Activation of Cannabinoid Receptors by the Pentacyclic Triterpene a,B-Amyrin

w w w . e l s e v i e r . c o m / l o c a t e / p a i n

PAIN�

152 (2011) 1872–1887

Activation of cannabinoid receptors by the pentacyclic triterpene a,b-amyrininhibits inflammatory and neuropathic persistent pain in mice

Kathryn A.B. Simão da Silva, Ana F. Paszcuk, Giselle F. Passos, Eduardo S. Silva, Allisson Freire Bento,Flavia C. Meotti, João B. Calixto ⇑Departamento de Farmacologia, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Campus Universitário Trindade, Bloco D, CCB, Caixa Postal 476,CEP 88049-900, Florianópolis, Santa Catarina, Brazil

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

a r t i c l e i n f o a b s t r a c t

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

Keywords:a,b-AmyrinCannabinoidsCB1RCB2RHypernociceptionNeuropathyInflammation

0304-3959/$36.00 � 2011 International Associationdoi:10.1016/j.pain.2011.04.005

⇑ Corresponding author. Tel.: +55 48 3721 9491; faE-mail addresses: [email protected], calixto@f

In this study, we report that a,b-amyrin, a plant-derived pentacyclic triterpene, reduced persistentinflammatory and neuropathic hyperalgesia in mice by a direct activation of the CB1 and CB2 cannabinoidreceptors (CB1R and CB2R). The oral treatment with a,b-amyrin (30 mg/kg) significantly reduced mechan-ical and thermal hyperalgesia and inflammation induced by complete Freund’s adjuvant (CFA) and bypartial sciatic nerve ligation (PSNL). The pretreatment with either CB1R or CB2R antagonists and theknockdown gene of the receptors significantly reverted the antinociceptive effect of a,b-amyrin. Of note,binding studies showed that a,b-amyrin directly bound with very high affinity to CB1R (Ki = 0.133 nM)and with a lower affinity to CB2R (Ki = 1989 nM). Interestingly, a,b-amyrin, ACEA (CB1R agonist), orJWH-133 (CB2R agonist), at doses that caused antinociception, failed to provoke any behavioral distur-bance, as measured in the tetrad assay. In addition, a,b-amyrin largely decreased interleukin-1b (IL-1b), 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 fac-tors: 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 demon-strated that a,b-amyrin exhibits long-lasting antinociceptive and anti-inflammatory properties in 2 mod-els of persistent nociception via activation of cannabinoid receptors and by inhibiting the production ofcytokines 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.

1. Introduction

Chronic pain is a public health problem that causes personaland social afflictions. Depending on its origin, chronic pain canbe classified as inflammatory or neuropathic. Inflammatory painis usually initiated by damage in tissues or by inflammation,whereas neuropathic pain is caused by a primary injury ordysfunction in the peripheral or central nervous system. Clinicalinvestigations have revealed that opiates, nonsteroidal anti-inflammatory drugs, anticonvulsants, and antidepressants constitutethe most commonly used pharmacotherapies for the treatment ofchronic pain [24]. However, these drugs present several adverseeffects and have limited therapeutic efficacy in some patients[31]. Chronic inflammatory and neuropathic pain differs fromacute pain in time duration, threshold for stimulation, and plasticalterations in the tissue of injury and in the dorsal horn of the

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x: +55 48 3337 5479.armaco.ufsc.br (J.B. Calixto).

spinal cord [29,56,61]. These substantial differences could explainwhy some analgesic drugs that are employed for treatment ofacute pain may not be effective against chronic pain.

Recently, cannabinoids have emerged as attractive potentialalternatives or supplemental therapies for the treatment of chronicpain. Two human cannabinoid receptors have been identified, CB1

and CB2 [39,42]. Both of these receptors are members of the G pro-tein-coupled receptor (GPCR) super family. The CB1 receptors(CB1R) are primarily distributed in the peripheral and central ner-vous system (CNS) [42], whereas the CB2 receptors (CB2R) areextensively localized in cells of the immune system [4,20]. Thebinding of agonist to CB1R and CB2R inhibits adenylyl cyclase bya Gai/o-dependent mechanism and activate Gbc-dependent mito-gen-activate protein kinases (MAPK) cascades [9,10]. Numerousstudies have demonstrated the antinociceptive effects of someselective and nonselective cannabinoid receptor agonists in differ-ent models of inflammatory and neuropathic pain [7].

In this context, the search for new substances that activate thecannabinoid system and that lack serious side effects is of great

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relevance for chronic pain management. Earlier studies have re-ported that the isomeric mixture of a-amyrin and b-amyrin(Fig. 1) exhibits antinociceptive and anti-inflammatory propertiesin rodents [6,32,43]. a,b-Amyrin is a pentacyclic triterpene andconstitutes the main bioactive component of the resin of Protiumkleinii and Protium heptaphyllum [44,45]. Moreover, a,b-amyrin ex-erted a pronounced anti-inflammatory effect in acute models ofinflammation [40,45,54]. Likewise, this triterpene presented antin-ociceptive activity when administered at peripheral, spinal, orsupraspinal sites in acute nociception [44]. However, it is still un-known whether a,b-amyrin is effective in counteracting chronicpain. In addition, the precise mechanism of action of this com-pound is not completely understood.

Therefore, the present study investigated, through functionaland molecular assays, the mechanisms by which a,b-amyrin exertsits antinociceptive and anti-inflammatory properties in 2 models ofpersistent pain, the intraplantar injection of complete Freund’sadjuvant (CFA) and the partial sciatic nerve ligation (PSNL). Fur-thermore, we investigated the ability of a,b-amyrin to specificallybind to CB1R and CB2R. In addition, the effects of a,b-amyrin onCB1R and CB2R mRNA expression, pro-inflammatory cytokines lev-els, myeloperoxidase (MPO) activity, cyclooxygenase 2 (COX-2)expression, and activation of transcriptional factors nuclear factorjB (NF-jB) and cyclic adenosine monophosphate response ele-ment binding (CREB) were also investigated.

2. Materials and methods

2.1. Animals

The experiments were carried out in male Swiss mice (20 to30 g) or male Wistar rats (200 to 250 g) that were kept in a roomwith controlled temperature (22 ± 1�C) and humidity (50% to80%), under a 12 hour:12 hour light-dark cycle (lights on at06:00 am) with food and water ad libitum. The animals were accli-matized to the laboratory settings for at least 1 hour before testingand were used only once throughout the experiments. All of theprocedures used in the present study were approved by the Insti-tutional Ethics Committee of the Universidade Federal de SantaCatarina (PP00148) and were carried out in accordance with the‘‘Principles of Laboratory Animal Care’’ from National Institutes ofHealth publication No. 85–23. In addition, the experimental proce-dures were in agreement with the current guidelines for the care oflaboratory animals and the ethical guidelines for investigations ofexperimental pain in conscious animals as previously specified[60]. The number of animals and the intensity of noxious stimuliused were the minimum necessary to demonstrate consistent ef-fects of the drug treatment.

