Effect of moxibustion on M2 and P2X3 receptors of bladder tissue in rats with neurogenic bladder of detrusor areflexia after lumbar-sacral spinal cord injury / 中国针灸

OBJECTIVE@#To observe the effect of moxibustion at "Guanyuan" (CV 4) and "Shenque" (CV 8) on acetylcholine (Ach), adenosine triphosphate (ATP) and muscarinic-type choline receptor (M2) and purine receptor P2X3 in bladder tissue in the rats with neurogenic bladder (NB) of detrusor areflexia after lumbar-sacral spinal cord injury and explore the underlying mechanism of moxibustion for promoting detrusor contraction.@*METHODS@#Sixty SD rats were randomly divided into a model preparation group (n=45) and a sham-operation group (n=15). In the model preparation group, the modified Hassan Shaker spinal cord transection method was used to prepare the model of NB. In the sham-operation group, the spinal cord transection was not exerted except laminectomy and spinal cord exposure. Among the rats with successfully modeled, 30 rats were selected and divided randomly into a model group and a moxibustion group, with 15 rats in each one. On the 15th day after the operation, moxibustion was applied at "Guanyuan" (CV 4) and "Shenque" (CV 8) in the moxibustion group, 10 min at each acupoint, once a day. The consecutive 7-day treatment was as one course and the intervention for 2 courses was required. Urodynamic test was adopted to evaluate bladder function in rats. Using HE staining, the morphological changes in bladder tissue were observed. The content of Ach and ATP in bladder tissue was measured with biochemical method, and the protein and mRNA expression levels of M2 and P2X3 receptors in bladder tissue were detected with Western blot and real-time fluorescence quantification PCR method.@*RESULTS@#Compared with the sham-operation group, the maximum bladder capacity, leakage point pressure and bladder compliance were increased in the rats of the model group (P<0.05). Compared with the model group, the maximum bladder capacity, the leakage point pressure and bladder compliance were decreased in the rats of the moxibustion group (P<0.05). In the model group, the detrusor fibres were arranged irregularly, bladder epithelial tissues were not tightly connected and cell arrangement was disordered, combined with a large number of vacuolar cells. In the moxibustion group, compared with the model group, the detrusor fibres were arranged regularly, bladder epithelial cells were well distributed and vacuolar cells were reduced. Compared with the sham-operation group, the content of Ach and ATP in bladder tissue was decreased (P<0.05), the protein and mRNA expression levels of M2 and P2X3 receptors were reduced (P<0.05) in the model group. In the moxibustion group, the content of Ach and ATP in bladder tissue was increased (P<0.05) and the protein and mRNA expression levels of M2 and P2X3 receptors were increased (P<0.05) as compared with the model group.@*CONCLUSION@#Moxibustion at "Guanyuan" (CV 4) and "Shenque" (CV 8) may effectively improve bladder function in the rats with NB of detrusor areflexia after lumbar-sacral spinal cord injury and its underlying mechanism is related to promoting the release of Ach and up-regulating the expression of M2 receptor, thereby enhancing the release of ATP and increasing the expression of P2X3 receptor. Eventually, detrusor contraction is improved.

Pulsed radiofrequency inhibits expression of P2X3 receptors and alleviates neuropathic pain induced by chronic constriction injury in rats / 中华医学杂志(英文版)

