- Email: [email protected]
Effect of puerarin on P2X3 receptor involved in hyperalgesia after burn injury in the rat
Brain Research Bulletin 80 (2009) 341–346
Contents lists available at ScienceDirect
Brain Research Bulletin journal homepage: www.elsevier.com/locate/brainresbull
Research report
Effect of puerarin on P2X3 receptor involved in hyperalgesia after burn injury in the rat Changshui Xu 1 , Guilin Li 1 , Yun Gao, Shuangmei Liu, Jiari Lin, Jun Zhang, Xin Li, Han Liu, Shangdong Liang ∗ Department of Physiology, Medical College of Nanchang University, Nanchang, Jiangxi 330006, PR China
a r t i c l e
i n f o
Article history: Received 5 July 2009 Received in revised form 30 August 2009 Accepted 31 August 2009 Available online 8 September 2009 Keywords: P2X3 receptor Puerarin Burn injury pain Hyperalgesia
a b s t r a c t The study investigated the effects of puerarin on P2X3 receptor involved in hyperalgesia after burn injury in the rat. Superficial second degree burn injury models were adopted. Mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were measured and the P2X3 receptor expressions in dorsal root ganglion (DRG) from burn injury models rats were detected by immunohistochemistry, in situ hybridization, RT-PCR and western blot. MWL and TWL in untreated superficial second paw burn rats were reduced. MWL and TWL of puerarin-treated superficial second paw burn rats showed significant increase compared with untreated superficial second paw burn rats. Puerarin can decrease the hyperalgesia after burn injury. At day 3 post-burn, the expressions of P2X3 protein and mRNA in DRG neurons in untreated superficial second degree back burn group were increased significantly compared with sham back burn group, puerarin-treated back unburned control group, blank back control group, while in puerarin-treated superficial second degree back burn group, the P2X3 protein and mRNA expressions were decreased markedly. There is no significant difference in sham back burn group, puerarin-treated back unburned control group, blank back control group. Therefore, puerarin may reduce the nociceptive transmission of burn injury pain mediated by P2X3 receptor and alleviate P2X3 receptor involved in hyperalgesia after burn injury in the rats. © 2009 Elsevier Inc. All rights reserved.
1. Introduction Burn is a severe injury, and burn pain is a very important pain [40]. Management of pain after burn injury is an unresolved clinical issue. Acute burn injury is usually associated with pain in the injured and nearby areas. Burns are classified by thickness and area affected, yet pain does not always correlate accordingly. Afferent nerve destruction associated with deeper burns theoretically reduces the amount of pain experienced, but in clinical practice this is not a reliable predictor [32]. It is necessary to combine antinociceptive treatment with antineuropathic treatment for the method of burn pain management [35]. Regular, ongoing pain assessment is essential to guide the dynamic analgesic regime necessary to cope with the evolving nature of burn pain and its response to medication. Opioids are the cornerstone of burn pain control. They are effective and the variety of drugs available provides a range of potencies, methods of administration and duration of actions. The effects of opioids are wide ranging and include clinically relevant side effects such as respiratory depression, itch, nausea and vomit-
∗ Corresponding author. Tel.: +86 0791 6360552; fax: +86 0791 6360552. E-mail address: [email protected] (S. Liang). 1 Joint first authors. 0361-9230/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.brainresbull.2009.08.027
ing. Tolerance is defined as increasing doses of a particular opioid being required to achieve the same analgesic effect and applies equally to side effects [1,35,39]. So it is a focal point to search a new mechanism of pain formation and analgesic drug. ATP injected into the skin causes moderate pain that becomes intense when the skin is inflamed [19,20]. It has been postulated that the pain of tissue damage is conveyed by the release of cytosolic ATP onto receptors expressed by nociceptive sensory neurons (nociceptors) [5]. Further evidence was demonstrated in an in vitro model that rupture of a single cell could elicit action potentials in adjacent sensory neurons, and that ATP was the principal excitatory component of cytoplasm mediating action potential firing [12]. Seven subtypes of P2X receptors (P2X1–7 ) have been cloned [11,23,33], in which much more attention has been paid to the P2X3 receptor in the case of nociceptive mechanisms, because P2X3 receptors were expressed in a subset of predominantly small (presumed nociceptive) sensory neurons, including their central terminals. It has been reported that the P2X receptors in sensory neurons are involved in pain transmission [11,15,23,28,29]. ATP released from damaged or stressed cells could play a role in activating nerve P2X3 receptor, evoke action potential transmitting to central nervous system along neural axis [11,23,38,47,49]. Therefore, the P2X3 receptor is important in mediating both acute pain and chronic pain [7,10,13,48].
342
C. Xu et al. / Brain Research Bulletin 80 (2009) 341–346
Puerarin is a major active ingredient extracted from the traditional Chinese medicine Ge-gen (Radix Puerariae, RP), which is one of three major isoflavonoid compounds and has been widely used in treatment of myocardial and cerebral ischemia, glaucoma, and sudden deafness in clinical setting in China for thousands of years [17,51–53,56]. It has also been reported that Ge-gen could inhibit the inflammatory effect and oxidative damage [41,50]. Puerarin can relieve chest pain of angina pectoris [36]. Thus it was suggested puerarin might bring out the antinociceptive response. The present studies investigated the effects of puerarin on the nociceptive transmission of burn pain mediated by P2X3 receptor. 2. Materials and methods 2.1. Animals Male Sprague–Dawley rats (180–230 g) were provided by the Center of Laboratory Animal Science of Nanchang University. The animals were housed in plastic boxes in a group of three at 21–25 ◦ C. 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 were followed. All animals were treated in accordance with ARVO Statement for the use of Animals in Ophthalmic and Vision Research in China. 2.2. Antibodies and reagents Puerarin was the product of Kangenbei Pharmaceutical Limited Corporation, China (071106). P2X3 antibody was bought from Chemicon International Company of America. Array slides were obtained from Qiagen (Valencia, CA, USA). -Actin was from Advanced Immunochemicals, Long Beach, CA. Other antibodies and reagents were ascribed as following. 2.3. Animal groups Rats were divided into 10 groups randomly, each group containing 6 animals. The groups include sham paw burn group (SPB group), puerarin-treated paw unburned control group (PPC group), untreated superficial second degree paw burn group (PB group), puerarin-treated superficial second degree paw burn group (PPB group), blank paw control group (PC group), sham back burn group (SBB group), puerarin-treated back unburned control group (PBC group), untreated superficial second degree back burn group (BB group), puerarin-treated superficial second degree back burn group (PBB group) and blank back control group (BC group), respectively. Puerarin (100 mg/kg/day) was intraperitoneally (i.p.) injected to rats at 30 min before burn and used following 3 days. 2.4. Burn pain model Superficial second degree burn rat models were adopted. The establishment of burn injury model was judged according to the development of the different types in hot water for various periods. The hot water immersed for 8 s induced the similar skin changes at beginning, and then showed blister formation at about 2 h after injury. After baseline responses were measured, animals were lightly anesthetized with 2% isofluorane, and then the right hind paw or back was placed and held in 70 ◦ C water for 8 s, while rat right hind paw or back was placed in 37 ◦ C water for 8 s as sham. After burn injury was applied, the rat was returned to individual testing compartment where the rat recovered from anesthesia within 2–3 min.
2.6. Measurement of thermal withdrawal latency (TWL) Noxious heat stimulation for assessment of thermal hyperalgesia was applied by the Thermal Paw Stimulation System (BME-410C, Tianjin). Rats were placed in a transparent, square, bottomless acrylic box (22 cm × 12 cm × 22 cm), on a glass plate under which a light was located. Radiant heat stimuli were applied by directing a beam of light at the foot pad of each hind paw through the glass plate. The light beam was turned off automatically when the rat lifted the paw, allowing the measurement of time between the beginning of the light beam and the elevation of the foot. This time was designated as the paw withdrawal latency. The hind paws were tested alternately at 5 min intervals. The cut-off time for the heat stimulation was 30 s. 2.7. Immunohistochemistry Immunohistochemical staining was performed using SP-9001 kit (Beijing Zhongshan Biotech CO.) according to the manufacturer’s instruction. In briefly, on 4th day after burn, animals were anesthetized with penthiobarbital sodium, DRG neurons were dissected from the burn fields, about 6–10 samples were harvested each rat. DRG isolated from rats were washed by phosphate-buffered saline (PBS). After fixed with 4% paraformaldehyde (PFA) for 24 h, the ganglia were dehydrated by 20% sucrose for overnight at 4 ◦ C, and then ganglia were cut into 20 m thick on a cryostat. After washed by PBS for three times, the preparations were incubated in 3% H2 O2 for 10 min to block the endogenous peroxidase activity, then with 10% goat serum for 30 min at room temperature to block non-specific antigen. After rinsed and washed in PBS, the block sections were incubated with rabbit anti-P2X3 (1:2500 diluted in PBS; CHEMICON International, Inc., USA) for overnight at 4 ◦ C. After three rinses in PBS, the sections were incubated with biotinylated goat anti-rabbit secondary antibody (Beijing Zhongshan Biotech CO.) for 1 h at room temperature. The preparations were washed in PBS and then added streptavidin-horseradish peroxidase (Beijing Zhongshan Biotech CO.) for 30 min. After development of the diaminobenzidine chromogen for 2 min, the slides were washed with distilled water and cover slipped. After immunohistochemistry, image scanning analysis system (HMIV-2000, Wuhan) was used to analyze the changes in stain values (average optical density) of P2X3 in ganglia. Background was determined by averaging the optical density of 10 random areas. 2.8. In situ hybridization (ISH) In situ hybridization was performed using P2X3 receptors kit (Wuhan Boster Co., China) according to the manufacturer’s instruction. In briefly, on 4th day after burn, animals were anesthetized with penthiobarbital sodium, DRG were dissected from the burn fields, about 6–10 samples were harvested each rat. DRG were dissected immediately and fixed in 4% paraformaldehyde (PFA) for 2 h at room temperature, then transferred to 15% sucrose in 4% PFA overnight. Tissues were sectioned at 15 m at a cryostat and stored in 4% PFA at 4 ◦ C. Diethyl pyrocarbonate (DEPC) water was used for all solutions and appliances necessary for ISH. Sections were treated with 0.5% H2 O2 , followed by digestion with pepsin at 37 ◦ C for 1–2 min, terminated with 0.5 mol/L phosphate-buffered saline (PBS) and washed with it for 15 min. Then the sections were incubated in prehybridization for 2 h at 37 ◦ C and in hybridization overnight at 37 ◦ C. The sections were washed with gradient SSC (2× SSC 17.6 g sodium chloride, 8.8 g sodium citrate in 1000 mL distilled water) thoroughly, 2× SSC for 10 min, 0.5× SSC for 15 min and 0.2× SSC for 15 min to remove the background signals and followed by treatment biotinylated digoxim antibody at 37 ◦ C for 2 h. After strongly washed with PBS the sections were incubated with SABCPOD for 30 min and with biotinylated peroxidase (Beijing Zhongshan Biotech CO.) for 30 min at 37 ◦ C. The color was developed in DAB (Beijing Zhongshan Biotech CO.) substrate, then dehydrated and mounted with neutral gum. After immunohistochemistry, image scanning analysis system (HMIV-2000, Wuhan) was used to analyze the changes in stain values of P2X3 in ganglia. Background was determined by averaging the optical density of 10 random areas. 2.9. Western blot analysis
2.5. Measurement of mechanical withdrawal threshold (MWT) Noxious-pressure stimulation was used to evaluate mechanical hyperalgesia. Unrestrained rats were placed inside a clear plastic chamber (22 cm × 12 cm × 22 cm) on a stainless steel mesh floor and allowed to acclimate. Withdrawal responses to mechanical stimulation were determined using calibrated von Frey filaments (BME-403, Tianjin) applied through an opening in the stainless steel mesh floor of the cage (grid 1 cm × 1 cm) to an area adjacent to the paw. Each von Frey filament was applied once starting with 0.0044 g and continuing until a withdrawal response occurred or the force reached 1.4791 g (the cut-off value). The hind paws were tested alternately at 2 min intervals. Measurements of three times were taken using the up and down method on each side and the lowest value was taken as the threshold values. The filaments were applied in the order of increasing bending force (0.13, 0.20, 0.33, 0.60, 1.30, 3.60, 5.00, 7.30, 9.90, 20.1 g), with each applied 10 times at intervals of 15 s to different parts of the midplantar glabrous skin. The strength of the filaments in the series that evoked at least five positive responses among the 10 trials was designated the pain threshold.