2.2. CFA-induced inflammation and hypernociception

Mice were gently immobilized and received an intraplantar injec-tion of 20 lL CFA (1 mg/mL heat-killed Mycobacterium tuberculosis in

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

85% paraffin oil and 15% mannide mono-oleate). The control groupsreceived 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 nervewas performed by tying 1/3 to 1/2 dorsal area of the distal partof sciatic nerve, according to the procedure described by Malmbergand Basbaum [36]. In sham-operated mice, the sciatic nerve wasexposed without ligation.

2.4. Drug treatment protocols

2.4.1. CFA injection groupMechanical and thermal hypernociception and paw edema

were assessed in mice previously treated by oral gavage (p.o.) witha,b-amyrin (3 to 30 mg/kg) or vehicle (Tween/ethanol/PBS) 1 hourbefore CFA injection. The mechanical or thermal hypernociceptionand the paw volume were taken 1 hour from CFA injection. Toinvestigate the effects of long-term treatment with a,b-amyrin,mice received a second treatment with a,b-amyrin (30 mg/kgp.o.) 3 days after the CFA, when the hypernociceptive responsehad been reestablished. The treatment was repeated for 5 consec-utive days (days 3 to 7) after the CFA injection. After an interrup-tion of 11 days, the treatment was reinitiated on day 18th andmaintained until day 22nd after the CFA injection. The mechanicalhypernociception was measured daily, always 3 hours after thea,b-amyrin treatment. For the local treatment, a,b-amyrin(30 lg/paw) or vehicle (Tween/ethanol/PBS) were directly injectedin the ventral surface of the hind paw (in the hypodermis) 24 hoursafter the CFA injection. The mechanical hypernociception was eval-uated 30 minutes after the a,b-amyrin treatment.

2.4.2. PSNL groupa,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 evaluationof mechanical or thermal hypernociception started on the 4thday after surgery. To investigate the effects of long-term treatment,a,b-amyrin (30 mg/kg) or vehicle were administered orally to miceonce per day for 5 consecutive days (from the 4th to the 8th dayafter surgery). The treatment was interrupted and reinitiated onthe 13th day after the surgery and continued until the 17th day.The mechanical hypernociception was measured every day, always4 hours after the a,b-amyrin treatment.

2.4.3. Investigation of the CB1R and CB2R mechanismsThe role of cannabinoid receptors in the antihypernociceptive

action of a,b-amyrin was investigated by treating mice with theselective CB1R antagonist AM251 (1 mg/kg i.p.) [33] or the selec-tive CB2R antagonist AM630 (3 mg/kg i.p.) [48] 30 minutes beforea,b-amyrin (30 mg/kg p.o.). These treatments were performed24 hours after CFA injection or 4 days after PSNL. The positive con-trols experiments were performed using the selective CB1R agonistACEA (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 onprevious experiments from our group. The evaluation of themechanical hypernociception started 30 minutes after the treat-ment with a,b-amyrin.

To investigate the role of cannabinoid receptors in the anti-inflammatory action of a,b-amyrin, different groups of mice werepretreated with the selective CB1R antagonist AM251 (1 mg/kgi.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. Thirtyminutes after the treatment with a,b-amyrin, mice received 20 lL

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CFA intraplantarly. Tissues were collected in different time pointsafter a,b-amyrin treatment, as specified in Sections 2.12 and 2.13.

2.4.4. Intrathecal administration of antisense oligonucleotideTo further confirm the role of CB1R and CB2R on the antihypern-

ociceptive effect of a,b-amyrin, the antisense oligonucleotide(AS-ODN) was injected intrathecally (i.t.) into the subarachnoidspace (L3–L5) of anaesthetized mice 24 hours after the PSNL usinga modified version of a described method [5]. Spinal injections of5 lL of a specific AS-ODN for CB1R (50-GCCTGCTAGAATCGCATT-30), CB2R (50-CTGCTGAGCGCCCTGGAGAAGAAC-30), or a mismatchMM-ODN control (50-GCCTGCTAGAATCGCATT-30) were appliedfor 3 consecutive days at 12-hour intervals. The AS-ODNs werereconstituted in nuclease-free water to a final concentration of12.5 lg/mouse. On the 3rd day, the mice received an oral adminis-tration of a,b-amyrin (30 mg/kg) or vehicle 30 minutes after thelast 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 accessto the ventral surface of the right hind paw. The withdrawal re-sponse frequency was measured after 10 applications (3 secondsin duration) of the von Frey hairs (Stoelting, Chicago, IL). The ani-mals were acclimatized for at least 30 minutes before the behav-ioral test, and mechanical hypernociception was evaluated atseveral time points. The withdrawal threshold in naïve mice wastaken before CFA injection, PSNL procedure, or a,b-amyrin treat-ment. This measurement is presented in the graphs as the baseline(B). The pressure exerted by the von Frey hairs (0.6 g) produced abaseline withdrawal frequency of around 15%, which is consideredadequate for evaluation of mechanical hypernociception [8].

2.6. Measurement of thermal hyperalgesia

Thermal hyperalgesia was evaluated by measuring the latencyof paw withdrawal according to the method described byHargreaves et al. [26], with minor modifications. The mice wereplaced in clear plastic chambers (7 � 9 � 11 cm) on an elevatedsurface and allowed to acclimatize to the environment for 1 hour.The heat stimulus was directed to the plantar surface of each hindpaw near to the toes. The infrared intensity was adjusted to obtainbaseline paw-withdrawal latencies of �15 seconds. In graphs, thebaseline paw-withdrawal latencies are referred to as B. Anautomatic 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 inlL, as the difference between the right (CFA-injected paw) andthe 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-ODNwas assessed by immunohistochemistry analyses. The lumbar por-tions of the spinal cords were harvested 6 hours after the a,b-amyrin treatment, and the tissues were prepared as described inSection 2.11. The immunohistochemistry technique was carriedout in paraffin-embedded sections of the lumbar spinal cord, usingpolyclonal-rabbit anti-CB1R (1:500) or polyclonal-rabbit-anti-CB2R(1:150) antibodies (Cayman Chemical, Ann Arbor, Michigan). Theimmunohistochemistry technique was conducted as describedlater (Section 2.11).

