PAIN 152 (2011) 18721887

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Activation of cannabinoid receptors by the pentacyclic triterpene a,b-amyrin inhibits inammatory and neuropathic persistent pain in mice
Kathryn A.B. Simo da Silva, Ana F. Paszcuk, Giselle F. Passos, Eduardo S. Silva, Allisson Freire Bento, Flavia C. Meotti, Joo B. Calixto
Departamento de Farmacologia, Centro de Cincias Biolgicas, Universidade Federal de Santa Catarina, Campus Universitrio Trindade, Bloco D, CCB, Caixa Postal 476, CEP 88049-900, Florianpolis, Santa Catarina, Brazil

Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article.

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a b s t r a c t
In this study, we report that a,b-amyrin, a plant-derived pentacyclic triterpene, reduced persistent inammatory and neuropathic hyperalgesia in mice by a direct activation of the CB1 and CB2 cannabinoid receptors (CB1R and CB2R). The oral treatment with a,b-amyrin (30 mg/kg) signicantly reduced mechanical and thermal hyperalgesia and inammation induced by complete Freunds adjuvant (CFA) and by partial sciatic nerve ligation (PSNL). The pretreatment with either CB1R or CB2R antagonists and the knockdown gene of the receptors signicantly reverted the antinociceptive effect of a,b-amyrin. Of note, binding studies showed that a,b-amyrin directly bound with very high afnity to CB1R (Ki = 0.133 nM) and with a lower afnity to CB2R (Ki = 1989 nM). Interestingly, a,b-amyrin, ACEA (CB1R agonist), or JWH-133 (CB2R agonist), at doses that caused antinociception, failed to provoke any behavioral disturbance, as measured in the tetrad assay. In addition, a,b-amyrin largely decreased interleukin-1b (IL1b), tumor necrosis factor a (TNF-a), keratinocyte-derived chemokine (KC) and interleukin 6 (IL-6) levels, and myeloperoxidase activity. Likewise, a,b-amyrin prevented the activation of the transcriptional factors: nuclear factor jB (NF-jB) and cyclic adenosine monophosphate response element binding (CREB) and the expression of cyclooxygenase 2 in mice footpads and spinal cords. The present results demonstrated that a,b-amyrin exhibits long-lasting antinociceptive and anti-inammatory properties in 2 models of persistent nociception via activation of cannabinoid receptors and by inhibiting the production of cytokines and expression of NF-jB, CREB and cyclooxygenase 2. 2011 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.

Article history: Received 5 September 2010 Received in revised form 1 April 2011 Accepted 4 April 2011

Keywords: a,b-Amyrin Cannabinoids CB1R CB2R Hypernociception Neuropathy Inammation

1. Introduction Chronic pain is a public health problem that causes personal and social afictions. Depending on its origin, chronic pain can be classied as inammatory or neuropathic. Inammatory pain is usually initiated by damage in tissues or by inammation, whereas neuropathic pain is caused by a primary injury or dysfunction in the peripheral or central nervous system. Clinical investigations have revealed that opiates, nonsteroidal antiinammatory drugs, anticonvulsants, and antidepressants constitute the most commonly used pharmacotherapies for the treatment of chronic pain [24]. However, these drugs present several adverse effects and have limited therapeutic efcacy in some patients [31]. Chronic inammatory and neuropathic pain differs from acute pain in time duration, threshold for stimulation, and plastic alterations in the tissue of injury and in the dorsal horn of the
Corresponding author. Tel.: +55 48 3721 9491; fax: +55 48 3337 5479.
E-mail addresses: calixto3@terra.com.br, calixto@farmaco.ufsc.br (J.B. Calixto).

spinal cord [29,56,61]. These substantial differences could explain why some analgesic drugs that are employed for treatment of acute pain may not be effective against chronic pain. Recently, cannabinoids have emerged as attractive potential alternatives or supplemental therapies for the treatment of chronic pain. Two human cannabinoid receptors have been identied, CB1 and CB2 [39,42]. Both of these receptors are members of the G protein-coupled receptor (GPCR) super family. The CB1 receptors (CB1R) are primarily distributed in the peripheral and central nervous system (CNS) [42], whereas the CB2 receptors (CB2R) are extensively localized in cells of the immune system [4,20]. The binding of agonist to CB1R and CB2R inhibits adenylyl cyclase by a Gai/o-dependent mechanism and activate Gbc-dependent mitogen-activate protein kinases (MAPK) cascades [9,10]. Numerous studies have demonstrated the antinociceptive effects of some selective and nonselective cannabinoid receptor agonists in different models of inammatory and neuropathic pain [7]. In this context, the search for new substances that activate the cannabinoid system and that lack serious side effects is of great

0304-3959/$36.00 2011 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.pain.2011.04.005

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relevance for chronic pain management. Earlier studies have reported that the isomeric mixture of a-amyrin and b-amyrin (Fig. 1) exhibits antinociceptive and anti-inammatory properties in rodents [6,32,43]. a,b-Amyrin is a pentacyclic triterpene and constitutes the main bioactive component of the resin of Protium kleinii and Protium heptaphyllum [44,45]. Moreover, a,b-amyrin exerted a pronounced anti-inammatory effect in acute models of inammation [40,45,54]. Likewise, this triterpene presented antinociceptive activity when administered at peripheral, spinal, or supraspinal sites in acute nociception [44]. However, it is still unknown whether a,b-amyrin is effective in counteracting chronic pain. In addition, the precise mechanism of action of this compound is not completely understood. Therefore, the present study investigated, through functional and molecular assays, the mechanisms by which a,b-amyrin exerts its antinociceptive and anti-inammatory properties in 2 models of persistent pain, the intraplantar injection of complete Freunds adjuvant (CFA) and the partial sciatic nerve ligation (PSNL). Furthermore, we investigated the ability of a,b-amyrin to specically bind to CB1R and CB2R. In addition, the effects of a,b-amyrin on CB1R and CB2R mRNA expression, pro-inammatory cytokines levels, myeloperoxidase (MPO) activity, cyclooxygenase 2 (COX-2) expression, and activation of transcriptional factors nuclear factor jB (NF-jB) and cyclic adenosine monophosphate response element binding (CREB) were also investigated. 2. Materials and methods 2.1. Animals The experiments were carried out in male Swiss mice (20 to 30 g) or male Wistar rats (200 to 250 g) that were kept in a room with controlled temperature (22 1C) and humidity (50% to 80%), under a 12 hour:12 hour light-dark cycle (lights on at 06:00 am) with food and water ad libitum. The animals were acclimatized to the laboratory settings for at least 1 hour before testing and were used only once throughout the experiments. All of the procedures used in the present study were approved by the Institutional Ethics Committee of the Universidade Federal de Santa Catarina (PP00148) and were carried out in accordance with the Principles of Laboratory Animal Care from National Institutes of Health publication No. 8523. In addition, the experimental procedures were in agreement with the current guidelines for the care of laboratory animals and the ethical guidelines for investigations of experimental pain in conscious animals as previously specied [60]. The number of animals and the intensity of noxious stimuli used were the minimum necessary to demonstrate consistent effects of the drug treatment. 2.2. CFA-induced inammation and hypernociception Mice were gently immobilized and received an intraplantar injection of 20 lL CFA (1 mg/mL heat-killed Mycobacterium tuberculosis in