Background@#Pulsed radiofrequency (PRF) is a minimally invasive interventional technique that provides a novel and effective treatment strategy for neuropathic pain (NP). PRF is advantageous because it does not damage nerves and avoids sensory loss after treatment. At present, animal studies have demonstrated that PRF is safe and effective for relieving the NP associated with sciatic nerve damage in rats with chronic constriction injury (CCI). However, the mechanism through which this effect occurs is unknown. An increasing body of evidence shows that the expression of the P2X ligand-gated ion channel 3 (P2X3) receptor is closely related to NP; this study was to investigate whether the expression of this receptor is involved in NP relief due to PRF.@*Methods@#A total of 36 healthy adult male Sprague-Dawley (SD) rats were randomly divided into three groups: Sham group, CCI group, and PRF group. The right sciatic nerve was ligated in CCI group and PRF group to establish a CCI model; the right sciatic nerve was separated but not ligated in Sham group. On day 14 after the operation, PRF was administered to the ligated sciatic nerve in PRF group (42°C, 45 V, 2 min). A non-live electrode was placed at the exposed sciatic nerve for the rats in Sham and CCI groups. The hindpaw withdrawal threshold (HWT) and thermal withdrawal latency (TWL) were measured at the right hindpaw at different time points before and after PRF or sham therapy. On day 28 after treatment, the dorsal root ganglion (DRG) and spinal dorsal horn of the right L4–6 were harvested from each group to determine the mRNA and protein levels of the P2X3 receptor.@*Results@#On day 28 after PRF treatment, the HWT (8.33 ± 0.67 g vs. 3.62 ± 0.48 g) and TWL (25.42 ± 1.90 s vs. 15.10 ± 1.71 s) were significantly higher in PRF group as compared to CCI group (P < 0.05). The mRNA expression of the P2X3 receptor in the DRG in PRF group was 23.7% lower than that in CCI group (P < 0.05), in the spinal dorsal horns in PRF group was 22.7% lower than that in CCI group (P < 0.05). The protein expression of the P2X3 receptor in the DRG in PRF group was 27.8% lower than that in CCI group (P < 0.05), in the spinal dorsal horns in PRF group was 35.6% lower than that in CCI group (P < 0.05).@*Conclusion@#PRF possibly reduces NP in CCI rats by inhibiting the expression of the P2X3 receptor in the L4–6 DRG and spinal dorsal horns.

Osthole inhibits HIV gpl20 induced neuropathic pain by down-regulating P2X3 receptor in DRG / 中国药理学通报

Aim To investigate whether osthole can alleviate neuropathic pain induced by HIV gpl20 and its mechanism. Methods The paw withdrawal threshold and the paw withdrawal latency were observed to assess pain behaviors in four groups of the rats, including sham group, sham combined with osthole treatment group, gpl20 treatment group, and gpl20 combined with osthole treatment group. The protein expression levels of the P2X3 receptor, tumor necrosis factor-a receptor (TNF-aR), ERK, p-ERK in the L4-L6 dorsal root ganglia (DRG) were measured by Western blot. The mRNA expression level of P2X3 receptor was assessed by real-time quantitative polymerase chain reaction ( qPCR). The whole path clamp recording was used to measure HEK293 cell current activated by ATP. Results Osthole attenuated the mechanical and thermal hyperalgesia in gpl20 treated rats and down-regulated P2X3 receptor mRNA and protein expression in L4-L6 DRGs of gpl20 treated rats. Additionally, osthole simultaneously decreased the expression of TNF-ctR protein in L4-L6 DRGs and inhibited the phosphorylated ERK1/2 protein expression. Moreover, osthole reduced ATP activated current of HEK293 cells transfected with hP2X3R. Conclusion Osthole decreases the mechanical and thermal hyperalgesia induced by gpl20 through inhibiting P2X3R in DRG.

The functions of P2X3 receptor in inflammatory pain and it's influencial factors / 医学研究生学报

Inflammatory Pain is one of the most common clinical symptom with unclear pathogenesis,which causes difficulty in patien's work and daily life. Previous studies have shown that P2X3 receptor participates in the pathological process of acute and chronic inflammatory pain. This paper focuses on the functions of P2X3 receptor in inflammatory pain and its relationship with TRPV1 receptor,inflammatory mediator and Epac-PKC signal transduction pathway,thereby provides a new thought for research and treatment of this disease.

