PolyphyllinVI alleviates the spared nerve injury-induced neuropathic pain based on P2X3 receptor-mediated the release of inflammatory mediators.

ETHNOPHARMACOLOGICAL RELEVANCE: PolyphyllinVI (PPⅥ) 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 PPⅥ by using neuropathic pain (NPP) mice. MATERIALS AND METHODS: The potential targets and mechanisms of PPⅥ 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 PPⅥ on NPP and the expression of the P2X3 receptor. We identified the signaling pathways of PPⅥ 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 PPⅥ. PPⅥ 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, PPⅥ 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: PPⅥ 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 PPⅥ may be based on the AMPK/NF-κB pathway.

The ethanol extract of Scutellaria baicalensis Georgi attenuates complete Freund's adjuvant (CFA)-induced inflammatory pain by suppression of P2X3 receptor.

ETHNOPHARMACOLOGICAL RELEVANCE: Scutellaria baicalensis Georgi (SBG) is a perennial herb with anti-inflammatory, antibacterial, and antioxidant activities, which is traditionally used to treat inflammation of respiratory tract and gastrointestinal tract, abdominal cramps, bacterial and viral infections. Clinically, it is often used to treat inflammatory-related diseases. Research has shown that the ethanol extract of Scutellaria baicalensis Georgi (SGE) has anti-inflammatory effect, and its main components baicalin and baicalein have analgesic effects. However, the mechanism of SGE in relieving inflammatory pain has not been deeply studied. AIM OF THE STUDY: This study aimed to evaluate the analgesic effect of SGE on complete Freund's adjuvant (CFA)-induced inflammatory pain rats, and to investigate whether its effect on relieving inflammatory pain is associated with regulation of P2X3 receptor. MATERIALS AND METHODS: The analgesic effects of SGE on CFA-induced inflammatory pain rats were evaluated by measuring mechanical pain threshold, thermal pain threshold, and motor coordination ability. The mechanisms of SGE in relieving inflammatory pain were explored by detecting inflammatory factors levels, NF-κB, COX-2 and P2X3 expression, and were further verified by addition of P2X3 receptor agonist (α, ß me-ATP). RESULTS: Our results revealed that SGE can notably increase the mechanical pain threshold and thermal pain threshold of CFA-induced inflammatory pain rats, and markedly alleviate the pathological damage in DRG. SGE could suppress the release of inflammatory factors including IL-1ß, IL-6, TNF-α and restrain the expression of NF-κB, COX-2 and P2X3. Moreover, α, ß me-ATP further exacerbated the inflammatory pain of CFA-induced rats, while SGE could markedly raise the pain thresholds and relieve inflammatory pain. SGE could attenuate the pathological damage, inhibit P2X3 expression, inhibit the elevation of inflammatory factors caused by α, ß me-ATP. SGE can also inhibit NF-κB and ERK1/2 activation caused by α, ß me-ATP, and inhibit the mRNA expression of P2X3, COX-2, NF-κB, IL-1ß, IL-6 and TNF-α in DRG of rats induced by CFA coupled with α, ß me-ATP. CONCLUSIONS: In summary, our research indicated that SGE could alleviate CFA-induced inflammatory pain by suppression of P2X3 receptor.

Dorsal root ganglia P2X4 and P2X7 receptors contribute to diabetes-induced hyperalgesia and the downregulation of electroacupuncture on P2X4 and P2X7.

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.

Analgesic effect of electroacupuncture on bone cancer pain in rat model: the role of peripheral P2X3 receptor.

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.

[Effect of electroacupuncture on the expressions of TRPV1, P2X3 receptors in bladder of rats with interstitial cystitis].

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.

Moxibustion attenuates neurogenic detrusor overactivity in spinal cord injury rats by inhibiting M2/ATP/P2X3 pathway.

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.

[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].

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.

Estrogen inhibits bladder overactivity in rats with cyclophosphamide-induced cystitis via downregulating the expression of P2X3 receptors in bladder epithelium cells.

