European Journal of Pharmacology 727 (2014) 99–105
Contents lists available at ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Pulmonary, gastrointestinal and urogenital pharmacology
Dexamethasone alleviates motion sickness in rats in part by enhancing the endocannabinoid system Yan Zheng a,b,1, Xiao-Li Wang a,1, Feng-Feng Mo a, Min Li a,n a b
Department of Military Hygiene, Faculty of Naval Medicine, Second Military Medical University, 800 Xiang Yin Road, Shanghai, China Department of Nutrition, Tong Ren Hospital Affiliated to Shanghai JiaoTong University School of Medicine, 1111 Xian Xia Road, Shanghai, China
art ic l e i nf o
a b s t r a c t
Article history: Received 3 May 2013 Received in revised form 22 January 2014 Accepted 24 January 2014 Available online 4 February 2014
Low-dose dexamethasone has been widely used for the prevention of nausea and vomiting after chemotherapy and surgical procedures and to treat motion sickness due to its minimal adverse effects, but the mechanisms underlying its anti-motion sickness effects are poorly understood. Previous studies have demonstrated that the endocannabinoid system is suppressed by motion sickness but stimulated by dexamethasone. The aim of the present study was to determine whether dexamethasone has an antimotion sickness effect in rats and to elucidate the mechanism of this action. We used HPLC–MS/MS to measure the plasma concentrations of anandamide and 2-arachidonoylglycerolþ1-arachidonoylglycerol, and we employed real-time quantitative PCR (qRT-PCR) and/or Western blot analysis to assay the expression of N-acylphosphatidyl-ethanolamine hydrolyzing phospholipase D, sn-1-selective diacylglycerol lipase, fatty acid hydrolase, monoacylglycerol lipase and endocannabinoid CB1 receptor in the dorsal vagal complex and stomach of rats exposed to a motion sickness protocol. The results showed that dexamethasone lowered the motion sickness index and restored the levels of endogenous cannabinoids and the expression of the endocannabinoid CB1 receptor, which declined after the induction of motion sickness, in the dorsal vagal complex and stomach. & 2014 Elsevier B.V. All rights reserved.
Keywords: AEA 2-AG Glucocorticoid Nausea and vomiting
1. Introduction Motion sickness is a malady characterized by a combination of signs and symptoms, such as gasping, drowsiness, general inactivity and excessive vomiting, that accompany movement or perceived environmental movement (Oosterveld, 1995; Yates et al., 1998). Motion sickness can be triggered by many different stimuli, including traveling in automobiles, aircraft, spacecraft or boats and exposure to moving visual scenes (Money, 1970; Reason, 1978). A parabolic flight experiment showed that motion sickness was Abbreviations: AEA, anandamide; ACTH, adrenocorticotropin; ACN, acetonitrile; 2-AG, 2-arachidonylglycerol; AP, area postrema; AVP, Arginine vasopressin; CIV, chemotherapy induced vomiting; CNS, central neural system; CRH, corticotropin releasing hormone; DAGL-a, sn-1-selective diacylglycerol lipase; DMNV, dorsal motor nucleus of the vagus; FAAH, fatty acid hydrolase; GAPDH, glyceraldehyde phosphate dehydrogenase; PONV, postoperative nausea and vomiting; PVN, paraventricular nucleus; TRPV1, transient receptor potential vanilloid-1; MAGL, monoacylglycerol lipase; NAPE-PLD, N-acylphosphatidyl-ethanolamine hydrolyzing phospholipase D; NTS, nucleus tractus solitarius; NR, nucleus of raphe; HPA, hypothalamic-pituitary-adrenal; Δ9-THC, delta-9-tetrahydrocannabinol n Corresponding author. Tel.: þ 86 21 81871120. E-mail addresses: [email protected] (Y. Zheng), [email protected] (X.-L. Wang), [email protected] (F.-F. Mo), [email protected] (M. Li). 1 Yan Zheng and Xiao-Li Wang contributed equally to this work http://dx.doi.org/10.1016/j.ejphar.2014.01.047 0014-2999 & 2014 Elsevier B.V. All rights reserved.