Role of P2X3 receptors in development and maintenance of neuropathic pain in rats: the relationship with NF-κB in dorsal root ganglia / 中华麻醉学杂志

Objective To evaluate the role of P2X3 receptors in the development and mnaintenance of neuropathic pain (NP) and the relationship with nuclear factor kappa B (NF-κB) in dorsal root ganglia of rats.Methods Thirty-six SPF healthy male Sprague-Dawley rats,weighing 180-200 g,aged 4-6 weeks,in which intrathecal catheters were successfully implanted,were divided into 3 groups (n =12 each) using a random number table:sham operation group (group S),NP group and P2X3 receptor antagonist A-317491 group (group A).At 3 days after intrathecal catheters were successfully implanted,NP was induced by chronic constriction injury to the sciatic nerve.The sciatic nerve was only exposed but not occluded in group S.Intrathecal injection was performed twice a day for 14 consecutive days starting from 1 day after operation.Normal saline 20 μ l was intrathecally injected in group S and group NP,and A-317491 100 nmol/10 μ1 and normal saline 10 μ l were intrathecally injected in group A.Thermal paw withdrawal threshold (TWT) and mechanical paw withdrawal threshold (MWT) were measured on day 1 before operation and at 30 min after intrathecal injection on days 1,3,7,10 and 14 after operation.On days 7 and 14 after operation,the rats were sacrificed and the dorsal root ganglia of the lumbar segment were removed for determination of the expression of P2X3 receptors and NF-κB p65 by Western blot.Results Compared with group S,TWT and MWT were significantly decreased at each time point after operation,and thc expression of P2X3 receptors andi NF-κB p65 in dorsal root ganglia was up-regulated on days 7 and [4 after operation in group NP (P＜0.05 or 0.01).Compared with group NP,TWT was significantly increased at each time point after operation,MWT was increased on days 3,7,10 and 14 after operation,anl the expression of P2X3 receptors and NF-κB p65 in dorsal root ganglia was down-regulated on days 7 and 14 after operation in group A (P＜0.05).Conclusion The mechanism underlying the development and maintenance of NP is related to the up-regulation of P2X3 receptor expression and promotion of the expression of NF-κB in dorsal root ganglia of rats.

Current Pharmacologic Approaches in Painful Bladder Research: An Update / 대한배뇨장애요실금학회지

The symptoms of interstitial cystitis (IC)/bladder pain syndrome (BPS) may have multiple causes and involve many contributing factors. Traditional treatments (intravesical instillations) have had a primary focus on the bladder as origin of symptoms without adequately considering the potential influence of other local (pelvic) or systemic factors. Systemic pharmacological treatments have had modest success. A contributing factor to the low efficacy is the lack of phenotyping the patients. Individualized treatment based on is desirable, but further phenotype categorization is needed. There seems to be general agreement that IC is a unique disease and that BPS is a syndrome with multiple pathophysiologies, but this has so far not been not been well reflected in preclinical research with the aim of finding new pharmacological treatments. Current research approaches, including anti-nerve growth factor treatment, anti-tumor necrosis factor-α treatment, activation of SHIP1 (AQX-1125), and P2X3 receptor antagonists, and α1-adrenoceptor antagonists are potential systemic treatments, implying that not only the bladder is exposed to the administered drug, which may be beneficial if the IC/BPS is a bladder manifestation of a systemic disease, or negative (adverse effects) if it is a local bladder condition. Local treatment approaches such as the antagonism of Toll-like receptors (which still is only experimental) and intravesical liposomes (with positive proof-of-concept), may have the advantages of a low number of systemic adverse effects, but cannot be expected to have effects on symptoms generated outside the bladder. Assessment of which of the treatment approaches discussed in this review that can be developed into useful therapies requires further studies.

Effect of Electroacupuncture at Hegu (LI 4) on P2X2 and P2X3 Receptors in Trigeminal Ganglion in Pulpalgia Rats / 上海针灸杂志