85% parafn oil and 15% mannide mono-oleate). The control groups received the same volume of phosphate-buffered saline (PBS). 2.3. PSNL The mice were anaesthetized with 7% chloral hydrate (0.6 mL/ kg) intraperitoneally (i.p.). A partial ligation of the sciatic nerve was performed by tying 1/3 to 1/2 dorsal area of the distal part of sciatic nerve, according to the procedure described by Malmberg and Basbaum [36]. In sham-operated mice, the sciatic nerve was exposed without ligation. 2.4. Drug treatment protocols 2.4.1. CFA injection group Mechanical and thermal hypernociception and paw edema were assessed in mice previously treated by oral gavage (p.o.) with a,b-amyrin (3 to 30 mg/kg) or vehicle (Tween/ethanol/PBS) 1 hour before CFA injection. The mechanical or thermal hypernociception and the paw volume were taken 1 hour from CFA injection. To investigate the effects of long-term treatment with a,b-amyrin, mice received a second treatment with a,b-amyrin (30 mg/kg p.o.) 3 days after the CFA, when the hypernociceptive response had been reestablished. The treatment was repeated for 5 consecutive days (days 3 to 7) after the CFA injection. After an interruption of 11 days, the treatment was reinitiated on day 18th and maintained until day 22nd after the CFA injection. The mechanical hypernociception was measured daily, always 3 hours after the a,b-amyrin treatment. For the local treatment, a,b-amyrin (30 lg/paw) or vehicle (Tween/ethanol/PBS) were directly injected in the ventral surface of the hind paw (in the hypodermis) 24 hours after the CFA injection. The mechanical hypernociception was evaluated 30 minutes after the a,b-amyrin treatment. 2.4.2. PSNL group a,b-Amyrin (3 to 30 mg/kg) or vehicle (Tween/ethanol/PBS) were administered by gavage (p.o.) 1 hour before (pretreatment) or on the 4th day after surgery (posttreatment). The evaluation of mechanical or thermal hypernociception started on the 4th day after surgery. To investigate the effects of long-term treatment, a,b-amyrin (30 mg/kg) or vehicle were administered orally to mice once per day for 5 consecutive days (from the 4th to the 8th day after surgery). The treatment was interrupted and reinitiated on the 13th day after the surgery and continued until the 17th day. The mechanical hypernociception was measured every day, always 4 hours after the a,b-amyrin treatment. 2.4.3. Investigation of the CB1R and CB2R mechanisms The role of cannabinoid receptors in the antihypernociceptive action of a,b-amyrin was investigated by treating mice with the selective CB1R antagonist AM251 (1 mg/kg i.p.) [33] or the selective CB2R antagonist AM630 (3 mg/kg i.p.) [48] 30 minutes before a,b-amyrin (30 mg/kg p.o.). These treatments were performed 24 hours after CFA injection or 4 days after PSNL. The positive controls experiments were performed using the selective CB1R agonist ACEA (10 mg/kg i.p.) and the selective CB2R agonist JWH-133 (10 mg/kg i.p.). The selected doses of the agonists were based on previous experiments from our group. The evaluation of the mechanical hypernociception started 30 minutes after the treatment with a,b-amyrin. To investigate the role of cannabinoid receptors in the antiinammatory action of a,b-amyrin, different groups of mice were pretreated with the selective CB1R antagonist AM251 (1 mg/kg i.p.) or with the selective CB2R antagonist AM630 (3 mg/kg i.p.) 30 minutes before a,b-amyrin (30 mg/kg p.o.) treatment. Thirty minutes after the treatment with a,b-amyrin, mice received 20 lL

Fig. 1. Chemical structure of a-amyrin and b-amyrin isolated from Protium kleinii.

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CFA intraplantarly. Tissues were collected in different time points after a,b-amyrin treatment, as specied in Sections 2.12 and 2.13. 2.4.4. Intrathecal administration of antisense oligonucleotide To further conrm the role of CB1R and CB2R on the antihypernociceptive effect of a,b-amyrin, the antisense oligonucleotide (AS-ODN) was injected intrathecally (i.t.) into the subarachnoid space (L3L5) of anaesthetized mice 24 hours after the PSNL using a modied version of a described method [5]. Spinal injections of 5 lL of a specic AS-ODN for CB1R (50 -GCCTGCTAGAATCGCATT30 ), CB2R (50 -CTGCTGAGCGCCCTGGAGAAGAAC-30 ), or a mismatch MM-ODN control (50 -GCCTGCTAGAATCGCATT-30 ) were applied for 3 consecutive days at 12-hour intervals. The AS-ODNs were reconstituted in nuclease-free water to a nal concentration of 12.5 lg/mouse. On the 3rd day, the mice received an oral administration of a,b-amyrin (30 mg/kg) or vehicle 30 minutes after the last AS-ODN injection [17]. 2.5. Assessment of the mechanical hyperalgesia The mice were individually placed in clear Plexiglas boxes (9 7 11 cm) on an elevated wire mesh platform to allow access to the ventral surface of the right hind paw. The withdrawal response frequency was measured after 10 applications (3 seconds in duration) of the von Frey hairs (Stoelting, Chicago, IL). The animals were acclimatized for at least 30 minutes before the behavioral test, and mechanical hypernociception was evaluated at several time points. The withdrawal threshold in nave mice was taken before CFA injection, PSNL procedure, or a,b-amyrin treatment. This measurement is presented in the graphs as the baseline (B). The pressure exerted by the von Frey hairs (0.6 g) produced a baseline withdrawal frequency of around 15%, which is considered adequate for evaluation of mechanical hypernociception [8]. 2.6. Measurement of thermal hyperalgesia Thermal hyperalgesia was evaluated by measuring the latency of paw withdrawal according to the method described by Hargreaves et al. [26], with minor modications. The mice were placed in clear plastic chambers (7 9 11 cm) on an elevated surface and allowed to acclimatize to the environment for 1 hour. The heat stimulus was directed to the plantar surface of each hind paw near to the toes. The infrared intensity was adjusted to obtain baseline paw-withdrawal latencies of $15 seconds. In graphs, the baseline paw-withdrawal latencies are referred to as B. An automatic 20-second cut-off was used to prevent tissue damage. 2.7. Measurement of paw edema Edema was measured by use of a plethysmometer (Ugo Basile, Comerio, VA, Italy) at several time points and was expressed in lL, as the difference between the right (CFA-injected paw) and the left paw [16,18]. 2.8. CB1 and CB2 expression after AS-ODN treatment The expression of CB1R and CB2R in mice treated with AS-ODN was assessed by immunohistochemistry analyses. The lumbar portions of the spinal cords were harvested 6 hours after the a,bamyrin treatment, and the tissues were prepared as described in Section 2.11. The immunohistochemistry technique was carried out in parafn-embedded sections of the lumbar spinal cord, using polyclonal-rabbit anti-CB1R (1:500) or polyclonal-rabbit-anti-CB2R (1:150) antibodies (Cayman Chemical, Ann Arbor, Michigan). The immunohistochemistry technique was conducted as described later (Section 2.11).

