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Withania somnifera root extract prolongs analgesia and suppresses hyperalgesia in mice treated with morphine.

ABSTRACT

Previous studies demonstrated that Withania somnifera Dunal (WS), a safe medicinal plant, prevents the development of tolerance to the analgesic effect of morphine.

In the present study, we investigated whether WS extract (WSE) (100 mg/kg, i.p.) may also modulate the analgesic effect induced by acute morphine administration (2.5, 5,10 mg/kg, s.c.) in the tail-flick and in the hot plate tests, and if it may prevent the development of 2.5 mg/kg morphine-induced rebound hyperalgesia in the low intensity tail-flick test. Further, to characterize the receptor(s) involved in these effects, we studied, by receptor-binding assay, the affinity of WSE for opioid ([mu], [delta], k), cannabinoid ([CB.sub.1], [CB.sub.2]), glutamatergic (NMDA), GABAergic ([GABA.sub.A], [GABA.sub.B]), serotoninergic (5HT2A) and adrenergic ([[alpha].sub.2]) receptors.

The results demonstrated that (i) WSE alone failed to alter basal nociceptive threshold in both tests, (ii) WSE pre-treatment significantly protracted the antinociceptive effect induced by 5 and 10 mg/kg of morphine only in tail-flick test, (iii) WSE pre-treatment prevented morphine- induced hyperalgesia in the low intensity tail-flick test, and (iv) WSE exhibited a high affinity for the [GABA.sub.A] and moderate affinity for [GABA.sub.B], NMDA and [delta] opioid receptors.

WSE prolongs morphine-induced analgesia and suppresses the development of morphine-induced rebound hyperalgesia probably through involvement of [GABA.sub.A], [GABA.sub.B], NMDA and [delta] opioid receptors. This study suggests the therapeutic potential of WSE as a valuable adjuvant agent in opioid-sparing therapies.

Keywords:

Withania somnifera

Morphine

Antinociception

Hyperalgesia

Binding assay

Introduction

Pain has negative consequences on health status and life quality, and its relief is considered one of the major and complex medical concerns. To date, opioid analgesics remain the most efficacious pharmacological agents for the treatment of moderate to severe pain, but their therapeutic benefit is often hampered by the development of analgesic tolerance and hyperalgesia (Benyamin et al. 2008). Opioid-induced hyperalgesia (OIH), defined as a paradoxical increase in sensitivity to noxious stimuli (Pasero and McCaffery 2012), is a side effect difficult to recognize because its clinical manifestations are similar to those observed in opioid-induced analgesic tolerance (DuPen et al. 2007; Raffa and Pergolizzi 2011). Despite such difficulty, OIH incidence is growing as opioid prescriptions are increasing (Raffa and Pergolizzi 2011). A considerable effort has been then expended in the development of novel therapeutic strategies that may maintain adequate opioid-induced analgesia and, at the same time, mitigate the appearance of tolerance and OIH. Adjuvant pharmacological therapies are among the therapeutic approaches used to reach this purpose, and consist in the association of opioid analgesics with a second non-opioid agent (DuPen et al. 2007; Khan et al. 2011). This approach has been found to improve opioid-induced analgesia at different levels: enhancing pain relief (with a concurrent reduction of opioid dosage) and preventing, at the same time, the development of side effects, specifically tolerance and OIH (DuPen et al. 2007). The main adjuvant agents that are currently associated to opioids in the management of pain are NMDA receptor antagonists, [[alpha].sub.2] receptor agonists and cyclooxigenase-2 inhibitors (Low et al. 2012; Pasero and McCaffery 2012).

Medicinal plants are among the potential adjuvant agents that could be also evaluated in this therapeutic approach. Phytotherapy offers, in fact, a valid support to conventional medicine in a number of diseases (Patwardhan 2005) and this also applies to the management of pain (Low Dog 2008). Withania somnifera Dunal (WS, family: Solanaceae) also known as Ashwagandha or Winter Cherry is a plant commonly used in Ayurvedic medicine to treat several diseases (Alam et al. 2012). A growing number of pharmacological studies demonstrated that WS extract (WSE) has anticancer, anti-inflammatory, immunomodulatory, adaptogenic and neuroprotective properties (Alam et al. 2012). Interestingly, Kulkarni and Ninan (1997) demonstrated that, in mice chronically treated with morphine, WSE prevented the development of tolerance to the antinociceptive effect of morphine.

The molecular underpinnings responsible for the development of analgesic tolerance show similarities with those involved in OIH (DuPen et al. 2007; Raffa and Pergolizzi 2011), and are the consequence of plastic changes in neurotransmitter systems that contribute to mediate pain transmission and opioid-induced analgesia. Accordingly, pre-clinical studies demonstrated that several pharmacological agents prevent the development both of antinociceptive tolerance and OIH (Bryant et al. 2006; Gupta et al. 2011), such as NMDA antagonists, among others. Moreover, the same compounds have been found to prolong or increase the acute analgesic effect of opioids (Fischer et al. 2005).

