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Role of sodium ferulate in the nociceptive sensory facilitation of neuropathic pain injury mediated by P2X3 receptor
Neurochemistry International 53 (2008) 278–282
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Neurochemistry International journal homepage: www.elsevier.com/locate/neuint
Role of sodium ferulate in the nociceptive sensory facilitation of neuropathic pain injury mediated by P2X3 receptor Aixia Zhang 1, Changshui Xu 1, Shangdong Liang *, Yun Gao, Guilin Li, Jie Wei, Fang Wan, Shuangmei Liu, Jiari Lin Department of Physiology, Medical College of Nanchang University, Bayi Road 461, Nanchang 330006, PR China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 25 August 2008 Accepted 25 August 2008 Available online 30 August 2008
Neuropathic pain usually is persistent and no effective treatment. ATP plays an important role in the initiation of pain. P2X3 receptors are localized in the dorsal root ganglion (DRG) neurons and activated by extracellular ATP. Sodium ferulate (SF) is an active principle from Chinese herbal medicine and has antiinflammatory activities. This study observed the effects of SF on the nociceptive facilitation of the primary sensory afferent after chronic constriction injury (CCI) mediated by P2X3 receptor. In this study, the content of ATP in DRG neurons was measured by high-performance liquid chromatography (HPLC). P2X3 agonist-activated currents in DRG neurons was recorded by the whole-cell patch-clamp skill. The expression of P2X3 mRNA in DRG neurons was analyzed by in situ hybridization. The ATP content of DRG was increased after CCI. In CCI rats treated with SF, the content of ATP in DRG neurons was reduced. SF decreased the increment of P2X3 agonist-activated currents and P2X3 mRNA expression in DRG neurons during CCI. SF may inhibit the initiation of pain and primary afferent sensitization mediated by P2X3 receptor during CCI. ß 2008 Elsevier Ltd. All rights reserved.
Keywords: ATP P2X3 receptor Sodium ferulate Dorsal root ganglia Neuropathic pain
1. Introductory statement Purine receptors have been widely implicated in nociceptive processing (Burnstock, 2000; Khakh, 2001; Liu and Salter, 2005). Adenosine-50 -triphosphate (ATP) activates cation-permeable ion channels (P2X receptors) and G-protein-coupled receptors (P2Y receptors) (Burnstock, 2007; Lecca and Abbracchio, 2008). To date, 7 members of the P2X receptor family (P2X1-7R) have been cloned (Burnstock, 2007; Go´mez-Villafuertes et al., 2007). It has been postulated that the P2X3 receptor in sensory neurons plays a role in pain transmission (Burnstock, 2000). P2X3 receptor is most highly expressed in a subpopulation of small diameter primary afferent neurons in the pain pathway. P2X3 receptor plays a facilitatory role in nociception (Chizh and Illes, 2001; Jarvis, 2003; Kennedy, 2005; Kennedy et al., 2003; North, 2003). A human study applying ATP to the skin (Hamilton et al., 2001) and in vivo physiological studies using P2X receptor agonists and antagonists have suggested that activation of P2X3 by ATP depolarizes nociceptive neurons to evoke a sensation of pain (Bland-Ward and Humphrey, 2000; Jarvis et al., 2001).
Downregulation of P2X3 expression using antisense oligonucleotides or siRNA in rats (Dorn et al., 2004; Honore et al., 2002; Barclay et al., 2002) and disruption of the P2X3 gene in mice (Cockayne et al., 2000; Souslova et al., 2000; Zhong et al., 2001) have also strongly supported a pronociceptive role for this receptor, particularly after inflammation or injury. Sodium ferulate (SF) or 3-methoxy-4-hydroxy-cinamate sodium is an active principle from Chinese herbal medicine Angelica sinensis, Cimicifuga heracleifolia, Lignsticum chuangxiong. It has been used for treatment of cardiovascular and cerebrovascular diseases (Liu, 2005; Wang and Ou-Yang, 2005). SF has antithrombotic, platelet aggregation inhibitory activities in animals and humans. SF also expresses antioxidant and anti-inflammatory activities (Fang et al., 2006; Liu, 2005). In the present study, we investigated purinoceptors as the pharmacological target for the therapeutic treatment of neuropathic pain injury and characterized the effects of SF on the nociceptive facilitation of the primary sensory afferent after chronic constriction injury (CCI) mediated by P2X3 receptor. 2. Materials and methods 2.1. Animals and drugs
* Corresponding author. E-mail address: [email protected] (S. Liang). 1 Joint first authors. 0197-0186/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2008.08.008
Male Sprague–Dawley rats (180–230 g) were provided by the Center of Laboratory Animal Science of Nanchang University. Use of the animals was reviewed and approved by the Animal Care and Use Committee of Medical College of Nanchang University. The IASP’s ethical guidelines for pain research in animals
A. Zhang et al. / Neurochemistry International 53 (2008) 278–282 were followed. The animals were housed in plastic boxes in a group of three at 21– 25 8C. Rats were divided into 5 groups randomly. There are 6 rats in each group for every experimental method. The chronic constriction injury rat model was as the neuropathy pain model (Bennett and Xie, 1988; Novakovic et al., 1999). Five groups were group I [sham + normal salin (NS) group], II (sham group), III (SF group), IV (CCI + SF group) and V (CCI group) respectively. SF (Suzhou Lixin Medical Company) was administrated by intraperitoneal injection in group IV after CCI operation (50 mg/kg) or group III. Adenosine 50 -triphosphate disodium (ATP) and a,b-methylen-ATP (a,b-meATP) were obtained from Sigma. All drugs were dissolved and diluted in 0.9% saline. 2.2. Chronic constriction injury model We prepared the CCI model as a neuropathy pain model (Bennett and Xie, 1988; Gao et al., 2008; Novakovic et al., 1999). Each rat was anesthetized with penthiobarbital sodium (Shanghai Xingya Medical Company) during surgical procedures. The sciatic nerve was exposed at the middle level of rat thigh. Proximal to the sciatic trifurcation, four ligatures (4–0 chromic gut) were performed loosely with microsurgical techniques. Intervals among ligatures were about 1 mm. The same investigator created CCI animals to avoid variation. In the sham-operated group, the sciatic nerve was exposed and was not ligatured by chromic gut.
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H2O2, followed by digestion with pepsin at 37 8C for 1–2 min, terminated with 0.5 m mol/L phosphate-buffered saline (PBS) and washed with it for 15 min. Then the preparations were incubated in prehybridization solution for 2 h at 37 8C and in hybridization solution overnight at 37 8C. To remove the background signals, the preparations were washed with gradient saline sodium citrate (SSC) (20 SSC 17.6 g sodium chloride, 8.8 g sodium citrate in 1000 ml distilled water) thoroughly, namely 2 SSC for 10 min, 0.5 SSC for 15 min and 0.2 SSC for 15 min, and followed by treatment with biotinylated digoxin antibody for 2 h at 37 8C. After strongly washed with PBS, the preparations were incubated with SABC-POD for 30 min and with biotinylated peroxidase for 30 min at 37 8C. The color was developed in DAB substrate, then dehydrated and mounted with neutral gum. The in situ hybridization kit for P2X3 receptor was purchased from Boster (Wuhan in China). 3,30 -Diaminobenzidine (DAB) was purchased from ZhongShan (Beijing in China). The gray scale of P2X3 receptor expression was determines with an image analysis system (HMIAS-2000). 2.7. Statistical analysis Data for the experiments are expressed as mean S.E. Single factor analysis of variance (ANOVA) was used. The significance was assured when the p-value was less than 0.05.