AIMS: The therapeutic effect of estrogen on interstitial cystitis/bladder pain syndrome is unclear. We aim to explore the effect of estrogen on bladder overactivity in rats with cyclophosphamide-induced cystitis and its underlying mechanism. METHODS: In vivo cystometry was used to determine the effect of estrogen on bladder excitability. The effect of estrogen on the expression of P2X3 receptors in bladder epithelium was detected by real-time polymerase chain reaction and western blot. Effect of P2X3 receptors in bladder urothelium on stretch-released adenosine triphosphate was performed by a Flexcell FX5000 Compression system and an Enzyme-Linked Immunosorbent Assay Kit. RESULTS: Estrogen deprivation significantly increased the urinary frequency, while supplementation with diarylpropionitrile (DPN), an estrogen receptor ß (ERß) agonist, alleviated the urinary frequency. 17ß-Estradiol and DPN decreased the expression of P2X3 receptors in urothelium cells which was partially inhibited by ERß antagonist 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol. Meanwhile, inhibiting the expression of P2X3 receptors by ERß agonist or antagonizing the function of P2X3 receptors by selective P2X3 receptor antagonist AF-353 or A-317491 significantly reduced the stretch-released ATP from urothelium cells. CONCLUSIONS: Estrogen has a direct effect on the regulation of bladder overactivity in rats with cyclophosphamide-induced cystitis by downregulating the expression of bladder epithelial P2X3 receptors through ERß and reducing the adenosine triphosphate released from urothelium during bladder filling, thereby inhibiting the generation of the micturition reflex.

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.

SZAP exerts analgesic effects on rheumatalgia in CIA rats by suppressing pain hyperalgesia and inhibiting TRPV1 and P2X3.

ETHNOPHARMACOLOGICAL RELEVANCE: ShexiangZhuifeng Analgesic Plaster (SZAP) is a traditional Chinese medicine and transdermal formulation composed of many Chinese herbs and active compounds. SZAP was recently approved by the China Food and Drug Administration for the treatment of pain associated with osteoarticular diseases and is preferred by most rheumatoid arthritis patients in China. However, its mechanism has not been elucidated in detail. AIM OF THE STUDY: We sought to determine the analgesic effect of SZAP in collagen-induced arthritis (CIA) rats and explore the underlying mechanisms of pain transmission, such as via the TRPV1 and P2X3 receptors. METHODS: After CIA was established, rats were treated with SZAP for 7 days. Paw thickness, arthritis score, and haematoxylin and eosin staining were used to evaluate the effectiveness of SZAP. Paw withdrawal threshold (PWT) and tail-flick latency (TFL) were used to estimate the analgesic effect of SZAP. The levels of PGE2, BK, 5-HT, SP, and CGRP in the serum and synovium were determined using ELISA kits, and ATP in the synovium was measured using HPLC. The expression of TRPV1 and P2X3 in the DRG was detected using western blotting and immunofluorescence. TRPV1 and P2X3 agonists were further used to determine the analgesic effects of SZAP on CIA rats based on PWT and TFL. RESULTS: SZAP not only significantly ameliorated arthritis scores and paw thickness by improving the pathological damage of synovial joints, but also remarkably alleviated pain in CIA rats. Further, treatment with SZAP significantly reduced peripheral 5-HT, PGE2 BK, SP, CGRP, and ATP. Additionally, the expression of TRPV1 and P2X3 in the DRG was markedly downregulated by SZAP. Interestingly, the analgesic effect of SZAP was weakened (reduction of PWT and TFL) when TRPV1 and P2X3 were activated by capsaicin or α,ß-meATP, respectively. CONCLUSION: SZAP ameliorates rheumatalgia by suppressing hyperalgesia and pain transmission through the inhibition of TRPV1 and P2X3 in the DRG of CIA rats.

Eliapixant is a selective P2X3 receptor antagonist for the treatment of disorders associated with hypersensitive nerve fibers.

ATP-dependent P2X3 receptors play a crucial role in the sensitization of nerve fibers and pathological pain pathways. They are also involved in pathways triggering cough and may contribute to the pathophysiology of endometriosis and overactive bladder. However, despite the strong therapeutic rationale for targeting P2X3 receptors, preliminary antagonists have been hampered by off-target effects, including severe taste disturbances associated with blocking the P2X2/3 receptor heterotrimer. Here we present a P2X3 receptor antagonist, eliapixant (BAY 1817080), which is both highly potent and selective for P2X3 over other P2X subtypes in vitro, including P2X2/3. We show that eliapixant reduces inflammatory pain in relevant animal models. We also provide the first in vivo experimental evidence that P2X3 antagonism reduces neurogenic inflammation, a phenomenon hypothesised to contribute to several diseases, including endometriosis. To test whether eliapixant could help treat endometriosis, we confirmed P2X3 expression on nerve fibers innervating human endometriotic lesions. We then demonstrate that eliapixant reduces vaginal hyperalgesia in an animal model of endometriosis-associated dyspareunia, even beyond treatment cessation. Our findings indicate that P2X3 antagonism could alleviate pain, including non-menstrual pelvic pain, and modify the underlying disease pathophysiology in women with endometriosis. Eliapixant is currently under clinical development for the treatment of disorders associated with hypersensitive nerve fibers.