Animals were killed and tissue collection was performed as described above, except tissues were quick frozen in tubes on dry ice during collection. In briefly, on 4th day after burn, animals in SBB group, PBC group, BB group, PBB group and BC group were anesthetized with penthiobarbital sodium, DRG were dissected from the burn fields, about 6–10 samples were harvested each rat. DRG were isolated immediately and rinsed in ice-cold phosphate-buffered saline (PBS). Ganglia were homogenized by mechanical disruption in lysis buffer containing the following: 50 mmol/L Tris–Cl, pH 8.0, 150 mmol/L NaCl, 0.1% sodium dodecyl sulfate (SDS), 1% Nonidet P-40, 0.02% sodium deoxycholate, 100 g/mL phenylmethylsulfonyl fluoride, 1 g/mL aprofinin, and incubated on ice for 50 min. Homogenate was then pelleted at 12,000 rpm for 10 min and supernatant was collected. Using Lowry method, the quantity of total protein was determined in the supernatant. After diluted with sample buffer (250 mmol/L Tris–Cl, 200 mmol/L dithiothreitol, 10% sodium dodecyl sulfate (SDS), 0.5% bromophenol blue, 50% glycerol) and heated to 95 ◦ C for 10 min, samples containing equal amounts of protein (20 g) were separated by SDS-polyacrylamide gel electrophoresis by using Bio-Rad system and 10% gel. In the wake of electrophoretic transfer onto nitrocellulose (NC) membrane using
C. Xu et al. / Brain Research Bulletin 80 (2009) 341–346
343
the same system, the membrane was blocked with 5% non-fat dry milk in 25 mmol/L Tris buffered saline, pH 7.2, plus 0.05% Tween 20 (TBST) for 3 h at room temperature, and incubated with primary antibodies in blocking buffer for 2 h at room temperature or overnight at 4 ◦ C. The membranes were washed (twice with TBST and twice with TBS or three times with PBS) and incubated (1 h, room temperature) with horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit IgG (1:3000, Beijing Zhongshan Biotech CO.) in blocking buffer. After another wash cycle, labeled proteins were visualized by enhanced chemiluminescence on highperformance film (Shanghai Pufei Biotech CO.). Chemiluminescent signals were collected on autoradiography film, and the quantity of band intensity was carried out using AlphaImager 2200 software. The primary antibodies and dilutions used were the following: rabbit polyclonal anti-P2X3 (1:1000; Chemicon International Company of America), and monoclonal -actin (1:10,000; Advanced Immunochemicals, Long Beach, CA). Band densities were normalized to each -actin internal control. 2.10. Statistical analysis Statistical analyses of the data were performed on computer (SPSS 11.5). All results were expressed as mean ± SE. Statistical significance was determined by one factor analysis of variance (ANOVA) followed by the Fisher post hoc test for multiple comparisons. p < 0.05 was considered significant. Sigmaplot 11.0 software was used for painting. Statistical analyses of the data were performed on computer.
3. Results 3.1. Effect of puerarin on mechanical hyperalgesia of burn rats At hour 1 after operation, the mechanical withdrawal threshold (MWT) in PB group and PPB group were lower than those in SPB group, PPC group and PC group [p < 0.01; F(4, 25) = 19.752], and there is no statistically significance among SPB group, PPC group and PC group (p > 0.05; F(2, 15) = 0.294). After 24 h, MWT in PPB group increased significantly compared with that in PPC group (p < 0.01). After 72 or 96 h, MWT in PPB group had no difference compared with the SPB group, PPC group and PC group [p > 0.05; F(3, 20) = 1.934 for 72 h; F(3, 20) = 1.626 for 96 h], while MWL in PB group is lower than those in the SPB group, PPC group and PC group [p < 0.01; F(3, 20) = 10.008 for 72 h; F(3, 20) = 9.651 for 96 h] (Fig. 1). 3.2. Effect of puerarin on thermal hyperalgesia of burn rats At hour 1, 24, 48, 72 after operation, the thermal withdrawal latency (TWL) in PB group and PPB group were lower than those in SPB group, PPC group and PC group [p < 0.01; F(4, 25) = 89.204 for 1 h; F(4, 25) = 35.439 for 24 h; F(4, 25) = 25.573 for 48 h; F(4, 25) = 13.525 for 72 h], and there is no statistical significance among
Fig. 2. Effect of puerarin on TWL (thermal withdrawal latency) in superficial second degree paw burn rats. Each group consisted of six rats (n = 6 per group). Data present mean ± SE. The significant difference was denoted as * when p < 0.05 and ** when p < 0.01 compared with SPB group, PPC group and PC group. The significant difference was denoted as # when p < 0.05 and ## when p < 0.01 compared with PB group.
SPB group, PPC group and PC group [p > 0.05; F(2, 15) = 2.91 for 1 h; F(2, 15) = 0.438 for 24 h; F(2, 15) = 0.372 for 48 h; F(2, 15) = 0.224 for 72 h]. At hour 24 after operation, although TWL in PPB group is still lower than those in the SPB group, PPC group and PC group [p < 0.01; F(3, 20) = 7.670], TWL in PPB group increased significantly compared with those in PB group (p < 0.01). After hour 144, TWL in the PPB group had no difference with those in SPB group, PPC group and PC group [p > 0.05; F(3, 20) = 1.973], while TWL in the PB group was still lower than those in the SPB group, PPC group and PC group (p < 0.05; F(3, 20) = 4.064) (Fig. 2). 3.3. Effect of puerarin on the expression of P2X3 immunoreactivity in DRG of burn rats P2X3 receptor immunoreactivities in the DRG were detected using immunohistochemistry. The stain values (average optical density) of P2X3 receptor expression in SBB group, PBC group, BB group, PBB group and BC group were 127.2 ± 3.69, 124.2 ± 2.43, 158.6 ± 3.14, 135.9 ± 4.53, 125.1 ± 1.88, respectively (n = 10 for each group). The average optical density of P2X3 receptor expression in BB group was significantly higher than those in SBB group, PBC group, PBB group and BC group [p < 0.01; F(4, 45) = 19.445]. No difference was found in the intensity of P2X3 receptor expression of DRG among SBB group, PBC group, and BC group [p > 0.05; F(2, 27) = 0.309]. The average optical density of P2X3 receptor expression in PBB group was higher than those in SBB group, PBC group and BC group, but had no significant difference [p > 0.05; F(3, 36) = 2.635] and it was lower than that in BB group (p < 0.05) (Fig. 3). 3.4. Effect of puerarin on the expression of P2X3 mRNA in DRG of burn rats by ISH
Fig. 1. Effect of puerarin on MWT (mechanical withdrawal threshold) in superficial second degree paw burn rats. Each group consisted of six rats (n = 6 per group). Data present mean ± SE. The significant difference was denoted as * when p < 0.05 and ** when p < 0.01 compared with SPB group, PPC group and PC group. The significant difference was denoted as # when p < 0.05 and ## when p < 0.01 compared with PB group.
The levels of P2X3 mRNA expression in the DRG were studied by in situ hybridization (ISH). By image analysis, the stain values (average optical density) of P2X3 mRNA expression in SBB group, PBC group, BB group, PBB group and BC group were 111.4 ± 2.03, 105.4 ± 0.72, 129.1 ± 1.15, 101.7 ± 1.76, 108.5 ± 3.56 respectively (n = 10 for each group). The average optical density of P2X3 mRNA expression in BB group was significantly higher than those in SBB group, PBC group, PBB group and BC group [p < 0.05; F(4, 45) = 25.972]. No difference was found in the intensity of P2X3 mRNA expression of DRG among SBB group, PBC group and
344
C. Xu et al. / Brain Research Bulletin 80 (2009) 341–346
Fig. 3. Effect of puerarin on the expression of P2X3 receptor in DRG neurons of rats after superficial second degree back burn by immunohistochemistry. P2X3 receptor expression in SBB group (A), PBC group (B), BB group (C), PBB group (D) and BC group (E). The expression level of P2X3 receptor in PBB group (D) was lower than that in BB group (C) (p < 0.05). (Arrows indicate the immunostaining neurons; scale bars, 50 m.)
Fig. 4. Effect of puerarin on the expression of P2X3 mRNA in DRG neurons of rats after superficial second degree back burn by in situ hybridization. The stain values of P2X3 mRNA expression in SBB group (A), PBC group (B), BB group (C), PBB group (D) and BC group (E). The stain values of P2X3 mRNA expression in PBB group (D) was lower than that in BB group (C) (p < 0.01). (Arrows indicate the immunostaining neurons; scale bars, 100 m.)