2.9. Binding assay

Binding to cannabinoid receptors was carried out as describedpreviously [27] with minor modifications. Membranes were pre-pared from rat brain (containing CB1R) or rat spleen (containingCB2R). The rats were killed by decapitation; the brain and spleenwere quickly removed and homogenized in ice-cold buffer contain-ing 50 mM Tris–HCl, 3 mM MgCl2, 1 mM EDTA, and 320 mM sucrose,pH 7.4. The homogenate was first centrifuged for 10 minutes at3600 rpm at 4�C. The low-speed pellets were discarded, and thesupernatants were further centrifuged for 1 hour at 18,000 rpm at4�C. The resulting high-speed pellets were suspended in 1 mL ofice-cold buffer containing 50 mM Tris–HCl, 3 mM MgCl2, and1 mM EDTA, pH 7.4. The protein content was measured as describedby Lowry et al. [34]. Membranes (50 lg/protein) were incubated in50 mM Tris–HCl, 3 mM MgCl2, 1 mM EDTA, 1 mM PMSF, 0.2% bovineserum albumin buffer, pH 7.4. The brain or spleen membranes wereincubated with 0.5 nM [3H] SR141716A (specific activity 52 Ci/mmol) or 0.8 nM [3H]CP-55,940 (specific activity 147.9 Ci/mmol),respectively. a,b-Amyrin (10�11 to 10�4 M) was added to the incuba-tion system. The samples were incubated at 30�C for 60 minutes. Thecontent was filtered using microfilter GF/B by vacuum filtration. Fil-ters were washed with ice-cold buffer: 50 mM Tris–HCl 50 mM,3 mM MgCl2, 1 mM EDTA, 0.2% bovine serum albumin buffer, pH7.4. The radioactivity retained on the filters was measured in a Pack-ard scintillation counter. Specific binding was calculated discount-ing the retained radioactivity in the presence of 100 lM WIN55.212-2 (nonspecific binding). The results were normalized be-tween 0% and 100% radioactivity specifically bound. All data wereobtained in duplicate of 4 independent experiments.

2.10. Tetrad evaluation

2.10.1. Locomotor suppression (rota-rod assay)To exclude the possible nonspecific 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 appara-tus (model-DS 37; Ugo Basille) consisted of a bar with a diameterof 2.5 cm, subdivided into 6 compartments by disks, 25 cm indiameter. The bar rotated at a constant speed of 22 rpm. The ani-mals were previously selected, eliminating those mice that didnot remain on the bar for 2 consecutive periods of 60 seconds.The performance of mice before any treatment is represented asbaseline (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/kgi.p.). The time that mice remained on the rotating bar (cut-off60 seconds) was recorded.

2.10.2. Tail-flick assayA radiant heat tail-flick analgesiometer was used to measure re-

sponse latencies according to the method described by D’Amourand Smith [15], with minor modifications. Mice tail was exposedto a focused beam of radiant heat of intensity of 14. Tail-flick laten-cies were defined as the interval between the onset of the thermalstimulus and withdrawal of the tail. A 20-second cut-off was usedto prevent tissue damage. Withdrawal latencies were measuredimmediately before treatment (baseline, B) and then, at severalpoints after drug or vehicle administration. The latency for tailremoving 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 reactiv-ity in the model.

2.10.3. Hypothermia assayThe 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. Temperaturemeasurements were performed in a temperature-controlled room(22�C ± 2�C), between 3:00 pm and 5:00 pm. Temperatures wererecorded 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 tem-perature was recorded 30 to 180 minutes after treatments [53].

2.10.4. CatalepsyCatalepsy was measured by placing the forepaws over a 0.5-cm-

diameter horizontal glass bar at a 4-cm height from the floor. Micewere previously submitted to evaluation before (baseline, B) andimmediately 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 thatmice remained with both paws on the bar was measured up to180 seconds.

2.11. Immunohistochemistry

For the immunohistochemical studies, different groups of micewere submitted to PSNL or were injected with CFA. At differenttime points after the procedures, the mice were anesthetized with7% chloral hydrate (10 mL/kg i.p.) and transcardially perfused withheparin (1000 U/mL) in physiological saline, followed by 4% para-formaldehyde diluted in 0.9% NaCl. The footpad and lumbar spinalcord (L3–L5) were rapidly removed and postfixed overnight in 4%paraformaldehyde. The immunohistochemistry technique was car-ried out in paraffin-embedded sections of footpads or lumbarspinal cords. The sectioned tissues were incubated overnight at4�C with the primary antibodies for phospho-p65 NF-jB (1:100),phospho-CREB (1:200), or COX-2 (1:500) (Cell Signaling Technol-ogy, Danvers, MA). To block the endogenous peroxidase activityand to eliminate the occurrence of nonspecific reactions, the tissuesections were incubated with 1.5% hydrogen peroxide in methanol(v/v) for 20 minutes. To recover the antigens enclosed by parafor-maldehyde and by the paraffin inclusion, we performed high-temperature antigen retrieval by immersion of the slides in a waterbath at 95�C to 98�C in 10 mM trisodium citrate buffer (pH 6.0) for45 minutes. The slides were then processed using the VectastainElite ABC reagent (Vector Laboratories, Burlingame, CA) accordingto the manufacturer’s instructions. The sections were covered withthe appropriate biotinylated secondary antibody for 90 minutes atroom temperature. Subsequently, the sections were washed in PBS,and visualization was performed using DAB (3,30-diaminobenzi-dine; Dako Cytomation) in chromogen solution and counterstain-ing with Harris hematoxylin. The control and experimentaltissues were placed on the same slides and processed under thesame conditions.

The immunostaining was assessed at the dermis region of thepaws or at the dorsal horn region of the lumbar spinal cords. Threeimages of the sections from each of these tissues were acquiredusing a Sight DS-5M-L1 digital camera (Nikon, Melville, NY) con-nected to an Eclipse 50i light microscope (Nikon). The thresholdoptical density that discriminated the staining from the back-ground 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 intensitywas determined, and the data are expressed as optical density(O.D.). All of the histological assessments were made by an exam-iner blind to the sample identities.