2.3. Measurement of ATP Sprague–Dawley (SD) rats were killed by CO2 inhalation. Rats were randomly divided into sham + normal salin group (n = 5), SF group (n = 5), sham group (n = 5), CCI + SF group (n = 5) and CCI group (n = 5). The dorsal root ganglia from different groups were rapidly excised and put in liquid nitrogen. The ganglion was taken in perchloric acid for homogenate and centrifuged for 10 min at 4000 g in a centrifuge (at 4 8C). The pellet was then removed and the supernatant was neutralised with KOH 3 M + Tris 1.5 M and then centrifuged at 4000 g for another 10 min. The supernatant was measured by high-performance liquid chromatography (HPLC: LC-10Atvp, Japan) combined with ultraviolet detection (UV: SPD-10Avp, Japan). 2.4. Isolation of DRG neurons Sprague–Dawley rats, 2–3 weeks old, of both sexes were decapitated after anesthetized with urethane [1.2 g/kg, intraperitoneally (i.p.)]. The thoracic and lumbar segments of the vertebral column were dissected and longitudinally divided into two halves along the median lines on both dorsal and ventral sides. The L4 and L5 lumbar DRGs together with dorsal and ventral roots and attached spinal nerves were taken out from the inner side of each half of the dissected vertebrae with fine dissecting forceps and transferred immediately into Dubecco’s modified Eagle’s medium (DMEM, Sigma) at pH 7.4 and 340 mOsmol/kg. After the removal of attached nerves and surrounding connective tissues, the DRGs were minced with dissecting spring scissors and incubated with trypsin (0.5 mg/ml; type III, Sigma), collagenase (1.0 mg/ml; type IA, sigma) and Dnase (0.1 mg/ml; type IV, sigma) in 5 ml DMEM at 35 8C in a shaking bath for 35–40 min, after which soybean trypsin inhibitor (1.25 mg/ml; type II-S, sigma) was added to stop the enzymatic digestion. The isolated neurons were transferred into a 35-mm culture dish and kept still for 30 min. Experiments were performed at room temperature (20–30 8C) [15,17].
3. Results 3.1. Effects of sodium ferulate on the ATP content in DRG of CCI rats The content of ATP was measured by HPLC from the DRG in group I (sham + normal salin group), II (sham group), III (SF group), IV (CCI + SF group) and V (CCI group). The data were 22.73 2.34 mg/g, 22.09 1.18 mg/g, 21.96 1.70 mg/g, 24.44 1.20 mg/g, 40.57 1.25 mg/g ganglion wet weight mean from above five group, respectively. The content of ATP in group V (CCI group) was higher than those in group I (sham + normal salin group), II (SF group), III (sham group), IV (CCI + SF group) [F(3.51) = 20.93, p < 0.01] (Fig. 1). No significant difference in the content of ATP had been observed in group I (sham + normal salin group) compared with those in group II (SF group), group III (sham group), or group IV (CCI + SF group) (p > 0.05). 3.2. Inhibition of sodium ferulate on P2X agonist-activated currents potentiated by CCI The majority of the DRG neurons isolated from group I (sham + normal salin group) 85.0% (17/20), group II (sham group) 81.2% (13/16), group III (SF group) 73.9% (17/23), group IV (CCI + SF group) 75.0% (15/20) and group V (CCI group) 72.0% (13/18) were responded to the external application of ATP (1–1000 mM) in a
2.5. Electrophysiological recordings The whole-cell patch-clamp recording (Liang et al., 2005) was carried out using a patch/whole-cell clamp amplifer (CEZ-2400, Nihon Kohden). The micropipette was filled with internal solution containing (in mM): KCl 140, MgCl2 2, HEPES 10, EGTA 11, ATP 5; its osmolarity was adjusted to 340 mOsmol/kg with sucrose and pH adjusted to 7.4 with KOH. The external solution contained (mM): NaCl 150, KCl 5, CaCl2 2.5, MgCl2 1, HEPES 10, D-glucose 10; its osmolarity was adjusted to 340 mOsmol with sucrose, pH was adjusted to 7.4 with NaOH. The resistance of recording electrodes were in the range of 1–4 MV. A small patch of membrane underneath the tip of the pipette was aspirated to form a seal (1–10 GV) and then a more negative pressure was applied to rupture it, thus a whole-cell mode was established. Membrane currents were filtered at 1 kHz (3 dB), data were recorded by a pen recorder (LMS-2B, Chengdu). The holding potential was set at 60 mV. The drugs were dissolved in external solution and delivered by gravity flow from an array of tubules (500 mm O.D., 200 mm I.D.) connected to a series of independent reservoirs. The distance from the tubule mouth to the cell examined was approximately 100 mm. Rapid solution-exchange was achieved by shifting the tubules horizontally with a micromanipulator. 2.6. In situ hybridization (ISH) The L4 and L5 lumbar DRG were dissected immediately and fixed in 4% paraformaldehyde for 2 h at room temperature, then transferred to 15% sucrose in 4% PFA overnight. Ganglia were sectioned at 15 mm with a cryostat and stored in 4% PFA at 4 8C until used. Diethyl pyrocarbonate (DEPC) water was used for all solutions and appliances necessary for ISH. Preparations were treated with 0.5%
Fig. 1. Effects of sodium ferulate on the ATP content of DRG after CCI. The bar histograms showed the content of ATP measured by HPLC from the DRG in group I (sham + normal salin group), II (sham group), III (SF group), IV (CCI + SF group) and V (CCI group). The content of ATP in group V (CCI group) was higher than those in group I (sham + normal salin group), II (SF group), III (sham group), or IV (CCI + SF group) [F(3.51) = 20.93, p < 0.01]. Compared with the results in other groups, the significant difference in group V was showed with asterisk. No significant difference in the content of ATP had been observed compared group IV (CCI + SF group) with group I (sham + normal salin group), group II (SF group), or group III (sham group) (p > 0.05).
