Evidence for the involvement of opioid and cannabinoid systems in the peripheral antinociception mediated by resveratrol

Cristina da Costa Oliveira, Marina Gomes Miranda e Castor, Camila Gomes Miranda e Castor, Ághata de França Costa, Renata Cristina Mendes Ferreira, Josiane Fernandes da Silva, Juliana Maria Navia Pelaez, Luciano dos Santos Aggum Capettini, Virginia Soares Lemos, Igor Dimitri Gama Duarte, Andrea de Castro Perez, Sérgio Henrique Sousa Santos, Thiago Roberto Lima Romero

ABSTRACT

Despite all the development of modern medicine, around 100 compounds derived from natural products were undergoing clinical trials only at the end of 2013. Among these natural substances in clinical trials, we found the resveratrol (RES), a pharmacological multi-target drug. RES analgesic properties have been demonstrated, although the bases of these mechanisms have not been fully elucidated. The aim of this study was to evaluate the involvement of opioid and cannabinoid systems in RES- induced peripheral antinociception. Paw withdrawal method was used and hyperalgesia was induced by carrageenan (200 μg/paw). All drugs were given by intraplantar injection in male Swiss mice (n=5). RES (100 μg/paw) administered in the right hind paw induced local antinociception that was antagonized by naloxone, non-selective opioid receptor antagonist, and clocinnamox, μOR selective antagonist. Naltrindole and nor-binaltorfimine, selective antagonists for δOR and kOR, respectively, did not reverse RES-induced peripheral antinociception. CB1R antagonist AM251, but not CB2R antagonist AM630, antagonized RES-induced peripheral antinociception. Peripheral antinociception of RES intermediate-dose (50 µg/paw) was increased by: (i) bestatin, inhibitor of endogenous opioid degradation involved-enzymes; (ii) MAFP, inhibitor of anandamide amidase; (iii) JZL184, inhibitor of 2-arachidonoylglycerol degradation involved-enzyme; (iv) VDM11, endocannabinoid reuptake inhibitor. Acute and peripheral administration of RES failed to affect the amount of µOR, CB1R and CB2R. Experimental data suggest that RES induces peripheral antinociception through μOR and CB1R activation by endogenous opioid and endocannabinoid releasing.

1. Introduction

Pain is a multifactorial process, being symptomatic of the most diverse pathological conditions, which makes pain treatment a complex medical issue (Apkarian et al., 2005; Brodal, 2017; Garland, 2013). It is the most commonly reported symptom in medical consultations and it leads to a multi-billion dollar investment in healthcare worldwide (Romanelli, 2017; Turk, 2002). Loss of productivity at work due to painful conditions generate an estimated cost of $61.2 billion per year in the EUA, where it is calculated that approximately 25 million people suffer from painful symptoms daily (Nahin, 2015; Stewart, 2003). Prescription of opioids has been increasing and, along with it, the concern about adverse effects, misuse and deaths. 28,647 Americans had opioid overdose-related death only in 2014 (Elsesser and Cegla, 2017; Han et al., 2017; Rudd et al., 2016). Taken together, these data emphasize that pain management needs to be redirected with the inclusion of new pharmacotherapeutic approaches. Plant-derived natural substances has been appeared as a pharmacological alternative for refractory patients to conventional treatments, including pain therapy (Almeida et al., 2001). Despite all the development of modern medicine, 100 compounds derived from natural products were undergoing clinical trials only at the end of 2013, indicating that nature is still a viable source of new drug candidates (Butler et al., 2014; Mishra and Tiwari, 2011). Among these natural substances in clinical trials, we found the resveratrol (trans-3,5,4’-trihydroxystilbene; RES; Figure 1), a nutraceutical compound with potential to become a pharmacological multi-target drug. This non-flavonoid polyphenol is found in about 72 plant species and it has been widely studied pharmacologically (Saiko et al., 2008; Tomé-Carneiro et al., 2013).