Photobiomodulation inhibits inflammation in the temporomandibular joint of rats.

Photobiomodulation (PBM) has been applied as a non-invasive technique for treating temporomandibular joint symptoms, especially on painful condition's relief, however the anti-inflammatory mechanism underlying the effect of PBM remains uncertain. This study aims to evaluate the mechanisms of action of PBM (808 nm) in a carrageenan-induced inflammation on temporomandibular joint (TMJ) of rats. In this study male Wistar rats were pre-treated with irradiation of a low-power diode laser for 15 s on TMJ (infra-red 808 nm, 100 mW, 50 J/cm2 and 1.5 J) 15 min prior an injection in the temporomandibular joint of carrageenan (100 µg/TMJ). 1 h after the TMJ treatments, the rats were terminally anesthetized for joint cavity wash and periarticular tissues collect. Samples analysis demonstrated that PBM inhibit leukocytes chemotaxis in the TMJ and significantly reduces amounts of TNF-α, IL-1ß and CINC-1. In addition, Western blotting analysis demonstrated that PBM significantly decreased the protein levels of P2X3 and P2X7 receptors in the periarticular tissues. On the other hand, PBM was able to increase protein level of IL-10 (anti-inflammatory cytokine). In summary, it is possible to suggest that PBM inhibit inflammatory chemotaxis, modulation the balance of the pro- and anti-inflammatory characteristics of inflammatory cells.

Electroacupuncture inhibits the interaction between peripheral TRPV1 and P2X3 in rats with different pathological pain.

Chronic pain is regarded to be one of the common and refractory diseases to cure in the clinic. One hundred Hz electroacupuncture (EA) is commonly used for inflammatory pain and 2 Hz for neuropathic pain possibly by modulating the transient receptor potential vanilloid subtype 1 (TRPV1) or the purinergic P2X3 related pathways. To clarify the mechanism of EA under various conditions of pathological pain, rats received a subcutaneous administration of complete Freund's adjuvant (CFA) for inflammatory pain and spared nerve injury (SNI) for neuropathic pain. The EA was performed at the bilateral ST36 and BL60 1 d after CFA or SNI being successfully established for 3 consecutive days. The mechanical hyperalgesia test was measured at baseline, 1 d after model establishment, 1 d and 3 d after EA. The co-expression changes, co-immunoprecipitation of TRPV1 and P2X3, and spontaneous pain behaviors (SPB) test were performed 3 d after EA stimulation. One hundred Hz EA or 2Hz EA stimulation could effectively down-regulate the hyperalgesia of CFA or SNI rats. The increased co-expression ratio between TRPV1 and P2X3 at the dorsal root ganglion (DRG) in two types of pain could be reduced by 100Hz or 2Hz EA intervention. While 100Hz or 2Hz EA was not able to eliminate the direct physical interaction between TRPV1 and P2X3. Moreover, EA could significantly inhibit the SPB induced by the co-activation of peripheral TRPV1 and P2X3. All results indicated that EA could significantly reduce the hyperalgesia and the SPB, which was partly related to inhibiting the co-expression and indirect interaction between peripheral TRPV1 and P2X3.

Effects of low- and high-frequency electroacupuncture on protein expression and distribution of TRPV1 and P2X3 in rats with peripheral nerve injury.