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concentration-dependent manner with an inward currents (IATP). The amplitudes of the currents in group V (CCI) were highest compared with those obtained in other groups after application of same concentration ATP (100 mM, P < 0.01) (Fig. 2). The amplitudes of IATP in group IV were reduced by SF administrated by intraperitoneal injection. The amplitudes of IATP in group IV compared with those in group I, group II, or group III were no significant difference (P > 0.05). P2X3 selective agonist a,b-meATP (0.3–100 mM) could induce the currents in DRG neurons. The responses in DRG neurons of CCI rats were more higher than those obtained in other groups (P < 0.01). The dose-response curves for a,b-meATP-activated current in different groups are showed in Fig. 3. The amplitude of a,b-meATP-activated currents in group IV was reduced by SF administrated by intraperitoneal injection. The amplitude of a,bmeATP-activated currents in group IV compared with those in group I, group II or group III was no significant difference (P > 0.05) (Fig. 3). 3.3. Effect of sodium ferulate on the expression of P2X3 mRNA in DRG of CCI rats The expression of P2X3 mRNA was tested by ISH within the L4 and L5 DRG neurons. The stain values of P2X3 mRNA expression
were 109.25 4.37 (group I), 108.48 2.91 (group II), 108.48 5.05 (group III), 110.41 4.37 (group IV) and 129.74 4.37 (group V), respectively (n = 6 for each group). The gray scale of P2X3 receptor expression in group V was significantly higher than those in group I, group II, group III, or group IV [p < 0.01, F(4.94) = 20.53] (Fig. 4). No significantly difference was found in the intensity of P2X3 mRNA expression compared DRG neurons of group IV with DRG neurons of group I, group II, or group III (p > 0.05). Thus, SF reduced P2X3 mRNA expression in DRG neurons enhanced by CCI. 4. Discussion Neuropathic pain due to nerve injury is a prevalent condition, for which currently there is no effective treatment (Saarto and Wiffen, 2005; Tha´n et al., 2007). It is a condition that is often refractory to opioids or requires larger doses that possess unacceptable side effects (Przewlocki and Przewlocka, 2005). Neuropathic pain usually is persistent, and nerve injury can produce sensory/motor deficits. Several rodent models of neuropathic pain have been used to explore the mechanisms and treatment of neuropathic pain states (Campbell and Meyer, 2006). Among these models, the sciatic nerve chronic constriction injury has been widely used, as it produces robust
Fig. 2. The inhibition of sodium ferulate on P2X agonist-activated currents potentiated by CCI, P2X agonist- (ATP or a, b-meATP) activated currents in the DRG neurons. The current traces were obtained from the same cell. ATP-activated currents were concentration-dependent. (A) Current traces showed P2X agonist-activated currents in the DRG neurons of group I (sham + normal salin group); (B) current traces showed P2X agonist-activated currents in the DRG neurons of group II (sham group); (C) current traces showed P2X agonist-activated currents in the DRG neurons of group III (SF group); (D) current traces showed P2X agonist-activated currents in the DRG neurons of group IV (CCI + SF group). SF administrated by intraperitoneal injection reduced the amplitudes of IATP in group IV; (E) current traces showed P2X agonist-activated currents in the DRG neurons of group V (CCI group). The amplitude of the currents in group V (CCI) was highest compared with those obtained in other groups after application of same concentration ATP (100 mM, P < 0.01).
A. Zhang et al. / Neurochemistry International 53 (2008) 278–282
Fig. 3. The dose-response curve of a,b-meATP-activated currents with or without pretreatment of SF. The graph shows that the dose-response curves for a,b-meATP (0.3–100 mM)-activated current in DRG neurons. The responses in DRG neurons of group V were more higher than those obtained in other groups (P < 0.01). Compared with other group, the significant difference in group V was showed with asterisk. The amplitude of a,b-meATP-activated currents after CCI was reduced by SF administrated by intraperitoneal injection. The amplitude of a,b-meATPactivated currents in group IV compared with those in group I, group II or group III was no significant difference (P > 0.05).
Wallerian degeneration with additional inflammation (Bennett and Xie, 1988). P2X receptors are localized in the central and peripheral nervous system. P2X receptors are ion channels that were activated by extracellular ATP (Burnstock, 2007; North, 2002) and play the important role in excitatory nociceptive processing (Liu and Salter, 2005; Burnstock, 2006). ATP released from the damaged peripheral nerve tissue might contribute to initiate neuron activation. As a result of cell damage, ATP plays an important role in the initiation of pain and in primary afferent sensitization after different kinds of acute nervous system injury
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(Agteresch et al., 1999; Schwiebert, 2000). A participation of extracellular ATP and P2 receptors in neuropathic pain has been reported (Inoue, 2006; North, 2003). The ATP content of DRG in CCI group was higher than those in other group. It was indicated that sensory impulses generated during nerve injury may increase the release of endogenous ATP. The content of ATP in CCI rats treated with SF was lower than that in CCI rats. SF may reduce the content of ATP in damaged DRG neuron during CCI. The somata of DRG neurons actively releases transmitters and play a crucial role in bidirectional communication between neurons and surrounding satellite glial cells. The neuronal somata also has a significant role in cell–cell signaling. P2X3 receptor agonist ATP and a,b-meATP can activate currents in DRG neurons. P2X3 receptor may be sensitized following tissue injury or inflammation. Our works showed that the amplitude of currents in DRG neurons of CCI rats was much larger than those in other group after application of same concentration ATP and a,bmeATP. In CCI rats treated with SF, P2X3 receptor agonist-activated currents were reduced. SF may decreased the sensitization of P2X3 receptor in DRG neurons of CCI rats. The sciatic nerve injury by chronic constriction results in upregulation of P2X3 receptor expression on DRG neurons (Burnstock, 2006; Novakovic et al., 1999). In our study, the expression of P2X3 mRNA in DRG neurons was increased after CCI. The results suggested that the nociceptive stimuli of neuropathic pain injury increased P2X3 receptor expression and enhanced P2X3 receptor sensitivity. SF administrated by intraperitoneal injection decreased the expression of P2X3 mRNA in CCI rats. SF might block primary afferent transmission mediated by P2X3 receptor and inhibit the activation of P2X3 receptor during chronic pain. It is possible that purinoceptors as the pharmacological target for the therapeutic treatment of neuropathic pain injury. The results indicate SF may antagonize P2X3 receptor to alleviate the chronic neuropathic pain injury. The main finding of our study is that SF reduced the content of ATP generated from sensory neurons during nerve injury, the expression of P2X3 and the sensitization of P2X3 receptor in DRG neurons of CCI rats; the effects were shown SF may inhibit primary afferent facilitation mediated by P2X3 receptor during CCI.
Fig. 4. The effects of sodium ferulate P2X3 mRNA expression in DRG neurons after CCI measured by in situ hybridization. Photos of P2X3 mRNA expression in L4/L5 DRG neurons measured by in situ hybridization (n = 6 each group). (A) The expression of P2X3 mRNA in L4/L5 DRG neurons of group I; (B) the expression of P2X3 mRNA in L4/L5 DRG neurons of group II; (C) the expression of P2X3 mRNA in L4/L5 DRG neurons of group III; (D) the expression of P2X3 mRNA in L4/L5 DRG neurons of group IV. (E) The expression of P2X3 mRNA in L4/L5 DRG neurons of group V (arrows indicate the immunostaining neurons; scale bars, 50 mm).
