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Yulangsan polysaccharide attenuates withdrawal symptoms and regulates the NO pathway in morphine-dependent rats
Neuroscience Letters 570 (2014) 63–68
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Yulangsan polysaccharide attenuates withdrawal symptoms and regulates the NO pathway in morphine-dependent rats Chunxia Chen, Zhihuan Nong, Jiangchun Huang, Zhaoni Chen, Shijun Zhang, Yang Jiao, Xiaoyu Chen, Renbin Huang ∗ Department of Pharmacology, Guangxi Medical University, Nanning, Guangxi 530021, PR China
h i g h l i g h t s
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a b s t r a c t
• YLSP attenuated morphine withdrawal symptoms.
• YLSP decreased brain NO levels and NOS activity in morphine dependent rats. • YLSP modulated the level of DA as well as NE in NAc, VTA, HIP, and PFC.
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Article history: Received 25 November 2013 Received in revised form 22 March 2014 Accepted 8 April 2014 Available online 18 April 2014 Keywords: Yulangsan polysaccharide Withdrawal symptoms Morphine NO pathway Monoaminergic neurotransmitters
a b s t r a c t Yulangsan polysaccharide (YLSP) has been utilized as a phytomedicine to managing nervous dysfunction in China. Thus, this study aimed to evaluate the potential YLSP-mediated detoxification role against morphine dependence in rats. The results indicated that the morphine dependence model significantly increased withdrawal symptoms, levels of NO and NOS (P < 0.05). Furthermore, monoaminergic neurotransmitters, including DA and NE, were detected at elevated levels in the ventral tegmental area (VTA), hippocampus (HIP) and prefrontal cortex (PFC), respectively, while the level of DA was decreased and NE was increased in the nucleus accumbens (NAc). Conversely, YLSP administration significantly reversed naloxone-induced withdrawal symptoms, expression of brain NO and NOS, and monoaminergic neurotransmitters (P < 0.05). Interestingly, YLSP shows an even more effective trend in attenuating withdrawal symptoms than does clonidine, although without a significant difference. These findings indicate that YLSP attenuation of the naloxone-induced withdrawal symptoms of morphine dependence may be mediated by regulation of the NO pathway and modulation of monoaminergic neurotransmitters. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Morphine dependence is a major health problem across the world, and it also is associated with high mortality [1]. In addition, Long-term morphine abuse can lead to nervous system impair-
∗ Corresponding author at: Department of Pharmacology, Guangxi Medical University, 22 Shuangyong Road, Nanning 530021, Guangxi Zhuang Autonomous Region, PR China. Tel.: +86 771 5339805; fax: +86 771 5358272. E-mail address: [email protected] (R. Huang). http://dx.doi.org/10.1016/j.neulet.2014.04.006 0304-3940/© 2014 Elsevier Ireland Ltd. All rights reserved.
ment and a series of physical disorders [22]. To date, there is no cure for the morphine dependence, but the conditions can be managed or controlled, such as: clonidine, methadone, Yi’an decoction [13,20,23]. Therefore, available methods are required to develop novel therapeutic strategies for detoxification treatment. Increasing evidence suggests that the NO pathway is associated with morphine dependence and withdrawal symptoms [32]. After activating soluble guanylate cyclase, the intracellular cyclic adenosine monophosphate (cAMP) is increased and then morphine withdrawal symptoms are aggravated [6]. NOS, as the key enzyme for NO formation, induces endogenous NO discharge when catalyzed
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by l-arginine. Meanwhile, NE and DA neurotransmitters are closely related to the formation of morphine dependence. DA catalyzes the generation of cAMP mainly by D1-receptors-cAMP pathway [19]. Moreover, NE and DA have been associated with multiple addiction-related regions in the CNS, including the nucleus accumbens (NAc) [7], ventral tegmental area (VTA) [24], hippocampus (HIP) [25], and prefrontal cortex (PFC) [34]. Yulangsan, also called Longyanshen, is a commonly used traditional Chinese medicine for the remedy of hypomnesia, immune disorders and mood disorders. Our previous study demonstrated that Yulangsan polysaccharide (YLSP), the major active component of Yulangsan, has neuroprotective effects that include reducing oxidative stress and NO in brain tissue, regulating the synthesis of NE, DA and 5-HT, and improving learning and memory [10–12]. Recently, it was found that YLSP inhibits depression by upregulating the neurotransmitters NE and 5-HT, prefrontal cortex adenylate cyclase activity and hippocampal brain-derived neurotrophic factor expression [17]. Based on data from our laboratory, we suggested that YLSP may be effective in attenuating the naloxone-induced withdrawal symptoms in morphine dependent animals. In this study, we explored the effect and the relevant mechanisms of YLSP on morphine dependence model induced by naloxone. 2. Materials and methods 2.1. Preparation of Yulangsan polysaccharide (YLSP) extract Yulangsan polysaccharide (YLSP) was prepared by the method described previously [12,18]. Next, the gas chromatography (GC) analysis report showed that YLSP was composed of d-glucose and d-arabinose in a molar ratio of 90.79% and 9.21%, with an average molecular weight of about 14,301 Da. 2.2. Animals and drug intervention A total of 140 male SD rats, weighing 180–220 g were purchased from the Experimental Animal Centre of Guangxi Medical University (Certificate No. SYXK 2009-0002) and were allowed to acclimate in quarantine for a week prior to experimentation. Rats were randomly divided into seven groups of 20 rats per group. For establishment of morphine dependent models according to the method of [36], groups II–VII were subcutaneously injected with morphine (Shenyang No. 1 Pharmaceutical, China; 20, 40, 60, 80, 100, 100, 100 mg/kg) twice daily (9:00 AM, 5:00 PM) for 7 days. Group II served as the morphine-dependent model group, Group III served as the morphine-sustained group and was treated with 10 continuous days of morphine. In addition to morphine, Group IV was intragastrically given clonidine (Shaanxi Xiyue Pharmaceutical, China; 0.4 mg/kg) twice daily starting with the day 5 to the day 10, 30 min before morphine injection, serving as the positive control. In addition to morphine administration, Groups V–VII were administered orally with YLSP (0.3, 0.45, and 0.6 g/kg, representing low, medium and high dosage, respectively) in parallel to Group IV. Group I served as a normal control, was subcutaneously given equivalent physiological saline daily for 7 days and received 10 ml/kg physiological saline intragastrically for 6 days. All rats, except the saline-treated normal control group, were treated with morphine (100 mg/kg) on day 8 (9:00 AM). Naloxone (Beijing Sihuan Pharmaceutical, China; 4 mg/kg, intraperitoneal) treatment was administered to all animals at 2 h after the final morphine treatment. The experimental animals were treated according to the Guidance Suggestions for the Care and Use of Laboratory Animals issued by the Ministry of Science and Technology of the People’s Republic of China.