accompanied by a significant decrease in the reactivity of the peripheral endocannabinoid system (Chouker et al., 2010). A study of metabolic differences between subjects who experience nausea/ vomiting upon acceleration and those who do not revealed that the level of arachidonic acid, the downstream metabolite of endocannabinoids, was significantly increased in nausea/vomiting subjects (Mo et al., 2012), suggesting that motion sickness may be associated with an impairment of endocannabinoid activity (Chouker et al., 2010). Dexamethasone is often administered to cancer patients to counteract chemotherapy-induced vomiting, postoperative nausea/vomiting and analgesic requirements after thyroidectomy (Fujii and Nakayama, 2007), but its mechanism of action has not been clearly demonstrated (Malik et al., 2007; Hesketh, 2008). In 1986, Kohl proposed the use of dexamethasone to modulate motion sickness due to its long-acting, slow tolerance and fewer adverse effects compared with amphetamine and scopolamine (Kohl, 1986). The endocannabinoid system comprises (i) cannabinoid receptors (cannabinoid type 1 receptor (CB1R), cannabinoid type 2 receptor and transient receptor potential vanilloid-1); (ii) their specific endogenous ligands (endocannabinoids), which include N-arachidonylethanolamine (anandamide, AEA), 2-arachidonoylglycerol (2-AG), noladin ether and virodhamine; and (iii) a number of biosynthetic and
100
Y. Zheng et al. / European Journal of Pharmacology 727 (2014) 99–105
degradation enzymes (De Petrocellis and Di Marzo, 2009). NAcylphosphatidyl-ethanolamine hydrolyzing phospholipase D (NAPEPLD) and sn-1-selective diacylglycerol lipase (DAGL-a) are responsible for the synthesis of AEA and 2-AG, respectively, whereas fatty acid hydrolase (FAAH) is responsible for the degradation of AEA, and monoacylglycerol lipase (MAGL) is responsible for the degradation of 2-AG (Pagano et al., 2008). There are bidirectional and functional correlations between glucocorticoids and the endocannabinoid system. Glucocorticoids recruit the endocannabinoid system to exert rapid negative feedback control of the hypothalamic–pituitary–adrenal (HPA) axis during stress conditions (Evanson et al., 2010). AEA and 2-AG, as endogenous endocannabinoids at the terminals of vagal afferents in the gastrointestinal tract, have been demonstrated to participate in the complex regulation of food intake as well as emesis induced by toxins (Hu et al., 2007). Recently, researchers have found that AEA and 2-AG play important roles in the pathophysiology of nausea and vomiting induced by conditions such as migraine and cancer chemotherapy drugs and the conditioned gaping response elicited by a lithium-paired context (Rossi et al., 2008; Parker et al., 2009). To understand the protective effect of dexamethasone against motion sickness in rats and determine whether this effect is mediated by stimulating the endocannabinoid system, we measured the levels of two major endocannabinoids AEA and 2-AG in plasma and their turnover enzymes in the dorsal vagal complex (DVC) and stomach. We analyzed the mRNA and protein levels of CB1R in these tissues to further elucidate the role of endocannabinoid signaling in the course of motion sickness.