Starting from these studies, since the administration of WSE blocked the development of tolerance to the analgesic effect of morphine (Kulkarni and Ninan 1997), we hypothesize that WSE could modulate additional aspects of morphine-induced analgesia. Therefore, we aimed to determine whether the combined administration of WSE with morphine could enhance or prolong the antinociceptive effect of morphine in the tail-flick and hot plate tests. Moreover, we investigated the potential ability of WSE to block the development of morphine-induced hyperalgesia using the low intensity tail-flick test, a validated model of OIH (Gupta et al. 2011). Finally, since little information exists regarding the sites of action of WSE in the brain and about the receptor(s) involved in its pharmacological and behavioural properties, performing competition studies we determined WSE affinity for opioid ([mu], [delta], k), cannabinoid ([CB.sub.1], [CB.sub.2]), glutamatergic (NMDA), GABAergic ([GABA.sub.A], [GABA.sub.B]), serotoninergic ([5HT.sub.2A]) and adrenergic ([[alpha].sub.2]) receptors.

Materials and methods

Subjects

Male CD1 mice (Charles River, Calco, Italy) 20-25 g were used for both behavioural and receptor binding studies. Animals were housed in an animal facility on a 12 h light/dark cycle (lights on from 07:00 AM), at a constant room temperature of 21 [+ or -] 1[degrees]C (relative humidity approximately 60%). Standard rodent chow and water were available ad libitum. Animals were allowed to adapt to the animal facility conditions for at least two weeks after arrival.

Procedures involving animals and their care were conducted in accordance with the institutional guidelines that are in compliance with national (D.L. 116/1992) and international laws and policies (EEC Council Directive 86/609, OJL 358, 1, December 12, 1987; Guide for the Care and Use of Laboratory Animals, U.S. National Research Council, 1996). Every effort was made to minimize animal pain and discomfort and to reduce the number of experimental subjects.

Drugs and chemicals

Morphine hydrochloride (Salars, Como, Italy) was dissolved in saline (NaCl 0.9%) and administered subcutaneously (s.c.) in a volume of 5 ml/kg. The standardized root methanolic extract of WS (Withania somnifera Dunal, Natural Remedies Pvt. Ltd., Bangalore, India) was dissolved in saline for analgesia experiments [administered intraperitoneally (i.p.) in a volume of 5 ml/kg] and in dimethyl sulfoxide (DMSO, Sigma-Aldrich, Milan, Italy) for binding assay. The dose of WSE for analgesia experiments was selected on the basis of previous studies (Kasture et al. 2009; Ruiu et al. 2013). [[sup.3]H]- DAMGO ([d-Ala2, N-Me-Phe4,Gly-ol5]-enkephalin), [[sup.3]H]-DPDPE ([D-[Pen.sup.2], D-[Pen.sup.5]]-enkephalin), [[sup.3]H]-U-69,593, [[sup.3]H]- CP55,950, [[sup.3]H]-MK801, [[sup.3]H]-Muscimol, [[sup.3]H]-Baclofen, [[sup.3]H]-Clonidine and [[sup.3]H]-Ketanserine were purchased from Perkin Elmer, Monza (MB), Italy. CP-55,940, Naloxone, U-69,593, muscimol, MK801, yohimbine and methysergide were obtained from Tocris Cookson Ltd. (Bristol, UK).

Plant materials

The standardized WSE was kindly provided by Natural Remedies Pvt. Ltd., Bangalore, India.

High-performance liquid chromatographic (HPLC) analysis

The WSE has been characterized by an HPLC-fingerprint analysis, as certified by Natural Remedies Pvt. Ltd., with identification of the main withanolides. This analysis with the necessary description of the technique has been published in Neurotoxicity Research by Kasture et al. (2009). An HPLC system (Shimadzu, LC 2010 A, Japan) equipped with UV detector, auto-injector, and column oven with class VP software was used. The stationary phase was an octadecylsilane column (Phenomenex-Luna, C18, 5 [micro]n, 250 mm x 4.6 mm). The mobile phase was a mixture of phosphate buffer (Solvent A) [prepared by dissolving 0.136g of K[H.sub.2]P[O.sub.4] in 900 ml of HPLC grade water and by adding 10% dilute aqueous H3P04, adjusting the pH to 2.8 [+ or -] 0.05 and making the volume of 1000 ml with water] and acetonitrile (Solvent B). The following withanolides were identified: withanoside-lV, 0.49%w/w; physagulin D, 0.11%w/w; 27-hydroxywithanone, 0.01 %w/w; withanoside-V, 0.33%w/w; withaferin-A, 0.11%w/w; 12-deoxy withastramonolide, 0.16%w/w; withanolide-A, 0.19%w/w; withanone 0.004%w/w, and withanolide-B, 0.03%w/w.

Analgesia experiments

The antinociceptive effects were quantified using the tail-flick test and the hot plate test (Ruiu et al. 2003). An automated device (model 7360, Ugo Basile, Italy) was used to determine the tail-flick latency, defined by the time (s) at which the animals withdraw the tail from a radiant heat source. Mice were held and gently restrained above the apparatus; the light beam was focused 1.5 cm from the tip of the ventral surface of the tail. The stimulus intensity was adjusted to result in a mean pre-drug control latency of 2-3 s, and a cut-off time of 12 s was applied to avoid tissue damage.