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This work was supported by grant (No. 30860086, 30460040 and 30660048) from National Natural Science Foundation of China and grant (No. 0640042) from Natural Science Foundation of Jiangxi Province to Dr. Shangdong Liang. References Agteresch, H.J., Dagnelie, P.C., van der Berg, J.W., Wilson, J.H., 1999. Adenosine triphosphate: established and potential clinical applications. Drugs 58, 211– 232. Barclay, J., Patel, S., Dorn, G., Wotherspoon, G., Moffatt, S., Eunson, L., Abdel’al, S., Natt, F., Hall, J., Winter, J., Bevan, S., Wishart, W., Fox, A., Ganju, P., 2002. Functional downregulation of P2X3 receptor subunit in rat sensory neurons reveals a significant role in chronic neuropathic and inflammatory pain. J. Neurosci. 22, 8139–8147. Bennett, G.J., Xie, Y.K., 1988. A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man. Pain 33, 87–107. Bland-Ward, P.A., Humphrey, P.P., 2000. P2X receptors mediate ATP-induced primary nociceptive neuron activation. J. Auton. Nerv. Syst. 81, 146–151. Burnstock, G., 2000. P2X receptors in sensory neurons. Br. J. Anaesth. 84, 476–488. Burnstock, G., 2007. Physiology and pathophysiology of purinergic neurotransmission. Physiol. Rev. 87, 659–797. Burnstock, G., 2006. Purinergic P2 receptors as targets for novel analgesics. Pharmacol. Ther. 110, 433–454. Campbell, J.N., Meyer, R.A., 2006. Mechanisms of neuropathic pain. Neuron 52, 77–92. Chizh, B.A., Illes, P., 2001. P2X receptors and nociception. Pharmacol. Rev. 53, 553– 568. Cockayne, D.A., Hamilton, S.G., Zhu, Q.M., Dunn, P.M., Zhong, Y., Novakovic, S., Malmberg, A.B., Cain, G., Berson, A., Kassotakis, L., Hedley, L., Lachnit, W.G., Burnstock, G., McMahon, S.B., Ford, A.P., 2000. Urinary bladder hyporeflexia and reduced pain-related behaviour in P2X3-deficient mice. Nature 407, 1011–1015. Dorn, G., Patel, S., Wotherspoon, G., Hemmings-Mieszczak, M., Barclay, J., Natt, F.J., Martin, P., Bevan, S., Fox, A., Ganju, P., Wishart, W., Hall, J., 2004. siRNA relieves chronic neuropathic pain. Nucleic Acids Res. 32, e49. Fang, C.H., Liu, S.P., Dong, W.G., Yu, J.P., 2006. Effects of sodium ferulate on function of macrophages in the colonic tissue of colitis rats. Chin. J. Gastroenterol. Hepatol. 15, 116–118. Gao, Y., Xu, C.S., Liang, S.D., Mu, S.N., Zhang, A.X., Wang, Y.X., Fang Wan, F., 2008. Effect of tetramethylpyrazine on primary afferent transmission mediated by P2X3 receptor in neuropathic pain states. Brain Res. Bull. 77, 27–32. Go´mez-Villafuertes, R., Sa´nchez-Nogueiro, J., Marı´n-Garcı´a, P., Miras-Portugal, M.T., 2007. Existence of high and low affinity dinucleotides pentaphosphate-induced calcium responses in individual synaptic terminals and lack of correlation with the distribution of P2X1-7 subunits. Neurochem. Int. 50, 628–641. Hamilton, S.G., McMahon, S.B., Lewin, G.R., 2001. Selective activation of nociceptors by P2X receptor agonists in normal and inflamed rat skin. J. Physiol. 534, 437–445. Honore, P., Kage, K., Mikusa, J., Watt, A.T., Johnston, J.F., Wyatt, J.R., Faltynek, C.R., Jarvis, M.F., Lynch, K., 2002. Analgesic profile of intrathecal P2X(3) antisense oligonucleotide treatment in chronic inflammatory and neuropathic pain states in rats. Pain 99, 11–19.
Inoue, K., 2006. The function of microglia through purinergic receptors: neuropathic pain and cytokine release. Pharmacol. Ther. 109, 210–226. Jarvis, M.F., Wismer, C.T., Schweitzer, E., Yu, H., van Biesen, T., Lynch, K.J., Burgard, E.C., Kowaluk, E.A., 2001. Modulation of BzATP and formalin induced nociception: attenuation by the P2X receptor antagonist, TNP-ATP and enhancement by the P2X(3) allosteric modulator, cibacron blue. Br. J. Pharmacol. 132, 259–269. Jarvis, M.F., 2003. Contributions of P2X3 homomeric and heteromeric channels to acute and chronic pain. Expert Opin. Ther. Targets 7, 513–522. Kennedy, C., 2005. P2X receptors: targets for novel analgesics? Neuroscientist 11, 345–356. Kennedy, C., Assis, T.S., Currie, A.J., Rowan, E.G., 2003. Crossing the pain barrier: P2 receptors as targets for novel analgesics. J. Physiol. 553, 683–694. Khakh, B.S., 2001. Molecular physiology of P2X receptors and ATP signalling at synapses. Nat. Rev. Neurosci. 2, 165–174. Lecca, D., Abbracchio, M.P., 2008. Deorphanisation of G protein-coupled receptors: a tool to provide new insights in nervous system pathophysiology and new targets for psycho-active drugs. Neurochem. Int. 52, 339–351. Liang, S.D., Xu, C.S., Zhou, T., Liu, H.Q., Gao, Y., Li, G.L., 2005. Tetramethylpyrazine inhibits ATP-activated currents in rat dorsal root ganglion neurons. Brain Res. 1040, 92–97. Liu, H.R., 2005. Pharmacological action and clinical application of sodium ferulate. China Pharm. 14, 78–79. Liu, X.J., Salter, M.W., 2005. Purines and pain mechanisms: recent developments. Curr. Opin. Investig. Drugs. 6, 65–75. North, R.A., 2002. Molecular physiology of P2X receptors. Physiol. Rev. 82, 1013– 1067. North, R.A., 2003. The P2X3 subunit: a molecular target in pain therapeutics. Curr. Opin. Investig. Drugs 4, 833–840. Novakovic, S.D., Kassotakis, L.C., Oglesby, I.B., Smith, J.A., Eglen, R.M., Ford, A.P., Hunter, J.C., 1999. Immunocytochemical localization of P2X3 purinoceptors in sensory neurons in naive rats and following neuropathic injury. Pain 80, 273– 282. Przewlocki, R., Przewlocka, B., 2005. Opioids in neuropathic pain. Curr. Pharm. Des. 11, 3013–3025. Saarto, T., Wiffen, P.J., 2005. Antidepressants for neuropathic pain. Cochrane Database Syst. Rev. 3 CD005454. Schwiebert, E.M., 2000. Extracellular ATP-mediated propagation of Ca2+ waves. Focus on ‘‘mechanical strain-induced Ca2+ waves are propagated via ATP release and purinergic receptor activation’’ Am. J. Physiol. Cell Physiol. 279, C281–C283. Souslova, V., Cesare, P., Ding, Y., Akopian, A.N., Stanfa, L., Suzuki, R., Carpenter, K., Dickenson, A., Boyce, S., Hill, R., Nebenuis-Oosthuizen, D., Smith, A.J., Kidd, E.J., Wood, J.N., 2000. Warm-coding deficits and aberrant inflammatory pain in mice lacking P2X3 receptors. Nature 407, 1015–1017. Tha´n, M., Kocsis, P., Tihanyi, K., Fodor, L., Farkas, B., Kova´cs, G., Kis-Varga, A., Szombathelyi, Z., Tarnawa, I., 2007. Concerted action of antiepileptic and antidepressant agents to depress spinal neurotransmission: possible use in the therapy of spasticity and chronic pain. Neurochem. Int. 50, 642–652. Wang, B.H., Ou-Yang, J.P., 2005. Pharmacological actions of sodium ferulate in cardiovascular system. Cardiovasc. Drug Rev. 23, 161–172. Zhong, Y., Dunn, P.M., Bardini, M., Ford, A.P., Cockayne, D.A., Burnstock, G., 2001. Changes in P2X receptor responses of sensory neurons from P2X3-deficient mice. Eur. J. Neurosci. 14, 1784–1792.