Objective To explore the effect of electroacupuncture at Hegu (LI 4) on the expressions of P2X2 and P2X3 receptors in experimental pulpalgia rats.Method Forty-two male SD rats were randomized into a normal group (group N), a control group (group C), a pulpalgia model group (group M), an antagonist group (group A), an electroacupuncture group (group E), and an antagonist+electroacupuncture group (group AE), 7 rats in each group. Group N didn’t receive any interventions; group C received injection of normal saline into pulp cavity of the same dose as the injection in group M, and the cavity was then blocked by dental fillings 5-6 min later; in group M, maxillary first and second molar teeth were drilled (drill bit of 1 mm in diameter) to expose pulp and lipopolysaccharide (LPS) solution 5μg/μL was injected into the holes (1~3μL for each hole), and the holes were then covered by dental fillings 5-6 min later; group A received the same modeling method as that in group M, but A-317491 was injected together with LPS (0.5 mg/kg); group E received electroacupuncture at bilateral Hegu (LI 4) with needles retained for 30 min, once a day, totally for 3 times; group AE received the same electroacupuncture intervention after receiving the same treatments as that in group A. The rats’ behaviors and weight were observed for 30 min after intervention each day. The rats were sacrificed on the 4th day, and the mRNA expressions of P2X2 and P2X3 receptors andβ-actin in trigeminal ganglion were detected by using RT-PCR. The mRNA expressions were then compared among the groups.Result The behavioral changes in group M, E, and AE were more significant than that in group C and N (P<0.01); the behavioral changes in group A, E, and AE were less significant than that in group M (P<0.01). The weight in group C was significantly lower than that in group N (P<0.01); the weights in group M, A, E and AE were significantly lower than that in group C and N (P<0.01); the weights in group E and AE were significantly higher than that in group M and A (P<0.01,P<0.05); the weight in group A was slightly higher than that in group M (P<0.05); the weights in group AE was significantly higher than that in group E (P<0.01). The mRNA expressions of P2X2 receptor in group M, A, and AE were significantly higher than that in group N and C (P<0.01); the mRNA expressions of P2X2 receptor in group A, E, and AE were lower than that in group M (P<0.05); the mRNA expression of P2X2 receptor in group A was lower than that in group E (P<0.05); the mRNA expression of P2X2 receptor in group E was higher than that in group AE (P<0.05). The mRNA expressions of P2X3 receptor in group M, A, and E were significantly higher than that in group C (P<0.05) and group N (P<0.01); the mRNA expressions of P2X3 receptor in group A, E, and AE were significantly lower than that in group M (P<0.01); the mRNA expression of P2X3 receptor in group AE was markedly lower than that in group E (P<0.01).Conclusion The expressions of P2X2 and P2X3 receptors in trigeminal ganglion were increased in LPS-induced pulpalgia rats. Electroacupuncture at Hegu (LI 4) and injection of A-317491 both can down-regulate the mRNA expressions of P2X2 and P2X3 receptors, which is plausibly the action mechanism of electroacupuncture at Hegu (LI 4) in analgesia.

Spatio-temporal expression of P2X3 receptor in rats with diabetic mechanical allodynia / 解剖学报

Objective To investigate the spatio-temporal expression of P2X3 receptor ( P2X3R) in rats with diabetic mechanical allodynia ( DMA ) .Methods DMA model in rats was induced by intraperitoneal injection of streptozocin ( STZ) .The von Frey filaments were applied to identify the changes of the paw withdrawal threshold ( PWT) in DMA rats.Immunofluorescence was employed to detect the spatio-temporal expression of P2X3R in the spinal dorsal horn (SDH), dorsal root ganglion (DRG) and hind paw skin on different time points after intraperitoneal injection of STZ , respectively.The protein expression of P2X3R in SDH and DRG was further semi-quantitatively analyzed by Western blotting.Results Compared with control group , intraperitoneal injection STZ induced significant mechanical allodynia indicated by the reduced PWT from 7 days, and which reached the peak on 14d and maintained to 28days (P<0.05). The expression of P2X3R in DRG neurons was significantly increased on 14 days and 21 days (P<0.05), while that in SDH and skin was markedly increased on 21 days and 28 days, compared with control group (P<0.05).Conclusion With the progress of DMA, the expression of P2X3R was significantly increased in the SDH, DRG and skin, which was almost parallel with the mechanical allodynia , but the changes in SDH and skin were 1 week later than that in DRG .These results suggest that P2X3R may play a key role in the maintenance of the DMA .