administration method were selected based on our previous experiments. During the 30 min rotation, the rats were given no food or tap water and were enclosed in a cuboidal Plexiglas box suspended on a metal frame, which revolved around an axis parallel to the floor as described by Cai et al. (2010). It has been demonstrated in our laboratory that this magnitude of stimulation can induce motion sickness in rats (Cai et al., 2010; Mo et al., 2012). Dexamethasone acetate was dissolved in distilled water. AM251 was dissolved in a vehicle composed of 2% DMSO plus 1% Tween-80 (Sigma-Aldrich, St. Louis, MO). Preliminary experiments showed that the motion sickness index induced by acceleration stimulation in gastric lavage-treated animals was not significantly different (P 40.05) from the response of animals in the control group. 2.2. Observation of motion sickness symptoms Because the rats had no emetic reaction, motion sickness symptoms were observed after rotation and recorded according to the motion sickness index, which reflects the severity of gastrointestinal symptoms caused by motion stimulation (Yu et al., 2007; Wei et al., 2011). The evaluation criteria of the motion sickness index were as follows: 1 point was given for each fecal granule, urination scored 1.2 points, severe piloerection scored 1.2 points, slight piloerection scored 0.6, and tremor scored 1.2 (Yu et al., 2007). For each criterion, 0 points were allotted if the symptom was not present. The motion sickness index was calculated by summing of all of these scores. 2.3. Tissue and blood preparation
2. Materials and methods 2.1. Animals and acceleration exposure Thirty-three six-week-old male Sprague-Dawley (SD) rats weighing 190–240 g were purchased from Sino-British SIPPR/BK Lab Animal Ltd. (Shanghai, China). The animal protocols and procedures were approved by the Animal Use and Care Committee for Research and Education of the Second Military Medical University (Shanghai, China), and experiments were carried out in accordance with the guidelines published by the National Institutes of Health (USA) regarding the care and use of animals for experimental procedures. The animals were housed in Plexiglas box cages in the colony room at an ambient temperature of 2272 1C with a 12 h light/12 h dark schedule (lights on at 8 a.m.) and were maintained on an ad libitum schedule of food and water. The rats were randomly assigned to four groups: the control group (n ¼8), which received an intra-gastric administration of water and, 30 min later, watched the acceleration apparatus and experienced the noise of the apparatus without experiencing actual acceleration; the acceleration model group (n¼ 8), which received an intra-gastric administration of water 30 min before acceleration exposure; the DEX group (n ¼9), which received an intra-gastric administration of 0.05 mg/kg dexamethasone acetate 30 min before acceleration exposure, ensuring that the drug was completely absorbed according to the criteria defined by the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China); and the AM-251/DEX group (n¼ 8), in which the animals were injected intraperitoneally with 5 mg/kg N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4methyl-1H-pyrazole-3-carboxamide (AM-251) (Tocris, Ballwin, MO) 15 min before the intra-gastric administration of 0.05 mg/kg dexamethasone acetate 30 min before acceleration exposure. Rats in the acceleration model, the DEX and AM-251/DEX groups were subjected to rotation. The dexamethasone acetate dosage and