2.3. Calculation of withdrawal symptoms scores and body weight change in rats On day 8, the rats (n = 10) were individually placed in a plastic cage, and the score of withdrawal symptoms was evaluated at 15 min after naloxone injection. Behaviors scored in this manner included: writhing (2), wet dog shakes (2), irritability (conflict-induced vocalization, 1 = touchness, 2 = nearness), teeth chatter (0.5 = discontinuity, 1 = continuity), sialorrhea (1 = mild, 2 = marked), diarrhea (4 = mild, 8 = marked), lacrimation (4). The body weight loss rate was similarly scored from 0 to 20 by multiples of 2 occurrences at 1 h of withdrawal, and was also included in overall withdrawal ratings. The quantifying of score above was guided by previous studies [28,35]. On day 9 and 10, the withdrawal symptoms scores and body weight change were recorded at the same time as day 8 (but had no naloxone injection). 2.4. Serum and brain regions tissue sampling The other 10 animals for each group were sacrificed by cervical dislocation. Serum samples were collected from the supraorbital vein and were kept frozen at −80 ◦ C. Brain samples were promptly removed and weighed. The right brain hemispheres were dissected on an ice-cold plate into four regions: nucleus accumbens (NAc) (+2.76 mm anterior, +1.0 mm lateral to midline and 5.5 mm ventral), ventral tegmental area (VTA) (−6.84 mm posterior, +0.2 mm lateral to midline and 8.5 mm ventral), hippocampus (HIP) (−3.60 mm posterior, +0.2 mm lateral to midline and 3.4 mm ventral) and prefrontal cortex (PFC) (+2.76 mm anterior, midline and 2.4 mm ventral) with respect to bregma as per the stereotaxic atlas by Paxinos and Watson [27]. The serum and four brain regions were properly conserved for NE and DA (Sigma, Germany) test. 2.5. Assay of NO and NOS in brain homogenate in rats Left brain homogenates were prepared in ice-cold physiological saline (10%, w/v) using a homogenizer (Ningbo, China). The homogenate was centrifuged (Hitachi, Japan) at 10,000 rpm for 10 min at 4 ◦ C. The supernatant was used immediately for the measurement of NO and NOS (Nanjing Jiancheng Bioengineering Institute, China). These enzymes were quantified according to the manufacturer’s instructions. 2.6. High performance liquid chromatography (HPLC) analysis of serum and brain regions levels of NE and DA Chromatography was performed on a Shimadzu LC-20AT System (Shimadzu Corp., Japan), equipped with a RF-20A fluorescence detector (Shimadzu Corp., Japan) and an automatic sample injector (Shimadzu Corp., Japan). The separation was achieved on an Ultimate AQ-C18 column (4.6 mm × 250 mm i.d., 5 m; Welch Materials, Inc.). The column temperature was maintained at 35 ◦ C. A mixture of 0.1 M phosphate buffer–methanol (Fisher Scientific, USA; 92:8, v/v) was used as the mobile phase, at a flow rate of 0.8 ml/min. The fluorescence detector of HPLC was set at 254 nm for the excitation wavelength and 338 nm for the emission wavelength. Peaks of NE and DA were indentified by comparing the retention time of each peak in the sample solution with that in the standard solution. The serum and four brain regions levels of DA and NE were quantified. 2.7. Statistical analysis The data are expressed as means ± SD. Statistical analysis was performed using Statistics Package for Social Science 13.0 software (SPSS Inc., USA). Withdrawal symptoms scores and body weight
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loss were performed using two-way analysis of variance (ANOVA), with treatment as between subject variable and days as within subject variable. The remaining data were analyzed by one-way analysis of variance (ANOVA). Post hoc analysis was carried out with LSD t-test. The level of significance was set at P < 0.05. 3. Results 3.1. Effects of YLSP on withdrawal symptoms scores and body weight loss in morphine-dependent rats Two-way ANOVA revealed significant effects of time [F(2, 126) = 244.889, P < 0.001], treatment [F(6, 63) = 87.461, P < 0.001], and time × treatment [F(12, 126) = 17.020, P < 0.001]. The post hoc analysis revealed that low-, medium- and high-dosage YLSP treatments were effective at reducing withdrawal symptoms scores, compared with the model group (P < 0.05). Clonidine significantly attenuated withdrawal symptoms scores, compared with the morphine-dependent model group (P < 0.05), which was similar to high-dose YLSP. However, the withdrawal symptoms score remained significantly higher in the morphine-sustained rats than in normal control animals (Fig. 1A). Two-way ANOVA revealed significant effects of time [F(3, 189) = 68.530, P < 0.001], treatment [F(6, 63) = 21.512, P < 0.001], and time × treatment [F(18, 189) = 46.715, P < 0.001]. The rate of body weight change was markedly decreased in the animals treated with morphine alone and reached its lowest level at 19 h (1 h after naloxone injection) [F(6, 63) = 24.644, P < 0.001]. The post hoc analysis revealed that the body weight loss was significantly reduced following administration of low, medium, and high dosages of YLSP (P < 0.05). Clonidine was less effective at relieving body weight change (P < 0.05) than all YLSP groups (Fig. 1B). 3.2. Effects of YLSP on brain NO levels and NOS activity in morphine-dependent rats As indicated in Fig. 1C, NO and NOS concentration increased in the brains of morphine-dependent rats [NO: F(6, 63) = 19.016, P < 0.001; NOS: F(6, 63) = 22.756, P < 0.001, vs. normal control group], but were significantly decreased by low, medium and high dosages of YLSP, as a favorable dose-response relationship in rats (P < 0.05) by post hoc analysis. High NO and NOS levels were also relieved by clonidine treatment in morphine-dependent rats.
Fig. 1. Effects of YLSP on withdrawal symptoms scores (A) and body weight loss (B) in morphine-dependent rats. Effects of YLSP on brain NO levels and NOS activity in morphine-dependent rats (C). I: normal control; II: morphine-dependent model control; III: morphine-sustained control; IV: morphine + 0.4 mg/kg clonidine; V: morphine + 0.3 g/kg YLSP; VI: morphine + 0.45 g/kg YLSP; VII: morphine + 0.6 g/kg YLSP. Results are presented as the mean ± SD (n = 10). a P < 0.05 compared with normal control group, b P < 0.05 compared with morphine-dependent model group. Withdrawal symptoms were scored for 15 min after injecting naloxone in (A). The times are given in hour (h) with reference to base body weight from day 7 (5:00 PM) (B).
3.3. Effects of YLSP on NE and DA content in serum and four brain regions of morphine-dependent rats HPLC analysis revealed that serum NE levels were increased in the morphine-dependent model group compared to the normal control group [F(6, 63) = 15.898, P < 0.001]. However, rats in the morphine-sustained group showed a remarkable decrease compared to normal control rats. The post hoc analysis revealed that administration of 0.3, 0.45, 0.6 g/kg bw YLSP to morphine-dependent rats resulted in a significant decrease in NE compared to the morphine-dependent model group (P < 0.05). The amount of serum DA increased considerably after morphine administration [F(6, 63) = 5.673, P < 0.001], but the increase was attenuated after treatment with YLSP or clonidine by post hoc analysis (P < 0.05) (Fig. 2A and B). Four brain regions results demonstrated that endogenous monoaminergic neurotransmitters expressions are related to nerve cell activities. As shown in Fig. 2C, the concentration of NE in NAc, VTA, HIP and PFC rose to a higher level in morphine-dependent model rats
[NAc: F(6, 63) = 67.790, P < 0.001; VTA: F(6, 63) = 36.577, P < 0.001; HIP: F(6, 63) = 9.930, P < 0.001; PFC: F(6, 63) = 20.861, P < 0.001]. Subsequently, the NE concentration was rapidly decreased in morphine-sustained group, even below normal control. Compared with the morphine-dependent model group, NE level was markedly decreased after YLSP or clonidine treatment by post hoc analysis (P < 0.05). DA levels in VTA, HIP and PFC in the morphine-dependent model group were significantly higher than in the normal control group [VTA: F(6, 63) = 49.810, P < 0.001; HIP: F(6, 63) = 18.730, P < 0.001; PFC: F(6, 63) = 1.388, P < 0.001], but DA levels were decreased in the NAc [F(6, 63) = 21.579, P < 0.001]. However, compared with the normal control group, levels of DA in the morphine-sustained group were markedly increased, by 1.2-, 2.1-, 1.8- and 2.4-fold in the NAc, VTA, HIP and PFC, respectively. The post hoc analysis revealed that treatment with 0.45, 0.6 g/kg bw YLSP effectively increased DA levels in the NAc, while DA decreased in the VTA, HIP and PFC (P < 0.05). In contrast to the NAc, treatment with clonidine had little influence on the DA levels in the VTA, HIP and PFC (Fig. 2D).