A semi-automated device (model 7280, Ugo Basile, Italy) was used to determine the reaction of mice placed on the hot plate, defined by the time (s) at which mice exhibited a nociceptive response or discomfort (licking or fanning the paws, jumping). A 50 cm high Plexiglas cylinder was suspended over the hot plate and the temperature was maintained at 55 [+ or -] 0.2[degrees]C; to avoid skin damage, after 15 s mice were removed from the hot plate. Mice were pre-treated with saline or WSE 100mg/kg 30 min before saline or morphine treatment; increasing doses of morphine were used (2.5, 5 and 10mg/kg). Basal algesia was assessed right before saline or WSE pre-treatment (baseline). The effects of the drugs were evaluated 30,60,120,240 and 360 min after morphine treatment.

Morphine-induced hyperalgesia experiment

The ability of WSE to inhibit the development of morphine-induced hyperalgesia was evaluated using the low-intensity tail-flick test according Gupta et al. (2011) with few modifications. Briefly, the stimulus intensity was adjusted to result in a mean pre-drug control latency of 5-7 s, and a cut-off time of 10s was applied. Mice were pre-treated with saline or WSE 100 mg/kg 30 min before saline or morphine treatment (2.5 mg/kg). Basal algesia was assessed right before saline or WSE pre-treatment (baseline). The effects of the drugs were evaluated 30, 60, 120, 240, 300, 360 and 420 min after morphine treatment.

Spontaneous and morphine-induced motor activity experiments

In order to evaluate the ability of WSE to modulate further pharmacological effects induced by morphine, spontaneous and morphine-induced motor activity experiments have been carried out (Ruiu et al. 2013). Mice were tested in chambers made of transparent Plexiglas (40Lcm x 30Wcm x 40H cm) interfaced to a computer equipped with a TSE software (TSE Systems, Bad Homburg, Germany). The distance travelled (m) by mice inside the chambers was detected by 8 horizontal photocells. Mice were habituated to the chambers for 30 min. Immediately after habituation, mice were treated with saline or WSE 100 mg/Kg and 30 min later were injected with saline or morphine (5 or 10 mg/Kg). Motor activity was evaluated for 360 min after morphine administration.

Receptor binding experiments

[[sup.3]H]-DAMGO--[[sup.3]H]-DPDPE--[[sup.3]H]-U69,593. Ligand binding assays were carried out according to the procedure described by Ruiu et al. (2013). Briefly, the whole brain minus cerebellum was homogenized in Tris-HCl (pH 7.4), centrifuged at 48,000 x g, resuspended in the same buffer solution, and incubated at 37[degrees]C. After a further centrifugation step at 48,000xg, the final pellet was re-suspended in the same buffer solution. Brain membranes were incubated with [[sup.3]H]-DAMGO, [[sup.3]H]-DPDPE or [[sup.3]H]-U69,593 in Tris-HCl buffer at 25[degrees]C for 60 min in the absence or presence of naloxone (1 [micro]M) (for [mu] and [delta] receptors) or U 69,593 (10 [micro]M) (for k receptors).

[[sup.3]H]-CP55,940. The whole brain minus cerebellum and spleen were homogenized in TME buffer (50 mM Tris-HCl, 1 mM EDTA and 3.0 mM Mg[Cl.sub.2], pH 7.4), centrifuged at 1086 x g for 10 min and the resulting supernatants were centrifuged at 45,000 x g. [[sup.3]H]-CP55,940 binding was performed by the method previously described by Ruiu et al. (2003). Briefly, the membranes were incubated with [[sup.3]H]-CP55,940 for 1 h at 30[degrees]C in TME buffer containing 5 mg/ml of fatty acid-free bovine serum albumin (BSA). Non-specific binding was estimated in the presence of 10 [micro]M of CP55.940.

[[sup.3]H]-Muscimol. Ligand binding assays were carried out according to the procedure described by Beaumont et al. (1978) with slight modifications. Briefly, the cerebral cortex were homogenized in 0.32 M sucrose, centrifuged at 1000 x g and the resulting supernatants were centrifuged at 20,000 x g. The resulting pellet was suspended in ice-cold water, homogenized and centrifuged at 8000 x g. The supernatant together with the buffy layer on the pellet was then centrifuged at 48,000 x g. The resulting pellet was re-suspended in water, and once more centrifuged at 48,000 x g. The final pellet was frozen and stored at -80[degrees]C. On the day of analysis, membrane pellet was allowed to thaw at 4[degrees]C before re- suspension in 50 mM Tris-citrate buffer, pH 7.1, containing 0.05% Triton X-100 and incubated for 30 min at 37[degrees]C. Following incubation, the suspension was centrifuged at 48,000 x g. The washing step was repeated three more times and the final pellet was then re-suspended in the binding buffer. Non-specific binding was estimated in the presence of 200 [micro]M of GABA.