2.9. Binding assay Binding to cannabinoid receptors was carried out as described previously [27] with minor modications. Membranes were prepared from rat brain (containing CB1R) or rat spleen (containing CB2R). The rats were killed by decapitation; the brain and spleen were quickly removed and homogenized in ice-cold buffer containing 50 mM TrisHCl, 3 mM MgCl2, 1 mM EDTA, and 320 mM sucrose, pH 7.4. The homogenate was rst centrifuged for 10 minutes at 3600 rpm at 4C. The low-speed pellets were discarded, and the supernatants were further centrifuged for 1 hour at 18,000 rpm at 4C. The resulting high-speed pellets were suspended in 1 mL of ice-cold buffer containing 50 mM TrisHCl, 3 mM MgCl2, and 1 mM EDTA, pH 7.4. The protein content was measured as described by Lowry et al. [34]. Membranes (50 lg/protein) were incubated in 50 mM TrisHCl, 3 mM MgCl2, 1 mM EDTA, 1 mM PMSF, 0.2% bovine serum albumin buffer, pH 7.4. The brain or spleen membranes were incubated with 0.5 nM [3H] SR141716A (specic activity 52 Ci/ mmol) or 0.8 nM [3H]CP-55,940 (specic activity 147.9 Ci/mmol), respectively. a,b-Amyrin (1011 to 104 M) was added to the incubation system. The samples were incubated at 30C for 60 minutes. The content was ltered using microlter GF/B by vacuum ltration. Filters were washed with ice-cold buffer: 50 mM TrisHCl 50 mM, 3 mM MgCl2, 1 mM EDTA, 0.2% bovine serum albumin buffer, pH 7.4. The radioactivity retained on the lters was measured in a Packard scintillation counter. Specic binding was calculated discounting the retained radioactivity in the presence of 100 lM WIN 55.212-2 (nonspecic binding). The results were normalized between 0% and 100% radioactivity specically bound. All data were obtained in duplicate of 4 independent experiments. 2.10. Tetrad evaluation 2.10.1. Locomotor suppression (rota-rod assay) To exclude the possible nonspecic muscle-relaxant or sedative effects of a,b-amyrin, the mice were tested in the rota-rod test, which was used to measure motor performance [52]. The apparatus (model-DS 37; Ugo Basille) consisted of a bar with a diameter of 2.5 cm, subdivided into 6 compartments by disks, 25 cm in diameter. The bar rotated at a constant speed of 22 rpm. The animals were previously selected, eliminating those mice that did not remain on the bar for 2 consecutive periods of 60 seconds. The performance of mice before any treatment is represented as baseline (B) in the graph. Mice were treated with a,b-amyrin (30 mg/kg p.o.), JWH-133 (10 mg/kg i.p.) or with ACEA (10 mg/kg i.p.). The time that mice remained on the rotating bar (cut-off 60 seconds) was recorded. 2.10.2. Tail-ick assay A radiant heat tail-ick analgesiometer was used to measure response latencies according to the method described by DAmour and Smith [15], with minor modications. Mice tail was exposed to a focused beam of radiant heat of intensity of 14. Tail-ick latencies were dened as the interval between the onset of the thermal stimulus and withdrawal of the tail. A 20-second cut-off was used to prevent tissue damage. Withdrawal latencies were measured immediately before treatment (baseline, B) and then, at several points after drug or vehicle administration. The latency for tail removing was recorded for animals treated with a,b-amyrin (30 mg/kg p.o.), JWH-133 (10 mg/kg i.p.) or ACEA (10 mg/kg i.p.). Mice were selected 24 hours previously according to their reactivity in the model. 2.10.3. Hypothermia assay The body temperature was measured by using a commercially available thermometer (Pro-check, Guangdong, China), which

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was gently inserted (0.5 cm) into the mouse rectum. Temperature measurements were performed in a temperature-controlled room (22C 2C), between 3:00 pm and 5:00 pm. Temperatures were recorded before any treatment as the baseline (B) measurement. Different groups of mice received a,b-amyrin (30 mg/kg p.o.), JWH-133 (10 mg/kg i.p.), or ACEA (10 mg/kg i.p.). The body temperature was recorded 30 to 180 minutes after treatments [53]. 2.10.4. Catalepsy Catalepsy was measured by placing the forepaws over a 0.5-cmdiameter horizontal glass bar at a 4-cm height from the oor. Mice were previously submitted to evaluation before (baseline, B) and immediately after treatment with a,b-amyrin (30 mg/kg p.o.), JWH-133 (10 mg/kg i.p.), or ACEA (10 mg/kg i.p.). The time that mice remained with both paws on the bar was measured up to 180 seconds. 2.11. Immunohistochemistry For the immunohistochemical studies, different groups of mice were submitted to PSNL or were injected with CFA. At different time points after the procedures, the mice were anesthetized with 7% chloral hydrate (10 mL/kg i.p.) and transcardially perfused with heparin (1000 U/mL) in physiological saline, followed by 4% paraformaldehyde diluted in 0.9% NaCl. The footpad and lumbar spinal cord (L3L5) were rapidly removed and postxed overnight in 4% paraformaldehyde. The immunohistochemistry technique was carried out in parafn-embedded sections of footpads or lumbar spinal cords. The sectioned tissues were incubated overnight at 4C with the primary antibodies for phospho-p65 NF-jB (1:100), phospho-CREB (1:200), or COX-2 (1:500) (Cell Signaling Technology, Danvers, MA). To block the endogenous peroxidase activity and to eliminate the occurrence of nonspecic reactions, the tissue sections were incubated with 1.5% hydrogen peroxide in methanol (v/v) for 20 minutes. To recover the antigens enclosed by paraformaldehyde and by the parafn inclusion, we performed hightemperature antigen retrieval by immersion of the slides in a water bath at 95C to 98C in 10 mM trisodium citrate buffer (pH 6.0) for 45 minutes. The slides were then processed using the Vectastain Elite ABC reagent (Vector Laboratories, Burlingame, CA) according to the manufacturers instructions. The sections were covered with the appropriate biotinylated secondary antibody for 90 minutes at room temperature. Subsequently, the sections were washed in PBS, and visualization was performed using DAB (3,30 -diaminobenzidine; Dako Cytomation) in chromogen solution and counterstaining with Harris hematoxylin. The control and experimental tissues were placed on the same slides and processed under the same conditions. The immunostaining was assessed at the dermis region of the paws or at the dorsal horn region of the lumbar spinal cords. Three images of the sections from each of these tissues were acquired using a Sight DS-5M-L1 digital camera (Nikon, Melville, NY) connected to an Eclipse 50i light microscope (Nikon). The threshold optical density that discriminated the staining from the background was obtained using the NIH ImageJ 1.36b imaging software (National Institutes of Health, Bethesda, MD). For phospho-CREB, phospho-p65 NF-jB, or COX-2 analyses, the total pixel intensity was determined, and the data are expressed as optical density (O.D.). All of the histological assessments were made by an examiner blind to the sample identities. 2.12. Myeloperoxidase activity assay MPO activity was carried out in the footpad and in the lumbar spinal cord segment (L3L5) as previously described [11]. The mice were treated with a,b-amyrin (30 mg/kg p.o.) or vehicle (Tween/eth-

anol/PBS) 1 hour before the intraplantar injection of CFA (20 lL) or the PSNL. The subcutaneous paw tissue and the lumbar spinal cord segment were removed 6 hours after the CFA injection, and the lumbar spinal cord segment was removed 4 days after the PSNL. The tissues were homogenized at 5% (w/v) in EDTA/NaCl buffer (pH 4.7) and centrifuged at 10,000 rpm for 15 minutes at 4C. The pellet was resuspended in 0.5% hexadecyltrimethyl ammonium bromide buffer (pH 5.4) and the samples were frozen and thawed 3 times in liquid nitrogen. The samples were centrifuged (10,000 rpm, 15 minutes, 4C), and 25 lL of the supernatant was used for the MPO assay. The enzymatic reaction was assessed with 1.6 mM tetramethylbenzidine, 80 mM sodium phosphate buffer pH 7.2, and 0.3 mM hydrogen peroxide. The absorbance was measured at 650 nm. The results are expressed as the O.D. per milligram of tissue. 2.13. Determination of cytokine levels The levels of cytokines were determined in the plantar tissue and lumbar spinal cord of CFA-injected mice and in the lumbar spinal cord of PSNL mice. The tissues were collected at different time points after the CFA injection: 3 hours for keratinocytederived chemokine (KC); 6 hours for interleukin-1b (IL-1b) and interleukin-6 (IL-6) or after the PSNL: 6 hours for KC; 4 hours for tumor necrosis factor a (TNF-a); 7 days for IL-1b, and 3 days for IL-6. These time points were chosen based on preliminary experiments (data not shown). The subcutaneous paw tissue or the tissue from the lumbar area of the spinal cord (L3L5) were removed from each mouse and homogenized in PBS containing 0.05% Tween 20, 0.1 mM phenylmethylsulphonyl uoride, 0.1 mM benzamethonium chloride, 10 mM EDTA, and 20 UI aprotinin A and centrifuged at 3000g for 10 minutes. The supernatant was stored at 70C until analysis. KC, TNF-a, and IL-1b levels were measured using ELISA kits (R&D Systems, Minneapolis, MN) according to the manufacturers instructions. For the determination of IL-6 levels, an enzyme-linked immunosorbent assay (ELISA) kit from eBioscience (San Diego, CA) was used. 2.14. RNA extraction and real-time PCR For mRNA quantication, the lumbar spinal cords (L3L5) were collected on the 4th day after PSNL. In another set of experiments, the lumbar spinal cords and footpads were harvested 24 hours and 4 days after CFA injection. Tissues were collected 6 hours after a,b-amyrin (30 mg/kg p.o.) treatment. The groups were divided into: (1) nave; (2) sham; (3) a,b-amyrin (30 mg/kg p.o.); (4) injured (CFA or PSNL); (5) injured (CFA or PSNL) plus a,b-amyrin (30 mg/kg p.o.). mRNA quantication in mice that received only a,b-amyrin (30 mg/kg p.o.) was also performed in the cortical area of the brain. For RNA extraction, tissues were homogenate in TRIzol (Invitrogen, So Paulo, Brazil). The concentration of total RNA was determined by a NanoDrop 1100 (Beckman Coulter; Fullerton, CA). One microgram of the total RNA was used for cDNA synthesis by the SuperScript reverse transcriptase (Invitrogen) protocol according to the manufacturers instructions. The cDNA (100 ng) was amplied in duplicate using a TaqMan Universal PCR Master Mix Kit with specic TaqMan Gene expression target genes; 30 quencher MGB- and FAM-labeled probes were used for mouse CB1R (Mm01212171_s1) and CB2R (Mm00438286_m1), and Glyceraldehyde 3-phosphate dehydrogenase ((GAPDH) (NM_0080842) was used as an endogenous control for normalization (Applied Biosystems, Foster City, CA). The amplications were carried out in a thermal cycler (StepOne Plus, Applied Biosystems) for 54 cycles; the uorescence was collected for each amplication cycle, and the data were analyzed using the 2DDCt method for relative quantication of expression. The expression of the target genes was calibrated against conditions found in nave/sham animals, ie, without treatment.