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Fig. 2. Effects of YLSP on NE and DA content in serum (A, B) and four brain regions (C, D) of morphine-dependent rats. (A, B) I: normal control; II: morphine-dependent model control; III: morphine-sustained control; IV: morphine + 0.4 mg/kg clonidine; V: morphine + 0.3 g/kg YLSP; VI: morphine + 0.45 g/kg YLSP; VII: morphine + 0.6 g/kg YLSP. Results are presented as the mean ± SD (n = 6). a P < 0.05 compared with normal control group, b P < 0.05 compared with morphine-dependent model group.
4. Discussion The present study partly investigated the effects of YLSP, as a Yulangsan extract, on the attenuating properties of naloxoneprecipitated withdrawal symptoms and its possible mechanism in rats using a morphine-dependent model. In our study, we observed that rats treated with naloxone during the morphinedependent phase showed a significant increase in some behavioral symptoms and body weight loss, when compared to the normal control group, which was consistent with recent studies [2,33].
Conversely, administration of YLSP attenuates the development of morphine dependence by reducing the naloxone-induced behavioral withdrawal symptoms in a dose-dependent manner. YLSP even shows a more effective trend in regulating withdrawal symptom than clonidine, although it does not significantly reverse body weight change. The ability of YLSP to reduce the incidence of withdrawal symptoms is extremely important because withdrawal symptoms were historically believed to have a major role in the relapse to drug-taking behavior after drug abstinence [15].
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There is accumulating evidence indicating that NO, which is produced through NOS, might have a key role in withdrawal processing. Accordingly, withdrawal symptoms was reversed by the administration of selective NOS and guanylate cyclase inhibitors and was attenuated or abolished in NOS knockout animals [8,9]. For Drug dependence was exhibited as compulsive drug administration with consecutive craving behaviors, NO signaling was likely involved in learning and memory functions [37,38]. In a recent study, NO was shown to be mediated via the classical vasodilation process and was also involved in learning and memory [31]. Moreover, NO is a highly reactive oxidant and acts as a neurotransmitter as well as a defense against tumor cells, parasites and bacteria in the immune system [21]. In our study, there were significant increases in NO production and NOS level after morphine administration. Conversely, both NO and NOS levels were down regulated after treatment with YLSP. Thereby, the possible mechanism of YLSP in counteracting naloxone-induced withdrawal symptoms may be associated with regulation of the NO pathway. The release of monoaminergic neurotransmitters such as DA and NE may be an important mechanism underlying the development of morphine dependence. In particular, the mesolimbic dopaminergic system has a critical role in mechanisms related to memory and is involved in the establishment of the morphine-dependent model [16,30]. Administration of D1 and D2 receptor agonists has been shown to attenuate somatic withdrawal signs [4]. The HIP is recognized as an important region involved in morphine dependence [25,26]. Moreover, previous studies have indicated that a strong increase of DA and NE release takes place in the medial prefrontal cortex during naloxone-precipitated withdrawal [29,34]. The present results confirm previous observations showing that naloxone-induced abstinence symptoms in morphine dependent rats is associated with a dramatic and marked increase in both DA and NE release in the VTA, HIP and PFC. Importantly, in this study, DA content was decreased in the NAc after naloxone-induced withdrawal, suggesting that there is an inverse relationship between the mesocortical and mesolimbic dopaminergic systems, as proposed by other authors [3]. In contrast, the levels of these neurotransmitters were reversed after treatment with YLSP, which appears to act as a modulator of monoaminergic neurotransmitters. Besides, we also found that NE was reduced in brain regions in rats following morphine-sustained treatment. Neuronal plasticity induced by dependence might be associated with changes in pain sensitivity resulting from the adaptive response of neurons [39]. The other potential neuronal processes underlying the effects of YLSP on naloxone-precipitated withdrawal symptoms might include depression inhibition. Several studies have highlighted the efficacies of medicinal herbs on morphine withdrawal, due to their antidepressant and anxiolytic properties [5,14]. Previous studies have shown that administration of YLSP can produce an increase in neurotransmitters such as 5-HT and NE, primarily in brain areas closely related to reward processes: the PFC and the HIP [17]. YLSP, as a traditional medicine, is widely used for the treatment of mood disorders, such as anxiety and depression, which appear to be related to the same reward mechanisms. In summary, our study demonstrates that YLSP attenuates withdrawal symptoms of morphine dependence by regulating the NO pathway and modulating monoaminergic neurotransmitters. Further research to elucidate the underlying mechanism is important and will provide additional evidence for the exploitation of the broader remedial usage of YLSP.
Conflict of interest The authors declare that there are no conflicts of interest.
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Acknowledgments This work was financially supported by the National Natural Science Fund (30960504), Guangxi Scientific Research and Technology Development research projects (0630002-2A), Guangxi Natural Science Foundation (2011GXNSFD018030), Nanning Science and Technology Research and Production of new products (No. 201102084C). References [1] L. Brådvik, M. Berglund, A. Frank, A. Lindgren, P. Löwenhielm, Number of addictive substances used related to increased risk of unnatural death: a combined medico-legal and case-record study, BMC Psychiatry 9 (2009) 48. [2] H.B. Cai, Z.X. Mo, X. Li, Efects of caulis sinomenii and sinomenine on withdrawal syndrome in morphine-dependent mice, Beijing J. Tradit. Chin. Med. 27 (2008) 652–655. [3] E.H. Chartoff, M.F. Barhight, S.D. Mague, A.M. Sawyer, W.A. Carlezon, Anatomically dissociable effects of dopamine D1 receptor agonists on reward and relief of withdrawal in morphine-dependent rats, Psychopharmacology (Berl.) 204 (2009) 227–239. [4] E.H. Chartoff, S.D. Mague, M.F. Barhight, A.M. Smith, W.A. Carlezon Jr., Behavioral and molecular effects of dopamine D1 receptor stimulation during naloxone-precipitated morphine withdrawal, J. Neurosci. 