[[sup.3]H]-MK801. Ligand binding assays were carried out according to the procedure described by Wong et al. (1986) with slight modifications. Briefly, the cerebral cortex were homogenized in 0.32 M sucrose, centrifuged at 1000 x g for 10 min at 4[degrees]C and the resulting supernatants were centrifuged at 20,000 x g. The resulting pellet was re-suspended in ice-cold water and centrifuged at 8000 x g. The supernatant together with the buffy layer on the pellet was then centrifuged at 48,000 x g. The resulting pellet was re-suspended in 5mM Tris-HCl buffer (pH 7.4) containing 5mM EDTA and incubated at 37[degrees]C for 30 min prior to centrifugation at 48,000 x g. The washing step was repeated once more times and the final pellet was frozen and stored at -80 C. On the day of analysis, membrane pellet was allowed to thaw at 4[degrees]C before re-suspension in 5 mM Tris-HCl buffer and incubated at 37[degrees]C. Following incubation, the suspension was centrifuged at 48,000 x g. The resulting pellet was re-suspended in ice-cold water, and once more centrifuged at 48,000xg. The washing step was repeated once more times and the final pellet was then re-suspended in 5 mM Tris-HCl buffer. Nonspecific binding was defined by 100 [micro]M unlabeled MK-801.

[[sup.3]H]-Clonidine. Ligand binding assays were carried out according to the procedure described by Barturen and Garcia-Sevilla (1992) with slight modifications. Briefly, the cerebral cortex were homogenized in Tris-HCl containing 0.32 M sucrose, centrifuged at 1000 x g for 10 min and the resulting supernatants were centrifuged at 31,000 x g. Cerebral cortex membranes were incubated with [[sup.3]H]-Clonidine in Tris-HCl buffer containing 10 mM Mg[Cl.sub.2] and 250 [micro]M ascorbic acid at 25[degrees]C for 60 min in the absence or presence of yohimbine (50 p.M).

[[sup.3]H]-Badofen.Ligand binding assays were carried out according to the procedure described by Ruiu et al. (2013). Briefly, the whole brain minus cerebellum was homogenized in 0.32 M sucrose containing 1 mM EDTA. The homogenate was centrifuged at 10,000 x g and the supernatant collected and re-centrifuged at 20,000 x g. The pellet was re-suspended in ice-cold water, homogenized and centrifuged at 8000 x g. The supernatant together with the buffy layer on the pellet was then centrifuged at 45,000 x g. The resulting pellet was re-suspended in ice-cold water and once more centrifuged at 45,000 x g. The final pellet was frozen and stored at -80[degrees]C. To this end, membrane pellets were allowed to thaw at 4 C before re-suspension in 50 mM Tris-HCl. The suspension was incubated at 20[degrees]C before centrifugation at 7000 x g. The washing step was repeated three more times allowing 15 min incubation with each addition of the same buffer to remove the endogenous ligand GABA. The final pellet was then re-suspended in appropriate binding buffer. The incubation buffer was: 50 mM Tris-HCl (pH 7.4) containing 2.5 mM Ca[Cl.sub.2]. GABA (100 [micro]M) was used to define non-specific binding.

[[sup.3]H]-Ketanserin. The assays for [[sup.3]H]-ketanserin binding were performed as previously described (Gauffre et al. 1997) with slight modifications. Briefly, the cerebral cortex were homogenized in 50 mM Tris-HCl buffer and centrifuged at 48,000 x g. Pellets were washed twice by resuspension in the same buffer followed by centrifugation at 48,000 x g. The final pellets were then stored at -80[degrees]C. When needed, pellets were suspended in Tris-HCl buffer and the membranes were incubated in the presence of [[sup.3]H]-ketanserin at 37[degrees]C. Non-specific binding was assessed by adding 10 [micro]M of methysergide.

Analysis of samples. Displacement curves were carried out using serial dilutions ranging from 2 mg/ml to 0.001 mg/ml of the unlabelled compound. To avoid possible undesired effects on radioligand binding, DMSO concentration in the different assays never exceeded 1% (v/v). The binding reaction were stopped by rapid filtration under vacuum through glass-fibre filters (Whatman) using a Brandell 36-sample harvester (Gaithesburg, MD, USA). Filter-bound radioactivity was counted in a liquid scintillation counter (Tricarb 2900; PerkinElmer Life Sciences, Boston, MA, USA) using Ultima Gold (Packard, USA) as scintillation fluid.

Data analysis

Treatment-induced variations in tail-flick and hot plate response were calculated as the percentage of maximal possible effect (MPE) according to the following formula: MPE [%]: [(T1-T0)/(T2-T0)] x 100, where TO and T1 are the latency before (baseline) and after treatment, and T2 is the cut-off time (12 s in the tail-flick, 15 s in the hot plate and 10 s in the low-intensity tail-flick tests). Data from behavioural experiments are expressed as mean [+ or -] standard error (S.E.M.) of %MPE. The %MPE in tail-flick and hot plate experiments were analyzed separately by repeated measure three-way analysis of variance (ANOVA) with pre-treatment (saline and WSE) and treatment (saline and morphine) as between-subjects factors, and time as within-subjects factor (repeated measures).