2.12. Myeloperoxidase activity assay

MPO activity was carried out in the footpad and in the lumbarspinal cord segment (L3–L5) as previously described [11]. The micewere treated witha,b-amyrin (30 mg/kg p.o.) or vehicle (Tween/eth-

anol/PBS) 1 hour before the intraplantar injection of CFA (20 lL) orthe PSNL. The subcutaneous paw tissue and the lumbar spinal cordsegment were removed 6 hours after the CFA injection, and the lum-bar spinal cord segment was removed 4 days after the PSNL. The tis-sues were homogenized at 5% (w/v) in EDTA/NaCl buffer (pH 4.7) andcentrifuged at 10,000 rpm for 15 minutes at 4�C. The pellet wasresuspended in 0.5% hexadecyltrimethyl ammonium bromide buffer(pH 5.4) and the samples were frozen and thawed 3 times in liquidnitrogen. The samples were centrifuged (10,000 rpm, 15 minutes,4�C), and 25 lL of the supernatant was used for the MPO assay.The enzymatic reaction was assessed with 1.6 mM tetramethyl-benzidine, 80 mM sodium phosphate buffer pH 7.2, and 0.3 mMhydrogen peroxide. The absorbance was measured at 650 nm. Theresults 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 tissueand lumbar spinal cord of CFA-injected mice and in the lumbarspinal cord of PSNL mice. The tissues were collected at differenttime points after the CFA injection: 3 hours for keratinocyte-derived chemokine (KC); 6 hours for interleukin-1b (IL-1b) andinterleukin-6 (IL-6) or after the PSNL: 6 hours for KC; 4 hours fortumor necrosis factor a (TNF-a); 7 days for IL-1b, and 3 days forIL-6. These time points were chosen based on preliminary experi-ments (data not shown). The subcutaneous paw tissue or the tissuefrom the lumbar area of the spinal cord (L3–L5) were removedfrom each mouse and homogenized in PBS containing 0.05% Tween20, 0.1 mM phenylmethylsulphonyl fluoride, 0.1 mM benzametho-nium chloride, 10 mM EDTA, and 20 UI aprotinin A and centrifugedat 3000g for 10 minutes. The supernatant was stored at�70�C untilanalysis. KC, TNF-a, and IL-1b levels were measured using ELISAkits (R&D Systems, Minneapolis, MN) according to the manufac-turer’s instructions. For the determination of IL-6 levels, an en-zyme-linked immunosorbent assay (ELISA) kit from eBioscience(San Diego, CA) was used.

2.14. RNA extraction and real-time PCR

For mRNA quantification, the lumbar spinal cords (L3–L5) werecollected on the 4th day after PSNL. In another set of experiments,the lumbar spinal cords and footpads were harvested 24 hoursand 4 days after CFA injection. Tissues were collected 6 hours aftera,b-amyrin (30 mg/kg p.o.) treatment. The groups were dividedinto: (1) naïve; (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/kgp.o.). mRNA quantification 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,São Paulo, Brazil). The concentration of total RNA was determinedby a NanoDrop 1100 (Beckman Coulter; Fullerton, CA). One micro-gram of the total RNA was used for cDNA synthesis by the Super-Script reverse transcriptase (Invitrogen) protocol according to themanufacturer’s instructions. The cDNA (100 ng) was amplified induplicate using a TaqMan Universal PCR Master Mix Kit with spe-cific TaqMan Gene expression target genes; 30 quencher MGB- andFAM-labeled probes were used for mouse CB1R (Mm01212171_s1)and CB2R (Mm00438286_m1), and Glyceraldehyde 3-phosphatedehydrogenase ((GAPDH) (NM_0080842) was used as an endoge-nous control for normalization (Applied Biosystems, Foster City,CA). The amplifications were carried out in a thermal cycler(StepOne Plus, Applied Biosystems) for 54 cycles; the fluorescencewas collected for each amplification cycle, and the data were ana-lyzed using the 2�DDCt method for relative quantification of expres-sion. The expression of the target genes was calibrated againstconditions found in naïve/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 theresin of Protium kleinii as previously described [44], with a puritydegree 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 theselective CB2R antagonist AM630 and the selective CB1R or CB2Ragonists, ACEA and JWH-133, or nonselective agonist WIN55,212-3 were purchased from Tocris Bioscience (Ellisville,Missouri). The AS-ODN specific for CB1R (50-GCCTGCTAGAATCGC-ATT-30), the AS-ODN specific for CB2R (50-CTGCTGAGCGCCCTGGA-GAAGAAC-30), and a mismatch MM-ODN control (50-GCCTGCTAGAATCGCATT-30) were purchased from Prodimol Biotecnologia (BeloHorizonte, Brazil). The radiolabel [3H]CP-55,940 (specific activity147.9 Ci/mmol) was purchased from PerkinElmer (Waltham,Massachusetts) and [3H]SR141716A specific activity 52 Ci/mmol)was purchased from Amersham Life Science (Buckinghamshire,England). The ELISA kits for mouse IL-1b, TNF-a, and KC werepurchased from R&D Systems (Minneapolis, MN). The IL-6 mouseELISA kit was purchased from Bioscience (San Diego, CA). Thechloral 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, poly-clonal rabbit anti-COX-2, and polyclonal rabbit anti-phospho-CREBwere purchased from Cell Signaling Technology. The secondaryantibody Envision Plus, streptavidin–horseradish peroxidase re-agent, and 3,3-diaminobenzidine chromogen were purchased fromDako Cytomation (Carpinteria, CA). The drugs were prepared insaline solution (0.9% NaCl) with the exception of the a,b-amyrin,which was prepared in 5% Tween 80, 5% ethanol, and 90% PBS justbefore use, and the solutions were adjusted to 10 mL/kg of bodyweight [44].

2.16. Statistical analyses

The results are presented as the mean ± SEM of 3 to 6 mice pergroup. The percentages of inhibition were calculated using theareas under the time-response curve (AUC). The statistical compar-ison of the data was performed using a one-way analysis of variance(ANOVA), with repeated measures when necessary. These analyseswere followed by the Bonferroni post hoc test. Values of P < .05were considered significant. 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% confidentintervals were determined by nonlinear regression analysis usingGraphPad PRISM from 4 independent experiments. The inhibitionsconstants (Ki) of competitor compounds were calculated by usingthe Cheng-Prusoff equation [Ki = IC50/(1 + L/KD)] [14].