BC group [p > 0.05; F(2, 27) = 1.561]. Compared with P2X3 mRNA expression in BB group, the average optical density of P2X3 mRNA expression in PBB group was lower (p < 0.01). No difference was found among PBB group, SBB group, PBC group and BC group [p > 0.05; F(3, 36) = 2.397] (Fig. 4). 3.5. Effect of puerarin on the expression of P2X3 protein in DRG of burn rats by western blotting P2X3 expression in protein level was analyzed by western blotting. By image analysis, the stain values (average optical density) of P2X3 protein expression (normalized to each -actin internal control) in SBB group, PBC group, BB group, PBB group and BC group were 0.99 ± 0.07, 0.97 ± 0.02, 1.23 ± 0.06, 0.93 ± 0.02, 0.98 ± 0.03 respectively (n = 10 for each group). The average optical density of P2X3 protein expression in BB group was significantly higher than those in SBB group, PBC group, PBB group and BC group [p < 0.01; F(4, 45) = 7.250]. No difference was found in the intensity of P2X3 protein expression of DRG among SBB group, PBC group and BC group [p > 0.05; F(2, 27) = 0.034]. Compared with P2X3 protein expression in BB group, the average optical density of P2X3 protein expression in PBB group was significantly decreased (p < 0.01). The average optical density of P2X3 protein expression in PBB group had no significant difference than those in SBB group, PBC group and BC group [p > 0.05; F(3, 36) = 0.466] (Fig. 5).
4. Discussion The skin is a complex laminar tissue that serves both as a protective barrier and as the body’s largest sensory organ. The outer region of the skin, the epidermis, is extensively innervated by axons arising from sensory neurons of the dorsal root ganglion (DRG), which convey sensory input to the central nervous system [32]. Pain is perceived at the time and site of burn, due to stimulation of local nociceptors and transmission of the nerve impulse in A␦ and C fibres thereby relaying the pain message to the dorsal horn of the spinal cord. Primary hyperalgesia arises at the site of tissue injury, and responses to both mechanical and thermal stimuli are typically enhanced [31,45]. Our results show that the mechanical withdrawal threshold (MWL) and thermal withdrawal latency (TWL) in untreated superficial second paw burn rats were reduced. MWL and TWL of puerarin-treated superficial second paw burn rats showed significant increase compared with untreated superficial second paw burn rats. So after burn injury, the mechanical withdrawal threshold and thermal withdrawal latency were significantly increased. Puerarin can decrease the hyperalgesia after burn injury. Which mechanism is responsible for puerarin inhibiting hyperalgesia of burn injury rats? ATP can facilitate nociceptive sensitivity after tissue injury. P2X receptor activation by ATP stimulates cellular excitability, initiates nociceptive responses. P2X receptor agonists to elicit nociceptive
Fig. 5. Effect of puerarin on the expression of P2X3 receptor protein in DRG neurons of rats after superficial second degree back burn measured by western blotting. Compared with P2X3 protein expression in BB group, the average optical density of P2X3 protein expression in PBB group was significantly decreased (p < 0.01).
C. Xu et al. / Brain Research Bulletin 80 (2009) 341–346
responses are increased in situations of peripheral inflammationinduced neuronal sensitization [20,34]. Application of ATP or its analogs to the soma of acutely dissociated or cultured DRG neurons can result in depolarization or inward current due to the activation of P2X receptors [7,14]. P2X3 receptor is most highly expressed in a subpopulation of small diameter primary afferent neurons in the pain pathway. Systemic administration of P2X3 antagonist A-317491 effectively reduced nociception in inflammatory and neuropathic pain models [24]. P2X3 receptors may account for fast currents in small-diameter neurons that are capsaicin sensitive and isolectin B4-positive [4,46]. The peripheral terminals of primary afferent neurons are one of the most important sites where sensory signals are regulated [9,42]. The neurons expressing P2X3 are dramatically more responsive to ATP with even small increases in temperature [26]. It has been shown that hyperalgesia, an exaggerated painful response to noxious stimulus, can be induced following intrathecal application of P2X3 receptor agonist ␣,-meATP in the mouse spinal cord in vivo [42]. Therefore, P2X3 receptor plays an important role in the heat hyperalgesia [37]. Our previous works showed that sodium ferulate, a tradition Chinese medicine, reduced the nociceptive sensory facilitation of neuropathic pain injury mediated by P2X3 receptor [55]. Other study in our laboratory observed that puerarin decreased currents activated by P2X3 receptor agonists (ATP or ␣,-meATP) in acute dissociated dorsal root ganglion neuron (data not shown). Perhaps puerarin decreased the hyperalgesia after burn injury via inhibiting the nociceptive sensory facilitation of burn injury mediated by P2X3 receptor. P2X receptors may be activated by endogenously released ATP under certain pathological conditions [3,18]. The blockade of P2X3 receptors can reduce nociception mediated by both small and larger diameter sensory neurons in pain states. MWL and TWL are increased after superficial second paw burn rat treated with puerarin. The expression level of P2X3 is upregulated or the activity of P2X3 is sensitized under different conditions of acute or chronic inflammation models [22,30]. Purinergic sensitivity develops in sensory neurons after chronic peripheral nerve injury [6–8,11,16,25,27]. P2X3 selective antagonist A-317491 reduced both thermal hyperalgesia and tactile allodynia in the CCI neuropathic pain model [24]. Additionally, in some neuropathic pain models like sciatic nerve injury, P2X3 knockdown with siRNA or antisense reduces pain sensitivity [2,22]. Keratinocytes in the skin were considered as primary sites that release ATP because keratinocytes form the epidermis that is innervated by sensory nerve terminals. A co-culture system of sensory neurons and keratinocytes and successfully demonstrated that ATP is released from the cytosol of damaged keratinocytes and elicits P2X-like current response in the vicinal sensory neurons [12]. P2X3 receptor on DRG neurons increases their activity after inflammation and contributes to the hypersensitivity to mechanical stimulation in the inflammatory state. Puerarin may decrease the signal transmission of burn injury pain mediated by P2X3 receptor. The present study showed that P2X3 protein and mRNA expression in DRG had been upregulated after burn injury. It means the activation of P2X3 receptors contributes to pain and hyperalgesia of burn injury rats. Mechanical allodynia caused by surgical injury has been considered to involve local release of ATP in the tissue injury area and its action on P2X nociceptive receptors [43,44,54]. ATP and ␣,-meATP activate nociceptive sensory nerve terminals in the skin, which increase in magnitude in inflammatory conditions due to increase in number and responsiveness of P2X3 receptors [19,21]. Sensory impulses generated during nerve injury may increase the release of endogenous ATP and the sensitization of P2X3 receptor in DRG and peripheral nerve terminal of burn injury rats. In order to observe the mechanism of tetramethylpyrazine reducing the hyperalgesia in the burn injury rats, other work in our laboratory showed
345
that P2X3 selective antagonist A-317491 reduced both mechanical and thermal hyperalgesia in the burn injury model. Present study observes puerarin can significantly reduce the expression of P2X3 protein and mRNA in DRG of burn injury rats. Together to reduce P2X3 receptor agonist activated currents, puerarin may decrease the upregulated expression of P2X3 receptor and then decrease the sensitization of P2X3 receptor in DRG and peripheral nerve terminal of burn injury rats. Therefore, puerarin can depress the activation of P2X3 receptor during burn injury pain and decrease the primary afferent transmission mediated by P2X3 receptor to reduce hyperalgesia in burn injury pain states. 5. Conclusions In short word, P2X3 receptor is involved in burn injury pain. Puerarin can decrease the upregulated expression of P2X3 receptor protein and mRNA in DRG neurons after burn injury rats and reduce the primary afferent transmission of P2X3 receptor activation in DRG neurons of burn injury rats. Afterwards, puerarin has the effects to increase the threshold of thermal and mechanical hypersensitivity in burn injury rats. Therefore, puerarin can decrease the sensitization of P2X3 receptor involved in hyperalgesia after burn injury in the rats. Conflict of interest None. Acknowledgements This work was supported by the grant (Nos. 30860086, 30860333 and 30660048) from National Natural Science Foundation of China, the grant (No. 20070403007) from Doctoral Fund of Ministry of Education of China and the grant (Nos. 0640042 and 2008GZY0029) from Natural Science Foundation of Jiangxi Province, the grant (No. 2007-60 and GJJ08049) from the Educational Department of Jiangxi Province, the grant (YBP08A01) from Jiangxi Province Excellent Ph.D. Students Foundation and the grant (YC08B009) from the Innovation Foundation of Graduate School of Nanchang University. References [1] M.A. Ashburn, Burn pain: the management of procedure-related pain, J. Burn Care Rehab. 16 (3) (1995) 365–371. [2] J. Barclay, S. Patel, G. Dorn, G. Wotherspoon, S. Moffatt, L. Eunson, S. Abdel’al, F. Natt, J. Hall, J. Winter, S. Bevan, W. Wishart, A. Fox, P. Ganju, Functional downregulation of P2X3 receptor subunit in rat sensory neurons reveals a significant role in chronic neuropathic and inflammatory pain, J. Neurosci. 22 (2002) 8139–8147. [3] P. Bodin, G. Burnstock, Purinergic signalling: ATP release, Neurochem. Res. 26 (2001) 959–969. [4] E.C. Burgard, W. Niforatos, T. van Biesen, K.J. Lynch, E. Touma, R.E. Metzger, E.A. Kowaluk, M.F. Jarvis, P2X receptor-mediated ionic currents in dorsal root ganglion neurons, J. Neurophysiol. 82 (3) (1999) 1590–1598. [5] G. Burnstock, A unifying purinergic hypothesis for the initiation of pain, Lancet 347 (1996) 1604–1605. [6] G. Burnstock, Pathophysiology and therapeutic potential of purinergic signaling, Pharmacol. Rev. 58 (1) (2006) 58–86. [7] G. Burnstock, Physiology and pathophysiology of purinergic neurotransmission, Physiol. Rev. 87 (2) (2007) 659–797. [8] G. Burnstock, P2X receptors in sensory neurons, Br. J. Anaesth. 84 (4) (2000) 476–488. [9] R.C. Calvert, C.S. Thompson, G. Burnstock, ATP release from the human ureter on distension and P2X(3) receptor expression on suburothelial sensory nerves, Purin. Signal 4 (4) (2008) 377–381. [10] Y. Chen, Y. Shu, Z. Zhao, Ectopic purinergic sensitivity develops at sites of chronic nerve constriction injury in rat, Neuroreport 10 (13) (1999) 2779–2782. [11] B.A. Chizh, P. Illes, P2X receptors and nociception, Pharmacol. Rev. 53 (4) (2001) 553–568. [12] S.P. Cook, E.W. McCleskey, Cell damage excites nociceptors through release of cytosolic ATP, Pain 95 (2002) 41–47.