BACKGROUND: Whether electroacupuncture (EA) stimulation at different frequencies has a similar effect on spared nerve injury (SNI) as other neuropathic pain models, and how EA at different frequencies causes distinct analgesic effects on neuropathic pain is still not clear. METHODS: Adult male Sprague-Dawley rats were randomly divided into sham SNI, SNI, 2 Hz, 100 Hz and sham EA groups. Paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) were measured. EA was performed once a day on days 1 to 14 after SNI. The expressions of transient receptor potential cation subfamily V member 1 (TRPV1) and peripheral purinergic P2X receptor 3 (P2X3) were determined by western blotting and immunofluorescence. TRPV1 siRNA and P2X3 siRNA were administered by intrathecal injection. TRPV1 or P2X3 agonists were combined with EA. RESULTS: There were significant decreases in PWT, but no changes in PWL in the 14 days after SNI. EA using 2- or 100-Hz stimulation similarly increased PWT at every time point. The cytosol protein expression of P2X3 in the L4-L6 dorsal root ganglia (DRG) increased, but the expression of TRPV1 decreased in the SNI model. Both these effects were ameliorated by EA, with 2-Hz stimulation having a stronger effect than 100-Hz stimulation. Blocking either TRPV1 or P2X3 specific siRNAs attenuated the decreased PWT induced by SNI. Administration of either a TRPV1 or P2X3 agonist inhibited EA analgesia. CONCLUSION: 2- and 100-Hz EA similarly induced analgesic effects in SNI. This effect was related to up-regulation and down-regulation, respectively, of cytosol protein expression of P2X3 and TRPV1 in L4-L6 DRG, with 2 Hz having a better effect than 100 Hz.

Electroacupuncture alleviates diabetic neuropathic pain in rats by suppressing P2X3 receptor expression in dorsal root ganglia.

Diabetic neuropathic pain (DNP) is a troublesome diabetes complication all over the world. P2X3 receptor (P2X3R), a purinergic receptor from dorsal root ganglion (DRG), has important roles in neuropathic pain pathology and nociceptive sensations. Here, we investigated the involvement of DRG P2X3R and the effect of 2 Hz electroacupuncture (EA) on DNP. We monitored the rats' body weight, fasting blood glucose level, paw withdrawal thresholds, and paw withdrawal latency, and evaluated P2X3R expression in DRG. We found that P2X3R expression is upregulated on DNP, while 2 Hz EA is analgesic against DNP and suppresses P2X3R expression in DRG. To evaluate P2X3R involvement in pain modulation, we then treated the animals with A317491, a P2X3R specific antagonist, or α ß-me ATP, a P2X3R agonist. We found that A317491 alleviates hyperalgesia, while α ß-me ATP blocks EA's analgesic effects. Our findings indicated that 2 Hz EA alleviates DNP, possibly by suppressing P2X3R upregulation in DRG.

Therapeutic effects and modulatory mechanism of Alpiniae oxyphyllae Fructus in chronic intermittent hypoxia induced enuresis in rats.

OBJECTIVE: The objective of this study was to explore the effect of Alpiniae oxyphyllae Fructus (AOF) on a rat model of chronic intermittent hypoxia (CIH)-induced enuresis. Findings of this study may help identify therapeutic targets in children with nocturnal enuresis (NE). METHODS: Female rats were randomly divided into a control group (saline gavage, 4 weeks of normal air), CIH group (saline gavage, 4 weeks of CIH), and AOF group (AOF gavage, 4 weeks of CIH). The variables measured in this study included water intake, urine output, bladder leak point pressure (BLPP), malondialdehyde (MDA) levels, and superoxide dismutase (SOD) activity. The expression levels of the purinergic P2X3 receptor, muscarinic M3 receptor, and ß3-adrenergic receptor (ß3-AR) in the bladder were also measured. The bladder was subjected to haematoxylin and eosin (HE) and Weigert staining, and histological changes were observed under a light microscope to evaluate the morphological changes in the bladder in each group. RESULTS: Compared with the control group, urine output was increased, and the BLPP was decreased in the CIH group, but AOF administration decreased urine output and increased BLPP. In addition, the serum MDA level increased and the SOD activity decreased in the CIH group compared with the control group. Administration of AOF decreased the MDA level and increased the SOD activity. Additionally, compared with the control group, HE and Weigert staining in the CIH group showed that the bladder detrusor muscle bundles were disordered and loose, some muscle bundles were broken, the content of collagen fibres in the gap was reduced, and the gap was significantly widened. However, following the administration of AOF, the bladder detrusor muscle bundles were neatly arranged, and the content of collagen fibres in the gap was increased. Furthermore, compared with the control group, the purinergic P2X3 receptor and muscarinic M3 receptor were expressed at higher levels, and ß3-AR was expressed at lower levels in the CIH group, but AOF administration decreased the expression of the purinergic P2X3 receptor and muscarinic M3 receptor and increased the expression of the ß3-AR. CONCLUSIONS: AOF improves enuresis by inhibiting oxidative stress and regulating the expression of the purinergic P2X3 receptor, muscarinic M3 receptor, and ß3 adrenergic receptor.