3. Results

3.1. Effect of a,b-amyrin on CFA-induced mechanical and thermalhyperalgesia

The intraplantar injection of CFA induced a marked and long-lasting enhancement of response frequency to the von Frey hairapplication and decreased the latency to paw withdrawal duringa thermal stimulus in comparison to noninjured mice (Fig. 2Aand B). The oral pretreatment with 3, 10, or 30 mg/kg of a,b-amyrinsignificantly inhibited the mechanical hyperalgesia (Fig. 2A). Thecalculation of the area under the curve revealed that a,b-amyrindid not cause a dose-response effect. However, the dose of30 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-amyrinadministration (Fig. 2A). In addition, the oral pretreatment with30 mg/kg a,b-amyrin reduced the thermal hypersensitivity in-duced by CFA. The latency to response increased by 52%, and theeffect was significant for up to 6 hours after the CFA injection(Fig. 2B).

The daily oral treatment with a,b-amyrin (30 mg/kg) presenteda continuous antihyperalgesic effect. The measurement was alwaystaken 3 hours after a,b-amyrin treatment, and the repeated treat-ment for 5 consecutive days resulted in a long-lasting effect, reduc-ing the hyperalgesia for 9 days after the interruption of thetreatment. On the 18th day, a new repeated treatment was initi-ated (once per day for 5 days), and again there was a significant de-crease in mechanical hyperalgesia. The AUC calculation of the totalperiod revealed an inhibition of 45% (P < .05; Fig. 2C). The pretreat-ment with a,b-amyrin (30 mg/kg p.o.) was also able to decrease thepaw edema induced by CFA. The effect of a,b-amyrin was observedup to 24 hours after the treatment (Fig. 2D).

To verify the local effect of a,b-amyrin, the triterpene was di-rectly administered into the plantar area 24 hours after CFA injec-tion. The treatment with 30 lg/paw a,b-amyrin decreased themechanical hyperalgesia induced by CFA from 30 minutes up to2 hours after the treatment (Fig. 2E).

3.2. Effect of a,b-amyrin on the mechanical and thermal hyperalgesiainduced by PSNL

The PSNL produced a substantial and long-lasting mechanicaland thermal hyperalgesia when compared with the sham group(Fig. 3). When administered on the 4th day after surgery (post-treatment), the doses of 3 and 30 mg/kg p.o. of a,b-amyrin signifi-cantly reduced the mechanical hyperalgesia induced by PSNL.However, the dose of 10 mg/kg did not cause any significant antin-ociceptive effect (Fig. 3A). This result clearly shows that the effectsof a,b-amyrin are not dose dependent. Similar to the results foundin the CFA-injected mice, the dose of 30 mg/kg was the most effec-tive to reduce the hyperalgesia induced by the PSNL. The AUC cal-culation revealed an inhibition of 52% for 30 mg/kg a,b-amyrin,and this significant effect lasted for 7 hours (Fig. 3A). The dose of30 mg/kg a,b-amyrin also decreased the thermal hyperalgesia afterPSNL. This effect was observed during the whole testing period (upto 48 hours). There was a 72% inhibition according to the AUC(Fig. 3B).

In the long-term treatment, a,b-amyrin was administered dailyfor 5 days. The treatment significantly reduced the mechanicalhyperalgesia (inhibition of 60%; Fig. 3C), and the assessments werealways taken 4 hours after the treatment. When the treatment wasinterrupted, the mice immediately exhibited a reestablishment ofthe mechanical hyperalgesia. On the 13th day, the treatment wasreinitiated, and once again, a significant inhibition of the mechanicalhypernociception 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 ofmechanical hyperalgesia, an effect that was observed during alltested periods (inhibition of 83%; Fig. 3D). This long-lasting antihy-peralgesic effect of a,b-amyrin demonstrates that the compoundmay prevent some initial component of nociceptive transmission,and therefore, prevent hyperalgesia during the whole testingperiod.

3.3. Role of cannabinoid receptors in the antihyperalgesic effect causedby a,b-amyrin

Because a,b-amyrin presented a great effectiveness in reducinghyperalgesia in both models of chronic pain, we designed newexperiments to determine whether or not its effects were related

Fig. 2. Effect of the treatment with a,b-amyrin on the mechanical and thermal hypernociception and paw edema induced by complete Freund’s 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). Mechanicalhypernociception 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/kgp.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,b-amyrin (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 andreinitiated 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,b-amyrin (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 thevertical lines indicate the SEM. Statistical analysis were performed by one-way analysis of variance, with repeated measures, followed by the Bonferroni test. The symbolsdenote significant difference: #P < .05 from PBS intraplantarly injected mice, and ⁄P < .05 from vehicle (p.o.) plus CFA intraplantarly injected mice. B (baseline withdrawalthreshold) refers to the measurement before CFA and a,b-amyrin treatment. p.o. = orally.


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to the cannabinoid system. The previous treatment with the CB1Rantagonist AM251 (Fig. 4A and E) or with the CB2R antagonistAM630 (Fig. 4B and F) significantly prevented the antihyperalgesiceffect of a,b-amyrin (30 mg/kg p.o.) in CFA-injected mice. Interest-ingly, the antihyperalgesic effects of a,b-amyrin were quite similarto 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, pretreatmentwith the CB1R antagonist AM251 (Fig. 5A and C) or with the CB2R

antagonist AM630 (Fig. 5B and D) significantly prevented the anti-hyperalgesic effect of a,b-amyrin in PSNL mice.

3.4. Further evidence of the contribution of cannabinoid receptors inthe antihyperalgesia caused by a,b-amyrin

Because both CB1R and CB2R antagonists displayed a similareffect in preventing the antihyperalgesic effect of a,b-amyrin,which could be explained by the lack of selectivity of the

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) inmice. 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 micereceived 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 mixturewhen administered 1 hour before surgery (pretreatment). The mechanical hypernociception was evaluated by using von Frey hairs (0.6 g), and thermal hypernociception wasassessed 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-wayanalysis of variance, with repeated measures, followed by the Bonferroni test. The symbols denote significantly 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.


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antagonists, we carried out additional experiments using anti-sense oligonucleotides for both cannabinoid receptors in micesubjected to the PSNL. The knockdown gene of either CB1R(Fig. 6A and B) or CB2R (Fig. 6D and E) fully prevented the anti-hyperalgesic effect of a,b-amyrin in PSNL mice, suggesting thatboth receptors are important to the antihyperalgesic effect ofa,b-amyrin.