Animal model studies have attributed several therapeutic properties to RES, such as: (i) anticancer, through the inhibition of metastasis and angiogenesis (Ganapathy et al., 2010; He et al., 2017; Lee et al., 2012); (ii) antihypertensive, associated with endothelium-dependent vascular relaxation, enhanced eNOS activity and increased glutathione levels (Akar et al., 2012; Alturfan et al., 2012; Bhatt et al., 2011); (iii) hypolipidemic, possibly by regulating the hepatic lipoprotein/apolipoprotein secretion, hepatic and adipocytic lipid metabolism (Jeon et al., 2014); (iv) anti-inflammatory, reducing levels of TNFα, IL-1β, IL-6, COX-2 and iNOS (Gómez-Zorita et al., 2013; Inanaga et al., 2009; Prata et al., 2017); (v) lifespan prolonger, probably involving the SIRT1 induction (Bhullar and Hubbard, 2015). Despite the abundant preclinical studies and ongoing clinical trials, many issues related to the RES pharmacological actions remain unclear. In this context, the media and advertisements seem to be moving faster than the scientific research (Espín et al., 2007; Vang et al., 2011). Clinical trials conducted so far have shown the RES cardioprotective effects through the improvement of inflammatory marker profile (Tomé-Carneiro et al., 2013). Since pain is one of the four cardinal signs of inflammation (Lawrence et al., 2002), some animal studies have been directed to the RES analgesic actions (Bazzo et al., 2013; Bertelli et al., 2008; Wang et al., 2017).

It was shown that systemically administered RES, in rats, induces analgesia with possible involvement of the opioid system, considering that it has been reversed by naloxone, a nonspecific opioid antagonist (Gupta et al., 2004; Kokuba et al., 2017). In addition, one study demonstrated that intraperitoneally injected RES, also in rats, increases brain levels of endocannabinoids, suggesting a potential for treatment of neuropsychiatric disorders (Hassanzadeh et al., 2016). A major drawback to RES therapy is its rapid intestinal metabolism; the term “resveratrol paradox” was even created, referring to the antagonism between high bioactivity and low bioavailability (Azorín-Ortuño et al., 2011). Given these circumstances, we propose further evaluating the involvement of opioid pathway and analyzing the participation of endocannabinoids, substances widely reported as analgesics (Savage et al., 2016), in the peripheral antinociceptive action of locally administered RES in mice.

2. Materials and methods

2.1. Animals

9 weeks old male Swiss mice (30-40g), obtained from the Bioterism Center of Federal University of Minas Gerais (CEBIO-UFMG), were used. The mice were housed in standard cages and kept in a temperature-controlled room (23 ± 2°C) with an automatic 12-hour light/dark cycle (06:00-18:00 hours). The tests were performed during the light phase. Food and tap water were freely provided. Immediately after the testing procedures, the animals were euthanized by an intraperitoneal injection of general anesthetic agents (300 mg/kg of ketamine hydrochloride and 15 mg/kg of xylazine hydrochloride, both Sigma-Aldrich, USA). The experimental protocol was approved by the “Ethics Committee in Animal Experimentation at the Federal University of Minas Gerais” in 13/03/2017 (protocol number 278/2016) average was considered the final value. Hyperalgesia was considered as the difference between the averages three hours prior to and after the paw carrageenan injection and expressed in grams (Δ of nociceptive threshold). In order to reduce the possibility of paw damage, a cutoff value of 200 g was used.