346
C. Xu et al. / Brain Research Bulletin 80 (2009) 341–346
[13] D. Donnelly-Roberts, S. McGaraughty, C.C. Shieh, P. Honore, M.F. Jarvis, Painful purinergic receptors, J. Pharmacol. Exp. Ther. 324 (2008) 409–415. [14] P.M. Dunn, Y. Zhong, G. Bunstock, P2X receptors in peripheral neurons, Prog. Neurobiol. 65 (2) (2001) 107–134. [15] G. Dussora, H.R. Koerbere, A.L. Oaklanderb, F.L. Ricec, D.C. Molliverd, Nucleotide signaling and cutaneous mechanisms of pain transduction, Brain Res. Rev. 60 (2009) 24–35. [16] Y. Gao, C.S. Xu, S.D. Liang, A.X. Zhang, S.N. Mu, Y.X. Wang, F. Wan, Effect of tetramethylpyrazine on primary afferent transmission mediated receptor in neuropathic pain states by P2X, Brain Res. Bull. 77 (2008) 27–32. [17] Q. Gao, B. Yang, Z.G. Ye, J. Wang, I.C. Bruce, Q. Xia, Opening the calcium-activated potassium channel participates in the cardioprotective effect of puerarin, Eur. J. Pharmacol. 574 (2007) 179–184. [18] C. Grelik, G.J. Bennett, A. Ribeiro-da-Silva, Autonomic fibre sprouting and changes in nociceptive sensory innervation in the rat lower lip skin following chronic constriction injury, Eur. J. Neurosci. 21 (2005) 2475–2487. [19] S.G. Hamilton, S.B. McMahon, G.R. Lewin, Selective activation of nociceptors by P2X receptor agonists in normal and inflamed rat skin, J. Physiol. 534 (2001) 437–445. [20] S.G. Hamilton, J. Warburton, A. Bhattacharjee, J. Ward, S.B. McMahon, ATP in human skin elicits a dose-related pain response which is potentiated under conditions of hyperalgesia, Brain 123 (Part 6) (2000) 1238–1246. [21] M. Hilliges, C. Weidner, M. Schmelz, R. Schmidt, K. Ørstavik, E. Torebjörk, H. Handwerker, ATP responses in human C nociceptors, Pain 98 (2002) 59–68. [22] S.W. Hwang, U. Oh, Current concepts of nociception: nociceptive molecular sensors in sensory neurons, Curr. Opin. Anaesth. 20 (2007) 427–434. [23] M.F. Jarvis, Contributions of P2X3 homomeric and heteromeric channels to acute and chronic pain, Expert. Opin. Ther. Targets 7 (4) (2003) 513–522. [24] M.F. Jarvis, E.C. Burgard, S. McGaraughty, P. Honore, K. Lynch, T.J. Brennan, A. Subieta, T. Van Biesen, J. Cartmell, B. Bianchi, W. Niforatos, K. Kage, H. Yu, J. Mikusa, C.T. Wismer, C.Z. Zhu, K. Chu, C.H. Lee, A.O. Stewart, J. Polakowski, B.F. Cox, E. Kowaluk, M. Williams, J. Sullivan, C. Faltynek, A-317491, a novel potent and selective non-nucleotide antagonist of P2X3 and P2X2/3 receptors, reduces chronic inflammatory and neuropathic pain in the rat, Proc. Natl. Acad. Sci. U.S.A. 99 (2002) 17179–17184. [25] C. Kennedy, T.S. Assis, A.J. Currie, E.G. Rowan, Crossing the pain barrier: P2 receptors as targets for novel analgesics, J. Physiol. 553 (Part 3) (2003) 683–694. [26] V. Khmyz, O. Maximyuk, V. Teslenko, A. Verkhratsky, O. Krishtal, P2X3 receptor gating near normal body temperature, Pflug. Arch. 456 (2008) 339–347. [27] S.D. Liang, Y. Gao, C.S. Xu, B.H. Xu, S.N. Mu, Effect of tetramethylpyrazine on acute nociception mediated by signaling of P2X receptor activation in rat, Brain Res. 995 (2) (2004) 247–252. [28] H. Merskrey, Logic, truth and language in concepts of pain, Qual. Life Res. 3 (Suppl.) (1994) 69–76. [29] S.D. Novakovic, L.C. Kassotakis, I.B. Oglesby, J.A. Smith, R.M. Eglen, A.P. Ford, J.C. Hunter, Immunocytochemical localization of P2X3 purinoceptors in sensory neurons in naive rats and following neuropathic injury, Pain 80 (1/2) (1999) 273–282. [30] M. Paukert, R. Osteroth, H.S. Geisler, U. Brandle, E. Glowatzki, J.P. Ruppersberg, S. Gründer, Inflammatory mediators potentiate ATP-gated channels through the P2X(3) subunit, J. Biol. Chem. 276 (2001) 21077–21082. [31] S.N. Raja, J.N. Campbell, R.A. Meyer, Evidence for different mechanisms of primary and secondary hyperalgesia following heat injury to the glabrous skin, Brain 107 (Part 4) (1984) 1179–1188. [32] P. Richardson, L. Mustard, The management of pain in the burns unit, Burns (2009), doi:10.1016/j.burns.2009.03.003. [33] J. Sánchez-Nogueiro, P. Marín-GarcíaMustard, D. León, M. León-Otegui, R. Gómez-Villafuertes, J. Gualix, M.T. Miras-Portugal, Axodendritic fibres of mouse cerebellar granule neurons exhibit a diversity of functional P2X receptors, Neurochem. Int. 26 (2009) [Epub ahead of print]PMID: 19560503. [34] J. Sawynok, Adenosine and ATP receptors, Handb. Exp. Pharmacol. 177 (2007) 309–328.
[35] J.C. Schneider, N.L. Harris, A. El Shami, R.L. Sheridan, J.T. Schulz 3rd., M.L. Bilodeau, C.M. Ryan, A descriptive review of neuropathic-like pain after burn injury, J. Burn Care Res. 27 (4) (2006) 524–528. [36] E. Shantsila, T. Watson, G.Y.H. Lip, Endothelial progenitor cells in cardiovascular disorders, J. Am. Coll. Cardiol. 49 (2007) 741–752. [37] M. Shinoda, K. Kawashima, N. Ozaki, H. Asai, K. Nagamine, Y. Sugiura, P2X3 receptor mediates heat hyperalgesia in a rat model of trigeminal neuropathic pain, J. Pain 8 (7) (2007) 588–597. [38] B. Sperlagh, E.S. Vizi, Neuronal synthesis, storage and release of ATP, Semin. Neurosci. 8 (1996) 175–186. [39] C. Steina, J.D. Clarkb, U. Ohc, M.R. Vaskod, G.L. Wilcoxe, A.C. Overlande, T.W. Vanderahf, R.H. Spencerg, Peripheral mechanisms of pain and analgesia, Brain Res. Rev. 60 (2009) 90–113. [40] G.J. Summer, K.A. Puntillo, C. Miaskowski, P.G. Green, J.D. Levine, Burn injury pain: the continuing challenge, J. Pain 8 (7) (2007) 533–548. [41] K.Y. Tan, M. Wu, Pharmacological effect and adverse effect analysis on puerarin, China Pharma. 16 (3) (2007) 60–61. [42] M. Tsuda, S. Hasegawa, K. Inoue, P2X receptors-mediated cytosolic phospholipase A2 activation in primary afferent sensory neurons contributes to neuropathic pain, J. Neurochem. 103 (4) (2007) 1408–1416. [43] M. Tsuda, S. Koizumi, K. Inoue, Role of endogenous ATP at the incision area in a rat model of postoperative pain, Neuroreport 12 (2001) 1701–1704. [44] K. Tsuzuki, A. Ase, P. Seguela, T. Nakatsuka, C.Y. Wang, J.X. She, J.G. Gu, TNPATP-resistant P2X ionic currents on the central terminals and somata of rat primary sensory neurons, J. Neurophysiol. 89 (6) (2003) 3235–3242. [45] H. Uchida, M. Matsumoto, H. Ueda, Profiling of BoNT/C3-reversible gene expression induced by lysophosphatidic acid: ephrinB1 gene up-regulation underlying neuropathic hyperalgesia and allodynia, Neurochem. Int. 54 (3–4) (2009) 215–221. [46] S. Ueno, M. Tsuda, T. Iwanaga, K. Inoue, Cell type-specific ATP activated responses in rat dorsal root ganglion neurons, Br. J. Pharmacol. 126 (2) (1999) 429–436. [47] E.S. Vizi, S.D. Liang, B. Sperlagh, A. Kittel, Z. Jurányi, Studies on the release and extracellular metabolism of endogenous ATP in rat superior cervical ganglion: support for neurotransmitter role of ATP, Neuroscience 79 (1997) 893–903. [48] L. Vulchanova, U. Arvidsson, M. Riedl, J. Wang, G. Buell, A. Surprenant, R.A. North, R. Elde, Differential distribution of two ATP-gated channels (P2X receptors) determined by immunocytochemistry, Proc. Natl. Acad. Sci. U.S.A. 93 (15) (1996) 8063–8067. [49] K. Wirkner, B. Sperlagh, P. Illes, P2X3 receptor involvement in pain states, Mol. Neurobiol. 36 (2007) 165–183. [50] L.Z. Xiao, Z. Huang, S.C. Ma, Z. Zen, B. Luo, X. Lin, X. Xu, Study on the effect and mechanism of puerarin on the size of infarction in patients with acute myocardial infarction, Chin. J. Integr. Tradit. Western Med. 24 (9) (2004) 790–792. [51] R.Q. Xie, J. Du, Y.M. Hao, Myocardial protection and mechanism of puerarin injection on patients of coronary heart disease with ischemia/reperfusion, Chin. J. Integr. Tradit. Western Med. 23 (12) (2003) 895–897. [52] X. Xu, S. Zhang, L. Zhang, W. Yan, X. Zheng, The neuroprotection of puerarin against cerebral ischemia is associated with the prevention of apoptosis in rats, Planta Med. 71 (2005) 585–591. [53] D.K.Y. Yeung, S.W.S. Leung, Y.C. Xu, P.M. Vanhoutte, R.Y. Man, Puerarin, an isoflavonoid derived from Radix puerariae, potentiates endotheliumindependent relaxation via the cyclic AMP pathway in porcine coronary artery, Eur. J. Pharmacol. 552 (2006) 105–111. [54] X. Zhang, Y. Chen, C. Wang, L.Y.M. Huang, Neuronal somatic ATP release triggers neuron–satellite glial cell communication in dorsal root ganglia, Proc. Natl. Acad. Sci. U.S.A. 104 (23) (2007) 9864–9869. [55] A.X. Zhang, C.S. Xu, S.D. Liang, Y. Gao, G.L. Li, J. Wei, F. Wan, S.M. Liu, J.R. Lin, Role of sodium ferulate in the nociceptive sensory facilitation of neuropathic pain injury mediated by P2X(3) receptor, Neurochem. Int. 53 (6–8) (2008) 278–282. [56] J. Zhou, S.P. Fang, Y. Qi, H.R. Huo, T.L. Jiang, Q. Shi, Effect of GeGen decoction on inflammatory media in joint of adjuvant arthritis rats, Chin. J. Exp. Trad. Med. Form. 7 (2001) 29–31.