In the spontaneous and morphine-stimulated motor activity experiments, the average distance travelled (m) after WSE pretreatment and morphine treatment was analyzed by repeated measures three-way ANOVA, with pre-treatment (pre-treat) (saline and WSE) and treatment (treat) (saline and morphine) as between-subjects factors and time (t) (min) as within-subjects factor (repeated measures).

For each of the statistical analysis described above, when appropriate, post hoc comparisons were done using Tukey's test.

Data from radioligand inhibition experiments were analyzed by nonlinear regression analysis of a Sigmoid Curve using GraphPad Prism program (Graph Pad Software, Inc., San Diego, CA, USA). 1C50 values were derived from the calculated curves and converted to K, values as described previously (Cheng and Prusoff 1973). All receptor binding experiments were performed in triplicate and results were confirmed in at least four independent experiments.

Results

Effects of WSE on morphine-induced analgesia

The effects of WSE (100 mg/kg) pre-treatment on morphine-induced analgesia were evaluated using the tail-flick (Fig. 1) and the hot plate tests (Fig. 2). WSE alone failed to alter the nociceptive reaction time. Morphine (2.5, 5 and 10mg/kg) elicited a dose-dependent antinociceptive effect in both tests, although its efficacy was higher when the tail-flick test was used. The peak in the analgesic effect was reached 60 min after morphine administration. Co-administration of WSE with morphine modulated its analgesic activity depending on the dose of morphine and on the behavioural test used.

In the tail-flick test (Fig. 1) morphine alone at the dose of 2.5 mg/kg increased the tail-flick latency along 60 min after its administration [[F.sub.treat](1.30) = 46.16, p<0.0001; [F.sub.treat x t](4,120) = 8.35, p<0.0001; Tukey's test, p< 0.05 vs. saline + saline-treated mice], and along 120 min following 5mg/kg [[F.sub.treat](1.26) =135.99, p<0.0001; [F.sub.treat x t](4,104)= 10.97, p< 0.0001; Tukey's test, p<0.05 vs. saline + saline-treated mice] or lOmg/kg morphine administration [[F.sub.treat](1,31) = 260.48, P<0.0001; [F.sub.treat x t](4,124) = 5.61, p<0.005; Tukey's test, p<0.05 vs. saline + saline-treated mice]. Interestingly, although WSE co-administration failed to modulate the antinociceptive effect elicited by 2.5 mg/kg morphine [[F.sub.pre-treat](1.30) = 0.78, not significant (N.S.); [F.sub.pre-treat x treat](1.30) = 2.28, N.S.; [F.sub.pre-treat] x t(4,120) = 0.66, N.S.; [F.sub.pre-treat x treat x t](4,120) = 1.19, N.S.], it protracted the analgesic effect induced by 5 mg/kg morphine [[F.sub.pre-treat](1.26) = 5.76, p<0.05; [F.sub.pre-treat x treat](1,26) = 7.66, p<0.05; [F.sub.pre-treat x t](4, 104) = 2.82, p < 0.05; [F.sub.pre-treat x treat x t](4,104) = 2.66, p<0.05], and 10mg/kg morphine [[F.sub.pre-treat](1.31) = 2.10, N.S.; [F.sub.pre-treat x treat](1.31) = 3.57, N.S.; [F.sub.pre-treat x t](4,124)= 19.54, p<0.0001; [F.sub.pre-treat x treat x t](4,124) = 2.85, p<0.05). In fact, the analgesic effect was still evident 240 min after 5 mg/kg morphine treatment in mice pre-treated with WSE (78 [+ or -] 11%MPE) compared to mice pre-treated with saline (16 [+ or -] 7%MPE) (Tukey's test, p<0.05 vs. saline + saline-treated mice and vs. saline + morphine-treated mice). Likewise, analgesia was still evident at 240 min (55 [+ or -] 7%MPE) and 360 min (45 [+ or -] 11%MPE) after 10mg/kg morphine treatment, in mice pre-treated with WSE when compared to mice pre-treated with saline, in which the %MPE was respectively 20 [+ or -] 4% and 10 [+ or -] 3% (Tukey's test, p < 0.05 vs. saline + saline- treated mice and vs. saline + morphine-treated mice).