Animals in each group were anesthetized by an intraperitoneal (i.p.) injection of 10% 0.3 g/kg chloral hydrate, and blood was drawn by cardiac puncture followed by a trans-cardiac perfusion of 60 ml chilled saline. A blood sample (5 ml) was collected by cardiac puncture using a BD-Falcon vacutainer. Blood samples were centrifuged at 3000 rpm for 20 min at 4 1C, and the plasma layer was collected and stored at 80 1C. The brain was immediately dissected and cooled in iced saline for 1 min before the dorsal vagal complex was carefully removed. The stomach was also removed for analysis. The dissected tissues were immediately frozen in liquid nitrogen and stored at 80 1C for further use. 2.4. HPLC–MS/MS This analysis was performed on an LC/MS/MS system consisting of a Triple Quad™ 5500 (Applied Biosystems, Concord, ON, Canada), a SIL-HTc gradient pump (Shimadzu, Kyoto, Japan), two LC-20AD transfer pumps (Shimadzu, Kyoto, Japan), a DGU-20A3 vacuum pump (Shimadzu, Kyoto, Japan), a Gemini C18 chromatographic column (2.0 50 mm, 5 μm, Phenomenex, USA) and a C18 guard column (4.0 3.0 mm, 5 μm, Phenomenex, USA). Data acquisition and analysis were performed using Analyst software (version 1.5.1; Applied Biosystems/MDS Sciex). AEA, 2-AG, AEA d8 and 2-AG d8 were purchased from Cayman Chemicals (Nottingham, UK). Stock solutions were prepared at 0.1 mg/ml in acetonitrile for 2-AG, 2.5 μg/ml in acetonitrile for 2-AG d8, 0.5 mg/ml in methanol for AEA and 10 μg/ml in methanol for AEA d8. Stock and working solutions were stored at 80 1C or 20 1C. During sample preparation, all steps were carried out at 4 1C to prevent the isomerization or degradation of the compounds. After spiking with the internal standard, 300 ml of the plasma sample/calibration sample was treated by the liquid–liquid extraction method with 1 ml hexane/ ethyl acetate (1:1, v/v) by vortex shaking (approximately 1500 moves/min) for 10 min followed by centrifugation at 15,000 rpm
Y. Zheng et al. / European Journal of Pharmacology 727 (2014) 99–105
for 5 min at 4 1C. The upper organic phase was evaporated to dryness at room temperature, reconstituted with 50 ml of a 40% acetonitrile/water mixture, centrifuged at 15,000 rpm for 3 min at 4 1C and injected (5 ml) into the LC/MS/MS system. Multiple reactions monitoring of the product ions from the precursor ions of the analyte of interest was carried out, and the results were quantified. Details of the ions monitored (m/z) are as follows: 379.3287.4 amu for 2-AG and 1-AG, 387.4-294.4 amu for 2-AG-d8 and 1-AG-d8, 348.3-62.1 amu for AEA and 356.3-62.1 amu for AEAd8. Calibration curves (r2 ¼ 0.9988 for AEA and r2 ¼0.9980 for 2-AG) were constructed for both 2-AG and AEA. Because 1-AG and 2-AG undergo rapid isomerization, it is difficult to report with certainty the quantities of each species originating from tissues; thus, the results are reported as the sum of the individual peaks of 2-AGþ1AG (Zoerner et al., 2011).
101
peroxidase-labeled secondary antibodies (1:2000, anti-rabbit IgG, Jackson Immunoresearch, West Grove, PA, USA) at room temperature. The bands were then visualized with an ECL system (Millipore Corp, Bedford, MA, USA). The density of the bands was analyzed with a Gel Doc image system (Bio-Rad, San Diego, CA, USA). Signal intensities were normalized against the internal control (GAPDH). 2.7. Data analysis Statistical analysis was performed using the SPSS v16.0 statistical program. Data were tested with a one-way analysis of variance (ANOVA) followed by least significant difference (LSD) pair-wise comparison tests. All data are presented as mean values 7S.E.M. P values less than 0.05 were considered significant in all tests.
2.5. Real-time quantitative PCR 3. Results Tissue samples were homogenized in TRIzol reagent (Invitrogen, USA), and total RNA was extracted. Complementary DNA (cDNA) was synthesized using oligo dT primers and the PrimeScript RT Reagent Kit (Takara, Japan) according to the manufacture’s protocol. Reverse transcription of total RNA was performed at 95 1C for 5 min. Real-time PCR reactions were carried out in a Rotor-Gene PCR machine (RG-3000A, Corbett Research) as follows: an initial denaturation step at 95 1C for 5 s followed by 40 cycles of 95 1C for 5 s, annealing at 60 1C for 20 s and extension at 72 1C for 30 s. The amount of cDNA per sample