Effect of reactive oxygen species on dorsal root ganglion P2X3 receptor-induced pain signal / 第二军医大学学报

Objective To investigate the effect of reactive oxygen species(ROS) on P2X3 receptor-mediated neuropathic pain. Methods Neuropathic pain model was induced in female Sprague Dawley rats by chronic compression of dorsal root ganglia (CCD), and the CCD rats were randomly divided into 4 groups (each group containing 10 rats): Saline group (NS group); PBN 100 mg/kg treatment group; PBN 30 mg/kg treatment group; and PBN 10 mg/kg treatment group. The specific agonist of P2X3 receptor, α, β-meATP (50 nmol in 50 μL), was subcutaneously injected into the plantar surface of the right hind paws of each rat 30 minafter PBN or NS injection. The spontaneous paw flinching times and withdrawing time were observed for 15 min after injection and the paw lift time per minute (PLTPM) in every 2 minutes was calculated. Results Pre- treatmentwith PBN inhibited α, β-meATP-induced spontaneous pain in a dose-dependent manner. Compared with the NS group, PBN 100 mg/kg group significantly inhibited flinching response during the first 6 min (P<0. 05), while the rats in PBN 30 mg/kg group only had significantly attenuated flinching response during the second to the fourth minute compared with NS group(P<0. 05). Conclusion Oxygen free radical scavenger can effectively alleviate the neuropathic pain caused by CCD and P2X3 receptor agonist-induced spontaneous pain. ROS may act as a messenger in P2X3 receptor-mediated pain signaling transmission.

P2X3 receptor expression in DRG and bladder detrusor of an animal model of neurogenic bladder and ure-thra / 中华物理医学与康复杂志

Objective To investigate the P2X3 receptor expression in L6-S1 dorsal root ganglion (DRG)and bladder detrusor in a rat model of neurogenic bladder and urethra. Methods Eighty Sprague-Dawley rats wererecruited and randomly divided into a sacral injury group, a suprasacral injury group and a control group. Spinal tran-section was performed to establish the animal model of neurogenic bladder and urethra in rats of the sacral injurygroup and suprasacral injury group. Check the P2X3 receptor expression in DBG and bladder detrusor among thethree groups by Western blot test at 20 days after model establishment. Results P2X3 receptor expression in L6-S1DRG of sacral injury group was significantly less than that of the suprasacral injury group, which was in turn signifi-cantly higher than that of the control group. P2X3 receptor expression in bladder detrusor of sacral injury group wassignificantly lower than that of the suprasacral injury group, which was in turn significantly higher than that of thecontrol group. Conclusion There was close relationship between P2X3 receptor expression and dysfunction of blad-der and urethra.

Distribution of Purinergic Receptor and Vanilloid Receptor Participating in Micturition and Reciprocal Relationship between Them / 대한해부학회지

Two of the synaptic receptors involved in the regulation of micturition, P2X(3) receptor, which is operated by ATP, and vanilloid receptor 1 (VR1), which is operated by capsaicin, are regarded as newcomers. To investigate the possibility that these receptors act as therapeutic targets for treatment of an overactive bladder, we investigated their distribution and reciprocal relationship. Eight-week-old Sprague Dawley rats were injected with retrograde nerve tracer within the bladder wall, and 15 rats were injected with 0.5% acetic acid inside the bladder. After a week, the animals were killed, and their dorsal root ganglia (DRG) at the levels of L6 and S1 were harvested. Immunohistochemistry or Western blot analysis of P2X(3) and VR1 were performed on the DRG. The DRG neurons with afferent fibers from the bladder had increased expression of VR1 and downregulated P2X(3) receptors. The P2X(3) receptor and VR1 seemed to account for the important parts of the hypersensitivity of the inflammatory bladder. We conclude that the simultaneous modulations of both P2X(3) receptor and VR1 may have a synergic effect on the treatment of overactive bladder and may produce greater response rates.