2.3. Drug Administration

All drugs were administered using an injected volume of 20 μL/paw. Resveratrol (3,4′,5-trihydroxy-trans-stilbene; purity > 99%; Sigma-Aldrich, USA) was diluted in ethanol 0.5%; whereas hyperalgesic agent carrageenan (Sigma-Aldrich, USA) was dissolved in aqueous solution of sodium chloride (NaCl) sterile 0.9% (sterile saline solution). The broad spectrum opioid antagonist naloxone ([5α]-4,5-Epoxy-3,14- dihydro-17-[2-propenyl]morphinan-6-one; purity > 99%; Tocris Bioscience, UK), the irreversible μ antagonist clocinnamox (14β-[p-Chlorocinnamoylamino]-7,8-dihydro-N- cyclopropylmethylmorphinone; purity > 99%; Tocris Bioscience, UK), the selective non-peptide δ antagonist naltrindole (17-[Cyclopropylmethyl]-6,7 dehydro-4,5α-epoxy- 3,14-dihydroxy-6,7-2′,3′-indolomorphinan; purity > 99%; Tocris Bioscience, UK), the κ selective antagonist nor-binaltorphimine (17,17′-(Dicyclopropylmethyl)-6,6′,7,7′-6,6′- imino- 7,7′-binorphinan-3,4′,14,14′-tetrol; purity > 98%; Tocris Bioscience, UK) and the aminopeptidase inhibitor bestatin ( N-[(2S,3R)-3-Amino-2-hydroxy-1-oxo-4- phenylbutyl]-L-leucine; purity > 99%; Tocris Bioscience, UK) were also dissolved in sterile saline solution. The CB1 cannabinoid antagonist AM251 (N-[Piperidin-1-yl]-5- [4-iodophenyl]-1-[2,4-dichlorophenyl]-4-methyl-1H-pyrazole-3-carboxamide; purity > 99%; Tocris Bioscience, UK) and the CB2 cannabinoid antagonist AM630 (6-Iodo-2- methyl-1-[2-{4-morpholinyl}ethyl]-1H-indol-3-yl [4-ethoxyphenyl] methanone; purity > 98%; Tocris Bioscience, UK) were dissolved in dimethyl sulfoxide (DMSO) 10%. MAFP ([5Z,8Z,11Z,14Z]-5,8,11,14-eicosatetraenyl-methyl ester phosphonofluoridic acid; purity > 98%; Tocris Bioscience, UK), a selective inhibitor of fatty acid amide hydrolase (FAAH), enzyme responsible for anandamide hydrolysis, was dissolved in ethanol 3%. JZL184 (4-[Bis{1,3-benzodioxol-5-yl}hydroxymethyl]-1- piperidinecarboxylic acid 4-nitrophenyl ester; purity > 98%; Tocris Bioscience, UK), a selective inhibitor of monoacylglycerol lipase (MAGL), enzyme responsible for 2- arachidonyl glycerol (2-AG) hydrolyses, was dissolved in DMSO 10%. VDM11 ([5Z,8Z,11Z,14Z]-N-[4-Hydroxy-2-methylphenyl]-5,8,11,14-eicosatetraenamide; purity >98%; Tocris Bioscience, UK), a selective inhibitor of the anandamide membrane transporter, was dissolved in Tocrisolve™ 10%.

2.4. Experimental Protocol

Carrageenan was administered in the right hind paw and the measurements were performed immediately prior to and 180 minutes after carrageenan intraplantar injection. Resveratrol (RES) was administered 165 minutes after the carrageenan administration (except for the time-response curve protocol, in which RES was administered at 180 min. time). The opioid drugs naloxone, clocinnamox, naltrindole, nor-binaltorphimine and bestatin were administered 30 min prior to the administration of RES. The cannabinoid drugs AM630, AM251, MAFP, JZL184 and VDM11 were administered 10 minutes prior to the RES intraplantar injection. In the protocol used to determine whether RES was acting in the central nervous system or the contralateral paw, carrageenan was injected into both hind paws, whereas RES and vehicle were administered into the right and left hind paws, respectively, and the nociceptive threshold was measured in both hind paws. Protocols concerning the time of administration and dose of each drug used in this study were obtained through pilot experiments and literature data (Ferreira et al., 2017; Oliveira et al., 2017; Pacheco et al., 2008; Veloso et al., 2014).