The immunohistochemical assay confirmed the reduction ofCB1R and CB2R protein expression in the dorsal horn of the spinalcord after AS-ODN treatment. Compared with the control (MM-ODN treatment), AS-ODN treatment decreased by 76% the CB1Rand by 97% the CB2R expression. Interestingly, a,b-amyrin treat-ment prevented the upregulation of CB1R and CB2R in PSNL miceby 30% and 60%, respectively (Fig. 6C and F, SupplementaryFig. 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 concentration-dependent displacement of the [3H]SR141716A and [3H]CP-55,940 binding to CB1R and CB2R from rat brain and spleen mem-branes, respectively. Of high interest, a,b-amyrin presented a muchhigher affinity by CB1R than by CB2R. The calculated inhibitionsconstants (Ki) were 0.133 nM [IC50 = 0.55 (0.18 to 1.64) nM] forCB1R and 1989 nM [IC50 = 6070 (263 to 1397) nM] for CB2R. Fromthese results it is possible to infer that a,b-amyrin binds to CB1Rwith a very high potency, and it is likely to be one of the mostpotent binding competitors to CB1R that has been described inthe literature. a,b-amyrin presented a nearly 15,000-fold higheraffinity to CB1R than CB2R.

Fig. 4. Evidence for the involvement of cannabinoid receptors in the antihypernociceptive effect of a,b-amyrin mixture on mechanical hypernociception induced by completeFreund’s 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 -CFAinjection. 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) CB2Ragonist 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. Eachpoint 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 repeatedmeasures when appropriated, followed by the Bonferroni test. Symbols denote significant difference: #P < .05 from the CFA group, ⁄P < .05 from a,b-amyrin or CB1R and CB2Ragonist-treated group. B (baseline withdrawal threshold) refers to the measurement before CFA and drug treatments. p.o. = orally; N = naïve; Veh = vehicle.


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3.6. a,b-amyrin effects on tetrad assay

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

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

Fig. 5. Evidence for the involvement of cannabinoid receptors in the antihypernociception caused by a,b-amyrin treatment on mechanical hypernociception induced bypartial 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 onthe 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 thecurve 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-wayanalysis of variance, with repeated measures when appropriated, followed by the Bonferroni test. A significant difference is defined by the symbols #P < .05 from control PSNLand ⁄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.


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(Fig. 8C) and failed to cause catalepsy-like behavior (Fig. 8D).Similarly, the treatment with either CB1R or CB2R agonists, ACEAor JWH-133, in the dose that produced antihypernociception(10 mg/kg) did not alter the parameters evaluated in the tetradassay (Fig. 8A–D).

3.7. mRNA expression of CB1R and CB2R in the spinal cord after partialsciatic nerve ligation or CFA injection

Regarding the role of the cannabinoid system in the antihyper-algesic effect of a,b-amyrin, we next evaluated whether a,b-amyrin interferes with the expression of either CB1R or CB2R. Wefound a constitutive expression of both CB1R and CB2R mRNA inthe lumbar cord of naïve mice. However, CB1R was more predom-inantly expressed in this tissue compared with CB2R (data notshown). The intraplantar injection with CFA did not significantlychange CB1R or CB2R mRNA expression 24 hours (Fig. 9) and 4 daysafter the CFA injection (Supplementary Fig. 2). Interestingly, micethat were treated with a,b-amyrin (30 mg/kg p.o.) previously toCFA presented a significant upregulation of CB1R and CB2R mRNAexpression (Fig. 9A and B). Because a,b-amyrin was able to changethe mRNA expression of cannabinoid receptors in CFA-injectedmice, we investigated whether the compound could affect theexpression of these receptors. The treatment with a,b-amyrin inthe absence of any injury resulted in a significant increase inCB1R mRNA expression. In contrast, a,b-amyrin alone decreasedCB2R mRNA expression.

Different from the results obtained when CFA was injectedintraplantarly, the PSNL caused an upregulation of both CB1R andCB2R mRNA expression in the spinal cord when assessed 4 daysafter the injury. The treatment with a,b-amyrin (30 mg/kg p.o.)prevented the upregulation of CB2R mRNA but did not significantlyaffect CB1R mRNA upregulation (Fig. 9A and B).

To find out whether a,b-amyrin would alter the expression ofcannabinoid receptors in the mouse cortex, the mRNA for CB1Rand CB2R were quantified in this tissue. Differently from the spinalcord region, the oral treatment with a,b-amyrin (30 mg/kg) did notsignificantly change the cortical levels of both CB1R and CB2RmRNA (Supplementary Fig. 3).

3.8. a,b-Amyrin treatment reduces the synthesis/release of pro-inflammatory cytokines and myeloperoxidase activity induced by CFAinjection and PSNL procedure

PSNL caused a significant increase in the levels of TNF-a(4 hours after surgery), IL-1b (7 days after surgery), IL-6 (3 daysafter surgery), and KC (6 hours after surgery) and in the activityof myeloperoxidase (4 days after surgery) in the lumbar area ofthe mouse spinal cord. The treatment of mice with a,b-amyrin(30 mg/kg p.o. 1 hour before PSNL) significantly prevented thePSNL-induced release or synthesis of these cytokines and pre-vented the increase in myeloperoxidase activity. The inhibitionvalues were 75% for TNF-a, 60% for IL-1b, 64% for IL-6, 33% forKC, and 76% for MPO activity (Fig. 10A–E, respectively).

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 (AS-ODN; 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-hourintervals. 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 quantification 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 6animals, and the vertical lines indicate the SEM. Statistical analyses were performed by one-way analysis of variance, with repeated measures when appropriated, followed bythe Bonferroni test. A significant difference is defined by the symbols #P < .05 from PSNL in missense oligonucleotide (MM-ODN) plus vehicle (Veh) and ⁄P < .05 from PSNL inMM-ODN plus a,b-amyrin. B (baseline withdrawal threshold) refers to the measurement before PSNL procedure and AS-ODN treatment. N = naïve; Sh = sham.

Fig. 7. a,b-amyrin binding displacement of [3H]SR141716A and [3H]CP55,940 in ratbrain (CB1R) or spleen (CB2R) membrane preparations. Binding assays were carriedout at 30�C using 0.5 nM [3H]SR141716A or 0.8 nM [3H]CP55,940 and increasedconcentrations of a,b-amyrin. Data are the mean of 4 independent experimentsalways performed in duplicate. Vertical bars represent the standard error of mean.The asterisks denote a significant difference, ⁄P < .05 in the absence of a,b-amyrin.