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2.15. Drugs and reagents The triterpene a,b-amyrin (1:1 mixture) was isolated from the resin of Protium kleinii as previously described [44], with a purity degree higher than 95%. The primers and probes for mouse CB1R, CB2R, and GAPDH were purchased from Applied BioSystems (Washington, UK). The selective CB1R antagonist AM251 or the selective CB2R antagonist AM630 and the selective CB1R or CB2R agonists, ACEA and JWH-133, or nonselective agonist WIN 55,212-3 were purchased from Tocris Bioscience (Ellisville, Missouri). The AS-ODN specic for CB1R (50 -GCCTGCTAGAATCGCATT-30 ), the AS-ODN specic for CB2R (50 -CTGCTGAGCGCCCTGGAGAAGAAC-30 ), and a mismatch MM-ODN control (50 -GCCTGCTAG AATCGCATT-30 ) were purchased from Prodimol Biotecnologia (Belo Horizonte, Brazil). The radiolabel [3H]CP-55,940 (specic activity 147.9 Ci/mmol) was purchased from PerkinElmer (Waltham, Massachusetts) and [3H]SR141716A specic activity 52 Ci/mmol) was purchased from Amersham Life Science (Buckinghamshire, England). The ELISA kits for mouse IL-1b, TNF-a, and KC were purchased from R&D Systems (Minneapolis, MN). The IL-6 mouse ELISA kit was purchased from Bioscience (San Diego, CA). The chloral hydrate was purchased from Vetec (Rio de Janeiro, Brazil). The CFA, hydrogen peroxide (H2O2), Tween 20, EDTA, aprotinin, and PBS tablets were purchased from Sigma Chemical Co. (St. Louis, MO). The monoclonal mouse anti-phospho-p65 NF-jB, polyclonal rabbit anti-COX-2, and polyclonal rabbit anti-phospho-CREB were purchased from Cell Signaling Technology. The secondary antibody Envision Plus, streptavidinhorseradish peroxidase reagent, and 3,3-diaminobenzidine chromogen were purchased from Dako Cytomation (Carpinteria, CA). The drugs were prepared in saline solution (0.9% NaCl) with the exception of the a,b-amyrin, which was prepared in 5% Tween 80, 5% ethanol, and 90% PBS just before use, and the solutions were adjusted to 10 mL/kg of body weight [44]. 2.16. Statistical analyses The results are presented as the mean SEM of 3 to 6 mice per group. The percentages of inhibition were calculated using the areas under the time-response curve (AUC). The statistical comparison of the data was performed using a one-way analysis of variance (ANOVA), with repeated measures when necessary. These analyses were followed by the Bonferroni post hoc test. Values of P < .05 were considered signicant. A t-test was used when appropriate. All of the tests were carried out using the GraphPad Software (GraphPad Software, San Diego, CA). IC50 with their 95% condent intervals were determined by nonlinear regression analysis using GraphPad PRISM from 4 independent experiments. The inhibitions constants (Ki) of competitor compounds were calculated by using the Cheng-Prusoff equation [Ki = IC50/(1 + L/KD)] [14]. 3. Results 3.1. Effect of a,b-amyrin on CFA-induced mechanical and thermal hyperalgesia The intraplantar injection of CFA induced a marked and longlasting enhancement of response frequency to the von Frey hair application and decreased the latency to paw withdrawal during a thermal stimulus in comparison to noninjured mice (Fig. 2A and B). The oral pretreatment with 3, 10, or 30 mg/kg of a,b-amyrin signicantly inhibited the mechanical hyperalgesia (Fig. 2A). The calculation of the area under the curve revealed that a,b-amyrin did not cause a dose-response effect. However, the dose of 30 mg/kg caused the highest inhibition among the 3 tested doses (inhibition of 69%). Of note, the antihyperalgesic effect caused by

the dose of 30 mg/kg was observed up to 24 hours after a,b-amyrin administration (Fig. 2A). In addition, the oral pretreatment with 30 mg/kg a,b-amyrin reduced the thermal hypersensitivity induced by CFA. The latency to response increased by 52%, and the effect was signicant for up to 6 hours after the CFA injection (Fig. 2B). The daily oral treatment with a,b-amyrin (30 mg/kg) presented a continuous antihyperalgesic effect. The measurement was always taken 3 hours after a,b-amyrin treatment, and the repeated treatment for 5 consecutive days resulted in a long-lasting effect, reducing the hyperalgesia for 9 days after the interruption of the treatment. On the 18th day, a new repeated treatment was initiated (once per day for 5 days), and again there was a signicant decrease in mechanical hyperalgesia. The AUC calculation of the total period revealed an inhibition of 45% (P < .05; Fig. 2C). The pretreatment with a,b-amyrin (30 mg/kg p.o.) was also able to decrease the paw edema induced by CFA. The effect of a,b-amyrin was observed up to 24 hours after the treatment (Fig. 2D). To verify the local effect of a,b-amyrin, the triterpene was directly administered into the plantar area 24 hours after CFA injection. The treatment with 30 lg/paw a,b-amyrin decreased the mechanical hyperalgesia induced by CFA from 30 minutes up to 2 hours after the treatment (Fig. 2E). 3.2. Effect of a,b-amyrin on the mechanical and thermal hyperalgesia induced by PSNL The PSNL produced a substantial and long-lasting mechanical and thermal hyperalgesia when compared with the sham group (Fig. 3). When administered on the 4th day after surgery (posttreatment), the doses of 3 and 30 mg/kg p.o. of a,b-amyrin signicantly reduced the mechanical hyperalgesia induced by PSNL. However, the dose of 10 mg/kg did not cause any signicant antinociceptive effect (Fig. 3A). This result clearly shows that the effects of a,b-amyrin are not dose dependent. Similar to the results found in the CFA-injected mice, the dose of 30 mg/kg was the most effective to reduce the hyperalgesia induced by the PSNL. The AUC calculation revealed an inhibition of 52% for 30 mg/kg a,b-amyrin, and this signicant effect lasted for 7 hours (Fig. 3A). The dose of 30 mg/kg a,b-amyrin also decreased the thermal hyperalgesia after PSNL. This effect was observed during the whole testing period (up to 48 hours). There was a 72% inhibition according to the AUC (Fig. 3B). In the long-term treatment, a,b-amyrin was administered daily for 5 days. The treatment signicantly reduced the mechanical hyperalgesia (inhibition of 60%; Fig. 3C), and the assessments were always taken 4 hours after the treatment. When the treatment was interrupted, the mice immediately exhibited a reestablishment of the mechanical hyperalgesia. On the 13th day, the treatment was reinitiated, and once again, a signicant inhibition of the mechanical hypernociception was observed (inhibition of 65%; Fig. 3C). Notably, the oral administration with 30 mg/kg a,b-amyrin, 1 hour before the PSNL, markedly reduced the development of mechanical hyperalgesia, an effect that was observed during all tested periods (inhibition of 83%; Fig. 3D). This long-lasting antihyperalgesic effect of a,b-amyrin demonstrates that the compound may prevent some initial component of nociceptive transmission, and therefore, prevent hyperalgesia during the whole testing period. 3.3. Role of cannabinoid receptors in the antihyperalgesic effect caused by a,b-amyrin Because a,b-amyrin presented a great effectiveness in reducing hyperalgesia in both models of chronic pain, we designed new experiments to determine whether or not its effects were related