A different picture was observed in the hot plate test (Fig. 2) in which morphine, at the dose of 2.5 mg/kg had no analgesic effect [[F.sub.treat](1,30) = 0.35, N.S.; [F.sub.treat x t](4,120) = 5.4, p< 0.0001]; on the contrary, 5 and 10mg/kg increased the reaction time respectively along the first 60 min [[F.sub.treat](1.26) = 46.53, p<0.0001; [F.sub.treat x t](4,104) = 5.61, p<0.0001; Tukey's test, p<0.05 vs. saline + saline-treated mice], and along the first 120min [[F.sub.treat](1,31) = 58.72, P<0.0001; [F.sub.treat x t](4,124)= 10.27, p<0.0001; Tukey's test, p<0.05 vs. saline + saline-treated mice]. WSE pre-treatment failed to modulate the analgesic effect induced by 2.5 mg/kg [[F.sub.pre-treat](1.30) = 0.41, N.S,; [F.sub.pre-treat x treat](1.30) = 0.17, N.S.], [F.sub.pre-treat x t](4,120) = 0.17, N.S.; [F.sub.pre-treat x treat x t](4,120) = 0.28, N.S.], 5 mg/kg [[F.sub.pre-treat](1.26) = 0.0003, N.S.; [F.sub.pre-treat x treat](1.26) = 0.27, N.S.; [F.sub.pre-treat x t](4,104) = 0.82, N.S.; [F.sub.pre-treat x treat x t](4,104) = 0.48, N.S.] and 10mg/kg morphine [[F.sub.pre-treat](1.31) = 0.07, N.S.; [F.sub.pre-treat x treat](1.31) = 0.30, N.S.; [F.sub.pre-treat x t](4,124) = 1.18, N.S.; [F.sub.pre-treat x treat x t](4,124) = 0.55, N.S.].

Effect of WSE on morphine-induced hyperalgesia

The effects induced by WSE (100 mg/kg) pre-treatment on morphine-induced hyperalgesia were evaluated using the low-intensity tail-flick test (Fig. 3). WSE alone failed to alter the nociceptive reaction time. In agreement with Gupta et al. (2011), we found that a single injection of a low dose of morphine (2.5 mg/kg) elicited a biphasic effect in the nociceptive response of CD1 mice. In fact, analgesia was reached at 30 and 60 after morphine administration [[F.sub.treat](1.32) = 13.91, p < 0.001. [F.sub.treat x t](6,192) = 2.98, p < 0.01; Tukey's test, p < 0.05 vs. saline + saline-treated mice]. This effect was followed by a gradual appearance of hyperalgesia; Tukey's test revealed that morphine administration reduced the nociceptive response (-65 [+ or -] 13%MPE) when compared to the nociceptive response of control mice (-4[+ or -] 6%MPE) 360 min after morphine administration (p<0.05). Consistently with the results shown in Fig. 1A, pre-treatment with WSE did not potentiate the analgesic effect of morphine during the first 120 min, however, it significantly prevented morphine-induced hyperalgesia [[F.sub.pre-treat](1,32) = 7.25, p < 0.05; [F.sub.pre-treat x treat](1.32) = 9.40, p < 0.005; [F.sub.pre-treat x t](6.192) = 2.98, p < 0.001; [F.sub.pre-treat x treat x t](6,192) = 1.67, N.S.]. Tukey's test revealed that at 240, 300, 360 and 420 min after morphine administration the nociceptive threshold of mice pre-treated with WSE was higher when compared with the one observed in mice pre-treated with saline (p<0.05).

Effect of WSE on morphine-induced hyper-locomotion

Fig. 4 shows the effects of WSE on spontaneous motor activity and on morphine-induced (5 and 10 mg/kg) hyperlocomotion. Repeated measures three-way ANOVA of the average distances travelled revealed that morphine increased motor activity in a dose dependent manner [[F.sub.treat](2,38)= 18.43, p<0.0001; [F.sub.treat x t](8,152)= 19.61, p<0.0001; Tukey's test, p<0.05 vs. saline + saline-treated mice], WSE pre-treatment failed to alter both spontaneous motor activity and morphine-induced hyperlocomotion [[F.sub.pre-treat](1,38) = 0.09, N.S.J [F.sub.pre-treat x treat](2,38) = 0.18, N.S.; [F.sub.pre-treat x t](4,152) = 0.11, N.S.; [F.sub.pre-treat x treat x t](8,152) = 0.60, N.S.]. Specifically, Tukey's post hoc analysis revealed that 10 mg/kg morphine, alone and in combination with WSE increased motor activity 120, 240 and 360 min after administration (Tukey's test, p<0.05 vs. saline + saline-treated mice), but WSE pre-treatment did not affect morphine-induced hyper-locomotion.

Characterization of WSE binding properties

We evaluated whether the behavioural effects of WSE observed in the present study could be associated to the receptors involved in pain regulation. Therefore, we examined the affinity of WSE towards opioid ([mu], [delta], k), cannabinoid (CBlt CB2), glutamatergic (NMDA), GABAergic ([GABA.sub.A], [GABA.sub.B]), serotoninergic (5HT2A) and adrenergic ([[alpha].sub.2]) receptors using radioligand receptor binding assays. As shown in Table 1, WSE exhibits the highest affinity for the [GABA.sub.A] receptors. It is worth noting that WSE binds to [GABA.sub.A] with an affinity 10, 13 and 15 times greater than that for [GABA.sub.B], NMDA and 8 opioid receptors respectively. In contrast, WSE showed a weak binding affinity to pt and k opioid receptors and to [CB.sub.1] receptor and no binding affinity for the [CB.sub.2], [5HT.sub.2A] and [[alpha].sub.2] adrenergic receptors.