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Neurochemistry International journal homepage: www.elsevier.com/locate/neuint
Role of sodium ferulate in the nociceptive sensory facilitation of neuropathic pain injury mediated by P2X3 receptor Aixia Zhang 1, Changshui Xu 1, Shangdong Liang *, Yun Gao, Guilin Li, Jie Wei, Fang Wan, Shuangmei Liu, Jiari Lin Department of Physiology, Medical College of Nanchang University, Bayi Road 461, Nanchang 330006, PR China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 25 August 2008 Accepted 25 August 2008 Available online 30 August 2008
Neuropathic pain usually is persistent and no effective treatment. ATP plays an important role in the initiation of pain. P2X3 receptors are localized in the dorsal root ganglion (DRG) neurons and activated by extracellular ATP. Sodium ferulate (SF) is an active principle from Chinese herbal medicine and has antiinflammatory activities. This study observed the effects of SF on the nociceptive facilitation of the primary sensory afferent after chronic constriction injury (CCI) mediated by P2X3 receptor. In this study, the content of ATP in DRG neurons was measured by high-performance liquid chromatography (HPLC). P2X3 agonist-activated currents in DRG neurons was recorded by the whole-cell patch-clamp skill. The expression of P2X3 mRNA in DRG neurons was analyzed by in situ hybridization. The ATP content of DRG was increased after CCI. In CCI rats treated with SF, the content of ATP in DRG neurons was reduced. SF decreased the increment of P2X3 agonist-activated currents and P2X3 mRNA expression in DRG neurons during CCI. SF may inhibit the initiation of pain and primary afferent sensitization mediated by P2X3 receptor during CCI. ß 2008 Elsevier Ltd. All rights reserved.
Keywords: ATP P2X3 receptor Sodium ferulate Dorsal root ganglia Neuropathic pain
1. Introductory statement Purine receptors have been widely implicated in nociceptive processing (Burnstock, 2000; Khakh, 2001; Liu and Salter, 2005). Adenosine-50 -triphosphate (ATP) activates cation-permeable ion channels (P2X receptors) and G-protein-coupled receptors (P2Y receptors) (Burnstock, 2007; Lecca and Abbracchio, 2008). To date, 7 members of the P2X receptor family (P2X1-7R) have been cloned (Burnstock, 2007; Go´mez-Villafuertes et al., 2007). It has been postulated that the P2X3 receptor in sensory neurons plays a role in pain transmission (Burnstock, 2000). P2X3 receptor is most highly expressed in a subpopulation of small diameter primary afferent neurons in the pain pathway. P2X3 receptor plays a facilitatory role in nociception (Chizh and Illes, 2001; Jarvis, 2003; Kennedy, 2005; Kennedy et al., 2003; North, 2003). A human study applying ATP to the skin (Hamilton et al., 2001) and in vivo physiological studies using P2X receptor agonists and antagonists have suggested that activation of P2X3 by ATP depolarizes nociceptive neurons to evoke a sensation of pain (Bland-Ward and Humphrey, 2000; Jarvis et al., 2001).
Downregulation of P2X3 expression using antisense oligonucleotides or siRNA in rats (Dorn et al., 2004; Honore et al., 2002; Barclay et al., 2002) and disruption of the P2X3 gene in mice (Cockayne et al., 2000; Souslova et al., 2000; Zhong et al., 2001) have also strongly supported a pronociceptive role for this receptor, particularly after inflammation or injury. Sodium ferulate (SF) or 3-methoxy-4-hydroxy-cinamate sodium is an active principle from Chinese herbal medicine Angelica sinensis, Cimicifuga heracleifolia, Lignsticum chuangxiong. It has been used for treatment of cardiovascular and cerebrovascular diseases (Liu, 2005; Wang and Ou-Yang, 2005). SF has antithrombotic, platelet aggregation inhibitory activities in animals and humans. SF also expresses antioxidant and anti-inflammatory activities (Fang et al., 2006; Liu, 2005). In the present study, we investigated purinoceptors as the pharmacological target for the therapeutic treatment of neuropathic pain injury and characterized the effects of SF on the nociceptive facilitation of the primary sensory afferent after chronic constriction injury (CCI) mediated by P2X3 receptor. 2. Materials and methods 2.1. Animals and drugs
* Corresponding author. E-mail address: [email protected] (S. Liang). 1 Joint first authors. 0197-0186/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2008.08.008
Male Sprague–Dawley rats (180–230 g) were provided by the Center of Laboratory Animal Science of Nanchang University. Use of the animals was reviewed and approved by the Animal Care and Use Committee of Medical College of Nanchang University. The IASP’s ethical guidelines for pain research in animals
A. Zhang et al. / Neurochemistry International 53 (2008) 278–282 were followed. The animals were housed in plastic boxes in a group of three at 21– 25 8C. Rats were divided into 5 groups randomly. There are 6 rats in each group for every experimental method. The chronic constriction injury rat model was as the neuropathy pain model (Bennett and Xie, 1988; Novakovic et al., 1999). Five groups were group I [sham + normal salin (NS) group], II (sham group), III (SF group), IV (CCI + SF group) and V (CCI group) respectively. SF (Suzhou Lixin Medical Company) was administrated by intraperitoneal injection in group IV after CCI operation (50 mg/kg) or group III. Adenosine 50 -triphosphate disodium (ATP) and a,b-methylen-ATP (a,b-meATP) were obtained from Sigma. All drugs were dissolved and diluted in 0.9% saline. 2.2. Chronic constriction injury model We prepared the CCI model as a neuropathy pain model (Bennett and Xie, 1988; Gao et al., 2008; Novakovic et al., 1999). Each rat was anesthetized with penthiobarbital sodium (Shanghai Xingya Medical Company) during surgical procedures. The sciatic nerve was exposed at the middle level of rat thigh. Proximal to the sciatic trifurcation, four ligatures (4–0 chromic gut) were performed loosely with microsurgical techniques. Intervals among ligatures were about 1 mm. The same investigator created CCI animals to avoid variation. In the sham-operated group, the sciatic nerve was exposed and was not ligatured by chromic gut.
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H2O2, followed by digestion with pepsin at 37 8C for 1–2 min, terminated with 0.5 m mol/L phosphate-buffered saline (PBS) and washed with it for 15 min. Then the preparations were incubated in prehybridization solution for 2 h at 37 8C and in hybridization solution overnight at 37 8C. To remove the background signals, the preparations were washed with gradient saline sodium citrate (SSC) (20 SSC 17.6 g sodium chloride, 8.8 g sodium citrate in 1000 ml distilled water) thoroughly, namely 2 SSC for 10 min, 0.5 SSC for 15 min and 0.2 SSC for 15 min, and followed by treatment with biotinylated digoxin antibody for 2 h at 37 8C. After strongly washed with PBS, the preparations were incubated with SABC-POD for 30 min and with biotinylated peroxidase for 30 min at 37 8C. The color was developed in DAB substrate, then dehydrated and mounted with neutral gum. The in situ hybridization kit for P2X3 receptor was purchased from Boster (Wuhan in China). 3,30 -Diaminobenzidine (DAB) was purchased from ZhongShan (Beijing in China). The gray scale of P2X3 receptor expression was determines with an image analysis system (HMIAS-2000). 2.7. Statistical analysis Data for the experiments are expressed as mean S.E. Single factor analysis of variance (ANOVA) was used. The significance was assured when the p-value was less than 0.05.