26 (2006) 6450–6457. [5] A. Feily, N. Abbasi, The inhibitory effect of Hypericum perforatum extract on morphine withdrawal syndrome in rat and comparison with clonidine, Phytother. Res. 23 (2009) 1549–1552. [6] B.H. Gabra, E.A. Afify, T.T. Daabees, M.S. Abou Zeit-Har, The role of the NO/NMDA pathways in the development of morphine withdrawal induced by naloxone in vitro, Pharmacol. Res. 51 (2005) 319–327. [7] A. Haghparast, A. Omranifard, R. Arezoomandan, M. Ghalandari-Shamami, Z. Taslimi, A.A. Vafaei, A. Rashidy-Pour, Involvement of dopaminergic receptors of the rat nucleus accumbens in decreasing the conditioned place preference induced by lateral hypothalamus stimulation, Neurosci. Lett. 556 (2013) 10–14. [8] E.L. Heinzen, G.M. Pollack, The development of morphine antinociceptive tolerance in nitric oxide synthase-deficient mice, Biochem. Pharmacol. 67 (2004) 735–741. [9] A. Hervera, R. Negrete, S. Leánez, J. Martín-Campos, O. Pol, Peripheral effects of morphine and expression of -opioid receptors in the dorsal root ganglia during neuropathic pain: nitric oxide signaling, Mol. Pain 7 (2011) 25. [10] Z.S. Huang, Y. Jiao, S.J. Zhang, X. Lin, R.B. Huang, Improvement of LYSP on learning and memory abilities in the elderly aging SAMP8 mice model, Chin. J. Gerontol. 28 (2008) 2086–2088. [11] Z.S. Huang, J. Li, S.J. zhang, Y. Jiao, R.B. Huang, Effects of Longyanshen polysaccharides on brain NO, SOD and MDA in d-galactose-induced dementia mice, Chin. J. Gerontol. 25 (2005) 176–177. [12] Z.S. Huang, H.Y. Xie, S.J. Zhang, Y. Jiao, W.Z. Jiang, R.B. Huang, Effects of Longyanshen polysaccharides on free radical metabolism in senescence accelerated-prone mice, Neural Regen. Res. 3 (2008) 1099–1102. [13] H. Joseph, S. Stancliff, J. Langrod, Methadone Maintenance Treatment (MMT), Mount Sinai J. Med. (2000). [14] S. Kasture, S. Vinci, F. Ibba, A. Puddu, M. Marongiu, B. Murali, A. Pisanu, D. Lecca, G. Zernig, E. Acquas, Withania somnifera prevents morphine withdrawalinduced decrease in spine density in nucleus accumbens shell of rats: a confocal laser scanning microscopy study, Neurotox. Res. 16 (2009) 343–355. [15] G.F. Koob, L. Stinus, M.L. Moal, F.E. Bloom, Opponent process theory of motivation: neurobiological evidence from studies of opiate dependence, Neurosci. Biobehav. Rev. 13 (1989) 135–140. [16] G.F. Koob, N.D. Volkow, Neurocircuitry of addiction, Neuropsychopharmacology 35 (2010) 217–238. [17] S. Liang, R.B. Huang, X. Lin, J.C. Huang, Z.S. Huang, H.G. Liu, Effects of Yulangsan polysaccharide on monoamine neurotransmitters, adenylate cyclase activity and brain-derived neurotrophic factor expression in a mouse model of depression induced by unpredictable chronic mild stress, Neural Regen. Res. 7 (2012) 191–196. [18] X. Lin, Z. Huang, X. Chen, Y. Rong, S. Zhang, Y. Jiao, Q. Huang, R. Huang, Protective effect of Millettia pulchra polysaccharide on cognitive impairment induced by d-galactose in mice, Carbohydr. Polym. (2013). [19] A. Lintas, N. Chi, N.M. Lauzon, S.F. Bishop, S. Gholizadeh, N. Sun, H. Tan, S.R. Laviolette, Identification of a dopamine receptor-mediated opiate reward memory switch in the basolateral amygdala-nucleus accumbens circuit, J. Neurosci. 31 (2011) 11172–11183. [20] R. Liu, G. Xu, L. Duan, X. Lu, W. Gao, Y. An, Evaluation on phase IV clinical trail of Yi An oral liquor for detoxification of heroin addicts, Chin. J. Drug Depend. 10 (2001) 200–203. [21] C.J. Lowenstein, S.H. Snyder, Nitric oxide, a novel biologic messenger, Cell 70 (1992) 705–707. [22] L. Lu, J.D. Shepard, F.S. Hall, Y. Shaham, Effect of environmental stressors on opiate and psychostimulant reinforcement, reinstatement and discrimination in rats: a review, Neurosci. Biobehav. Rev. 27 (2003) 457–491. [23] T.R. Macedo, J. Relvas, C.A. Fontes Ribiero, F. Pacheco, M.T. Morgadinho, C.M. Pinto, P.C. Gomes, M. Ventura, V. Henriques, S.V. Nunes, Plasma catecholamines
68
[24]
[25]
[26] [27] [28]
[29]
[30]
[31]
C. Chen et al. / Neuroscience Letters 570 (2014) 63–68 during an ultrarapid heroin detoxification, Ann. N.Y. Acad. Sci. 914 (2000) 303–310. F.J. Meye, R. van Zessen, M.P. Smidt, R.A.H. Adan, G.M.J. Ramakers, Morphine withdrawal enhances constitutive -opioid receptor activity in the ventral tegmental area, J. Neurosci. 32 (2012) 16120–16128. J.A. Morón, N.S. Abul-Husn, R. Rozenfeld, G. Dolios, R. Wang, L.A. Devi, Morphine administration alters the profile of hippocampal postsynaptic density-associated proteins a proteomics study focusing on endocytic proteins, Mol. Cell. Proteom. 6 (2007) 29–42. E.J. Nestler, Common molecular and cellular substrates of addiction and memory, Neurobiol. Learn. Mem. 78 (2002) 637–647. G. Paxinos, C. Watson, The Rat Brain in Stereotaxic Coordinates, Academic Press, New York, 2007. K. Rasmussen, D.B. Beitner-Johnson, J.H. Krystal, G.K. Aghajanian, E.J. Nestler, Opiate withdrawal and the rat locus coeruleus: behavioral, electrophysiological, and biochemical correlates, J. Neurosci. 10 (1990) 2308–2317. Z.L. Rossetio, G. Longu, G. Mercuro, G.L. Gessa, Extraneuronal noradrenaline in the prefrontal cortex of morphine-dependent rats: tolerance and withdrawal mechanism, Brain Res. 609 (1993) 316–320. T.S. Shippenberg, R. Bals-Kubik, A. Herz, Examination of the neurochemical substrates mediating the motivational effects of opioids: role of the mesolimbic dopamine system and D-1 vs. D-2 dopamine receptors, J. Pharmacol. Exp. Ther. 265 (1993) 53–59. A.J. Susswein, A. Katzoff, N. Miller, I. Hurwitz, Nitric oxide and memory, Neuroscientist 10 (2004) 153–162.
[32] N. Toda, S. Kishioka, Y. Hatano, H. Toda, Modulation of opioid actions by nitric oxide signaling, Anesthesiology 110 (2009) 166–181. [33] K. Ueno, T. Maeda, N. Kiguchi, Y. Kobayashi, M. Ozaki, S. Kishioka, Availability of serum corticosterone level for quantitative evaluation of morphine withdrawal in mice, Drug Discov. Ther. 5 (2011) 71. [34] R. Ventura, A. Alcaro, S. Puglisi-Allegra, Prefrontal cortical norepinephrine release is critical for morphine-induced reward, reinstatement and dopamine release in the nucleus accumbens, Cereb. Cortex 15 (2005) 1877– 1886. [35] E. Wei, Assessment of precipitated abstinence in morphine-dependent rats, Psychopharmacologia 28 (1973) 35–44. [36] H.Y. Xie, Z. Yang, Y. Li, X.Q. Qiu, Q.F. LI, R.B. Huang, F.Q. Ruan, J.M. Fan, Liang Y.G., Chen T.P., Effect of cedemex on three monoamine neurotransmitters in different brain regions of morphine-dependent rats, Chin. J. New Drug 17 (2008) 2022–2026. [37] Y.X. Xue, Y.X. Luo, P. Wu, H.S. Shi, L.F. Xue, C. Chen, W.L. Zhu, Z.B. Ding, Y. Bao, J. Shi, A memory retrieval-extinction procedure to prevent drug craving and relapse, Science 336 (2012) 241–245. [38] Z. Yang, R.B. Huang, J.C. Huang, S.J. Zhang, X. Lin, Y. Jiao, W.Z. Jiang, Sidiming attenuates morphine withdrawal syndrome and nitric oxide (synthase) levels in morphine-dependent rats and rhesus monkeys, Neural Regen. Res. 6 (2011) 2589–2593. [39] Y. Zhang, F.M. Zhang, C.X. Yang, H.B. Jin, Y.B. Yang, M.Y. Xu, Dopamine affects the change of pain-related electrical activity induced by morphine dependence, Neurochem. Res. 37 (2012) 977–982.