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Likewise, the treatment of mice with CFA induced a significantincrease in IL-1b, IL-6, and KC levels and in the myeloperoxidaseactivity in the footpad and spinal cord at 6, 6, 3, or 6 hours afterthe CFA injection, respectively. The dose of 30 mg/kg a,b-amyrinp.o., given 30 minutes before the CFA, significantly prevented theincrease in the levels of these pro-inflammatory cytokines in mice

footpads with inhibitions of 65% for IL-1b, 42% for IL-6, 58% for KC,and 61% for myeloperoxidase activity (Fig. 11A–D, respectively). Inthe lumbar area of the spinal cord, the treatment with a,b-amyrincaused an inhibition of 88% in IL-1b, 60% in IL-6, 62% in KC, and 78%in myeloperoxidase activity (Fig. 11E–H, respectively).

The anti-inflammatory effect of a,b-amyrin was significantlyprevented by the pretreatment with the selective antagonists forboth 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 ofcannabinoid receptors also contribute to the anti-inflammatoryaction of a,b-amyrin.

3.9. a,-amyrin inhibits the activation of CREB and NF-jB and the overexpression of COX-2 induced by CFA injection or PSNL procedure

We found a marked activation of the transcription factors CREBand NF-jB in mice footpads when assessed 6 hours after the CFAinjection and in the lumbar spinal cord 7 days after PSNL. Theseevents were accompanied by an upregulation of COX-2, 24 hoursafter the CFA injection and 21 days after PSNL (Fig. 12 andSupplementary Figs. 4 and 5). All time points were selected basedon the activation peak of each target. These time points were pre-liminarily determined in our laboratory. CREB and NF-jB activa-tion as well as COX-2 upregulation were significantly preventedin mice pretreated with a,b-amyrin (30 mg/kg p.o.), as assessedin the footpads of mice injected with CFA. The inhibitions were100% for CREB, 78% for NF-jB, and 28% for COX-2 (Fig. 12A–C).

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 wereperformed by one-way analysis of variance, with repeated measures, followed by the Bonferroni test. B (baseline withdrawal threshold) refers to the evaluation performedbefore drugs treatment.

Fig. 9. mRNA expression of CB1R and CB2R in spinal cord after complete Freund’s adjuvant (CFA) injection or partial sciatic nerve ligation (PSNL). mRNA was quantified inspinal 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 tonormalize the relative amount of mRNA. Each column represents the mean of 3 samples performed in duplicate. The vertical lines show the SEM. Statistical analyses wereperformed 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 naïvemice. A significant difference is designed by #P < .05 from naïve (N) or sham (Sh) group. ⁄P < .05 from vehicle (Veh) in CFA or vehicle (Veh) in PSNL. p.o. = orally.


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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 levelslower than those found in the sham group (Fig. 12D–F); this effectof a,b-amyrin might explain the reduction in the progress ofinflammation in the injured tissue.

4. Discussion

In this study we have reported that the antihyperalgesic andanti-inflammatory effects of the pentacyclic triterpene a,b-amyrinagainst chronic and inflammatory pain are directly related to theability of a,b-amyrin to interact with cannabinoid receptors CB1

and CB2. Studies in neuropathic and inflammatory pain have

demonstrated that the activation of both CB1R and CB2R confersa valuable protection against persistent pain [19,41,46]. In fact,endocannabinoids and inhibitors of endocannabinoids transportor degradation exert pronounced analgesic effects [25]. Further-more, plant-derived substances defined as phytocannabinoidscan bind to cannabinoid receptors and induce analgesia and anti-inflammatory effects [21]. In the present study, we extended therelevance of the cannabinoid system in the control of inflammatorypain because both CB1R and CB2R agonists, ACEA and JWH-133, sig-nificantly inhibited the mechanical hyperalgesia induced by CFA.

Of high interest, the oral treatment with a,b-amyrin displayed asimilar antihyperalgesic effect when compared with ACEA andJWH-133. In addition, the pretreatment with the CB1R and CB2R

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 spinalcord. 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 theBonferroni test. The symbols denote a significant difference #P < .05 compared with the sham (Sh) group; ⁄P < .05 compared with the PSNL in vehicle (Veh) group. p.o. = orally.


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antagonists AM251 and AM630 equally prevented the hyperalgesiaand the production or release of inflammatory cytokines inducedby CFA, demonstrating that the protective effects afforded bya,b-amyrin are closely related to the activation of the cannabinoidsystem.

It is well recognized that the injuries caused by CFA or PSNLpromote profound alterations in the superficial (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 sciaticnerve ligation models revealed that they induce different tissueplasticity in the spinal cord. Indeed the sciatic nerve ligation up-regulated CB2R expression in the spinal cord, an event that was ab-sent in CFA-injected mice [58]. In agreement with this report, wealso found a significant increase in CB1R and CB2R mRNA in thespinal cords of mice that had the sciatic nerve constricted, butnot in those that were injected with CFA. Considering that these2 models present different profiles in the regulation of cannabinoidreceptor expression, we also investigated whether the antagonismof CB1R and CB2R would affect the antihyperalgesic effect of a,b-amyrin in PSNL injury as well as it affected the CFA injection mod-el. In this set of experiments, the systemic treatment with eitherCB1R or CB2R antagonists equally prevented the antihyperalgesic

effect of a,b-amyrin. Comparatively, the antinociceptive effects ofa,b-amyrin in CFA and PSNL were not affected by the differencein the expression of the cannabinoid receptors in the spinal cord.

Concerning a possible lack of selectivity of the antagonists uponCB1R or CB2R, we next used a knockdown strategy to better identifythe target for a,b-amyrin. Interestingly, the reduction of CB1R andCB2R expression by antisense oligonucleotides was more effectivethan the pharmacological use of antagonists in preventing theantinociception of a,b-amyrin. However, the knockdown gene ofCB1R or CB2R displayed a very similar profile in preventing a,b-amyrin effects.

The effects of a,b-amyrin in the cannabinoid system could bedue to the binding to cannabinoid receptors, to the prevention inendocannabinoids degradation, or via an indirect mechanism thatpromotes the activation of cannabinoid system. Therefore, weperformed an in vitro binding assay to determine whether or nota,b-amyrin could directly bind to CB1R, CB2R, or both. Remarkably,a,b-amyrin potently inhibited the radiolabel binding to CB1R andwith a lower potency it decreased the radiolabel binding to CB2R.Taking this into account, we can suggest that a,b-amyrin exertsmost of its antihyperalgesic effects by a direct stimulation of bothCB1R and CB2R.

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 Freund’sadjuvant (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 treatmentwith 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) keratinocyte-derived 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. Eachcolumn 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 Bonferronitest. The symbols denote a significant 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.