2.3. Measurement of ATP Sprague–Dawley (SD) rats were killed by CO2 inhalation. Rats were randomly divided into sham + normal salin group (n = 5), SF group (n = 5), sham group (n = 5), CCI + SF group (n = 5) and CCI group (n = 5). The dorsal root ganglia from different groups were rapidly excised and put in liquid nitrogen. The ganglion was taken in perchloric acid for homogenate and centrifuged for 10 min at 4000 g in a centrifuge (at 4 8C). The pellet was then removed and the supernatant was neutralised with KOH 3 M + Tris 1.5 M and then centrifuged at 4000 g for another 10 min. The supernatant was measured by high-performance liquid chromatography (HPLC: LC-10Atvp, Japan) combined with ultraviolet detection (UV: SPD-10Avp, Japan). 2.4. Isolation of DRG neurons Sprague–Dawley rats, 2–3 weeks old, of both sexes were decapitated after anesthetized with urethane [1.2 g/kg, intraperitoneally (i.p.)]. The thoracic and lumbar segments of the vertebral column were dissected and longitudinally divided into two halves along the median lines on both dorsal and ventral sides. The L4 and L5 lumbar DRGs together with dorsal and ventral roots and attached spinal nerves were taken out from the inner side of each half of the dissected vertebrae with fine dissecting forceps and transferred immediately into Dubecco’s modified Eagle’s medium (DMEM, Sigma) at pH 7.4 and 340 mOsmol/kg. After the removal of attached nerves and surrounding connective tissues, the DRGs were minced with dissecting spring scissors and incubated with trypsin (0.5 mg/ml; type III, Sigma), collagenase (1.0 mg/ml; type IA, sigma) and Dnase (0.1 mg/ml; type IV, sigma) in 5 ml DMEM at 35 8C in a shaking bath for 35–40 min, after which soybean trypsin inhibitor (1.25 mg/ml; type II-S, sigma) was added to stop the enzymatic digestion. The isolated neurons were transferred into a 35-mm culture dish and kept still for 30 min. Experiments were performed at room temperature (20–30 8C) [15,17].
3. Results 3.1. Effects of sodium ferulate on the ATP content in DRG of CCI rats The content of ATP was measured by HPLC from the DRG in group I (sham + normal salin group), II (sham group), III (SF group), IV (CCI + SF group) and V (CCI group). The data were 22.73 2.34 mg/g, 22.09 1.18 mg/g, 21.96 1.70 mg/g, 24.44 1.20 mg/g, 40.57 1.25 mg/g ganglion wet weight mean from above five group, respectively. The content of ATP in group V (CCI group) was higher than those in group I (sham + normal salin group), II (SF group), III (sham group), IV (CCI + SF group) [F(3.51) = 20.93, p < 0.01] (Fig. 1). No significant difference in the content of ATP had been observed in group I (sham + normal salin group) compared with those in group II (SF group), group III (sham group), or group IV (CCI + SF group) (p > 0.05). 3.2. Inhibition of sodium ferulate on P2X agonist-activated currents potentiated by CCI The majority of the DRG neurons isolated from group I (sham + normal salin group) 85.0% (17/20), group II (sham group) 81.2% (13/16), group III (SF group) 73.9% (17/23), group IV (CCI + SF group) 75.0% (15/20) and group V (CCI group) 72.0% (13/18) were responded to the external application of ATP (1–1000 mM) in a
2.5. Electrophysiological recordings The whole-cell patch-clamp recording (Liang et al., 2005) was carried out using a patch/whole-cell clamp amplifer (CEZ-2400, Nihon Kohden). The micropipette was filled with internal solution containing (in mM): KCl 140, MgCl2 2, HEPES 10, EGTA 11, ATP 5; its osmolarity was adjusted to 340 mOsmol/kg with sucrose and pH adjusted to 7.4 with KOH. The external solution contained (mM): NaCl 150, KCl 5, CaCl2 2.5, MgCl2 1, HEPES 10, D-glucose 10; its osmolarity was adjusted to 340 mOsmol with sucrose, pH was adjusted to 7.4 with NaOH. The resistance of recording electrodes were in the range of 1–4 MV. A small patch of membrane underneath the tip of the pipette was aspirated to form a seal (1–10 GV) and then a more negative pressure was applied to rupture it, thus a whole-cell mode was established. Membrane currents were filtered at 1 kHz (3 dB), data were recorded by a pen recorder (LMS-2B, Chengdu). The holding potential was set at 60 mV. The drugs were dissolved in external solution and delivered by gravity flow from an array of tubules (500 mm O.D., 200 mm I.D.) connected to a series of independent reservoirs. The distance from the tubule mouth to the cell examined was approximately 100 mm. Rapid solution-exchange was achieved by shifting the tubules horizontally with a micromanipulator. 2.6. In situ hybridization (ISH) The L4 and L5 lumbar DRG were dissected immediately and fixed in 4% paraformaldehyde for 2 h at room temperature, then transferred to 15% sucrose in 4% PFA overnight. Ganglia were sectioned at 15 mm with a cryostat and stored in 4% PFA at 4 8C until used. Diethyl pyrocarbonate (DEPC) water was used for all solutions and appliances necessary for ISH. Preparations were treated with 0.5%
Fig. 1. Effects of sodium ferulate on the ATP content of DRG after CCI. The bar histograms showed the content of ATP measured by HPLC from the DRG in group I (sham + normal salin group), II (sham group), III (SF group), IV (CCI + SF group) and V (CCI group). The content of ATP in group V (CCI group) was higher than those in group I (sham + normal salin group), II (SF group), III (sham group), or IV (CCI + SF group) [F(3.51) = 20.93, p < 0.01]. Compared with the results in other groups, the significant difference in group V was showed with asterisk. No significant difference in the content of ATP had been observed compared group IV (CCI + SF group) with group I (sham + normal salin group), group II (SF group), or group III (sham group) (p > 0.05).