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Yulangsan polysaccharide attenuates withdrawal symptoms and regulates the NO pathway in morphine-dependent rats Chunxia Chen, Zhihuan Nong, Jiangchun Huang, Zhaoni Chen, Shijun Zhang, Yang Jiao, Xiaoyu Chen, Renbin Huang ∗ Department of Pharmacology, Guangxi Medical University, Nanning, Guangxi 530021, PR China
h i g h l i g h t s
g r a p h i c a l
a b s t r a c t
• YLSP attenuated morphine withdrawal symptoms.
• YLSP decreased brain NO levels and NOS activity in morphine dependent rats. • YLSP modulated the level of DA as well as NE in NAc, VTA, HIP, and PFC.
a r t i c l e
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Article history: Received 25 November 2013 Received in revised form 22 March 2014 Accepted 8 April 2014 Available online 18 April 2014 Keywords: Yulangsan polysaccharide Withdrawal symptoms Morphine NO pathway Monoaminergic neurotransmitters
a b s t r a c t Yulangsan polysaccharide (YLSP) has been utilized as a phytomedicine to managing nervous dysfunction in China. Thus, this study aimed to evaluate the potential YLSP-mediated detoxification role against morphine dependence in rats. The results indicated that the morphine dependence model significantly increased withdrawal symptoms, levels of NO and NOS (P < 0.05). Furthermore, monoaminergic neurotransmitters, including DA and NE, were detected at elevated levels in the ventral tegmental area (VTA), hippocampus (HIP) and prefrontal cortex (PFC), respectively, while the level of DA was decreased and NE was increased in the nucleus accumbens (NAc). Conversely, YLSP administration significantly reversed naloxone-induced withdrawal symptoms, expression of brain NO and NOS, and monoaminergic neurotransmitters (P < 0.05). Interestingly, YLSP shows an even more effective trend in attenuating withdrawal symptoms than does clonidine, although without a significant difference. These findings indicate that YLSP attenuation of the naloxone-induced withdrawal symptoms of morphine dependence may be mediated by regulation of the NO pathway and modulation of monoaminergic neurotransmitters. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Morphine dependence is a major health problem across the world, and it also is associated with high mortality [1]. In addition, Long-term morphine abuse can lead to nervous system impair-
∗ Corresponding author at: Department of Pharmacology, Guangxi Medical University, 22 Shuangyong Road, Nanning 530021, Guangxi Zhuang Autonomous Region, PR China. Tel.: +86 771 5339805; fax: +86 771 5358272. E-mail address: [email protected] (R. Huang). http://dx.doi.org/10.1016/j.neulet.2014.04.006 0304-3940/© 2014 Elsevier Ireland Ltd. All rights reserved.
ment and a series of physical disorders [22]. To date, there is no cure for the morphine dependence, but the conditions can be managed or controlled, such as: clonidine, methadone, Yi’an decoction [13,20,23]. Therefore, available methods are required to develop novel therapeutic strategies for detoxification treatment. Increasing evidence suggests that the NO pathway is associated with morphine dependence and withdrawal symptoms [32]. After activating soluble guanylate cyclase, the intracellular cyclic adenosine monophosphate (cAMP) is increased and then morphine withdrawal symptoms are aggravated [6]. NOS, as the key enzyme for NO formation, induces endogenous NO discharge when catalyzed
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by l-arginine. Meanwhile, NE and DA neurotransmitters are closely related to the formation of morphine dependence. DA catalyzes the generation of cAMP mainly by D1-receptors-cAMP pathway [19]. Moreover, NE and DA have been associated with multiple addiction-related regions in the CNS, including the nucleus accumbens (NAc) [7], ventral tegmental area (VTA) [24], hippocampus (HIP) [25], and prefrontal cortex (PFC) [34]. Yulangsan, also called Longyanshen, is a commonly used traditional Chinese medicine for the remedy of hypomnesia, immune disorders and mood disorders. Our previous study demonstrated that Yulangsan polysaccharide (YLSP), the major active component of Yulangsan, has neuroprotective effects that include reducing oxidative stress and NO in brain tissue, regulating the synthesis of NE, DA and 5-HT, and improving learning and memory [10–12]. Recently, it was found that YLSP inhibits depression by upregulating the neurotransmitters NE and 5-HT, prefrontal cortex adenylate cyclase activity and hippocampal brain-derived neurotrophic factor expression [17]. Based on data from our laboratory, we suggested that YLSP may be effective in attenuating the naloxone-induced withdrawal symptoms in morphine dependent animals. In this study, we explored the effect and the relevant mechanisms of YLSP on morphine dependence model induced by naloxone. 2. Materials and methods 2.1. Preparation of Yulangsan polysaccharide (YLSP) extract Yulangsan polysaccharide (YLSP) was prepared by the method described previously [12,18]. Next, the gas chromatography (GC) analysis report showed that YLSP was composed of d-glucose and d-arabinose in a molar ratio of 90.79% and 9.21%, with an average molecular weight of about 14,301 Da. 2.2. Animals and drug intervention A total of 140 male SD rats, weighing 180–220 g were purchased from the Experimental Animal Centre of Guangxi Medical University (Certificate No. SYXK 2009-0002) and were allowed to acclimate in quarantine for a week prior to experimentation. Rats were randomly divided into seven groups of 20 rats per group. For establishment of morphine dependent models according to the method of [36], groups II–VII were subcutaneously injected with morphine (Shenyang No. 1 Pharmaceutical, China; 20, 40, 60, 80, 100, 100, 100 mg/kg) twice daily (9:00 AM, 5:00 PM) for 7 days. Group II served as the morphine-dependent model group, Group III served as the morphine-sustained group and was treated with 10 continuous days of morphine. In addition to morphine, Group IV was intragastrically given clonidine (Shaanxi Xiyue Pharmaceutical, China; 0.4 mg/kg) twice daily starting with the day 5 to the day 10, 30 min before morphine injection, serving as the positive control. In addition to morphine administration, Groups V–VII were administered orally with YLSP (0.3, 0.45, and 0.6 g/kg, representing low, medium and high dosage, respectively) in parallel to Group IV. Group I served as a normal control, was subcutaneously given equivalent physiological saline daily for 7 days and received 10 ml/kg physiological saline intragastrically for 6 days. All rats, except the saline-treated normal control group, were treated with morphine (100 mg/kg) on day 8 (9:00 AM). Naloxone (Beijing Sihuan Pharmaceutical, China; 4 mg/kg, intraperitoneal) treatment was administered to all animals at 2 h after the final morphine treatment. The experimental animals were treated according to the Guidance Suggestions for the Care and Use of Laboratory Animals issued by the Ministry of Science and Technology of the People’s Republic of China.