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Fig. 2. Effect of the treatment with a,b-amyrin on the mechanical and thermal hypernociception and paw edema induced by complete Freunds adjuvant (CFA) in mice. (A) Pretreatment with a,b-amyrin mixture (3, 10, and 30 mg/kg p.o.) or vehicle were performed 1 hour before CFA intraplantar injection (20 lL/paw). Mechanical hypernociception was measured by using von Frey hairs (0.6 g), and the assessment was initiated 1 hour after CFA injection. (B) Mice were treated with a,b-amyrin (30 mg/kg p.o.) or its vehicle 1 hour before CFA injection, and thermal hypernociception was assessed 1 hour from CFA injection by the Hargreaves apparatus. (C) The effect of a,bamyrin (30 mg/kg p.o.) in a long-term treatment schedule was assayed by a daily treatment with a,b-amyrin. The treatment was performed from the 3rd to the 7th day and reinitiated on the 18th up to the 22nd. The mechanical hypernociception was always evaluated 3 hours after a,b amyrin treatment. (D) Effect of the pretreatment with 30 mg/ kg a,b- amyrin on the paw edema induced by CFA. Paw edema was measured by digital plethysmometer 1 hour from CFA injection. (E) Effect of local treatment with a,bamyrin (30 lg/paw) in the mechanical hypernociception. a,b-Amyrin was given 24 hours after CFA injection. Each point represents the mean of 5 to 6 animals, and the vertical lines indicate the SEM. Statistical analysis were performed by one-way analysis of variance, with repeated measures, followed by the Bonferroni test. The symbols denote signicant difference: #P < .05 from PBS intraplantarly injected mice, and P < .05 from vehicle (p.o.) plus CFA intraplantarly injected mice. B (baseline withdrawal threshold) refers to the measurement before CFA and a,b-amyrin treatment. p.o. = orally.

to the cannabinoid system. The previous treatment with the CB1R antagonist AM251 (Fig. 4A and E) or with the CB2R antagonist AM630 (Fig. 4B and F) signicantly prevented the antihyperalgesic effect of a,b-amyrin (30 mg/kg p.o.) in CFA-injected mice. Interestingly, the antihyperalgesic effects of a,b-amyrin were quite similar to the effects caused by both the CB1R agonist ACEA (Fig. 4C and E) and the CB2R agonist JWH-133 (Fig. 4D and F). Similar to the results found in CFA-injected mice, pretreatment with the CB1R antagonist AM251 (Fig. 5A and C) or with the CB2R

antagonist AM630 (Fig. 5B and D) signicantly prevented the antihyperalgesic effect of a,b-amyrin in PSNL mice. 3.4. Further evidence of the contribution of cannabinoid receptors in the antihyperalgesia caused by a,b-amyrin Because both CB1R and CB2R antagonists displayed a similar effect in preventing the antihyperalgesic effect of a,b-amyrin, which could be explained by the lack of selectivity of the

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Fig. 3. Therapeutic and preventive effect of oral treatment with a,b-amyrin on mechanical and thermal hypernociception induced by partial sciatic nerve ligation (PSNL) in mice. a,b-Amyrin or its vehicle were administered on the 4th day after surgery and tested for (A) mechanical and (B) thermal hypernociception. (C) A separate group of mice received a,b-amyrin mixture or vehicle once per day from the 4th to 8th days and from the 13th to 17th days and (D) the antihypernociceptive effect of a,b-amyrin mixture when administered 1 hour before surgery (pretreatment). The mechanical hypernociception was evaluated by using von Frey hairs (0.6 g), and thermal hypernociception was assessed by the Hargreaves test. Each point represents the mean of 5 to 6 animals, and the vertical lines indicate the SEM. Statistical analyses were performed by one-way analysis of variance, with repeated measures, followed by the Bonferroni test. The symbols denote signicantly difference: #P < .05 from sham group and P < .05 from vehicle (p.o.) in PSNL mice. B (baseline withdrawal threshold) refers to the measurement before PSNL procedure and a,b-amyrin treatment. p.o. = orally.

antagonists, we carried out additional experiments using antisense oligonucleotides for both cannabinoid receptors in mice subjected to the PSNL. The knockdown gene of either CB1R (Fig. 6A and B) or CB2R (Fig. 6D and E) fully prevented the antihyperalgesic effect of a,b-amyrin in PSNL mice, suggesting that both receptors are important to the antihyperalgesic effect of a,b-amyrin. The immunohistochemical assay conrmed the reduction of CB1R and CB2R protein expression in the dorsal horn of the spinal cord after AS-ODN treatment. Compared with the control (MMODN treatment), AS-ODN treatment decreased by 76% the CB1R and by 97% the CB2R expression. Interestingly, a,b-amyrin treatment prevented the upregulation of CB1R and CB2R in PSNL mice by 30% and 60%, respectively (Fig. 6C and F, Supplementary Fig. 1).

3.5. Effect of the a,b-amyrin in the binding assay of [3H]SR141716A or [3H]CP55,940 in CB1R and CB2R Fig. 7 reveals that a,b-amyrin caused a concentrationdependent displacement of the [3H]SR141716A and [3H]CP55,940 binding to CB1R and CB2R from rat brain and spleen membranes, respectively. Of high interest, a,b-amyrin presented a much higher afnity by CB1R than by CB2R. The calculated inhibitions constants (Ki) were 0.133 nM [IC50 = 0.55 (0.18 to 1.64) nM] for CB1R and 1989 nM [IC50 = 6070 (263 to 1397) nM] for CB2R. From these results it is possible to infer that a,b-amyrin binds to CB1R with a very high potency, and it is likely to be one of the most potent binding competitors to CB1R that has been described in the literature. a,b-amyrin presented a nearly 15,000-fold higher afnity to CB1R than CB2R.

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Fig. 4. Evidence for the involvement of cannabinoid receptors in the antihypernociceptive effect of a,b-amyrin mixture on mechanical hypernociception induced by complete Freunds adjuvant (CFA) in mice. In (A) the CB1R antagonist AM251 (1 mg/kg i.p.) or (B) the CB2R antagonist AM630 (3 mg/kg i.p.) were administered 24 hours after -CFA injection. Thirty minutes after antagonist treatment, mice received a,b-amyrin mixture (30 mg/kg p.o.). In (C) the effect of CB1R agonist ACEA (10 mg/kg i.p.) or (D) CB2R agonist JWH-133 (10 mg/kg i.p.) on the hypernociception induced by CFA injection. (E) The areas under the curve of A and C. (F) The areas under the curve of B and D. Each point represents the mean of 5 to 6 animals, and the vertical lines indicate the SEM. Statistical analyses were performed by one-way analysis of variance, with repeated measures when appropriated, followed by the Bonferroni test. Symbols denote signicant difference: #P < .05 from the CFA group, P < .05 from a,b-amyrin or CB1R and CB2R agonist-treated group. B (baseline withdrawal threshold) refers to the measurement before CFA and drug treatments. p.o. = orally; N = nave; Veh = vehicle.