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The CB1R is predominantly expressed in neurons [2,37,51] anddirectly responds to neuronal changes. The binding of selectiveagonists to CB1R reduces inflammatory and neuropathic hyperalge-sia [12,19,38]. CB1R is distributed throughout the brain [35], where

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

Fig. 12. Effect of a,b-amyrin on the activation of transcriptional factors and cyclooxygenase 2 (COX-2) upregulation induced by complete Freund’s adjuvant (CFA) or partialsciatic 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 analysisfor (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 afterCFA), 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 (L3–L5) sections. Each column represents the mean of 4 animals, and vertical lines show the SEM. Statistical analyseswere performed by one-way analysis of variance followed by the Bonferroni test. Symbols denote a significant 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.


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resides in substances that do not cross the blood-brain barrier andthat exert their effects mainly in the peripheral nervous system. Infact, some studies revealed that CB1R present in the peripheral ner-vous system are also crucial to pain management, independently ofthe activation of CB1R from the central nervous system [1,3]. In linewith this evidence, we reported that local treatment with a,b-amyrin significantly prevented the hyperalgesia induced by CFA,suggesting that the activation of CB1R from peripheral sites bya,b-amyrin might be an important step in pain control.

Of high relevance, the oral treatment with a,b-amyrin did notcause any disturbance in the locomotor performance, body temper-ature, or sensitivity, or alter the catalepsy test, suggesting thata,b-amyrin is devoid of effects in brain areas. In addition, micechronically treated with a,b-amyrin were not susceptible to toler-ance because the antinociceptive effect of a,b-amyrin was main-tained even after repeated treatments and successive interruptions.

Previous studies from our group have shown that the adminis-tration of a,b-amyrin directly into the central nervous system, byintrathecal or intracerebroventricular injection, decreased thepro-nociceptive effects of formalin [44]. This same study foundno motor alteration by systemic treatment with a,b-amyrin. As aresult, it is likely that a,b-amyrin exerts central effects when it isdelivered directly into the brain, but it may not achieve the supra-spinal area after oral treatment. Corroborating this hypothesis, oraltreatment with a,b-amyrin was devoid of effect on the mRNAexpression of cannabinoid receptors in the brain cortex. On theother hand, the same treatment increased mRNA for CB1R and de-creased mRNA for CB2R in the lumbar spinal cord, which demon-strates that the effects displayed by a,b-amyrin on the lumbarspinal cord might not be extended to the forebrain. Therefore,the alterations caused by a,b-amyrin in mRNA expression of can-nabinoid receptors from the spinal cord may be an indirect effectof the compound due to the reduction of peripheral neuronal

activation and inflammation. These results suggest that a,b-amyrininduces antinociception by acting preferentially at the peripheralsites. However, some pharmacokinetic approaches are necessaryto specify the exact distribution of a,b-amyrin and its ability tocross the blood-brain barrier.

It is important to mention that, although a,b-amyrin presentednearly 15,000-fold more affinity to CB1R than CB2R, the CB2R antag-onists and the gene knockdown efficiently reverted the effects ofa,b-amyrin against pain and inflammation. Therefore, it is likelythat during inflammatory conditions, where there is an overexpres-sion 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, mac-rophages, microglia, mast and T-cells, and the activation of thisreceptor induces marked immunosuppression [23,49]. Indeed,a,b-amyrin greatly prevented the production/release of the pro-inflammatory cytokines TNF-a, IL-1b, IL-6, and KC, as well as theactivity of the pro-inflammatory enzyme myeloperoxidase. Thiseffect was clearly dependent on the activation of the cannabinoidsystem because the CB1R and CB2R antagonists significantlyprevented the anti-inflammatory effects of a,b-amyrin. Therefore,the antinociceptive effects of a,b-amyrin might be associated withanti-inflammatory properties of the compound.

Noteworthy is that the protective effects of a,b-amyrin are likelynot exclusively dependent on the activation of the cannabinoidreceptors. In fact, previous studies have demonstrated that a,b-amyrin inhibited intracellular protein kinase C, protein kinase A,mitogen-activated protein kinase, COX-2 expression, transcrip-tional factors NF-jB, and CREB activation [40,44,54]. In addition,a,b-amyrin effectively prevented the nociception induced by thereceptor 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 andTRPV1, glutamate, or bradykinin may be suggested, but whether


152 (2011) 1872–1887

or not these mechanisms are dependent on the activation of thecannabinoid receptors by a,b-amyrin requires further investigation.

Confirming previous studies [44,54], we found a significant inhibi-tion of the transcription factors NF-jB and CREB activation bya,b-amyrin in both CFA and PSNL models of chronic pain. The phos-phorylation of the p65 subunit of NF-jB and translocation to thenucleus promote the gene transcription of COX-2 and severalpro-inflammatory cytokines [22,30]. Additionally, the transcriptionfactor CREB binds to the promoter region in DNA (cyclic adenosinemonophosphate response elements) and also induces the transcrip-tion of COX-2 [50], growth factors, and other inflammatory mediators[47,59]. Taking this into account, the inhibition of NF-jB and CREBactivation likely contributes to the long-lasting antinociceptive effectof the a,b-amyrin because it greatly avoids the production of inflam-matory mediators responsible for the progress of inflammation.

In summary, our findings demonstrate that the main mecha-nism responsible for the a,b-amyrin antihyperalgesic and anti-inflammatory properties against persistent pain depends on itsability to bind to both CB1R and CB2R. Furthermore, the antinoci-ceptive effect of a,b-amyrin was also associated with the inhibitionof the levels of TNF-a, IL-1b, IL-6, and KC, the inhibition of the acti-vation of transcriptional factors NF-jB, CREB, upregulation of COX-2, and neutrophil migration/activation. As a,b-amyrin is quite safefor animals [54] and because there are few effective and safe drugsfor the treatment of persistent pain, especially neuropathic andinflammatory pain, the present study may have clinical relevanceand suggests that a,b-amyrin might be a potential molecule ofinterest for pain relief.

Conflict of interest statement

All authors declare that there are no conflicts of interest.

Acknowledgements

This work was supported by grants from the Conselho Nacionalde Desenvolvimento Científico e Tecnológico (CNPq), Coordenaçãode Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and Fun-dação de Apoio a Pesquisa do Estado de Santa Catarina (FAPESC), allfrom Brazil. K.A.B.S.S., A.F.P., G.F.P. and A.F.B. are Ph.D students inpharmacology receiving grants from CNPq. E.S.S. is a PhD studentin physiology. F.C.M. holds postdoctoral fellowships from CNPq.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.pain.2011.04.005.

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Activation of Cannabinoid Receptors by the Pentacyclic Triterpene a,B-Amyrin