2.3. Calculation of withdrawal symptoms scores and body weight change in rats On day 8, the rats (n = 10) were individually placed in a plastic cage, and the score of withdrawal symptoms was evaluated at 15 min after naloxone injection. Behaviors scored in this manner included: writhing (2), wet dog shakes (2), irritability (conflict-induced vocalization, 1 = touchness, 2 = nearness), teeth chatter (0.5 = discontinuity, 1 = continuity), sialorrhea (1 = mild, 2 = marked), diarrhea (4 = mild, 8 = marked), lacrimation (4). The body weight loss rate was similarly scored from 0 to 20 by multiples of 2 occurrences at 1 h of withdrawal, and was also included in overall withdrawal ratings. The quantifying of score above was guided by previous studies [28,35]. On day 9 and 10, the withdrawal symptoms scores and body weight change were recorded at the same time as day 8 (but had no naloxone injection). 2.4. Serum and brain regions tissue sampling The other 10 animals for each group were sacrificed by cervical dislocation. Serum samples were collected from the supraorbital vein and were kept frozen at −80 ◦ C. Brain samples were promptly removed and weighed. The right brain hemispheres were dissected on an ice-cold plate into four regions: nucleus accumbens (NAc) (+2.76 mm anterior, +1.0 mm lateral to midline and 5.5 mm ventral), ventral tegmental area (VTA) (−6.84 mm posterior, +0.2 mm lateral to midline and 8.5 mm ventral), hippocampus (HIP) (−3.60 mm posterior, +0.2 mm lateral to midline and 3.4 mm ventral) and prefrontal cortex (PFC) (+2.76 mm anterior, midline and 2.4 mm ventral) with respect to bregma as per the stereotaxic atlas by Paxinos and Watson [27]. The serum and four brain regions were properly conserved for NE and DA (Sigma, Germany) test. 2.5. Assay of NO and NOS in brain homogenate in rats Left brain homogenates were prepared in ice-cold physiological saline (10%, w/v) using a homogenizer (Ningbo, China). The homogenate was centrifuged (Hitachi, Japan) at 10,000 rpm for 10 min at 4 ◦ C. The supernatant was used immediately for the measurement of NO and NOS (Nanjing Jiancheng Bioengineering Institute, China). These enzymes were quantified according to the manufacturer’s instructions. 2.6. High performance liquid chromatography (HPLC) analysis of serum and brain regions levels of NE and DA Chromatography was performed on a Shimadzu LC-20AT System (Shimadzu Corp., Japan), equipped with a RF-20A fluorescence detector (Shimadzu Corp., Japan) and an automatic sample injector (Shimadzu Corp., Japan). The separation was achieved on an Ultimate AQ-C18 column (4.6 mm × 250 mm i.d., 5 m; Welch Materials, Inc.). The column temperature was maintained at 35 ◦ C. A mixture of 0.1 M phosphate buffer–methanol (Fisher Scientific, USA; 92:8, v/v) was used as the mobile phase, at a flow rate of 0.8 ml/min. The fluorescence detector of HPLC was set at 254 nm for the excitation wavelength and 338 nm for the emission wavelength. Peaks of NE and DA were indentified by comparing the retention time of each peak in the sample solution with that in the standard solution. The serum and four brain regions levels of DA and NE were quantified. 2.7. Statistical analysis The data are expressed as means ± SD. Statistical analysis was performed using Statistics Package for Social Science 13.0 software (SPSS Inc., USA). Withdrawal symptoms scores and body weight
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loss were performed using two-way analysis of variance (ANOVA), with treatment as between subject variable and days as within subject variable. The remaining data were analyzed by one-way analysis of variance (ANOVA). Post hoc analysis was carried out with LSD t-test. The level of significance was set at P < 0.05. 3. Results 3.1. Effects of YLSP on withdrawal symptoms scores and body weight loss in morphine-dependent rats Two-way ANOVA revealed significant effects of time [F(2, 126) = 244.889, P < 0.001], treatment [F(6, 63) = 87.461, P < 0.001], and time × treatment [F(12, 126) = 17.020, P < 0.001]. The post hoc analysis revealed that low-, medium- and high-dosage YLSP treatments were effective at reducing withdrawal symptoms scores, compared with the model group (P < 0.05). Clonidine significantly attenuated withdrawal symptoms scores, compared with the morphine-dependent model group (P < 0.05), which was similar to high-dose YLSP. However, the withdrawal symptoms score remained significantly higher in the morphine-sustained rats than in normal control animals (Fig. 1A). Two-way ANOVA revealed significant effects of time [F(3, 189) = 68.530, P < 0.001], treatment [F(6, 63) = 21.512, P < 0.001], and time × treatment [F(18, 189) = 46.715, P < 0.001]. The rate of body weight change was markedly decreased in the animals treated with morphine alone and reached its lowest level at 19 h (1 h after naloxone injection) [F(6, 63) = 24.644, P < 0.001]. The post hoc analysis revealed that the body weight loss was significantly reduced following administration of low, medium, and high dosages of YLSP (P < 0.05). Clonidine was less effective at relieving body weight change (P < 0.05) than all YLSP groups (Fig. 1B). 3.2. Effects of YLSP on brain NO levels and NOS activity in morphine-dependent rats As indicated in Fig. 1C, NO and NOS concentration increased in the brains of morphine-dependent rats [NO: F(6, 63) = 19.016, P < 0.001; NOS: F(6, 63) = 22.756, P < 0.001, vs. normal control group], but were significantly decreased by low, medium and high dosages of YLSP, as a favorable dose-response relationship in rats (P < 0.05) by post hoc analysis. High NO and NOS levels were also relieved by clonidine treatment in morphine-dependent rats.
Fig. 1. Effects of YLSP on withdrawal symptoms scores (A) and body weight loss (B) in morphine-dependent rats. Effects of YLSP on brain NO levels and NOS activity in morphine-dependent rats (C). I: normal control; II: morphine-dependent model control; III: morphine-sustained control; IV: morphine + 0.4 mg/kg clonidine; V: morphine + 0.3 g/kg YLSP; VI: morphine + 0.45 g/kg YLSP; VII: morphine + 0.6 g/kg YLSP. Results are presented as the mean ± SD (n = 10). a P < 0.05 compared with normal control group, b P < 0.05 compared with morphine-dependent model group. Withdrawal symptoms were scored for 15 min after injecting naloxone in (A). The times are given in hour (h) with reference to base body weight from day 7 (5:00 PM) (B).
3.3. Effects of YLSP on NE and DA content in serum and four brain regions of morphine-dependent rats HPLC analysis revealed that serum NE levels were increased in the morphine-dependent model group compared to the normal control group [F(6, 63) = 15.898, P < 0.001]. However, rats in the morphine-sustained group showed a remarkable decrease compared to normal control rats. The post hoc analysis revealed that administration of 0.3, 0.45, 0.6 g/kg bw YLSP to morphine-dependent rats resulted in a significant decrease in NE compared to the morphine-dependent model group (P < 0.05). The amount of serum DA increased considerably after morphine administration [F(6, 63) = 5.673, P < 0.001], but the increase was attenuated after treatment with YLSP or clonidine by post hoc analysis (P < 0.05) (Fig. 2A and B). Four brain regions results demonstrated that endogenous monoaminergic neurotransmitters expressions are related to nerve cell activities. As shown in Fig. 2C, the concentration of NE in NAc, VTA, HIP and PFC rose to a higher level in morphine-dependent model rats
[NAc: F(6, 63) = 67.790, P < 0.001; VTA: F(6, 63) = 36.577, P < 0.001; HIP: F(6, 63) = 9.930, P < 0.001; PFC: F(6, 63) = 20.861, P < 0.001]. Subsequently, the NE concentration was rapidly decreased in morphine-sustained group, even below normal control. Compared with the morphine-dependent model group, NE level was markedly decreased after YLSP or clonidine treatment by post hoc analysis (P < 0.05). DA levels in VTA, HIP and PFC in the morphine-dependent model group were significantly higher than in the normal control group [VTA: F(6, 63) = 49.810, P < 0.001; HIP: F(6, 63) = 18.730, P < 0.001; PFC: F(6, 63) = 1.388, P < 0.001], but DA levels were decreased in the NAc [F(6, 63) = 21.579, P < 0.001]. However, compared with the normal control group, levels of DA in the morphine-sustained group were markedly increased, by 1.2-, 2.1-, 1.8- and 2.4-fold in the NAc, VTA, HIP and PFC, respectively. The post hoc analysis revealed that treatment with 0.45, 0.6 g/kg bw YLSP effectively increased DA levels in the NAc, while DA decreased in the VTA, HIP and PFC (P < 0.05). In contrast to the NAc, treatment with clonidine had little influence on the DA levels in the VTA, HIP and PFC (Fig. 2D).