3.6. a,b-amyrin effects on tetrad assay The effect of the treatment with a,b-amyrin (30 mg/kg p.o.) in the tetrad assay is shown in Fig. 8. a,b-amyrin failed to alter the

body temperature (Fig. 8A). In addition, the compound did not alter the sensitivity threshold in healthy tissue, as measured by the withdrawal behavior against radiant heat in mice tails (Fig. 8B). a,b-Amyrin was also unable to alter the locomotor activity

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Fig. 5. Evidence for the involvement of cannabinoid receptors in the antihypernociception caused by a,b-amyrin treatment on mechanical hypernociception induced by partial sciatic nerve ligation (PSNL). (A) The CB1R selective antagonist AM251 (1 mg/kg i.p.) or (B) the CB2R selective antagonist AM630 (3 mg/kg i.p.) were administered on the 4th day after surgery. a,b-amyrin (30 mg/kg p.o.) or vehicle (Veh) were administered 30 minutes after antagonist treatment. Panels C and D represent the areas under the curve of panels A and B, respectively. Each point represents the mean of 5 to 6 animals, and the vertical lines indicate the SEM. Statistical analysis were performed by one-way analysis of variance, with repeated measures when appropriated, followed by the Bonferroni test. A signicant difference is dened by the symbols #P < .05 from control PSNL and P < .05 from PSNL in a,b-amyrin treated group. B (baseline withdrawal threshold) refers to the measurement before PSNL procedure and antagonists treatment. i.p. = intraperitoneally; p.o. = orally.

(Fig. 8C) and failed to cause catalepsy-like behavior (Fig. 8D). Similarly, the treatment with either CB1R or CB2R agonists, ACEA or JWH-133, in the dose that produced antihypernociception (10 mg/kg) did not alter the parameters evaluated in the tetrad assay (Fig. 8AD). 3.7. mRNA expression of CB1R and CB2R in the spinal cord after partial sciatic nerve ligation or CFA injection Regarding the role of the cannabinoid system in the antihyperalgesic effect of a,b-amyrin, we next evaluated whether a,bamyrin interferes with the expression of either CB1R or CB2R. We found a constitutive expression of both CB1R and CB2R mRNA in the lumbar cord of nave mice. However, CB1R was more predominantly expressed in this tissue compared with CB2R (data not shown). The intraplantar injection with CFA did not signicantly change CB1R or CB2R mRNA expression 24 hours (Fig. 9) and 4 days after the CFA injection (Supplementary Fig. 2). Interestingly, mice that were treated with a,b-amyrin (30 mg/kg p.o.) previously to CFA presented a signicant upregulation of CB1R and CB2R mRNA expression (Fig. 9A and B). Because a,b-amyrin was able to change the mRNA expression of cannabinoid receptors in CFA-injected mice, we investigated whether the compound could affect the expression of these receptors. The treatment with a,b-amyrin in the absence of any injury resulted in a signicant increase in CB1R mRNA expression. In contrast, a,b-amyrin alone decreased CB2R mRNA expression.

Different from the results obtained when CFA was injected intraplantarly, the PSNL caused an upregulation of both CB1R and CB2R mRNA expression in the spinal cord when assessed 4 days after the injury. The treatment with a,b-amyrin (30 mg/kg p.o.) prevented the upregulation of CB2R mRNA but did not signicantly affect CB1R mRNA upregulation (Fig. 9A and B). To nd out whether a,b-amyrin would alter the expression of cannabinoid receptors in the mouse cortex, the mRNA for CB1R and CB2R were quantied in this tissue. Differently from the spinal cord region, the oral treatment with a,b-amyrin (30 mg/kg) did not signicantly change the cortical levels of both CB1R and CB2R mRNA (Supplementary Fig. 3). 3.8. a,b-Amyrin treatment reduces the synthesis/release of proinammatory cytokines and myeloperoxidase activity induced by CFA injection and PSNL procedure PSNL caused a signicant increase in the levels of TNF-a (4 hours after surgery), IL-1b (7 days after surgery), IL-6 (3 days after surgery), and KC (6 hours after surgery) and in the activity of myeloperoxidase (4 days after surgery) in the lumbar area of the mouse spinal cord. The treatment of mice with a,b-amyrin (30 mg/kg p.o. 1 hour before PSNL) signicantly prevented the PSNL-induced release or synthesis of these cytokines and prevented the increase in myeloperoxidase activity. The inhibition values were 75% for TNF-a, 60% for IL-1b, 64% for IL-6, 33% for KC, and 76% for MPO activity (Fig. 10AE, respectively).

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Fig. 6. Effect of knockdown gene for CB1R and CB2R on the antihypernociceptive effect of a,b-amyrin in partial sciatic nerve ligation (PSNL). Antisense oligonucleotide (ASODN; 12.5 lg/5 lL) was administered by intrathecal (i.t.) route twice per day. The treatment started 24 hours after PSNL and was performed for 3 consecutive days at 12-hour intervals. Effect of a,b-amyrin in mechanical hypernociception in (A) CB1R or (D) CB2R knockdown gene mice. (B) and (E) represent the areas under the curve for A and D, respectively. The quantication of (C) CB1R and (F) CB2R protein expression in the dorsal horn of spinal cord after AS-ODN treatment. Each point represents the mean of 5 to 6 animals, and the vertical lines indicate the SEM. Statistical analyses were performed by one-way analysis of variance, with repeated measures when appropriated, followed by the Bonferroni test. A signicant difference is dened by the symbols #P < .05 from PSNL in missense oligonucleotide (MM-ODN) plus vehicle (Veh) and P < .05 from PSNL in MM-ODN plus a,b-amyrin. B (baseline withdrawal threshold) refers to the measurement before PSNL procedure and AS-ODN treatment. N = nave; Sh = sham.

footpads with inhibitions of 65% for IL-1b, 42% for IL-6, 58% for KC, and 61% for myeloperoxidase activity (Fig. 11AD, respectively). In the lumbar area of the spinal cord, the treatment with a,b-amyrin caused an inhibition of 88% in IL-1b, 60% in IL-6, 62% in KC, and 78% in myeloperoxidase activity (Fig. 11EH, respectively). The anti-inammatory effect of a,b-amyrin was signicantly prevented by the pretreatment with the selective antagonists for both CB1R (AM251; 1 mg/kg i.p.) and CB2R (AM630 3 mg/kg i.p.). The results depicted in Fig. 11 demonstrate that the activation of cannabinoid receptors also contribute to the anti-inammatory action of a,b-amyrin. 3.9. a,-amyrin inhibits the activation of CREB and NF-jB and the over expression of COX-2 induced by CFA injection or PSNL procedure
Fig. 7. a,b-amyrin binding displacement of [3H]SR141716A and [3H]CP55,940 in rat brain (CB1R) or spleen (CB2R) membrane preparations. Binding assays were carried out at 30C using 0.5 nM [3H]SR141716A or 0.8 nM [3H]CP55,940 and increased concentrations of a,b-amyrin. Data are the mean of 4 independent experiments always performed in duplicate. Vertical bars represent the standard error of mean. The asterisks denote a signicant difference, P < .05 in the absence of a,b-amyrin.

Likewise, the treatment of mice with CFA induced a signicant increase in IL-1b, IL-6, and KC levels and in the myeloperoxidase activity in the footpad and spinal cord at 6, 6, 3, or 6 hours after the CFA injection, respectively. The dose of 30 mg/kg a,b-amyrin p.o., given 30 minutes before the CFA, signicantly prevented the increase in the levels of these pro-inammatory cytokines in mice

We found a marked activation of the transcription factors CREB and NF-jB in mice footpads when assessed 6 hours after the CFA injection and in the lumbar spinal cord 7 days after PSNL. These events were accompanied by an upregulation of COX-2, 24 hours after the CFA injection and 21 days after PSNL (Fig. 12 and Supplementary Figs. 4 and 5). All time points were selected based on the activation peak of each target. These time points were preliminarily determined in our laboratory. CREB and NF-jB activation as well as COX-2 upregulation were signicantly prevented in mice pretreated with a,b-amyrin (30 mg/kg p.o.), as assessed in the footpads of mice injected with CFA. The inhibitions were 100% for CREB, 78% for NF-jB, and 28% for COX-2 (Fig. 12AC).