was determined using a SYBR Premix Ex Taq™ kit (Takara, Japan). The progression of the PCR reaction was assessed by changes in the amount of SYBR Green dye attached to double-stranded DNA. The target genes were normalized to the endogenous control gene, glyceraldehyde phosphate dehydrogenase (Gapdh). The primers used for real-time PCR were as follows: CB1R: sense primer 5'-TCAAAGCTGACGCCCTGAC-3', antisense primer 5'-GAACATAACGATGCGATACACG-3'; NAPE-PLD: sense primer 5'-TGATGGTGGAAATGGACGAG-3', antisense primer 5'-AATGTTGGGAGGGCGTGA-3'; FAAH: sense primer 5'-ATTGCCCAGTGGAAAGCG-3', antisense primer 5'-GGATGAACGACCCTCACGAT-3'; MAGL: sense primer 5'-AGAGGATGGTGGTATCGGACTT-3', antisense primer 5'-GCAGATGAGTGGGTCGGAGT-3'; DAGL-α: sense primer 5'-TGGAACCGTGGCAGAATG-30 , antisense primer 5'-AAGGAGACAGGGCTTTGGAT-3'; and GAPDH: sense primer 5'-GGCTCTCTGCTCCTCCCTGTTCTA-3', antisense primer 5'CGTCCGATACGGCCAAATCCGT-3'. 2.6. Western blot analysis Tissue samples were lysed in 1 ml tissue and cell lysis solution plus protease and phosphatase inhibitors (Keygen Biotech, Nanjing, China) and then centrifuged at 10,000g for 5 min at 4 1C. The supernatant was collected as the total protein extract, and the protein concentration was determined spectrophotometrically according to the bicinchoninic acid (BCA) method. Equal amounts of protein (60 μg per lane) from each sample were loaded on a 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). Following electrophoresis, the proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Biotech, Nanjing, China) using a Mini-Trans-Blot Electrophoresis Transfer Cell (Bio-Rad, Hercules, CA, USA). Nonspecific protein binding of antibodies was blocked by treating membranes in 5% nonfat milk in Tris-buffered saline containing Tween 20 (TBST; 10 mM Tris–HCl, 150 mM NaCl, 0.05% Tween-20). The membrane was incubated overnight at 4 1C with the CB1 rabbit polyclonal antibody (1:200; Abcam Ltd., Hong Kong) or anti-GAPDH (1:1000; Sigma-Aldrich, St. Louis, MO, USA) in TBST with 1% bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO, USA). The membrane was then washed with TBST buffer and incubated for 1 h with
3.1. Comparison of motion sickness severity among the four groups. The motion sickness index of the DEX group was significantly lower than that of the acceleration model group (4.4 71.36 vs. 12 73.31, P o0.001), whereas there was no significant difference between the control and DEX groups (3.2 71.0 vs. 4.4 71.36). AM251 (5 mg/kg) partially abolished the effect of DEX (P o0.001). The motion sickness index of the AM-251/DEX group was lower than that of the acceleration model group (8.53 71.18 vs. 12 73.31, Po 0.05) and higher than that of the control (P o0.001) and DEX groups (P o0.001) (Fig. 1). 3.2. Changes in plasma AEA and 2-AG þ 1-AG concentrations in rats AEA levels were similar across all groups (7.60 71.07 ng/ml in the control group vs. 6.19 70.98 ng/ml in the acceleration model group and 7.39 71.92 ng/ml in the DEX group) (Fig. 2A), whereas plasma 2-AG þ 1-AG levels were lower in the acceleration model group (32.39 710.91 ng/ml) than in the control and DEX groups (67 719.84 ng/ml and 54.28 720.28 ng/ml, respectively; P o0.05) (Fig. 2B). 3.3. Expression of endocannabinoid biosynthesis and degradation enzymes (NAPE-PLD, FAAH, DGL-a and MAGL) in the rat DVC and stomach Compared with the levels in the control group, the mRNA levels of NAPE-PLD and FAAH were decreased and increased, respectively, in the DVC after acceleration stimulation. These changes
Fig. 1. Comparison of motion sickness index in control group, acceleration model group, DEX group, and AM-251/DEX group. nStatistically significant when compared with control group (Po 0.001); #Statistically significant when compared with acceleration model group (P o0.05); Statistically significant when compared with DEX group(Po 0.001); and one-way ANOVA followed by LSD pairwise comparison tests, n¼ 8–9.