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Fig. 2. Effects of YLSP on NE and DA content in serum (A, B) and four brain regions (C, D) of morphine-dependent rats. (A, B) I: normal control; II: morphine-dependent model control; III: morphine-sustained control; IV: morphine + 0.4 mg/kg clonidine; V: morphine + 0.3 g/kg YLSP; VI: morphine + 0.45 g/kg YLSP; VII: morphine + 0.6 g/kg YLSP. Results are presented as the mean ± SD (n = 6). a P < 0.05 compared with normal control group, b P < 0.05 compared with morphine-dependent model group.
4. Discussion The present study partly investigated the effects of YLSP, as a Yulangsan extract, on the attenuating properties of naloxoneprecipitated withdrawal symptoms and its possible mechanism in rats using a morphine-dependent model. In our study, we observed that rats treated with naloxone during the morphinedependent phase showed a significant increase in some behavioral symptoms and body weight loss, when compared to the normal control group, which was consistent with recent studies [2,33].
Conversely, administration of YLSP attenuates the development of morphine dependence by reducing the naloxone-induced behavioral withdrawal symptoms in a dose-dependent manner. YLSP even shows a more effective trend in regulating withdrawal symptom than clonidine, although it does not significantly reverse body weight change. The ability of YLSP to reduce the incidence of withdrawal symptoms is extremely important because withdrawal symptoms were historically believed to have a major role in the relapse to drug-taking behavior after drug abstinence [15].
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There is accumulating evidence indicating that NO, which is produced through NOS, might have a key role in withdrawal processing. Accordingly, withdrawal symptoms was reversed by the administration of selective NOS and guanylate cyclase inhibitors and was attenuated or abolished in NOS knockout animals [8,9]. For Drug dependence was exhibited as compulsive drug administration with consecutive craving behaviors, NO signaling was likely involved in learning and memory functions [37,38]. In a recent study, NO was shown to be mediated via the classical vasodilation process and was also involved in learning and memory [31]. Moreover, NO is a highly reactive oxidant and acts as a neurotransmitter as well as a defense against tumor cells, parasites and bacteria in the immune system [21]. In our study, there were significant increases in NO production and NOS level after morphine administration. Conversely, both NO and NOS levels were down regulated after treatment with YLSP. Thereby, the possible mechanism of YLSP in counteracting naloxone-induced withdrawal symptoms may be associated with regulation of the NO pathway. The release of monoaminergic neurotransmitters such as DA and NE may be an important mechanism underlying the development of morphine dependence. In particular, the mesolimbic dopaminergic system has a critical role in mechanisms related to memory and is involved in the establishment of the morphine-dependent model [16,30]. Administration of D1 and D2 receptor agonists has been shown to attenuate somatic withdrawal signs [4]. The HIP is recognized as an important region involved in morphine dependence [25,26]. Moreover, previous studies have indicated that a strong increase of DA and NE release takes place in the medial prefrontal cortex during naloxone-precipitated withdrawal [29,34]. The present results confirm previous observations showing that naloxone-induced abstinence symptoms in morphine dependent rats is associated with a dramatic and marked increase in both DA and NE release in the VTA, HIP and PFC. Importantly, in this study, DA content was decreased in the NAc after naloxone-induced withdrawal, suggesting that there is an inverse relationship between the mesocortical and mesolimbic dopaminergic systems, as proposed by other authors [3]. In contrast, the levels of these neurotransmitters were reversed after treatment with YLSP, which appears to act as a modulator of monoaminergic neurotransmitters. Besides, we also found that NE was reduced in brain regions in rats following morphine-sustained treatment. Neuronal plasticity induced by dependence might be associated with changes in pain sensitivity resulting from the adaptive response of neurons [39]. The other potential neuronal processes underlying the effects of YLSP on naloxone-precipitated withdrawal symptoms might include depression inhibition. Several studies have highlighted the efficacies of medicinal herbs on morphine withdrawal, due to their antidepressant and anxiolytic properties [5,14]. Previous studies have shown that administration of YLSP can produce an increase in neurotransmitters such as 5-HT and NE, primarily in brain areas closely related to reward processes: the PFC and the HIP [17]. YLSP, as a traditional medicine, is widely used for the treatment of mood disorders, such as anxiety and depression, which appear to be related to the same reward mechanisms. In summary, our study demonstrates that YLSP attenuates withdrawal symptoms of morphine dependence by regulating the NO pathway and modulating monoaminergic neurotransmitters. Further research to elucidate the underlying mechanism is important and will provide additional evidence for the exploitation of the broader remedial usage of YLSP.
Conflict of interest The authors declare that there are no conflicts of interest.
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Acknowledgments This work was financially supported by the National Natural Science Fund (30960504), Guangxi Scientific Research and Technology Development research projects (0630002-2A), Guangxi Natural Science Foundation (2011GXNSFD018030), Nanning Science and Technology Research and Production of new products (No. 201102084C). References [1] L. Brådvik, M. Berglund, A. Frank, A. Lindgren, P. Löwenhielm, Number of addictive substances used related to increased risk of unnatural death: a combined medico-legal and case-record study, BMC Psychiatry 9 (2009) 48. [2] H.B. Cai, Z.X. Mo, X. Li, Efects of caulis sinomenii and sinomenine on withdrawal syndrome in morphine-dependent mice, Beijing J. Tradit. Chin. Med. 27 (2008) 652–655. [3] E.H. Chartoff, M.F. Barhight, S.D. Mague, A.M. Sawyer, W.A. Carlezon, Anatomically dissociable effects of dopamine D1 receptor agonists on reward and relief of withdrawal in morphine-dependent rats, Psychopharmacology (Berl.) 204 (2009) 227–239. [4] E.H. Chartoff, S.D. Mague, M.F. Barhight, A.M. Smith, W.A. Carlezon Jr., Behavioral and molecular effects of dopamine D1 receptor stimulation during naloxone-precipitated morphine withdrawal, J. Neurosci. 