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concentration-dependent manner with an inward currents (IATP). The amplitudes of the currents in group V (CCI) were highest compared with those obtained in other groups after application of same concentration ATP (100 mM, P < 0.01) (Fig. 2). The amplitudes of IATP in group IV were reduced by SF administrated by intraperitoneal injection. The amplitudes of IATP in group IV compared with those in group I, group II, or group III were no significant difference (P > 0.05). P2X3 selective agonist a,b-meATP (0.3–100 mM) could induce the currents in DRG neurons. The responses in DRG neurons of CCI rats were more higher than those obtained in other groups (P < 0.01). The dose-response curves for a,b-meATP-activated current in different groups are showed in Fig. 3. The amplitude of a,b-meATP-activated currents in group IV was reduced by SF administrated by intraperitoneal injection. The amplitude of a,bmeATP-activated currents in group IV compared with those in group I, group II or group III was no significant difference (P > 0.05) (Fig. 3). 3.3. Effect of sodium ferulate on the expression of P2X3 mRNA in DRG of CCI rats The expression of P2X3 mRNA was tested by ISH within the L4 and L5 DRG neurons. The stain values of P2X3 mRNA expression
were 109.25 4.37 (group I), 108.48 2.91 (group II), 108.48 5.05 (group III), 110.41 4.37 (group IV) and 129.74 4.37 (group V), respectively (n = 6 for each group). The gray scale of P2X3 receptor expression in group V was significantly higher than those in group I, group II, group III, or group IV [p < 0.01, F(4.94) = 20.53] (Fig. 4). No significantly difference was found in the intensity of P2X3 mRNA expression compared DRG neurons of group IV with DRG neurons of group I, group II, or group III (p > 0.05). Thus, SF reduced P2X3 mRNA expression in DRG neurons enhanced by CCI. 4. Discussion Neuropathic pain due to nerve injury is a prevalent condition, for which currently there is no effective treatment (Saarto and Wiffen, 2005; Tha´n et al., 2007). It is a condition that is often refractory to opioids or requires larger doses that possess unacceptable side effects (Przewlocki and Przewlocka, 2005). Neuropathic pain usually is persistent, and nerve injury can produce sensory/motor deficits. Several rodent models of neuropathic pain have been used to explore the mechanisms and treatment of neuropathic pain states (Campbell and Meyer, 2006). Among these models, the sciatic nerve chronic constriction injury has been widely used, as it produces robust
Fig. 2. The inhibition of sodium ferulate on P2X agonist-activated currents potentiated by CCI, P2X agonist- (ATP or a, b-meATP) activated currents in the DRG neurons. The current traces were obtained from the same cell. ATP-activated currents were concentration-dependent. (A) Current traces showed P2X agonist-activated currents in the DRG neurons of group I (sham + normal salin group); (B) current traces showed P2X agonist-activated currents in the DRG neurons of group II (sham group); (C) current traces showed P2X agonist-activated currents in the DRG neurons of group III (SF group); (D) current traces showed P2X agonist-activated currents in the DRG neurons of group IV (CCI + SF group). SF administrated by intraperitoneal injection reduced the amplitudes of IATP in group IV; (E) current traces showed P2X agonist-activated currents in the DRG neurons of group V (CCI group). The amplitude of the currents in group V (CCI) was highest compared with those obtained in other groups after application of same concentration ATP (100 mM, P < 0.01).
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Fig. 3. The dose-response curve of a,b-meATP-activated currents with or without pretreatment of SF. The graph shows that the dose-response curves for a,b-meATP (0.3–100 mM)-activated current in DRG neurons. The responses in DRG neurons of group V were more higher than those obtained in other groups (P < 0.01). Compared with other group, the significant difference in group V was showed with asterisk. The amplitude of a,b-meATP-activated currents after CCI was reduced by SF administrated by intraperitoneal injection. The amplitude of a,b-meATPactivated currents in group IV compared with those in group I, group II or group III was no significant difference (P > 0.05).
Wallerian degeneration with additional inflammation (Bennett and Xie, 1988). P2X receptors are localized in the central and peripheral nervous system. P2X receptors are ion channels that were activated by extracellular ATP (Burnstock, 2007; North, 2002) and play the important role in excitatory nociceptive processing (Liu and Salter, 2005; Burnstock, 2006). ATP released from the damaged peripheral nerve tissue might contribute to initiate neuron activation. As a result of cell damage, ATP plays an important role in the initiation of pain and in primary afferent sensitization after different kinds of acute nervous system injury
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(Agteresch et al., 1999; Schwiebert, 2000). A participation of extracellular ATP and P2 receptors in neuropathic pain has been reported (Inoue, 2006; North, 2003). The ATP content of DRG in CCI group was higher than those in other group. It was indicated that sensory impulses generated during nerve injury may increase the release of endogenous ATP. The content of ATP in CCI rats treated with SF was lower than that in CCI rats. SF may reduce the content of ATP in damaged DRG neuron during CCI. The somata of DRG neurons actively releases transmitters and play a crucial role in bidirectional communication between neurons and surrounding satellite glial cells. The neuronal somata also has a significant role in cell–cell signaling. P2X3 receptor agonist ATP and a,b-meATP can activate currents in DRG neurons. P2X3 receptor may be sensitized following tissue injury or inflammation. Our works showed that the amplitude of currents in DRG neurons of CCI rats was much larger than those in other group after application of same concentration ATP and a,bmeATP. In CCI rats treated with SF, P2X3 receptor agonist-activated currents were reduced. SF may decreased the sensitization of P2X3 receptor in DRG neurons of CCI rats. The sciatic nerve injury by chronic constriction results in upregulation of P2X3 receptor expression on DRG neurons (Burnstock, 2006; Novakovic et al., 1999). In our study, the expression of P2X3 mRNA in DRG neurons was increased after CCI. The results suggested that the nociceptive stimuli of neuropathic pain injury increased P2X3 receptor expression and enhanced P2X3 receptor sensitivity. SF administrated by intraperitoneal injection decreased the expression of P2X3 mRNA in CCI rats. SF might block primary afferent transmission mediated by P2X3 receptor and inhibit the activation of P2X3 receptor during chronic pain. It is possible that purinoceptors as the pharmacological target for the therapeutic treatment of neuropathic pain injury. The results indicate SF may antagonize P2X3 receptor to alleviate the chronic neuropathic pain injury. The main finding of our study is that SF reduced the content of ATP generated from sensory neurons during nerve injury, the expression of P2X3 and the sensitization of P2X3 receptor in DRG neurons of CCI rats; the effects were shown SF may inhibit primary afferent facilitation mediated by P2X3 receptor during CCI.
Fig. 4. The effects of sodium ferulate P2X3 mRNA expression in DRG neurons after CCI measured by in situ hybridization. Photos of P2X3 mRNA expression in L4/L5 DRG neurons measured by in situ hybridization (n = 6 each group). (A) The expression of P2X3 mRNA in L4/L5 DRG neurons of group I; (B) the expression of P2X3 mRNA in L4/L5 DRG neurons of group II; (C) the expression of P2X3 mRNA in L4/L5 DRG neurons of group III; (D) the expression of P2X3 mRNA in L4/L5 DRG neurons of group IV. (E) The expression of P2X3 mRNA in L4/L5 DRG neurons of group V (arrows indicate the immunostaining neurons; scale bars, 50 mm).