Contents lists available at ScienceDirect
Brain Research Bulletin journal homepage: www.elsevier.com/locate/brainresbull
Research report
Effect of puerarin on P2X3 receptor involved in hyperalgesia after burn injury in the rat Changshui Xu 1 , Guilin Li 1 , Yun Gao, Shuangmei Liu, Jiari Lin, Jun Zhang, Xin Li, Han Liu, Shangdong Liang ∗ Department of Physiology, Medical College of Nanchang University, Nanchang, Jiangxi 330006, PR China
a r t i c l e
i n f o
Article history: Received 5 July 2009 Received in revised form 30 August 2009 Accepted 31 August 2009 Available online 8 September 2009 Keywords: P2X3 receptor Puerarin Burn injury pain Hyperalgesia
a b s t r a c t The study investigated the effects of puerarin on P2X3 receptor involved in hyperalgesia after burn injury in the rat. Superficial second degree burn injury models were adopted. Mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were measured and the P2X3 receptor expressions in dorsal root ganglion (DRG) from burn injury models rats were detected by immunohistochemistry, in situ hybridization, RT-PCR and western blot. MWL and TWL in untreated superficial second paw burn rats were reduced. MWL and TWL of puerarin-treated superficial second paw burn rats showed significant increase compared with untreated superficial second paw burn rats. Puerarin can decrease the hyperalgesia after burn injury. At day 3 post-burn, the expressions of P2X3 protein and mRNA in DRG neurons in untreated superficial second degree back burn group were increased significantly compared with sham back burn group, puerarin-treated back unburned control group, blank back control group, while in puerarin-treated superficial second degree back burn group, the P2X3 protein and mRNA expressions were decreased markedly. There is no significant difference in sham back burn group, puerarin-treated back unburned control group, blank back control group. Therefore, puerarin may reduce the nociceptive transmission of burn injury pain mediated by P2X3 receptor and alleviate P2X3 receptor involved in hyperalgesia after burn injury in the rats. © 2009 Elsevier Inc. All rights reserved.
1. Introduction Burn is a severe injury, and burn pain is a very important pain [40]. Management of pain after burn injury is an unresolved clinical issue. Acute burn injury is usually associated with pain in the injured and nearby areas. Burns are classified by thickness and area affected, yet pain does not always correlate accordingly. Afferent nerve destruction associated with deeper burns theoretically reduces the amount of pain experienced, but in clinical practice this is not a reliable predictor [32]. It is necessary to combine antinociceptive treatment with antineuropathic treatment for the method of burn pain management [35]. Regular, ongoing pain assessment is essential to guide the dynamic analgesic regime necessary to cope with the evolving nature of burn pain and its response to medication. Opioids are the cornerstone of burn pain control. They are effective and the variety of drugs available provides a range of potencies, methods of administration and duration of actions. The effects of opioids are wide ranging and include clinically relevant side effects such as respiratory depression, itch, nausea and vomit-
∗ Corresponding author. Tel.: +86 0791 6360552; fax: +86 0791 6360552. E-mail address: [email protected] (S. Liang). 1 Joint first authors. 0361-9230/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.brainresbull.2009.08.027
ing. Tolerance is defined as increasing doses of a particular opioid being required to achieve the same analgesic effect and applies equally to side effects [1,35,39]. So it is a focal point to search a new mechanism of pain formation and analgesic drug. ATP injected into the skin causes moderate pain that becomes intense when the skin is inflamed [19,20]. It has been postulated that the pain of tissue damage is conveyed by the release of cytosolic ATP onto receptors expressed by nociceptive sensory neurons (nociceptors) [5]. Further evidence was demonstrated in an in vitro model that rupture of a single cell could elicit action potentials in adjacent sensory neurons, and that ATP was the principal excitatory component of cytoplasm mediating action potential firing [12]. Seven subtypes of P2X receptors (P2X1–7 ) have been cloned [11,23,33], in which much more attention has been paid to the P2X3 receptor in the case of nociceptive mechanisms, because P2X3 receptors were expressed in a subset of predominantly small (presumed nociceptive) sensory neurons, including their central terminals. It has been reported that the P2X receptors in sensory neurons are involved in pain transmission [11,15,23,28,29]. ATP released from damaged or stressed cells could play a role in activating nerve P2X3 receptor, evoke action potential transmitting to central nervous system along neural axis [11,23,38,47,49]. Therefore, the P2X3 receptor is important in mediating both acute pain and chronic pain [7,10,13,48].
342
C. Xu et al. / Brain Research Bulletin 80 (2009) 341–346
Puerarin is a major active ingredient extracted from the traditional Chinese medicine Ge-gen (Radix Puerariae, RP), which is one of three major isoflavonoid compounds and has been widely used in treatment of myocardial and cerebral ischemia, glaucoma, and sudden deafness in clinical setting in China for thousands of years [17,51–53,56]. It has also been reported that Ge-gen could inhibit the inflammatory effect and oxidative damage [41,50]. Puerarin can relieve chest pain of angina pectoris [36]. Thus it was suggested puerarin might bring out the antinociceptive response. The present studies investigated the effects of puerarin on the nociceptive transmission of burn pain mediated by P2X3 receptor. 2. Materials and methods 2.1. Animals Male Sprague–Dawley rats (180–230 g) were provided by the Center of Laboratory Animal Science of Nanchang University. The animals were housed in plastic boxes in a group of three at 21–25 ◦ C. 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 were followed. All animals were treated in accordance with ARVO Statement for the use of Animals in Ophthalmic and Vision Research in China. 2.2. Antibodies and reagents Puerarin was the product of Kangenbei Pharmaceutical Limited Corporation, China (071106). P2X3 antibody was bought from Chemicon International Company of America. Array slides were obtained from Qiagen (Valencia, CA, USA). -Actin was from Advanced Immunochemicals, Long Beach, CA. Other antibodies and reagents were ascribed as following. 2.3. Animal groups Rats were divided into 10 groups randomly, each group containing 6 animals. The groups include sham paw burn group (SPB group), puerarin-treated paw unburned control group (PPC group), untreated superficial second degree paw burn group (PB group), puerarin-treated superficial second degree paw burn group (PPB group), blank paw control group (PC group), sham back burn group (SBB group), puerarin-treated back unburned control group (PBC group), untreated superficial second degree back burn group (BB group), puerarin-treated superficial second degree back burn group (PBB group) and blank back control group (BC group), respectively. Puerarin (100 mg/kg/day) was intraperitoneally (i.p.) injected to rats at 30 min before burn and used following 3 days. 2.4. Burn pain model Superficial second degree burn rat models were adopted. The establishment of burn injury model was judged according to the development of the different types in hot water for various periods. The hot water immersed for 8 s induced the similar skin changes at beginning, and then showed blister formation at about 2 h after injury. After baseline responses were measured, animals were lightly anesthetized with 2% isofluorane, and then the right hind paw or back was placed and held in 70 ◦ C water for 8 s, while rat right hind paw or back was placed in 37 ◦ C water for 8 s as sham. After burn injury was applied, the rat was returned to individual testing compartment where the rat recovered from anesthesia within 2–3 min.
2.6. Measurement of thermal withdrawal latency (TWL) Noxious heat stimulation for assessment of thermal hyperalgesia was applied by the Thermal Paw Stimulation System (BME-410C, Tianjin). Rats were placed in a transparent, square, bottomless acrylic box (22 cm × 12 cm × 22 cm), on a glass plate under which a light was located. Radiant heat stimuli were applied by directing a beam of light at the foot pad of each hind paw through the glass plate. The light beam was turned off automatically when the rat lifted the paw, allowing the measurement of time between the beginning of the light beam and the elevation of the foot. This time was designated as the paw withdrawal latency. The hind paws were tested alternately at 5 min intervals. The cut-off time for the heat stimulation was 30 s. 2.7. Immunohistochemistry Immunohistochemical staining was performed using SP-9001 kit (Beijing Zhongshan Biotech CO.) according to the manufacturer’s instruction. In briefly, on 4th day after burn, animals were anesthetized with penthiobarbital sodium, DRG neurons were dissected from the burn fields, about 6–10 samples were harvested each rat. DRG isolated from rats were washed by phosphate-buffered saline (PBS). After fixed with 4% paraformaldehyde (PFA) for 24 h, the ganglia were dehydrated by 20% sucrose for overnight at 4 ◦ C, and then ganglia were cut into 20 m thick on a cryostat. After washed by PBS for three times, the preparations were incubated in 3% H2 O2 for 10 min to block the endogenous peroxidase activity, then with 10% goat serum for 30 min at room temperature to block non-specific antigen. After rinsed and washed in PBS, the block sections were incubated with rabbit anti-P2X3 (1:2500 diluted in PBS; CHEMICON International, Inc., USA) for overnight at 4 ◦ C. After three rinses in PBS, the sections were incubated with biotinylated goat anti-rabbit secondary antibody (Beijing Zhongshan Biotech CO.) for 1 h at room temperature. The preparations were washed in PBS and then added streptavidin-horseradish peroxidase (Beijing Zhongshan Biotech CO.) for 30 min. After development of the diaminobenzidine chromogen for 2 min, the slides were washed with distilled water and cover slipped. After immunohistochemistry, image scanning analysis system (HMIV-2000, Wuhan) was used to analyze the changes in stain values (average optical density) of P2X3 in ganglia. Background was determined by averaging the optical density of 10 random areas. 2.8. In situ hybridization (ISH) In situ hybridization was performed using P2X3 receptors kit (Wuhan Boster Co., China) according to the manufacturer’s instruction. In briefly, on 4th day after burn, animals were anesthetized with penthiobarbital sodium, DRG were dissected from the burn fields, about 6–10 samples were harvested each rat. DRG were dissected immediately and fixed in 4% paraformaldehyde (PFA) for 2 h at room temperature, then transferred to 15% sucrose in 4% PFA overnight. Tissues were sectioned at 15 m at a cryostat and stored in 4% PFA at 4 ◦ C. Diethyl pyrocarbonate (DEPC) water was used for all solutions and appliances necessary for ISH. Sections were treated with 0.5% H2 O2 , followed by digestion with pepsin at 37 ◦ C for 1–2 min, terminated with 0.5 mol/L phosphate-buffered saline (PBS) and washed with it for 15 min. Then the sections were incubated in prehybridization for 2 h at 37 ◦ C and in hybridization overnight at 37 ◦ C. The sections were washed with gradient SSC (2× SSC 17.6 g sodium chloride, 8.8 g sodium citrate in 1000 mL distilled water) thoroughly, 2× SSC for 10 min, 0.5× SSC for 15 min and 0.2× SSC for 15 min to remove the background signals and followed by treatment biotinylated digoxim antibody at 37 ◦ C for 2 h. After strongly washed with PBS the sections were incubated with SABCPOD for 30 min and with biotinylated peroxidase (Beijing Zhongshan Biotech CO.) for 30 min at 37 ◦ C. The color was developed in DAB (Beijing Zhongshan Biotech CO.) substrate, then dehydrated and mounted with neutral gum. After immunohistochemistry, image scanning analysis system (HMIV-2000, Wuhan) was used to analyze the changes in stain values of P2X3 in ganglia. Background was determined by averaging the optical density of 10 random areas. 2.9. Western blot analysis
2.5. Measurement of mechanical withdrawal threshold (MWT) Noxious-pressure stimulation was used to evaluate mechanical hyperalgesia. Unrestrained rats were placed inside a clear plastic chamber (22 cm × 12 cm × 22 cm) on a stainless steel mesh floor and allowed to acclimate. Withdrawal responses to mechanical stimulation were determined using calibrated von Frey filaments (BME-403, Tianjin) applied through an opening in the stainless steel mesh floor of the cage (grid 1 cm × 1 cm) to an area adjacent to the paw. Each von Frey filament was applied once starting with 0.0044 g and continuing until a withdrawal response occurred or the force reached 1.4791 g (the cut-off value). The hind paws were tested alternately at 2 min intervals. Measurements of three times were taken using the up and down method on each side and the lowest value was taken as the threshold values. The filaments were applied in the order of increasing bending force (0.13, 0.20, 0.33, 0.60, 1.30, 3.60, 5.00, 7.30, 9.90, 20.1 g), with each applied 10 times at intervals of 15 s to different parts of the midplantar glabrous skin. The strength of the filaments in the series that evoked at least five positive responses among the 10 trials was designated the pain threshold.