102
Y. Zheng et al. / European Journal of Pharmacology 727 (2014) 99–105
were significantly enhanced by DEX treatment. Compared with the levels in the control group, the mRNA levels of both DGL-a and MAGL were decreased and DGL-a was enhanced after motion stimulation. MAGL mRNA expression was markedly reduced by DEX administration (Fig. 3). Motion stimulation up-regulated FAAH, DGL-a and MAGL mRNA expression in the stomach relative to the levels in the control group, whereas DEX down-regulated the FAAH and MAGL levels compared with the those in the acceleration model group (Fig. 4).
3.4. Effect of acceleration exposure and DEX on the expression of endocannabinoid CB1 receptor mRNA and protein in the rat DVC and stomach mRNA levels of endocannabinoid CB1 receptor in the DVC were decreased in the acceleration model group compared with the control group (0.98 70.21 vs. 1.68 70.12) but were unaffected in the DEX group (1.78 70.55) (Fig. 5A). The levels of endocannabinoid CB1 receptor mRNA in the stomach were similarly decreased in the acceleration model group compared with the control group (1.04 70.20 vs. 1.87 70.17) and were unchanged in the DEX group (1.80 70.21) (Fig. 5B). Endocannabinoid CB1 receptor expression was further investigated by Western blotting (Fig. 6). To quantify protein expression levels, the optical density of the Western blot data was calculated and normalized to the GAPDH control. The protein levels of endocannabinoid CB1 receptor in the DVC were 0.046 70.015, 0.02 70.001 and 0.045 70.003 in the control, acceleration model and DEX groups, respectively (Fig. 6A,B), and 1.17 70.27, 0.84 70.18 and 1.16 70.20, respectively, in the stomach (Fig. 6C,D).
4. Discussion
Fig. 2. Comparison of plasma concentrations of endocannabinoid anandamide (A) and 2-AG (B) among control group, acceleration model group and DEX group. nn Indicates significantly lower 2-AG plasma concerntrations comparing with the control group, Po 0.01. One-way ANOVA followed by LSD pairwise comparison tests, n ¼8–9.
The motion sickness index is regarded as an appropriate measure for evaluating the severity of motion sickness in rodents (Yuet al., 2007; Wei et al., 2011; Mo et al., 2012). Motion sickness index scores were increased after motion stimulation in the control and acceleration model groups, whereas dexamethasone protected rats against motion sickness, confirming the previous finding that dexamethasone has an anti-motion sickness effect (Kohl, 1986). The ability of dexamethasone to modulate motion sickness-induced gastrointestinal symptoms was partially reversed by treatment with the selective endocannabinoid CB1 receptor antagonist AM-251. The incomplete reversal was most likely due to dexamethasone exerting its antiemesis effect via multiple mechanisms. Researchers have postulated that DEX may suppress the production of inflammatory autacoids, prostaglandins, thromboxane, leukotrienes and cytokines, thus decreasing histamine production and/or release from mast cells involved in mediating the emetic response (Rich et al., 1980; Harris,
Fig. 3. NAPE-PLD (A), FAAH (B), DGL-a (C), and MAGL (D) mRNA expression in rats dorsal vagal complex. nIndicates significant difference comparing with the control group, Po 0.01. One-way ANOVA followed by LSD pairwise comparison tests, n¼ 8–9.
Y. Zheng et al. / European Journal of Pharmacology 727 (2014) 99–105
103
Fig. 4. NAPE-PLD (A), FAAH (B), DGL-a (C), and MAGL (D) mRNA expression in rats stomach. nIndicates significantly higher mRNA expression comparing with the control group, P o 0.05. One-way ANOVA followed by LSD pairwise comparison tests, n¼ 8–9.