Discussion

Our results demonstrate that the administration of WSE, a methanolic root extract of Withania somnifera, prolongs the acute analgesic effect of morphine in the tail-flick test and prevents the development of hyperalgesia in a model of OIH. In addition, receptor binding assays suggest a possible involvement of [GABA.sub.A], [GABA.sub.B], NMDA and [delta] opioid receptors in the observed behavioural effects.

WSE was devoid of analgesic activity, in both the tail-flick and the hot plate tests, confirming previous results in which WSE (100 mg/kg) failed to increase the tail-flick latencies in mice 60 min after oral administration (Kulkarni and Ninan 1997). WSE also failed to strengthen the analgesic effect of morphine in the tail-flick test but, surprisingly, it prolonged the length of morphine action in a dose dependent manner. Indeed, while the analgesic effect induced by 5 and 10 mg/kg morphine alone declined 2 hours after morphine administration, it lasted respectively 4 and 6 hours when morphine was combined with WSE. Notably, this pharmacological effect appeared to be specific for morphine-elicited analgesia, since WSE pre-treatment failed to modify the hyper-locomotor activity induced by morphine. Interestingly, we found that WSE protracted the analgesic effect induced by morphine in the tail-flick test but not in the hot plate test. The tail-flick and hot plate tests are two behavioural models of acute pain thought to differ in the level of nociceptive information processing (Franklin and Abbott 1989). Accordingly, it was suggested that the response to the tail-flick test may predominantly reflect a spinally integrated nociceptive reflex, whereas the complex painful response assessed by the hot plate test is thought to require the involvement of higher brain functions. Thus, based on the present results it may be hypothesized that spinal rather than supraspinal mechanisms may account for the different effects induced by WSE in the hot plate and tail flick tests when associate with morphine.

Several hypotheses can be made to explain the ability of WSE to prolong morphine-induced analgesia in the tail flick test. One possible interpretation is that WSE might have altered morphine pharmacokinetics, as previously observed for L-type calcium channel blockers (Shimizu et al. 2004). However, Kasture et al. (2009) demonstrated that WSE administration induced statistically insignificant decrease, rather than increase, in plasma and brain morphine levels.

This observation brings us to conclude that the prolongation of morphine analgesia induced by WSE is pharmacodynamic in nature. An interesting hypothesis is that WSE might prolong morphine-induced analgesia by preventing the development of acute tolerance. Acute analgesic tolerance following opioids administration has been found to develop rapidly both in humans (Vinik and Kissin 1998) and in rodents (Kissin et al. 1991). Kissin et al. (1991) have shown in rats that, under continuous infusion or after a single subcutaneous injection, the analgesic effect of morphine rapidly declined despite brain levels of morphine have remained stable over time. The molecular mechanism underlying the development of analgesic tolerance following acute morphine administration is similar to that involved in chronic analgesic tolerance (Fairbanks and Wilcox 1997). Consistently, several classes of compounds, such as, NMDA receptor antagonists and [GABA.sub.A] modulators, have been found to prevent the development of analgesic tolerance following both acute (Kissin et al. 1997, 2000) and chronic opioid administration (Bryant et al. 2006; Tejwani et al. 1993). Since WSE administration has been found to prevent analgesic tolerance following chronic morphine administration (Kulkarni and Ninan 1997), the prolongation of morphine acute analgesic effect observed in the present study can be ascribed to a WSE-induced prevention of acute tolerance development.

Moreover, several studies suggest the involvement of OIH in the development of acute tolerance (Milne et al. 2013); more generally, although opioid tolerance and OIH should be considered distinct phenomena they partially share the same cellular mechanism, pathogenesis and therapeutic approach (DuPen et al. 2007). Consistently, using the low-intensity tail-flick test, that allows to magnify the development of hyperalgesia induced by a low morphine dose (Gupta et al. 2011), we found that WSE antagonized the progressive reduction on nociceptive threshold induced by 2.5 mg/kg morphine.

In the attempt to elucidate the mechanism(s) by which WSE modulates the effects induced by morphine, we studied the affinity of WSE for several receptors involved in pain transduction signalling and opioids-induced analgesia. These experiments brought us to restrict the search of potential molecular targets of WSE to [GABA.sub.A], [GABA.sub.B], [delta] opioid and NMDA receptors. WSE exhibited a high affinity for the [GABA.sub.A] and moderate affinity for [GABA.sub.B], NMDA and 8 opioid receptors, confirming and extending previous data showing that WSE interacts with [GABA.sub.A] and [GABA.sub.B] receptors (Mehta et al. 1991; Ruiu et al. 2013). Interestingly, GABA, NMDA and [delta] opioid receptors-mediated neurotransmission plays an important role in pain transduction signalling and is involved in the appearance of opioid-induced analgesic tolerance and hyperalgesia (Davis and Pasternack 2005; Enna and McCarson 2006). In particular, GABA transmission may have opposing effects on pain processing, depending upon its location within the central nervous system; its enhancement elicits analgesia at the spinal level whereas is pronociceptive at supraspinal level (Davis and Pasternack 2005; Giordano 2005). Systemic administration of [GABA.sub.A] and [GABA.sub.B] agonists, as well as benzodiazepines have been found to potentiate opioid-induced analgesia (Aley and Kulkarni 1989; Bergman et al. 1988); moreover, pre-treatment with benzodiazepines attenuated the development of tolerance following acute opioid treatment (Kissin et al. 1997). Thus, the observation that WSE binds the [GABA.sub.A] and [GABA.sub.B] receptors, combined with the finding that WSE contains a GABA-mimetic component (Mehta et al. 1991), suggests that the GABA system may be partially responsible for the behavioural effects observed in the present study.