Animals were killed and tissue collection was performed as described above, except tissues were quick frozen in tubes on dry ice during collection. In briefly, on 4th day after burn, animals in SBB group, PBC group, BB group, PBB group and BC group were anesthetized with penthiobarbital sodium, DRG were dissected from the burn fields, about 6–10 samples were harvested each rat. DRG were isolated immediately and rinsed in ice-cold phosphate-buffered saline (PBS). Ganglia were homogenized by mechanical disruption in lysis buffer containing the following: 50 mmol/L Tris–Cl, pH 8.0, 150 mmol/L NaCl, 0.1% sodium dodecyl sulfate (SDS), 1% Nonidet P-40, 0.02% sodium deoxycholate, 100 g/mL phenylmethylsulfonyl fluoride, 1 g/mL aprofinin, and incubated on ice for 50 min. Homogenate was then pelleted at 12,000 rpm for 10 min and supernatant was collected. Using Lowry method, the quantity of total protein was determined in the supernatant. After diluted with sample buffer (250 mmol/L Tris–Cl, 200 mmol/L dithiothreitol, 10% sodium dodecyl sulfate (SDS), 0.5% bromophenol blue, 50% glycerol) and heated to 95 ◦ C for 10 min, samples containing equal amounts of protein (20 g) were separated by SDS-polyacrylamide gel electrophoresis by using Bio-Rad system and 10% gel. In the wake of electrophoretic transfer onto nitrocellulose (NC) membrane using
C. Xu et al. / Brain Research Bulletin 80 (2009) 341–346
343
the same system, the membrane was blocked with 5% non-fat dry milk in 25 mmol/L Tris buffered saline, pH 7.2, plus 0.05% Tween 20 (TBST) for 3 h at room temperature, and incubated with primary antibodies in blocking buffer for 2 h at room temperature or overnight at 4 ◦ C. The membranes were washed (twice with TBST and twice with TBS or three times with PBS) and incubated (1 h, room temperature) with horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit IgG (1:3000, Beijing Zhongshan Biotech CO.) in blocking buffer. After another wash cycle, labeled proteins were visualized by enhanced chemiluminescence on highperformance film (Shanghai Pufei Biotech CO.). Chemiluminescent signals were collected on autoradiography film, and the quantity of band intensity was carried out using AlphaImager 2200 software. The primary antibodies and dilutions used were the following: rabbit polyclonal anti-P2X3 (1:1000; Chemicon International Company of America), and monoclonal -actin (1:10,000; Advanced Immunochemicals, Long Beach, CA). Band densities were normalized to each -actin internal control. 2.10. Statistical analysis Statistical analyses of the data were performed on computer (SPSS 11.5). All results were expressed as mean ± SE. Statistical significance was determined by one factor analysis of variance (ANOVA) followed by the Fisher post hoc test for multiple comparisons. p < 0.05 was considered significant. Sigmaplot 11.0 software was used for painting. Statistical analyses of the data were performed on computer.
3. Results 3.1. Effect of puerarin on mechanical hyperalgesia of burn rats At hour 1 after operation, the mechanical withdrawal threshold (MWT) in PB group and PPB group were lower than those in SPB group, PPC group and PC group [p < 0.01; F(4, 25) = 19.752], and there is no statistically significance among SPB group, PPC group and PC group (p > 0.05; F(2, 15) = 0.294). After 24 h, MWT in PPB group increased significantly compared with that in PPC group (p < 0.01). After 72 or 96 h, MWT in PPB group had no difference compared with the SPB group, PPC group and PC group [p > 0.05; F(3, 20) = 1.934 for 72 h; F(3, 20) = 1.626 for 96 h], while MWL in PB group is lower than those in the SPB group, PPC group and PC group [p < 0.01; F(3, 20) = 10.008 for 72 h; F(3, 20) = 9.651 for 96 h] (Fig. 1). 3.2. Effect of puerarin on thermal hyperalgesia of burn rats At hour 1, 24, 48, 72 after operation, the thermal withdrawal latency (TWL) in PB group and PPB group were lower than those in SPB group, PPC group and PC group [p < 0.01; F(4, 25) = 89.204 for 1 h; F(4, 25) = 35.439 for 24 h; F(4, 25) = 25.573 for 48 h; F(4, 25) = 13.525 for 72 h], and there is no statistical significance among
Fig. 2. Effect of puerarin on TWL (thermal withdrawal latency) in superficial second degree paw burn rats. Each group consisted of six rats (n = 6 per group). Data present mean ± SE. The significant difference was denoted as * when p < 0.05 and ** when p < 0.01 compared with SPB group, PPC group and PC group. The significant difference was denoted as # when p < 0.05 and ## when p < 0.01 compared with PB group.
SPB group, PPC group and PC group [p > 0.05; F(2, 15) = 2.91 for 1 h; F(2, 15) = 0.438 for 24 h; F(2, 15) = 0.372 for 48 h; F(2, 15) = 0.224 for 72 h]. At hour 24 after operation, although TWL in PPB group is still lower than those in the SPB group, PPC group and PC group [p < 0.01; F(3, 20) = 7.670], TWL in PPB group increased significantly compared with those in PB group (p < 0.01). After hour 144, TWL in the PPB group had no difference with those in SPB group, PPC group and PC group [p > 0.05; F(3, 20) = 1.973], while TWL in the PB group was still lower than those in the SPB group, PPC group and PC group (p < 0.05; F(3, 20) = 4.064) (Fig. 2). 3.3. Effect of puerarin on the expression of P2X3 immunoreactivity in DRG of burn rats P2X3 receptor immunoreactivities in the DRG were detected using immunohistochemistry. The stain values (average optical density) of P2X3 receptor expression in SBB group, PBC group, BB group, PBB group and BC group were 127.2 ± 3.69, 124.2 ± 2.43, 158.6 ± 3.14, 135.9 ± 4.53, 125.1 ± 1.88, respectively (n = 10 for each group). The average optical density of P2X3 receptor expression in BB group was significantly higher than those in SBB group, PBC group, PBB group and BC group [p < 0.01; F(4, 45) = 19.445]. No difference was found in the intensity of P2X3 receptor expression of DRG among SBB group, PBC group, and BC group [p > 0.05; F(2, 27) = 0.309]. The average optical density of P2X3 receptor expression in PBB group was higher than those in SBB group, PBC group and BC group, but had no significant difference [p > 0.05; F(3, 36) = 2.635] and it was lower than that in BB group (p < 0.05) (Fig. 3). 3.4. Effect of puerarin on the expression of P2X3 mRNA in DRG of burn rats by ISH
Fig. 1. Effect of puerarin on MWT (mechanical withdrawal threshold) in superficial second degree paw burn rats. Each group consisted of six rats (n = 6 per group). Data present mean ± SE. The significant difference was denoted as * when p < 0.05 and ** when p < 0.01 compared with SPB group, PPC group and PC group. The significant difference was denoted as # when p < 0.05 and ## when p < 0.01 compared with PB group.
The levels of P2X3 mRNA expression in the DRG were studied by in situ hybridization (ISH). By image analysis, the stain values (average optical density) of P2X3 mRNA expression in SBB group, PBC group, BB group, PBB group and BC group were 111.4 ± 2.03, 105.4 ± 0.72, 129.1 ± 1.15, 101.7 ± 1.76, 108.5 ± 3.56 respectively (n = 10 for each group). The average optical density of P2X3 mRNA expression in BB group was significantly higher than those in SBB group, PBC group, PBB group and BC group [p < 0.05; F(4, 45) = 25.972]. No difference was found in the intensity of P2X3 mRNA expression of DRG among SBB group, PBC group and
344
C. Xu et al. / Brain Research Bulletin 80 (2009) 341–346
Fig. 3. Effect of puerarin on the expression of P2X3 receptor in DRG neurons of rats after superficial second degree back burn by immunohistochemistry. P2X3 receptor expression in SBB group (A), PBC group (B), BB group (C), PBB group (D) and BC group (E). The expression level of P2X3 receptor in PBB group (D) was lower than that in BB group (C) (p < 0.05). (Arrows indicate the immunostaining neurons; scale bars, 50 m.)
Fig. 4. Effect of puerarin on the expression of P2X3 mRNA in DRG neurons of rats after superficial second degree back burn by in situ hybridization. The stain values of P2X3 mRNA expression in SBB group (A), PBC group (B), BB group (C), PBB group (D) and BC group (E). The stain values of P2X3 mRNA expression in PBB group (D) was lower than that in BB group (C) (p < 0.01). (Arrows indicate the immunostaining neurons; scale bars, 100 m.)