Exploring the molecular mechanism of the effect of puerarin on P2X3.

P2X3 is a ligand-gated nonselective cation channel and permeable to Na+, K+ and Ca2+. Adenosine triphosphate (ATP) activation of the P2X3 on primary sensory ganglion neurons is involved in nociceptive transmission. Puerarin is a major active ingredient extracted from the traditional Chinese medicine Ge-gen. Puerarin inhibits nociceptive signal transmission by inhibiting the P2X3 in the dorsal root ganglia (DRG) and sympathetic ganglia, but its molecular mechanism is unclear. The aim of this study was to explore the molecular mechanism of puerarin on the P2X3. Here, molecular docking results revealed that puerarin binds well to the human P2X3 protein in the vicinity of the ATP binding pocket. Protein-ligand docking showed that the V64A mutation reduced the effect of puerarin but had little effect on ATP. V64A site-directed mutagenesis of P2X3 was performed using an overlap extension PCR technique. The wild-type and V64A mutant pEGFP-C1-P2X3 recombinant plasmids were transfected into HEK 293 cells. The electrophysiology results demonstrated that puerarin exerted an obvious inhibitory effect on ATP-activated currents in HEK 293 cells transfected with the wild-type P2X3, while little inhibition was observed in HEK 293 cells transfected with the mutant P2X3. These studies suggest that puerarin inhibits the P2X3 by binding to V64A.

DT-0111: a novel drug-candidate for the treatment of COPD and chronic cough.

BACKGROUND: Extracellular adenosine 5'-triphosphate (ATP) plays important mechanistic roles in pulmonary disorders in general and chronic obstructive pulmonary disease (COPD) and cough in particular. The effects of ATP in the lungs are mediated to a large extent by P2X2/3 receptors (P2X2/3R) localized on vagal sensory nerve terminals (both C and Aδ fibers). The activation of these receptors by ATP triggers a pulmonary-pulmonary central reflex, which results in bronchoconstriction and cough, and is also proinflammatory due to the release of neuropeptides from these nerve terminals via the axon reflex. These actions of ATP in the lungs constitute a strong rationale for the development of a new class of drugs targeting P2X2/3R. DT-0111 is a novel, small, water-soluble molecule that acts as an antagonist at P2X2/3R sites. METHODS: Experiments using receptor-binding functional assays, rat nodose ganglionic cells, perfused innervated guinea pig lung preparation ex vivo, and anesthetized and conscious guinea pigs in vivo were performed. RESULTS: DT-0111 acted as a selective and effective antagonist at P2X2/3R, that is, it did not activate or block P2YR; markedly inhibited the activation by ATP of nodose pulmonary vagal afferents in vitro; and, given as an aerosol, inhibited aerosolized ATP-induced bronchoconstriction and cough in vivo. CONCLUSIONS: These results indicate that DT-0111 is an attractive drug-candidate for the treatment of COPD and chronic cough, both of which still constitute major unmet clinical needs. The reviews of this paper are available via the supplementary material section.

Electroacupuncture Stimulation Alleviates CFA-Induced Inflammatory Pain Via Suppressing P2X3 Expression.

Chronic inflammatory pain is one of the most common complaints that seriously affects patients' quality of life. Previous studies have demonstrated that the analgesic effect of electroacupuncture (EA) stimulation on inflammatory pain is related to its frequency. In this study, we focused on whether the analgesic effects of EA are related to the period of stimulation. Purinergic receptor P2X3 (P2X3) is involved in the pathological process underlying chronic inflammatory pain and neuropathic pain. We hypothesized that 100 Hz EA stimulation alleviated Freund's complete adjuvant (CFA) induced inflammatory pain via regulating P2X3 expression in the dorsal root ganglion (DRG) and/or spinal cord dorsal horn (SCDH). We also assumed that the analgesic effect of EA might be related to the period of stimulation. We found that both short-term (three day) and long-term (14 day) 100 Hz EA stimulation effectively increased the paw withdrawal threshold (PWT) and reversed the elevation of P2X3 in the DRG and SCDH of CFA rats. However, the analgesic effects of 100 Hz EA were not dependent on the period of stimulation. Moreover, P2X3 inhibition or activation may contribute to or attenuate the analgesic effects of 100 Hz EA on CFA-induced inflammatory pain. This result indicated that EA reduced pain hypersensitivity through P2X3 modulation.

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.