. ATP and α, 13 -meATP invoked amplitudes in the CCI ＋ EA groups were reduced. There was no significant difference between the CCI ＋ conEA group and the CCI ＋ ipsEA group （P〉0.05）. Conclusion： EA analgesia may be mediated by decreasing the response of P2X8 receptors to the agonists ATP and or, 13 -meATP in the DRG of rats with CCI. No pathological changes of the sciatic nerve of rats were observed after EA treatment.

CaMKIIα and caveolin-1 cooperate to drive ATP-induced membrane delivery of the P2X3 receptor

The P2X3 receptor plays a vital role in sensory processing and transmission. The assembly and trafficking of the P2X3 receptor are important for its function in primary sensory neurons. As an important inflammation mediator, ATP is released from different cell types around primary sensory neurons, especially under pathological pain conditions. Here, we showthat α, β-MeATP dramatically promoted membrane delivery of the P2X3 receptor both in HEK293T celts expressing recombinant P2X3 receptor and in rat primary sensory neurons. α, β-MeATP induced P2X3 receptor-mediated Ca^2＋ influx, which further activated Ca^2＋/calmodulin-dependent protein kinase Ilec （CaMKIIα）. The N terminus of the P2X3 receptor was responsible for CaMKIleα binding, whereas Thr38s in the C terminus was phosphorylated by CaMKIIα. Thr^388 phosphorylation increased P2X3 receptor binding to caveoUn-1. CaveoUn-1 knockdown abrogated the α, β-MeATP-induced membrane insertion of the P2X3 receptor. Moreover,α, β-MeATP drove the CaMKIlec-mediated membrane coinsertion of the P2X2 receptor with the P2X3 receptor. The increased P2X3 receptors on the cell membrane that are due to Thr388 phosphorytation facilitated P2X3 receptor-mediated signal transduction. Together, our data indicate that CaMKIIoL and caveoUn-1 cooperate to drive Ugand-induced membrane delivery of the P2X3 receptor and may provide a mechanism of P2X3 receptor sensitization in pain development.

Upper limb arthromyodynia can be treated with Notopterygium by down-regulating P2X3 to mediate PKC-induced inflammatory response

Rheumatoid arthritis(RA)is one of the most common refractory diseases in the world,and traditional Chinese medicine Notopterygium(NE)has been used in the treatment of upper limb pain for a long time.NE can significantly reduce the expression of inflammatory pain target P2X3 receptor in rats with upper-limb arthritis.To verify the relationship between the mechanism of NE for“upper limb paralysis”and the P2X3 receptor-mediated PKC inflammatory response pathway,UPLC was taken to measure the exact medicinal substance of ethyl acetate from NE.Sprague Dawley rats were randomly divided into a blank group,a model group,a live-action group,and a positive group.The joint cavity was removed after 21 d.Moreover,a model group,a live group,and a positive group were also set up with RA-FLS cells in our in vitro study.The expressions of P2X3 and PKC inflammation pathway indicators were detected by Western blotting analysis.A P2X3 inhibitor(A-317491)acted on RA-FLS cells,and a model group and a positive group were set.Then the protein expression of PKC was detected.NE reduced the expressions of P2X3,Rab7,PKC,and NF-κB at the protein level in both systems.NE and P2X3 receptor antagonists reduced the expressions of key proteins in the PKC pathway in RA-FLS cells to similar extents,and their effects were not additive.NE could effectively improve the“forelimb pain”of RA rats,with a mechanism closely related to the P2X3/Rab7/PKC/NF-κB pathway.

Effects of electroacupuncture at 2 and 100 Hz on rat type 2 diabetic neuropathic pain and hyperalgesia-related protein expression in the dorsal root ganglion

Objective： To investigate the analgesic effects of electroacupuncture （EA） at 2 and 100 Hz on type 2 diabetic neuropathic pain （DNP） and on the expressions of the P2X3 receptor and calcitonin gene-related peptide （CGRP） in the dorsal root ganglion （DRG）. Methods： Rat type 2 DNP was induced by a high calorie and high sugar diet fed for 7 weeks, plus a single intraperitoneal injection of streptozotocin （STZ） after 5 weeks. EA at 2 and 100 Hz was carried out once every day after 7 weeks for 7 consecutive days. Body weight, serum fasting insulin （FINS）, fasting blood glucose （FBG）, insulin sensitivity index （ISI）, and paw withdrawal latency （PWL） were measured. The expressions of L4-L6 DRG P2X3 receptors and CGRP were assessed by immunofluorescence. Results： Type 2 DNP was successfully induced as shown by the increased body weight, FINS, and FBG, as well as the reduced ISI and PWL. Expressions of P2X3 receptors and CGRP in L4-L6 DRGs increased. EA at both 2 and 100 Hz relieved type 2 DNP, but the analgesic effect of EA was stronger at 2 Hz. P2X3 receptor expression decreased in L4-L6 DRGs following EA at 2 Hz and in L5 and L6 DRGs following EA at 100 Hz. EA at both 2 and 100 Hz down-regulated CGRP overexpression in L4-L6 DRGs. Conclusions： These findings indicate that EA at 2 Hz is a good option for the management of type 2 DNP. The EA effect may be related to its down-regulation of the overexpressions of the DRG P2X3 receptors and CGRP in this condition.

Adult Stress Promotes Purinergic Signaling to Induce Visceral Pain in Rats with Neonatal Maternal Deprivation

Chronic visceral pain is one of the primary symptoms of patients with irritable bowel syndrome(IBS),which affects up to 15%of the population world-wide.The detailed mechanisms of visceral pain remain largely unclear.Our previous studies have shown that neonatal maternal deprivation(NMD)followed by adult multiple stress(AMS)advances the occurrence of visceral pain,likely due to enhanced norepinephrine(NE)-β2 adrenergic signaling.This study was designed to explore the roles of P2X3 receptors(P2X3Rs)in the chronic visceral pain induced by combined stress.Here,we showed that P2X3 Rs were co-expressed inβ2 adrenergic receptor(β2-AR)-positive dorsal root ganglion neurons and that NE significantly enhanced ATP-induced Ca2+signals.NMD and AMS not only significantly increased the protein expression of P2X3Rs,but also greatly enhanced the ATP-evoked current density,number of action potentials,and intracellular Ca2+concentration of colon-related DRG neurons.Intrathecal injection of the P2X3R inhibitor A317491 greatly attenuated the visceral pain and the ATP-induced Ca2+signals in NMD and AMS rats.Furthermore,theβ2-AR antagonist butoxamine significantly reversed the expression of P2X3Rs,the ATP-induced current density,and the number of action potentials of DRG neurons.Overall,our data demonstrate that NMD followed by AMS leads to P2X3R activation,which is most likely mediated by upregulation ofβ2 adrenergic signaling in primary sensory neurons,thus contributing to visceral hypersensitivity.