BC group [p > 0.05; F(2, 27) = 1.561]. Compared with P2X3 mRNA expression in BB group, the average optical density of P2X3 mRNA expression in PBB group was lower (p < 0.01). No difference was found among PBB group, SBB group, PBC group and BC group [p > 0.05; F(3, 36) = 2.397] (Fig. 4). 3.5. Effect of puerarin on the expression of P2X3 protein in DRG of burn rats by western blotting P2X3 expression in protein level was analyzed by western blotting. By image analysis, the stain values (average optical density) of P2X3 protein expression (normalized to each -actin internal control) in SBB group, PBC group, BB group, PBB group and BC group were 0.99 ± 0.07, 0.97 ± 0.02, 1.23 ± 0.06, 0.93 ± 0.02, 0.98 ± 0.03 respectively (n = 10 for each group). The average optical density of P2X3 protein expression in BB group was significantly higher than those in SBB group, PBC group, PBB group and BC group [p < 0.01; F(4, 45) = 7.250]. No difference was found in the intensity of P2X3 protein expression of DRG among SBB group, PBC group and BC group [p > 0.05; F(2, 27) = 0.034]. Compared with P2X3 protein expression in BB group, the average optical density of P2X3 protein expression in PBB group was significantly decreased (p < 0.01). The average optical density of P2X3 protein expression in PBB group had no significant difference than those in SBB group, PBC group and BC group [p > 0.05; F(3, 36) = 0.466] (Fig. 5).
4. Discussion The skin is a complex laminar tissue that serves both as a protective barrier and as the body’s largest sensory organ. The outer region of the skin, the epidermis, is extensively innervated by axons arising from sensory neurons of the dorsal root ganglion (DRG), which convey sensory input to the central nervous system [32]. Pain is perceived at the time and site of burn, due to stimulation of local nociceptors and transmission of the nerve impulse in A␦ and C fibres thereby relaying the pain message to the dorsal horn of the spinal cord. Primary hyperalgesia arises at the site of tissue injury, and responses to both mechanical and thermal stimuli are typically enhanced [31,45]. Our results show that the mechanical withdrawal threshold (MWL) and thermal withdrawal latency (TWL) in untreated superficial second paw burn rats were reduced. MWL and TWL of puerarin-treated superficial second paw burn rats showed significant increase compared with untreated superficial second paw burn rats. So after burn injury, the mechanical withdrawal threshold and thermal withdrawal latency were significantly increased. Puerarin can decrease the hyperalgesia after burn injury. Which mechanism is responsible for puerarin inhibiting hyperalgesia of burn injury rats? ATP can facilitate nociceptive sensitivity after tissue injury. P2X receptor activation by ATP stimulates cellular excitability, initiates nociceptive responses. P2X receptor agonists to elicit nociceptive
Fig. 5. Effect of puerarin on the expression of P2X3 receptor protein in DRG neurons of rats after superficial second degree back burn measured by western blotting. Compared with P2X3 protein expression in BB group, the average optical density of P2X3 protein expression in PBB group was significantly decreased (p < 0.01).
C. Xu et al. / Brain Research Bulletin 80 (2009) 341–346
responses are increased in situations of peripheral inflammationinduced neuronal sensitization [20,34]. Application of ATP or its analogs to the soma of acutely dissociated or cultured DRG neurons can result in depolarization or inward current due to the activation of P2X receptors [7,14]. P2X3 receptor is most highly expressed in a subpopulation of small diameter primary afferent neurons in the pain pathway. Systemic administration of P2X3 antagonist A-317491 effectively reduced nociception in inflammatory and neuropathic pain models [24]. P2X3 receptors may account for fast currents in small-diameter neurons that are capsaicin sensitive and isolectin B4-positive [4,46]. The peripheral terminals of primary afferent neurons are one of the most important sites where sensory signals are regulated [9,42]. The neurons expressing P2X3 are dramatically more responsive to ATP with even small increases in temperature [26]. It has been shown that hyperalgesia, an exaggerated painful response to noxious stimulus, can be induced following intrathecal application of P2X3 receptor agonist ␣,-meATP in the mouse spinal cord in vivo [42]. Therefore, P2X3 receptor plays an important role in the heat hyperalgesia [37]. Our previous works showed that sodium ferulate, a tradition Chinese medicine, reduced the nociceptive sensory facilitation of neuropathic pain injury mediated by P2X3 receptor [55]. Other study in our laboratory observed that puerarin decreased currents activated by P2X3 receptor agonists (ATP or ␣,-meATP) in acute dissociated dorsal root ganglion neuron (data not shown). Perhaps puerarin decreased the hyperalgesia after burn injury via inhibiting the nociceptive sensory facilitation of burn injury mediated by P2X3 receptor. P2X receptors may be activated by endogenously released ATP under certain pathological conditions [3,18]. The blockade of P2X3 receptors can reduce nociception mediated by both small and larger diameter sensory neurons in pain states. MWL and TWL are increased after superficial second paw burn rat treated with puerarin. The expression level of P2X3 is upregulated or the activity of P2X3 is sensitized under different conditions of acute or chronic inflammation models [22,30]. Purinergic sensitivity develops in sensory neurons after chronic peripheral nerve injury [6–8,11,16,25,27]. P2X3 selective antagonist A-317491 reduced both thermal hyperalgesia and tactile allodynia in the CCI neuropathic pain model [24]. Additionally, in some neuropathic pain models like sciatic nerve injury, P2X3 knockdown with siRNA or antisense reduces pain sensitivity [2,22]. Keratinocytes in the skin were considered as primary sites that release ATP because keratinocytes form the epidermis that is innervated by sensory nerve terminals. A co-culture system of sensory neurons and keratinocytes and successfully demonstrated that ATP is released from the cytosol of damaged keratinocytes and elicits P2X-like current response in the vicinal sensory neurons [12]. P2X3 receptor on DRG neurons increases their activity after inflammation and contributes to the hypersensitivity to mechanical stimulation in the inflammatory state. Puerarin may decrease the signal transmission of burn injury pain mediated by P2X3 receptor. The present study showed that P2X3 protein and mRNA expression in DRG had been upregulated after burn injury. It means the activation of P2X3 receptors contributes to pain and hyperalgesia of burn injury rats. Mechanical allodynia caused by surgical injury has been considered to involve local release of ATP in the tissue injury area and its action on P2X nociceptive receptors [43,44,54]. ATP and ␣,-meATP activate nociceptive sensory nerve terminals in the skin, which increase in magnitude in inflammatory conditions due to increase in number and responsiveness of P2X3 receptors [19,21]. Sensory impulses generated during nerve injury may increase the release of endogenous ATP and the sensitization of P2X3 receptor in DRG and peripheral nerve terminal of burn injury rats. In order to observe the mechanism of tetramethylpyrazine reducing the hyperalgesia in the burn injury rats, other work in our laboratory showed
345
that P2X3 selective antagonist A-317491 reduced both mechanical and thermal hyperalgesia in the burn injury model. Present study observes puerarin can significantly reduce the expression of P2X3 protein and mRNA in DRG of burn injury rats. Together to reduce P2X3 receptor agonist activated currents, puerarin may decrease the upregulated expression of P2X3 receptor and then decrease the sensitization of P2X3 receptor in DRG and peripheral nerve terminal of burn injury rats. Therefore, puerarin can depress the activation of P2X3 receptor during burn injury pain and decrease the primary afferent transmission mediated by P2X3 receptor to reduce hyperalgesia in burn injury pain states. 5. Conclusions In short word, P2X3 receptor is involved in burn injury pain. Puerarin can decrease the upregulated expression of P2X3 receptor protein and mRNA in DRG neurons after burn injury rats and reduce the primary afferent transmission of P2X3 receptor activation in DRG neurons of burn injury rats. Afterwards, puerarin has the effects to increase the threshold of thermal and mechanical hypersensitivity in burn injury rats. Therefore, puerarin can decrease the sensitization of P2X3 receptor involved in hyperalgesia after burn injury in the rats. Conflict of interest None. Acknowledgements This work was supported by the grant (Nos. 30860086, 30860333 and 30660048) from National Natural Science Foundation of China, the grant (No. 20070403007) from Doctoral Fund of Ministry of Education of China and the grant (Nos. 0640042 and 2008GZY0029) from Natural Science Foundation of Jiangxi Province, the grant (No. 2007-60 and GJJ08049) from the Educational Department of Jiangxi Province, the grant (YBP08A01) from Jiangxi Province Excellent Ph.D. Students Foundation and the grant (YC08B009) from the Innovation Foundation of Graduate School of Nanchang University. References [1] M.A. Ashburn, Burn pain: the management of procedure-related pain, J. Burn Care Rehab. 16 (3) (1995) 365–371. [2] J. Barclay, S. Patel, G. Dorn, G. Wotherspoon, S. Moffatt, L. Eunson, S. Abdel’al, F. Natt, J. Hall, J. Winter, S. Bevan, W. Wishart, A. Fox, P. Ganju, Functional downregulation of P2X3 receptor subunit in rat sensory neurons reveals a significant role in chronic neuropathic and inflammatory pain, J. Neurosci. 22 (2002) 8139–8147. [3] P. Bodin, G. Burnstock, Purinergic signalling: ATP release, Neurochem. Res. 26 (2001) 959–969. [4] E.C. Burgard, W. Niforatos, T. van Biesen, K.J. Lynch, E. Touma, R.E. Metzger, E.A. Kowaluk, M.F. Jarvis, P2X receptor-mediated ionic currents in dorsal root ganglion neurons, J. Neurophysiol. 82 (3) (1999) 1590–1598. [5] G. Burnstock, A unifying purinergic hypothesis for the initiation of pain, Lancet 347 (1996) 1604–1605. [6] G. Burnstock, Pathophysiology and therapeutic potential of purinergic signaling, Pharmacol. Rev. 58 (1) (2006) 58–86. [7] G. Burnstock, Physiology and pathophysiology of purinergic neurotransmission, Physiol. Rev. 87 (2) (2007) 659–797. [8] G. Burnstock, P2X receptors in sensory neurons, Br. J. Anaesth. 84 (4) (2000) 476–488. [9] R.C. Calvert, C.S. Thompson, G. Burnstock, ATP release from the human ureter on distension and P2X(3) receptor expression on suburothelial sensory nerves, Purin. Signal 4 (4) (2008) 377–381. [10] Y. Chen, Y. Shu, Z. Zhao, Ectopic purinergic sensitivity develops at sites of chronic nerve constriction injury in rat, Neuroreport 10 (13) (1999) 2779–2782. [11] B.A. Chizh, P. Illes, P2X receptors and nociception, Pharmacol. Rev. 53 (4) (2001) 553–568. [12] S.P. Cook, E.W. McCleskey, Cell damage excites nociceptors through release of cytosolic ATP, Pain 95 (2002) 41–47.