Fig. 5. Comparision of CB1R messenger RNA relative quantity in the dorsal vagal complex (DVC) (A) and stomach (B) among control group (solid bar), acceleration model group (open bar) and dexamethasone group (gray group). nSignificant difference (P o0.05) of acceleration model group from control group. nn Significant difference (P o 0.001) compares with control group. One-way ANOVA followed by LSD pairwise comparison tests, n¼ 4–5.
1982; Vane and Botting, 1996; Sam et al., 2001; Becker, 2010). In 1986, Kohl proposed that dexamethasone may exert its anti-motion sickness effect through genetic induction mechanisms requiring new protein synthesis (Kohl, 1986). We observed that plasma levels of 2-AG and AEA decreased during motion sickness and that this decrease was abrogated by dexamethasone treatment. We also evaluated the expression of endocannabinoid enzymes in the DVC and stomach. In the central nervous system, motion sickness stimulation primarily decreased
the levels of the enzymes that synthesize 2-AG, indicating that 2AG levels may decrease in the central neural system in response to motion stimulation. Dexamethasone treatment blocked this effect. In contrast, in the periphery, motion sickness stimulation primarily enhanced the expression of 2-AG and AEA degradation enzymes, thereby decreasing blood 2-AG and AEA levels. Dexamethasone treatment restored 2-AG and AEA levels in response to motion stimulation. To some extent, these findings suggest that changes in the peripheral expression of endocannabinoid enzymes coincide with changes in AEA and 2-AG plasma levels. We believe that 2-AG plays an important role in suppressing nausea and vomiting in the CNS, whereas in the periphery, both 2-AG and AEA appear to inhibit motion sickness-induced emesis. Plasma endocannabinoid levels may reflect, to some extent, a spill-over of endocannabinoids from peripheral tissues involved in nausea and vomiting (Caraceni et al., 2010). The reduction in plasma 2-AG observed in the acceleration model group suggests that the motion sickness stimulation may be accompanied by reductions in 2-AG. This observation is in accordance with the findings of Chouker et al. (2010). Motion-induced nausea/vomiting is known to be linked to a pronounced activation of the glucocorticoid- and sympatico-adrenergic stress response systems (Otto et al., 2006), and 2-AG may regulate the corticosterone response to stress (Storr and Sharkey, 2007). Our results show that dexamethasone treatment restored plasma endocannabinoid levels under acceleration stimulation. We thus postulate that motion sickness stimulation may influence AEA and 2-AG levels in the DVC and stomach, respectively. However, dexamethasone may exert its effect by increasing 2-AG levels in the DVC and stomach as well as AEA levels in the stomach. One limitation of this study is that we did not measure AEA and 2-AG levels in the DVC or stomach. Further study is needed to determine whether dexamethasone influences AEA and 2-AG levels in the DVC and stomach. We also present evidence that motion sickness down-regulated the expression of the endocannabinoid CB1 receptor. A recent study reported similar findings, namely, that endocannabinoid CB1 receptor expression levels differed between individuals with and without motion sickness (Chouker et al., 2010). We found that motion sickness decreased the expression of the endocannabinoid CB1 receptor in the DVC and stomach. Previous studies have
104
Y. Zheng et al. / European Journal of Pharmacology 727 (2014) 99–105
Fig. 6. Western-blot detection showed the expression levels of CB1R protein expression in dorsal vagal complex (A) and stomach (C). Quantitative analysis of western-blot corresponding to CB1 receptor in dorsal vagal complex (B) and stomach (D), respectively. nPo 0.05; nnPo 0.01, one-way ANOVA followed LSD pairwise comparison tests, n¼ 4–5.