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Fig. 8. The effect of the oral treatment with a,b-amyrin (30 mg/kg) in the tetrad assay. The tests included (A) thermal body measurement, (B) threshold sensitivity, (C) locomotor activity, and (D) catalepsy-like behavior. Each point represents the mean of 5 to 6 animals, and the vertical lines indicate the SEM. Statistical analyses were performed by one-way analysis of variance, with repeated measures, followed by the Bonferroni test. B (baseline withdrawal threshold) refers to the evaluation performed before drugs treatment.

Fig. 9. mRNA expression of CB1R and CB2R in spinal cord after complete Freunds adjuvant (CFA) injection or partial sciatic nerve ligation (PSNL). mRNA was quantied in spinal cord for (A) CB1R and (B) CB2R 24 hours after the CFA injection or on the 4th day after PSNL. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA was used to normalize the relative amount of mRNA. Each column represents the mean of 3 samples performed in duplicate. The vertical lines show the SEM. Statistical analyses were performed by one-way analysis of variance followed by the Bonferroni test. The effect of a,b-amyrin in noninjured mice was analyzed by t-test and compared with nave mice. A signicant difference is designed by #P < .05 from nave (N) or sham (Sh) group. P < .05 from vehicle (Veh) in CFA or vehicle (Veh) in PSNL. p.o. = orally.

Treatment with a,b-amyrin also decreased the levels of CREB, NF-jB, and COX-2 in the spinal cord of PSNL-injured mice to levels lower than those found in the sham group (Fig. 12DF); this effect of a,b-amyrin might explain the reduction in the progress of inammation in the injured tissue. 4. Discussion In this study we have reported that the antihyperalgesic and anti-inammatory effects of the pentacyclic triterpene a,b-amyrin against chronic and inammatory pain are directly related to the ability of a,b-amyrin to interact with cannabinoid receptors CB1 and CB2. Studies in neuropathic and inammatory pain have

demonstrated that the activation of both CB1R and CB2R confers a valuable protection against persistent pain [19,41,46]. In fact, endocannabinoids and inhibitors of endocannabinoids transport or degradation exert pronounced analgesic effects [25]. Furthermore, plant-derived substances dened as phytocannabinoids can bind to cannabinoid receptors and induce analgesia and antiinammatory effects [21]. In the present study, we extended the relevance of the cannabinoid system in the control of inammatory pain because both CB1R and CB2R agonists, ACEA and JWH-133, signicantly inhibited the mechanical hyperalgesia induced by CFA. Of high interest, the oral treatment with a,b-amyrin displayed a similar antihyperalgesic effect when compared with ACEA and JWH-133. In addition, the pretreatment with the CB1R and CB2R

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Fig. 10. Effect of a,b-amyrin on cytokine production/release and myeloperoxidase (MPO) activity induced by partial sciatic nerve ligation (PSNL). a,b-Amyrin (30 mg/kg p.o.) or vehicle were administered 1 hour before PSNL. The levels of (A) tumor necrosis factor (TNF)-a (4 hours after surgery), (B) interleukin (IL)-1b (7 days after surgery), (C) IL-6 (3 days after surgery), and (D) keratinocyte-derived chemokine (KC) (6 hours after surgery); (E) MPO activity (4 days after surgery) was measured in the mice lumbar spinal cord. Each column represents the mean of 4 animals, and vertical lines show the SEM. Statistical analyses were performed by one-way analysis of variance followed by the Bonferroni test. The symbols denote a signicant difference #P < .05 compared with the sham (Sh) group; P < .05 compared with the PSNL in vehicle (Veh) group. p.o. = orally.

antagonists AM251 and AM630 equally prevented the hyperalgesia and the production or release of inammatory cytokines induced by CFA, demonstrating that the protective effects afforded by a,b-amyrin are closely related to the activation of the cannabinoid system. It is well recognized that the injuries caused by CFA or PSNL promote profound alterations in the supercial (I and II) and deep (V and VI) laminal dorsal horn neurons that receive noxious inputs [13,56]. However, a comparative study between CFA and sciatic nerve ligation models revealed that they induce different tissue plasticity in the spinal cord. Indeed the sciatic nerve ligation upregulated CB2R expression in the spinal cord, an event that was absent in CFA-injected mice [58]. In agreement with this report, we also found a signicant increase in CB1R and CB2R mRNA in the spinal cords of mice that had the sciatic nerve constricted, but not in those that were injected with CFA. Considering that these 2 models present different proles in the regulation of cannabinoid receptor expression, we also investigated whether the antagonism of CB1R and CB2R would affect the antihyperalgesic effect of a,bamyrin in PSNL injury as well as it affected the CFA injection model. In this set of experiments, the systemic treatment with either CB1R or CB2R antagonists equally prevented the antihyperalgesic

effect of a,b-amyrin. Comparatively, the antinociceptive effects of a,b-amyrin in CFA and PSNL were not affected by the difference in the expression of the cannabinoid receptors in the spinal cord. Concerning a possible lack of selectivity of the antagonists upon CB1R or CB2R, we next used a knockdown strategy to better identify the target for a,b-amyrin. Interestingly, the reduction of CB1R and CB2R expression by antisense oligonucleotides was more effective than the pharmacological use of antagonists in preventing the antinociception of a,b-amyrin. However, the knockdown gene of CB1R or CB2R displayed a very similar prole in preventing a,bamyrin effects. The effects of a,b-amyrin in the cannabinoid system could be due to the binding to cannabinoid receptors, to the prevention in endocannabinoids degradation, or via an indirect mechanism that promotes the activation of cannabinoid system. Therefore, we performed an in vitro binding assay to determine whether or not a,b-amyrin could directly bind to CB1R, CB2R, or both. Remarkably, a,b-amyrin potently inhibited the radiolabel binding to CB1R and with a lower potency it decreased the radiolabel binding to CB2R. Taking this into account, we can suggest that a,b-amyrin exerts most of its antihyperalgesic effects by a direct stimulation of both CB1R and CB2R.

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Fig. 11. Effect of a,b-amyrin on cytokine production/release and myeloperoxidase activity in footpad and lumbar spinal cord of mice injected with complete Freunds adjuvant (CFA). The CB1R selective antagonist AM251 (1 mg/kg i.p.) or CB2R selective antagonist AM630 (3 mg/kg i.p.) were administered 30 minutes before the treatment with a,b-amyrin (30 mg/kg p.o.). The mice received CFA (20 lL/paw) 30 minutes after the a,b-amyrin treatment. In (A and E) IL-1b, (B and F) IL-6, (C and G) keratinocytederived chemokine (KC), and (D and H) MPO activity were measured in mice paw and lumbar spinal cord at 6, 6, 3, or 6 hours after the CFA injection, respectively. Each column represents the mean of 4 animals, and vertical lines show the SEM. Statistical analyses were performed by one-way analysis of variance followed by the Bonferroni test. The symbols denote a signicant difference #P < .05 from CFA plus vehicle (Veh) group; P < .05 from CFA plus a,b-amyrin treated group. i.p. = intraperitoneally; p.o. = orally.