26 (2006) 6450–6457. [5] A. Feily, N. Abbasi, The inhibitory effect of Hypericum perforatum extract on morphine withdrawal syndrome in rat and comparison with clonidine, Phytother. Res. 23 (2009) 1549–1552. [6] B.H. Gabra, E.A. Afify, T.T. Daabees, M.S. Abou Zeit-Har, The role of the NO/NMDA pathways in the development of morphine withdrawal induced by naloxone in vitro, Pharmacol. Res. 51 (2005) 319–327. [7] A. Haghparast, A. Omranifard, R. Arezoomandan, M. Ghalandari-Shamami, Z. Taslimi, A.A. Vafaei, A. Rashidy-Pour, Involvement of dopaminergic receptors of the rat nucleus accumbens in decreasing the conditioned place preference induced by lateral hypothalamus stimulation, Neurosci. Lett. 556 (2013) 10–14. [8] E.L. Heinzen, G.M. Pollack, The development of morphine antinociceptive tolerance in nitric oxide synthase-deficient mice, Biochem. Pharmacol. 67 (2004) 735–741. [9] A. Hervera, R. Negrete, S. Leánez, J. Martín-Campos, O. Pol, Peripheral effects of morphine and expression of -opioid receptors in the dorsal root ganglia during neuropathic pain: nitric oxide signaling, Mol. Pain 7 (2011) 25. [10] Z.S. Huang, Y. Jiao, S.J. Zhang, X. Lin, R.B. Huang, Improvement of LYSP on learning and memory abilities in the elderly aging SAMP8 mice model, Chin. J. Gerontol. 28 (2008) 2086–2088. [11] Z.S. Huang, J. Li, S.J. zhang, Y. Jiao, R.B. Huang, Effects of Longyanshen polysaccharides on brain NO, SOD and MDA in d-galactose-induced dementia mice, Chin. J. Gerontol. 25 (2005) 176–177. [12] Z.S. Huang, H.Y. Xie, S.J. Zhang, Y. Jiao, W.Z. Jiang, R.B. Huang, Effects of Longyanshen polysaccharides on free radical metabolism in senescence accelerated-prone mice, Neural Regen. Res. 3 (2008) 1099–1102. [13] H. Joseph, S. Stancliff, J. Langrod, Methadone Maintenance Treatment (MMT), Mount Sinai J. Med. (2000). [14] S. Kasture, S. Vinci, F. Ibba, A. Puddu, M. Marongiu, B. Murali, A. Pisanu, D. Lecca, G. Zernig, E. Acquas, Withania somnifera prevents morphine withdrawalinduced decrease in spine density in nucleus accumbens shell of rats: a confocal laser scanning microscopy study, Neurotox. Res. 16 (2009) 343–355. [15] G.F. Koob, L. Stinus, M.L. Moal, F.E. Bloom, Opponent process theory of motivation: neurobiological evidence from studies of opiate dependence, Neurosci. Biobehav. Rev. 13 (1989) 135–140. [16] G.F. Koob, N.D. Volkow, Neurocircuitry of addiction, Neuropsychopharmacology 35 (2010) 217–238. [17] S. Liang, R.B. Huang, X. Lin, J.C. Huang, Z.S. Huang, H.G. Liu, Effects of Yulangsan polysaccharide on monoamine neurotransmitters, adenylate cyclase activity and brain-derived neurotrophic factor expression in a mouse model of depression induced by unpredictable chronic mild stress, Neural Regen. Res. 7 (2012) 191–196. [18] X. Lin, Z. Huang, X. Chen, Y. Rong, S. Zhang, Y. Jiao, Q. Huang, R. Huang, Protective effect of Millettia pulchra polysaccharide on cognitive impairment induced by d-galactose in mice, Carbohydr. Polym. (2013). [19] A. Lintas, N. Chi, N.M. Lauzon, S.F. Bishop, S. Gholizadeh, N. Sun, H. Tan, S.R. Laviolette, Identification of a dopamine receptor-mediated opiate reward memory switch in the basolateral amygdala-nucleus accumbens circuit, J. Neurosci. 31 (2011) 11172–11183. [20] R. Liu, G. Xu, L. Duan, X. Lu, W. Gao, Y. An, Evaluation on phase IV clinical trail of Yi An oral liquor for detoxification of heroin addicts, Chin. J. Drug Depend. 10 (2001) 200–203. [21] C.J. Lowenstein, S.H. Snyder, Nitric oxide, a novel biologic messenger, Cell 70 (1992) 705–707. [22] L. Lu, J.D. Shepard, F.S. Hall, Y. Shaham, Effect of environmental stressors on opiate and psychostimulant reinforcement, reinstatement and discrimination in rats: a review, Neurosci. Biobehav. Rev. 27 (2003) 457–491. [23] T.R. Macedo, J. Relvas, C.A. Fontes Ribiero, F. Pacheco, M.T. Morgadinho, C.M. Pinto, P.C. Gomes, M. Ventura, V. Henriques, S.V. Nunes, Plasma catecholamines
68
[24]
[25]
[26] [27] [28]
[29]
[30]
[31]
C. Chen et al. / Neuroscience Letters 570 (2014) 63–68 during an ultrarapid heroin detoxification, Ann. N.Y. Acad. Sci. 914 (2000) 303–310. F.J. Meye, R. van Zessen, M.P. Smidt, R.A.H. Adan, G.M.J. Ramakers, Morphine withdrawal enhances constitutive -opioid receptor activity in the ventral tegmental area, J. Neurosci. 32 (2012) 16120–16128. J.A. Morón, N.S. Abul-Husn, R. Rozenfeld, G. Dolios, R. Wang, L.A. Devi, Morphine administration alters the profile of hippocampal postsynaptic density-associated proteins a proteomics study focusing on endocytic proteins, Mol. Cell. Proteom. 6 (2007) 29–42. E.J. Nestler, Common molecular and cellular substrates of addiction and memory, Neurobiol. Learn. Mem. 78 (2002) 637–647. G. Paxinos, C. Watson, The Rat Brain in Stereotaxic Coordinates, Academic Press, New York, 2007. K. Rasmussen, D.B. Beitner-Johnson, J.H. Krystal, G.K. Aghajanian, E.J. Nestler, Opiate withdrawal and the rat locus coeruleus: behavioral, electrophysiological, and biochemical correlates, J. Neurosci. 10 (1990) 2308–2317. Z.L. Rossetio, G. Longu, G. Mercuro, G.L. Gessa, Extraneuronal noradrenaline in the prefrontal cortex of morphine-dependent rats: tolerance and withdrawal mechanism, Brain Res. 609 (1993) 316–320. T.S. Shippenberg, R. Bals-Kubik, A. Herz, Examination of the neurochemical substrates mediating the motivational effects of opioids: role of the mesolimbic dopamine system and D-1 vs. D-2 dopamine receptors, J. Pharmacol. Exp. Ther. 265 (1993) 53–59. A.J. Susswein, A. Katzoff, N. Miller, I. Hurwitz, Nitric oxide and memory, Neuroscientist 10 (2004) 153–162.
[32] N. Toda, S. Kishioka, Y. Hatano, H. Toda, Modulation of opioid actions by nitric oxide signaling, Anesthesiology 110 (2009) 166–181. [33] K. Ueno, T. Maeda, N. Kiguchi, Y. Kobayashi, M. Ozaki, S. Kishioka, Availability of serum corticosterone level for quantitative evaluation of morphine withdrawal in mice, Drug Discov. Ther. 5 (2011) 71. [34] R. Ventura, A. Alcaro, S. Puglisi-Allegra, Prefrontal cortical norepinephrine release is critical for morphine-induced reward, reinstatement and dopamine release in the nucleus accumbens, Cereb. Cortex 15 (2005) 1877– 1886. [35] E. Wei, Assessment of precipitated abstinence in morphine-dependent rats, Psychopharmacologia 28 (1973) 35–44. [36] H.Y. Xie, Z. Yang, Y. Li, X.Q. Qiu, Q.F. LI, R.B. Huang, F.Q. Ruan, J.M. Fan, Liang Y.G., Chen T.P., Effect of cedemex on three monoamine neurotransmitters in different brain regions of morphine-dependent rats, Chin. J. New Drug 17 (2008) 2022–2026. [37] Y.X. Xue, Y.X. Luo, P. Wu, H.S. Shi, L.F. Xue, C. Chen, W.L. Zhu, Z.B. Ding, Y. Bao, J. Shi, A memory retrieval-extinction procedure to prevent drug craving and relapse, Science 336 (2012) 241–245. [38] Z. Yang, R.B. Huang, J.C. Huang, S.J. Zhang, X. Lin, Y. Jiao, W.Z. Jiang, Sidiming attenuates morphine withdrawal syndrome and nitric oxide (synthase) levels in morphine-dependent rats and rhesus monkeys, Neural Regen. Res. 6 (2011) 2589–2593. [39] Y. Zhang, F.M. Zhang, C.X. Yang, H.B. Jin, Y.B. Yang, M.Y. Xu, Dopamine affects the change of pain-related electrical activity induced by morphine dependence, Neurochem. Res. 37 (2012) 977–982.