2.5. Western Blotting Analysis

Following RES stimulation, performed as mentioned above, the animals were sacrificed, by cervical dislocation, and the plantar surfaces of the mouse paws were collected. These tissues were individually homogenized in 500 µl of RIPA buffer (180 mmol/L NaCl, 50 mmol/L TrisHCl, 0.5 mmol/L EDTA.2Na, 1 mmol/L MgCl2, 0.3% Triton X-100, 0.5% SDS; pH 7.4), containing a cocktail of protease inhibitors (SigmaFAST®; Sigma) plus 20 mmol/L NaF and 20 mmol/L PMSF. Thereafter, lysates were centrifuged at 16000x g for 20 min 4°C and protein content was determined by the use of Bradford method [Bradford, 1976]. Equal amounts of protein (50 µg) were denatured in the loading buffer at 100°C for 5 min and subjected to SDS-PAGE using 10% polyacrylamide gel. Proteins were transferred onto a 0.45 μm polyvinylidene fluoride membrane (PVDF; Immobilon P; Millipore, MA, USA). Blots were blocked at room temperature with 4% BSA in PBS enriched with 0.1% Tween 20 prior to incubation with rabbit polyclonal anti-µOR (diluted 1:1000; Abcam/ab10275, Cambridge, MA), rabbit polyclonal anti-CB1R (diluted 1:1000; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit polyclonal anti-CB2R (diluted 1:1000; Santa Cruz Biotechnology, Santa Cruz, CA), mouse monoclonal anti-GAPDH (diluted 1:1000; Novus Biologicals, Littleton, CO) and mouse monoclonal anti β-Actin (diluted 1:1000; Santa Cruz Biotechnology, Santa Cruz, CA). Blots were incubated overnight at 4°C with primary antibody followed by incubation with horseradish peroxidase (HRP)- conjugated secondary antibodies for 2.5h at room temperature. Immunocomplexes were detected by chemiluminescent reaction (Luminata™ Forte Western HRP Substrate, Millipore, MA) followed by densitometric analyses with software ImageJ 1.46r (Wayne Rasband, National Institutes of Health, Bethesda, MD).

2.6. Statistical analysis

Graph Pad Prism 5.0 Software® was used to analyze the data by analysis of variance (ANOVA) with the Bonferroni post-test. All results were expressed as mean ± S.E.M. Statistically significance was set at P<0.05.

3. Results

Intraplantar administration of RES (12.5, 25, 50 and 100 µg/paw), at the third hour after injection of carrageenan (CAR, 200 µg/paw), dose-dependently produced an antinociceptive response. RES inhibitory effect lasted for 20 minutes and it was reversible. The peak of activity was observed 15 minutes after RES injection. 12.5 µg/paw RES dose induced no effect, whereas a maximum antinociception was noticed by the 100 µg/paw. Despite highest RES dose almost completely reversed the CAR- induced inflammatory hypernociception, this dose without the presence of the nociceptive stimulus (CAR) did not alter significantly the basal nociceptive threshold (Figure 2). To exclude possible systemic effects, CAR was administered at time zero into both hind paws and RES (100 or 200 µg) was injected only in the right hind paw 15 minutes prior to the third hour following CAR administration. Nociceptive threshold measurements of both hind paws were made immediately prior to and 3h after CAR intraplantar injection and then the difference between measure averages was calculated (Δ of nociceptive threshold). RES at dose of 100 μg induced effect restricted to the treated paw, not altering the CAR-induced hyperalgesia in the contralateral paw, indicating that, at this dose, RES is effective only locally. Differently from 200 μg RES dose, which induced antinociception in both paws, even administered in only one (Figure 3).

The intraplantar administration of naloxone (NLX; 25, 50, and 100 μg/paw) antagonized the RES-induced peripheral antinociception in a dosage-dependent manner (Figure 4A). The µOR antagonist clocinnamox (CLOC; 5, 10, 20 and 40 µg/paw) dose- dependently inhibited RES peripheral antinociceptive response (Figure 4B). Differently from naltrindole (NTD; 120 µg/paw) and nor-binaltorphimine (nBNI; 100 µg/paw), respectively,