The CB1R is predominantly expressed in neurons [2,37,51] and directly responds to neuronal changes. The binding of selective agonists to CB1R reduces inammatory and neuropathic hyperalgesia [12,19,38]. CB1R is distributed throughout the brain [35], where

it modulates neuronal activity and mediates many of the known psychoactive and antinociceptive effects of the cannabinoids [28]. Therefore, some CB1R agonists may exhibit psychotropic side effects, which limit their use in pain treatment. A good alternative

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Fig. 12. Effect of a,b-amyrin on the activation of transcriptional factors and cyclooxygenase 2 (COX-2) upregulation induced by complete Freunds adjuvant (CFA) or partial sciatic nerve ligation (PSNL). a,b-Amyrin (30 mg/kg p.o.) or vehicle were administered 1 hour before CFA (20 L/paw) or 1 hour before surgery. Immunohistochemical analysis for (A) phospho-cyclic adenosine monophosphate response element binding (p-CREB) (6 hours after CFA), (B) phospho-p65 nuclear factor jB (p-p65 NF-jB) (6 hours after CFA), and (C) cyclooxygenase 2 (COX-2) (24 hours after CFA) was carried out in footpad tissue. In (D) p-CREB (7 days after surgery), (E) p-p65 NF-jB (7 days after surgery), (F) COX-2 (21 days after surgery) in lumbar spinal cord (L3L5) sections. Each column represents the mean of 4 animals, and vertical lines show the SEM. Statistical analyses were performed by one-way analysis of variance followed by the Bonferroni test. Symbols denote a signicant statistical difference #P < .05 from phosphate-buffered saline (PBS) or sham (Sh) group, and P < .05 from CFA or PSNL control group. Veh (vehicle). p.o. = orally.

resides in substances that do not cross the blood-brain barrier and that exert their effects mainly in the peripheral nervous system. In fact, some studies revealed that CB1R present in the peripheral nervous system are also crucial to pain management, independently of the activation of CB1R from the central nervous system [1,3]. In line with this evidence, we reported that local treatment with a,bamyrin signicantly prevented the hyperalgesia induced by CFA, suggesting that the activation of CB1R from peripheral sites by a,b-amyrin might be an important step in pain control. Of high relevance, the oral treatment with a,b-amyrin did not cause any disturbance in the locomotor performance, body temperature, or sensitivity, or alter the catalepsy test, suggesting that a,b-amyrin is devoid of effects in brain areas. In addition, mice chronically treated with a,b-amyrin were not susceptible to tolerance because the antinociceptive effect of a,b-amyrin was maintained even after repeated treatments and successive interruptions. Previous studies from our group have shown that the administration of a,b-amyrin directly into the central nervous system, by intrathecal or intracerebroventricular injection, decreased the pro-nociceptive effects of formalin [44]. This same study found no motor alteration by systemic treatment with a,b-amyrin. As a result, it is likely that a,b-amyrin exerts central effects when it is delivered directly into the brain, but it may not achieve the supraspinal area after oral treatment. Corroborating this hypothesis, oral treatment with a,b-amyrin was devoid of effect on the mRNA expression of cannabinoid receptors in the brain cortex. On the other hand, the same treatment increased mRNA for CB1R and decreased mRNA for CB2R in the lumbar spinal cord, which demonstrates that the effects displayed by a,b-amyrin on the lumbar spinal cord might not be extended to the forebrain. Therefore, the alterations caused by a,b-amyrin in mRNA expression of cannabinoid receptors from the spinal cord may be an indirect effect of the compound due to the reduction of peripheral neuronal

activation and inammation. These results suggest that a,b-amyrin induces antinociception by acting preferentially at the peripheral sites. However, some pharmacokinetic approaches are necessary to specify the exact distribution of a,b-amyrin and its ability to cross the blood-brain barrier. It is important to mention that, although a,b-amyrin presented nearly 15,000-fold more afnity to CB1R than CB2R, the CB2R antagonists and the gene knockdown efciently reverted the effects of a,b-amyrin against pain and inammation. Therefore, it is likely that during inammatory conditions, where there is an overexpression of CB2R [55,57], a,b-amyrin is also acting by binding to CB2R. The CB2R is expressed in immune cells [4,20], mainly B-cells, macrophages, microglia, mast and T-cells, and the activation of this receptor induces marked immunosuppression [23,49]. Indeed, a,b-amyrin greatly prevented the production/release of the proinammatory cytokines TNF-a, IL-1b, IL-6, and KC, as well as the activity of the pro-inammatory enzyme myeloperoxidase. This effect was clearly dependent on the activation of the cannabinoid system because the CB1R and CB2R antagonists signicantly prevented the anti-inammatory effects of a,b-amyrin. Therefore, the antinociceptive effects of a,b-amyrin might be associated with anti-inammatory properties of the compound. Noteworthy is that the protective effects of a,b-amyrin are likely not exclusively dependent on the activation of the cannabinoid receptors. In fact, previous studies have demonstrated that a,bamyrin inhibited intracellular protein kinase C, protein kinase A, mitogen-activated protein kinase, COX-2 expression, transcriptional factors NF-jB, and CREB activation [40,44,54]. In addition, a,b-amyrin effectively prevented the nociception induced by the receptor agonists: capsaicin, glutamate, and bradykinin. However, a,b-amyrin did not affect agonist binding to these receptors [44]. In this context, a cross-talk between cannabinoid receptors and TRPV1, glutamate, or bradykinin may be suggested, but whether

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K.A.B. Simo da Silva et al. / PAIN 152 (2011) 18721887 [7] Ashton JC, Milligan ED. Cannabinoids for the treatment of neuropathic pain: clinical evidence. Curr Opin Invest Drugs 2008;9:657. [8] Bortalanza LB, Ferreira J, Hess SC, Delle Monache F, Yunes RA, Calixto JB. Antiallodynic action of the tormentic acid, a triterpene isolated from plant, against neuropathic and inammatory persistent pain in mice. Eur J Pharmacol 2002;453:2038. [9] Bosier B, Muccioli GG, Hermans E, Lambert DM. Functionally selective cannabinoid receptor signaling: therapeutic implications and opportunities. Biochem Pharmacol 2010;80:112. [10] Bouaboula M, Poinot-Chazel C, Bourree B, Canat X, Calandra B, RinaldiCarmona M, Le Fur G, Casellas P. Activation of mitogen-activated protein kinases by stimulation of the central cannabinoid receptor CB1. Biochem J 1995;312:63741. [11] Bradley PP, Priebat DA, Christensen RD, Rothstein G. 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or not these mechanisms are dependent on the activation of the cannabinoid receptors by a,b-amyrin requires further investigation. Conrming previous studies [44,54], we found a signicant inhibition of the transcription factors NF-jB and CREB activation by a,b-amyrin in both CFA and PSNL models of chronic pain. The phosphorylation of the p65 subunit of NF-jB and translocation to the nucleus promote the gene transcription of COX-2 and several pro-inammatory cytokines [22,30]. Additionally, the transcription factor CREB binds to the promoter region in DNA (cyclic adenosine monophosphate response elements) and also induces the transcription of COX-2 [50], growth factors, and other inammatory mediators [47,59]. Taking this into account, the inhibition of NF-jB and CREB activation likely contributes to the long-lasting antinociceptive effect of the a,b-amyrin because it greatly avoids the production of inammatory mediators responsible for the progress of inammation. In summary, our ndings demonstrate that the main mechanism responsible for the a,b-amyrin antihyperalgesic and antiinammatory properties against persistent pain depends on its ability to bind to both CB1R and CB2R. Furthermore, the antinociceptive effect of a,b-amyrin was also associated with the inhibition of the levels of TNF-a, IL-1b, IL-6, and KC, the inhibition of the activation of transcriptional factors NF-jB, CREB, upregulation of COX2, and neutrophil migration/activation. As a,b-amyrin is quite safe for animals [54] and because there are few effective and safe drugs for the treatment of persistent pain, especially neuropathic and inammatory pain, the present study may have clinical relevance and suggests that a,b-amyrin might be a potential molecule of interest for pain relief. Conict of interest statement All authors declare that there are no conicts of interest. Acknowledgements This work was supported by grants from the Conselho Nacional de Desenvolvimento Cientco e Tecnolgico (CNPq), Coordenao de Aperfeioamento de Pessoal de Nvel Superior (CAPES), and Fundao de Apoio a Pesquisa do Estado de Santa Catarina (FAPESC), all from Brazil. K.A.B.S.S., A.F.P., G.F.P. and A.F.B. are Ph.D students in pharmacology receiving grants from CNPq. E.S.S. is a PhD student in physiology. F.C.M. holds postdoctoral fellowships from CNPq. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.pain.2011.04.005. References
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