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This work was supported by grant (No. 30860086, 30460040 and 30660048) from National Natural Science Foundation of China and grant (No. 0640042) from Natural Science Foundation of Jiangxi Province to Dr. Shangdong Liang. References Agteresch, H.J., Dagnelie, P.C., van der Berg, J.W., Wilson, J.H., 1999. Adenosine triphosphate: established and potential clinical applications. Drugs 58, 211– 232. Barclay, J., Patel, S., Dorn, G., Wotherspoon, G., Moffatt, S., Eunson, L., Abdel’al, S., Natt, F., Hall, J., Winter, J., Bevan, S., Wishart, W., Fox, A., Ganju, P., 2002. Functional downregulation of P2X3 receptor subunit in rat sensory neurons reveals a significant role in chronic neuropathic and inflammatory pain. J. Neurosci. 22, 8139–8147. Bennett, G.J., Xie, Y.K., 1988. A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man. Pain 33, 87–107. Bland-Ward, P.A., Humphrey, P.P., 2000. P2X receptors mediate ATP-induced primary nociceptive neuron activation. J. Auton. Nerv. Syst. 81, 146–151. Burnstock, G., 2000. P2X receptors in sensory neurons. Br. J. Anaesth. 84, 476–488. Burnstock, G., 2007. Physiology and pathophysiology of purinergic neurotransmission. Physiol. Rev. 87, 659–797. Burnstock, G., 2006. Purinergic P2 receptors as targets for novel analgesics. Pharmacol. Ther. 110, 433–454. Campbell, J.N., Meyer, R.A., 2006. Mechanisms of neuropathic pain. Neuron 52, 77–92. Chizh, B.A., Illes, P., 2001. P2X receptors and nociception. Pharmacol. Rev. 53, 553– 568. Cockayne, D.A., Hamilton, S.G., Zhu, Q.M., Dunn, P.M., Zhong, Y., Novakovic, S., Malmberg, A.B., Cain, G., Berson, A., Kassotakis, L., Hedley, L., Lachnit, W.G., Burnstock, G., McMahon, S.B., Ford, A.P., 2000. Urinary bladder hyporeflexia and reduced pain-related behaviour in P2X3-deficient mice. Nature 407, 1011–1015. Dorn, G., Patel, S., Wotherspoon, G., Hemmings-Mieszczak, M., Barclay, J., Natt, F.J., Martin, P., Bevan, S., Fox, A., Ganju, P., Wishart, W., Hall, J., 2004. siRNA relieves chronic neuropathic pain. Nucleic Acids Res. 32, e49. Fang, C.H., Liu, S.P., Dong, W.G., Yu, J.P., 2006. Effects of sodium ferulate on function of macrophages in the colonic tissue of colitis rats. Chin. J. Gastroenterol. Hepatol. 15, 116–118. Gao, Y., Xu, C.S., Liang, S.D., Mu, S.N., Zhang, A.X., Wang, Y.X., Fang Wan, F., 2008. Effect of tetramethylpyrazine on primary afferent transmission mediated by P2X3 receptor in neuropathic pain states. Brain Res. Bull. 77, 27–32. Go´mez-Villafuertes, R., Sa´nchez-Nogueiro, J., Marı´n-Garcı´a, P., Miras-Portugal, M.T., 2007. Existence of high and low affinity dinucleotides pentaphosphate-induced calcium responses in individual synaptic terminals and lack of correlation with the distribution of P2X1-7 subunits. Neurochem. Int. 50, 628–641. Hamilton, S.G., McMahon, S.B., Lewin, G.R., 2001. Selective activation of nociceptors by P2X receptor agonists in normal and inflamed rat skin. J. Physiol. 534, 437–445. Honore, P., Kage, K., Mikusa, J., Watt, A.T., Johnston, J.F., Wyatt, J.R., Faltynek, C.R., Jarvis, M.F., Lynch, K., 2002. Analgesic profile of intrathecal P2X(3) antisense oligonucleotide treatment in chronic inflammatory and neuropathic pain states in rats. Pain 99, 11–19.
Inoue, K., 2006. The function of microglia through purinergic receptors: neuropathic pain and cytokine release. Pharmacol. Ther. 109, 210–226. Jarvis, M.F., Wismer, C.T., Schweitzer, E., Yu, H., van Biesen, T., Lynch, K.J., Burgard, E.C., Kowaluk, E.A., 2001. Modulation of BzATP and formalin induced nociception: attenuation by the P2X receptor antagonist, TNP-ATP and enhancement by the P2X(3) allosteric modulator, cibacron blue. Br. J. Pharmacol. 132, 259–269. Jarvis, M.F., 2003. Contributions of P2X3 homomeric and heteromeric channels to acute and chronic pain. Expert Opin. Ther. Targets 7, 513–522. Kennedy, C., 2005. P2X receptors: targets for novel analgesics? Neuroscientist 11, 345–356. Kennedy, C., Assis, T.S., Currie, A.J., Rowan, E.G., 2003. Crossing the pain barrier: P2 receptors as targets for novel analgesics. J. Physiol. 553, 683–694. Khakh, B.S., 2001. Molecular physiology of P2X receptors and ATP signalling at synapses. Nat. Rev. Neurosci. 2, 165–174. Lecca, D., Abbracchio, M.P., 2008. Deorphanisation of G protein-coupled receptors: a tool to provide new insights in nervous system pathophysiology and new targets for psycho-active drugs. Neurochem. Int. 52, 339–351. Liang, S.D., Xu, C.S., Zhou, T., Liu, H.Q., Gao, Y., Li, G.L., 2005. Tetramethylpyrazine inhibits ATP-activated currents in rat dorsal root ganglion neurons. Brain Res. 1040, 92–97. Liu, H.R., 2005. Pharmacological action and clinical application of sodium ferulate. China Pharm. 14, 78–79. Liu, X.J., Salter, M.W., 2005. Purines and pain mechanisms: recent developments. Curr. Opin. Investig. Drugs. 6, 65–75. North, R.A., 2002. Molecular physiology of P2X receptors. Physiol. Rev. 82, 1013– 1067. North, R.A., 2003. The P2X3 subunit: a molecular target in pain therapeutics. Curr. Opin. Investig. Drugs 4, 833–840. Novakovic, S.D., Kassotakis, L.C., Oglesby, I.B., Smith, J.A., Eglen, R.M., Ford, A.P., Hunter, J.C., 1999. Immunocytochemical localization of P2X3 purinoceptors in sensory neurons in naive rats and following neuropathic injury. Pain 80, 273– 282. Przewlocki, R., Przewlocka, B., 2005. Opioids in neuropathic pain. Curr. Pharm. Des. 11, 3013–3025. Saarto, T., Wiffen, P.J., 2005. Antidepressants for neuropathic pain. Cochrane Database Syst. Rev. 3 CD005454. Schwiebert, E.M., 2000. Extracellular ATP-mediated propagation of Ca2+ waves. Focus on ‘‘mechanical strain-induced Ca2+ waves are propagated via ATP release and purinergic receptor activation’’ Am. J. Physiol. Cell Physiol. 279, C281–C283. Souslova, V., Cesare, P., Ding, Y., Akopian, A.N., Stanfa, L., Suzuki, R., Carpenter, K., Dickenson, A., Boyce, S., Hill, R., Nebenuis-Oosthuizen, D., Smith, A.J., Kidd, E.J., Wood, J.N., 2000. Warm-coding deficits and aberrant inflammatory pain in mice lacking P2X3 receptors. Nature 407, 1015–1017. Tha´n, M., Kocsis, P., Tihanyi, K., Fodor, L., Farkas, B., Kova´cs, G., Kis-Varga, A., Szombathelyi, Z., Tarnawa, I., 2007. Concerted action of antiepileptic and antidepressant agents to depress spinal neurotransmission: possible use in the therapy of spasticity and chronic pain. Neurochem. Int. 50, 642–652. Wang, B.H., Ou-Yang, J.P., 2005. Pharmacological actions of sodium ferulate in cardiovascular system. Cardiovasc. Drug Rev. 23, 161–172. Zhong, Y., Dunn, P.M., Bardini, M., Ford, A.P., Cockayne, D.A., Burnstock, G., 2001. Changes in P2X receptor responses of sensory neurons from P2X3-deficient mice. Eur. J. Neurosci. 14, 1784–1792.