Expression of P2X3 and its colocalization with trpv1 in the human dental pulp / 대한치과보존학회지

The purinoreceptor, P2X3 is a ligand-gated cation channel activated by extracellular ATP. It has been reported that ATP can be released during inflammation and tissue damage, which in turn may activate P2X3 receptors to initiate nociceptive signals. However, little is known about the contribution of P2X3 to the dental pain during pulpal inflammation. Therefore, the purpose of this study was to investigate the expression of P2X3 and its colocalization with TRPV1 to understand the mechanism of pain transmission through P2X3 in the human dental pulp with double labeling immunofluorescence method. In the human dental pulp, intense P2X3 immunoreactivity was observed throughout the coronal and radicular pulp. Of all P2X3-positive fibers examined, 79.4% coexpressed TRPV1. This result suggests that P2X3 along with TRPV1 may be involved in the transmission of pain and potentiation of noxious stimuli during pulpal inflammation.

Excitability changes of DRG neurons and expression of P2X_3 receptor in DRG neurons affected by prostatitis induced by CFA / 第三军医大学学报

Objective To explore excitability change of P2X receptor-mediated DRG neurons and expression of P2X_(3) receptor in the DRG neurons affected by prostatic inflammation.Methods ATP-evoked spontaneous firing and the expression of P2X_(3)receptor in the DRG neurons in the L6-S1 spinal cord were investigated by whole-cell patch clamp techniques,Western blotting on day 3,10 after Complete Freund's adjuvant(CFA) irritation of the rat prostate.Results Membrane potential of DRG neurons fluctuated spontaneously under current clamp.Membrane potential of neurons of control group and 3-day CFA irritation group were under threshold potential, and all recorded neurons had not action potential.However,4 of 39 neurons(10.26%) showed spontaneous action potential at membrane potential on day 10 after CFA irritation of prostate.Expression of P2X_(3) receptor increased significantly on day 10 after CFA irritation of the prostate by Western blotting.Conclusion The increase of expression and sensitivity of P2X_(3) receptor may play important roles in the development and maintenance of the pain owing to prostatic inflammation.

Effect of TMP on neuropathic pain mediated by P2X_3 receptor / 中国药理学通报

Aim To investigate effects of tetramethylpyrazine (TMP) on neuropathic pain induced by P2X_3 receptor. Methods Chronic constriction injury (CCI) model was adopted. Mechanical withdrawal threshold and thermal withdrawal latency were measured and P2X_3 immunoreactivity in L_4/L_5 spinal cord was detected by immunohistochemistry. Results At day 14 after operation, the mechanical withdrawal threshold and thermal withdrawal latency in group Ⅴ(CCI group) were lower than those in groupⅠ(NS group),Ⅱ(TMP group),Ⅲ(sham group) and Ⅳ (CCI+ TMP group)(P0.05). The expression of P2X_3 receptor in L_4/L_5 spinal cord of group Ⅳ was lower than that of group Ⅴ (P

Expression change of P2X_3 receptor in spinal dorsal horn and dorsal root ganglion in inflammatory pain rats / 第三军医大学学报

Objective To investigate the expression change of P2X3 receptor in spinal dorsal horn and dorsal root ganglion(DRG) in rats with experimental inflammatory pain.Methods Totally 30 SD rats received a subcutaneous injection of carrageenan in the right paw to establish inflammatory pain model,and then divided randomly and equally into 5 groups,that is 6,12,24,72 and 168 h after injection.Another 6 rats undergoing a subcutaneous injection of normal saline in the right paw served as control.Pain threshold was assayed by thermal radiation and von Frey filament in 2,4,6,12,24,72 and 168 h after injection.Expression of P2X3 receptor in spinal dorsal horn and DRG of every group was measured by immunohistochemical staining(IHC).Results After injection of carrageenan,thermal hyperalgesia and mechanical allodynia appeared in the rats.The thermal withdrawl latency(TWL) and 50% paw withdrawl threshold(PWT) were decreased obviously from 2 h,descended to the lowest at 12 h,then returned to the normal level after 72 h.The expression of P2X3 receptor in spinal dorsal horn was increased obviously from 24 h,while that at DRG was increased obviously from 12 h.Both of the expressions lasted for 72 h,and returned to the normal level at 168 h after injection.Conclusion Thermal hyperalgesia and mechanical allodynia appear in the rats after carrageenan injection,and the P2X3 receptor is activated and up-regulated in the DRG and spinal cord,suggesting that P2X3 receptor may play an important role in the incidence and development of inflammatory pain.