346
C. Xu et al. / Brain Research Bulletin 80 (2009) 341–346
[13] D. Donnelly-Roberts, S. McGaraughty, C.C. Shieh, P. Honore, M.F. Jarvis, Painful purinergic receptors, J. Pharmacol. Exp. Ther. 324 (2008) 409–415. [14] P.M. Dunn, Y. Zhong, G. Bunstock, P2X receptors in peripheral neurons, Prog. Neurobiol. 65 (2) (2001) 107–134. [15] G. Dussora, H.R. Koerbere, A.L. Oaklanderb, F.L. Ricec, D.C. Molliverd, Nucleotide signaling and cutaneous mechanisms of pain transduction, Brain Res. Rev. 60 (2009) 24–35. [16] Y. Gao, C.S. Xu, S.D. Liang, A.X. Zhang, S.N. Mu, Y.X. Wang, F. Wan, Effect of tetramethylpyrazine on primary afferent transmission mediated receptor in neuropathic pain states by P2X, Brain Res. Bull. 77 (2008) 27–32. [17] Q. Gao, B. Yang, Z.G. Ye, J. Wang, I.C. Bruce, Q. Xia, Opening the calcium-activated potassium channel participates in the cardioprotective effect of puerarin, Eur. J. Pharmacol. 574 (2007) 179–184. [18] C. Grelik, G.J. Bennett, A. Ribeiro-da-Silva, Autonomic fibre sprouting and changes in nociceptive sensory innervation in the rat lower lip skin following chronic constriction injury, Eur. J. Neurosci. 21 (2005) 2475–2487. [19] S.G. Hamilton, S.B. McMahon, G.R. Lewin, Selective activation of nociceptors by P2X receptor agonists in normal and inflamed rat skin, J. Physiol. 534 (2001) 437–445. [20] S.G. Hamilton, J. Warburton, A. Bhattacharjee, J. Ward, S.B. McMahon, ATP in human skin elicits a dose-related pain response which is potentiated under conditions of hyperalgesia, Brain 123 (Part 6) (2000) 1238–1246. [21] M. Hilliges, C. Weidner, M. Schmelz, R. Schmidt, K. Ørstavik, E. Torebjörk, H. Handwerker, ATP responses in human C nociceptors, Pain 98 (2002) 59–68. [22] S.W. Hwang, U. Oh, Current concepts of nociception: nociceptive molecular sensors in sensory neurons, Curr. Opin. Anaesth. 20 (2007) 427–434. [23] M.F. Jarvis, Contributions of P2X3 homomeric and heteromeric channels to acute and chronic pain, Expert. Opin. Ther. Targets 7 (4) (2003) 513–522. [24] M.F. Jarvis, E.C. Burgard, S. McGaraughty, P. Honore, K. Lynch, T.J. Brennan, A. Subieta, T. Van Biesen, J. Cartmell, B. Bianchi, W. Niforatos, K. Kage, H. Yu, J. Mikusa, C.T. Wismer, C.Z. Zhu, K. Chu, C.H. Lee, A.O. Stewart, J. Polakowski, B.F. Cox, E. Kowaluk, M. Williams, J. Sullivan, C. Faltynek, A-317491, a novel potent and selective non-nucleotide antagonist of P2X3 and P2X2/3 receptors, reduces chronic inflammatory and neuropathic pain in the rat, Proc. Natl. Acad. Sci. U.S.A. 99 (2002) 17179–17184. [25] C. Kennedy, T.S. Assis, A.J. Currie, E.G. Rowan, Crossing the pain barrier: P2 receptors as targets for novel analgesics, J. Physiol. 553 (Part 3) (2003) 683–694. [26] V. Khmyz, O. Maximyuk, V. Teslenko, A. Verkhratsky, O. Krishtal, P2X3 receptor gating near normal body temperature, Pflug. Arch. 456 (2008) 339–347. [27] S.D. Liang, Y. Gao, C.S. Xu, B.H. Xu, S.N. Mu, Effect of tetramethylpyrazine on acute nociception mediated by signaling of P2X receptor activation in rat, Brain Res. 995 (2) (2004) 247–252. [28] H. Merskrey, Logic, truth and language in concepts of pain, Qual. Life Res. 3 (Suppl.) (1994) 69–76. [29] S.D. Novakovic, L.C. Kassotakis, I.B. Oglesby, J.A. Smith, R.M. Eglen, A.P. Ford, J.C. Hunter, Immunocytochemical localization of P2X3 purinoceptors in sensory neurons in naive rats and following neuropathic injury, Pain 80 (1/2) (1999) 273–282. [30] M. Paukert, R. Osteroth, H.S. Geisler, U. Brandle, E. Glowatzki, J.P. Ruppersberg, S. Gründer, Inflammatory mediators potentiate ATP-gated channels through the P2X(3) subunit, J. Biol. Chem. 276 (2001) 21077–21082. [31] S.N. Raja, J.N. Campbell, R.A. Meyer, Evidence for different mechanisms of primary and secondary hyperalgesia following heat injury to the glabrous skin, Brain 107 (Part 4) (1984) 1179–1188. [32] P. Richardson, L. Mustard, The management of pain in the burns unit, Burns (2009), doi:10.1016/j.burns.2009.03.003. [33] J. Sánchez-Nogueiro, P. Marín-GarcíaMustard, D. León, M. León-Otegui, R. Gómez-Villafuertes, J. Gualix, M.T. Miras-Portugal, Axodendritic fibres of mouse cerebellar granule neurons exhibit a diversity of functional P2X receptors, Neurochem. Int. 26 (2009) [Epub ahead of print]PMID: 19560503. [34] J. Sawynok, Adenosine and ATP receptors, Handb. Exp. Pharmacol. 177 (2007) 309–328.
[35] J.C. Schneider, N.L. Harris, A. El Shami, R.L. Sheridan, J.T. Schulz 3rd., M.L. Bilodeau, C.M. Ryan, A descriptive review of neuropathic-like pain after burn injury, J. Burn Care Res. 27 (4) (2006) 524–528. [36] E. Shantsila, T. Watson, G.Y.H. Lip, Endothelial progenitor cells in cardiovascular disorders, J. Am. Coll. Cardiol. 49 (2007) 741–752. [37] M. Shinoda, K. Kawashima, N. Ozaki, H. Asai, K. Nagamine, Y. Sugiura, P2X3 receptor mediates heat hyperalgesia in a rat model of trigeminal neuropathic pain, J. Pain 8 (7) (2007) 588–597. [38] B. Sperlagh, E.S. Vizi, Neuronal synthesis, storage and release of ATP, Semin. Neurosci. 8 (1996) 175–186. [39] C. Steina, J.D. Clarkb, U. Ohc, M.R. Vaskod, G.L. Wilcoxe, A.C. Overlande, T.W. Vanderahf, R.H. Spencerg, Peripheral mechanisms of pain and analgesia, Brain Res. Rev. 60 (2009) 90–113. [40] G.J. Summer, K.A. Puntillo, C. Miaskowski, P.G. Green, J.D. Levine, Burn injury pain: the continuing challenge, J. Pain 8 (7) (2007) 533–548. [41] K.Y. Tan, M. Wu, Pharmacological effect and adverse effect analysis on puerarin, China Pharma. 16 (3) (2007) 60–61. [42] M. Tsuda, S. Hasegawa, K. Inoue, P2X receptors-mediated cytosolic phospholipase A2 activation in primary afferent sensory neurons contributes to neuropathic pain, J. Neurochem. 103 (4) (2007) 1408–1416. [43] M. Tsuda, S. Koizumi, K. Inoue, Role of endogenous ATP at the incision area in a rat model of postoperative pain, Neuroreport 12 (2001) 1701–1704. [44] K. Tsuzuki, A. Ase, P. Seguela, T. Nakatsuka, C.Y. Wang, J.X. She, J.G. Gu, TNPATP-resistant P2X ionic currents on the central terminals and somata of rat primary sensory neurons, J. Neurophysiol. 89 (6) (2003) 3235–3242. [45] H. Uchida, M. Matsumoto, H. Ueda, Profiling of BoNT/C3-reversible gene expression induced by lysophosphatidic acid: ephrinB1 gene up-regulation underlying neuropathic hyperalgesia and allodynia, Neurochem. Int. 54 (3–4) (2009) 215–221. [46] S. Ueno, M. Tsuda, T. Iwanaga, K. Inoue, Cell type-specific ATP activated responses in rat dorsal root ganglion neurons, Br. J. Pharmacol. 126 (2) (1999) 429–436. [47] E.S. Vizi, S.D. Liang, B. Sperlagh, A. Kittel, Z. Jurányi, Studies on the release and extracellular metabolism of endogenous ATP in rat superior cervical ganglion: support for neurotransmitter role of ATP, Neuroscience 79 (1997) 893–903. [48] L. Vulchanova, U. Arvidsson, M. Riedl, J. Wang, G. Buell, A. Surprenant, R.A. North, R. Elde, Differential distribution of two ATP-gated channels (P2X receptors) determined by immunocytochemistry, Proc. Natl. Acad. Sci. U.S.A. 93 (15) (1996) 8063–8067. [49] K. Wirkner, B. Sperlagh, P. Illes, P2X3 receptor involvement in pain states, Mol. Neurobiol. 36 (2007) 165–183. [50] L.Z. Xiao, Z. Huang, S.C. Ma, Z. Zen, B. Luo, X. Lin, X. Xu, Study on the effect and mechanism of puerarin on the size of infarction in patients with acute myocardial infarction, Chin. J. Integr. Tradit. Western Med. 24 (9) (2004) 790–792. [51] R.Q. Xie, J. Du, Y.M. Hao, Myocardial protection and mechanism of puerarin injection on patients of coronary heart disease with ischemia/reperfusion, Chin. J. Integr. Tradit. Western Med. 23 (12) (2003) 895–897. [52] X. Xu, S. Zhang, L. Zhang, W. Yan, X. Zheng, The neuroprotection of puerarin against cerebral ischemia is associated with the prevention of apoptosis in rats, Planta Med. 71 (2005) 585–591. [53] D.K.Y. Yeung, S.W.S. Leung, Y.C. Xu, P.M. Vanhoutte, R.Y. Man, Puerarin, an isoflavonoid derived from Radix puerariae, potentiates endotheliumindependent relaxation via the cyclic AMP pathway in porcine coronary artery, Eur. J. Pharmacol. 552 (2006) 105–111. [54] X. Zhang, Y. Chen, C. Wang, L.Y.M. Huang, Neuronal somatic ATP release triggers neuron–satellite glial cell communication in dorsal root ganglia, Proc. Natl. Acad. Sci. U.S.A. 104 (23) (2007) 9864–9869. [55] A.X. Zhang, C.S. Xu, S.D. Liang, Y. Gao, G.L. Li, J. Wei, F. Wan, S.M. Liu, J.R. Lin, Role of sodium ferulate in the nociceptive sensory facilitation of neuropathic pain injury mediated by P2X(3) receptor, Neurochem. Int. 53 (6–8) (2008) 278–282. [56] J. Zhou, S.P. Fang, Y. Qi, H.R. Huo, T.L. Jiang, Q. Shi, Effect of GeGen decoction on inflammatory media in joint of adjuvant arthritis rats, Chin. J. Exp. Trad. Med. Form. 7 (2001) 29–31.