Glutamate is the main excitatory neurotransmitter involved in the transmission of nociceptive stimuli at spinal level (Giordano 2005); moreover, NMDA receptor sensitization into spinal neurons has been found to play a pivotal role in the development of opioid-induced analgesic tolerance and OIH (DuPen et al. 2007). Consistently, co-administration of NMDA receptor antagonists has been found to potentiate opioid-induced analgesia (Fischer et al. 2005) and to prevent analgesic tolerance and OIH both in animals and in humans (DuPen et al. 2007). Moreover, Zhao and Joo (2008) found that the enhancement of NMDA transmission responsible for the development of analgesic tolerance and OIH is mediated by activation of [delta] opioid receptors. In this regard, our observation that WSE moderately binds to NMDA and 5 opioid receptors may suggest that they may contribute to prolong morphine analgesia and to prevent morphine-induced hyperalgesia. The involvement of NMDA receptors could also explain why we have observed a prolongation of morphine analgesia in the tail-flick but not in the hot plate test; accordingly, Kozela et al. (2001) demonstrated that low-affinity NMDA receptor antagonists preferentially potentiate morphine-induced analgesia recorded from the tail but not from the hind paw.

In conclusion, our results suggest that WSE could be a valuable adjuvant in opioid-sparing therapies, which aim to minimize opioid dosage while providing optimal analgesia and limiting the emergence of tolerance and hyperalgesia. The concurrent action of WSE on [GABA.sub.A], [GABA.sub.B], NMDA and [delta] opioid receptors could be responsible for the behavioural effects observed in the present study. Further studies are needed to evaluate the net contribution of each of these receptors, and this will be achieved by identifying the single phytochemical components of WSE and characterizing their intrinsic activity.

Source of funding

From Regione Autonoma della Sardegna (RAS) (CRP 26805-CUP B71J11001480002) to SR, EA, FC.

http://dx.doi.org/10.1016/j.phymed.2013.10.021

ARTICLE INFO

Article history:

Received 23 August 2013

Received in revised form 16 September 2013

Accepted 17 October 2013

Conflicts of interest

The authors declare they have no conflicts of interest.

Acknowledgements

The authors wish to thank Dr. Amit Agarwal (Natural Remedies Pvt. Ltd., Bangalore, India) for the generous gift of standardized Withania somnifera root extract. The authors are grateful to Mrs. Gabriella Manca and Mr. Antonio Pilleri for animal care.

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Alessandro Orru (a), *, Giorgio Marchese (a), Gianluca Casu (a), Maria Antonietta Casu (a), Sanjay Kasture (b), Filippo Cottiglia (c), Elio Acquas (d,e,f), Maria Paola Mascia (g), Nicola Anzanic, Stefania Ruiu (a), *

(a) CNR-lnstitute of Translational Pharmacology, U.O.S. of Cagliari, Science and Technology Park of Sardinia Polaris, Pula, Italy

(b) Sanjivani College of Pharmaceutical Education & Research, Kopargaon, India

(c) Department of Life and Environmental Sciences--Drug Sciences Section, University of Cagliari, Italy

(d) Department of Life and Environmental Sciences--Pharmaceutical, Pharmacological and Nutraceutical Sciences Section, University of Cagliari, Italy

(e) Centre of Excellence on Neurobiology of Addiction, University of Cagliari, Italy

(f) National Institute of Neuroscience--INN, University of Cagliari, Italy

(g) CNR-lnstitute of Neuroscience, Cagliari, Cittadella Universitaria, 09042 Monserrato, Italy

* Corresponding authors. Tel.: +39 070 9241057: fax: +39 070 9241057.

E-mail addresses: alessandro.orru@ift.cnr.it (A. Orru), stefania.miu@cnr.it (S. Ruiu).

Table 1
[K.sub.i] values for WSE were determined as described in details in
the Methods section. Results are mean [+ or -] SEM of four independent
experiments assayed in triplicate.

                  [K.sub.i] ([micro]g/ml)

[mu]              [delta]           k

385 [+ or -] 14   166 [+ or -] 11   775 [+ or -] 56

[CB.sub.1]        [CB.sub.2]        [GABA.sub.A]

837 [+ or -] 74   >1000             13 [+ or -] 2

[GABA.sub.B]      NMDA

130 [+ or -] 12   193 [+ or -] 21

[[alpha].sub.2]   5-[HT.sub.2A]

>1000             >1000
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Author:Orru, Alessandro; Marchese, Giorgio; Casu, Gianluca; Casu, Maria Antonietta; Kasture, Sanjay; Cottig
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Date:Apr 15, 2014
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