demonstrated that the functional endocannabinoid CB1 receptor, distributed in the DVC and the gastro-enteric tract, participates in intestinal motility, gastrointestinal transit, gastric secretion and colonic propulsion and mediates anti-emetic responses (Van Sickle et al., 2001; Casu et al., 2003; Derbenev et al., 2004; Storr et al., 2004; Roux et al., 2009). Other studies have found that delta-9tetrahydrocannabinol (Δ9-THC) and cannabinoids, both synthetic endocannabinoid CB1 receptor agonists, have anti-emesis effects (Van Sickle et al., 2001; Ray et al., 2009). Δ9-THC has been demonstrated to have an endocannabinoid CB1 receptor-dependent anti-emetic effect on motion-induced emesis in Suncus murinus (Cluny et al., 2008). In contrast, dexamethasone may lower the motion sickness index by enhancing the expression of endocannabinoid CB1 receptor. Both in vitro and in vivo studies have found that glucocorticoid can up-regulate the expression of endocannabinoid CB1 receptor via glucocorticoid receptor-dependent transcriptional and translational regulation (Wang et al., 2007; Wu et al., 2011; Ko et al., 2012). However, it should be pointed out that the glucocorticoid-mediated regulation of endocannabinoid CB1 receptor expression is not ubiquitous throughout the CNS. Hill et al. reported that corticosteroids did not affect endocannabinoid CB1 receptor density in the amygdala (Hill et al., 2005) and that glucocorticoids are responsible for the down-regulation of hippocampal endocannabinoid CB1 receptor expression (Hill et al., 2005). The endocannabinoid CB1 receptor may reduce emesis by acting on terminals of NTS neurons that project to the output neurons of the DMNV and by inhibiting transmitter release from interneurons of the NTS (Van Sickle et al., 2003). We thus conclude that dexamethasone may decrease the motion sickness index through both central and peripheral mechanisms, potentially by modulating the endocannabinoid system. Money et al. proposed that motion sickness responses could be divided into two categories: those related to stomach emptying and those related to “stress,” which may be secondary to stomach emptying (Money et al., 1996). Motion sickness-provoking conditions can thus be considered stressful stimulation. Resistant subjects present greater increases in stress-related hormones than susceptible subjects during stimulation (Li et al., 2005; Strewe
et al., 2012). Chouke et al. hypothesized that low levels of endocannabinoids accompanied by a low expression of the endocannabinoid CB1 receptor may indicate a failure to up-regulate endocannabinoid signaling during stress exposure in individuals with motion sickness (Chouker et al., 2010).
5. Conclusions Enhanced expression of the endocannabinoid CB1 receptor in the DVC and stomach and increased 2-AG levels suggest that dexamethasone may lower the motion sickness index by activating glucocorticoid receptors and influencing endocannabinoid levels, thereby stimulating endocannabinoid CB1 receptors to counteract stress and attenuate nausea and vomiting symptoms. Further studies are required to fully understand the role of the endocannabinoid system in the course of motion sickness and to identify other mechanisms underlying the anti-motion sickness effect of dexamethasone.
Acknowledgments This work was supported by (i) the Youth Grant of Second Millitary Medical University, China(2011QN03), (ii) the Military Special Research Project of Traditional Chinese Medicine (10ZYZ130), and (iii) the Shanghai Public Health funding for Construction of Key Disciplines.
References Becker, D.E., 2010. Nausea, vomiting, and hiccups: a review of mechanisms and treatment. Anesth. Prog. 57 (4), 150–156. Cai, Y.L., Wang, J.Q., et al., 2010. Decreased Fos protein expression in rat caudal vestibular nucleus is associated with motion sickness habituation. Neurosci. Lett. 480 (1), 87–91. Caraceni, P., Viola, A., et al., 2010. Circulating and hepatic endocannabinoids and endocannabinoid-related molecules in patients with cirrhosis. Liver Int. 30 (6), 816–825.
Y. Zheng et al. / European Journal of Pharmacology 727 (2014) 99–105
Casu, M.A., Porcella, A., et al., 2003. Differential distribution of functional cannabinoid CB1 receptors in the mouse gastroenteric tract. Eur. J. Pharmacol. 459 (1), 97–105. Chouker, A., Kaufmann, I., et al., 2010. Motion sickness, stress and the endocannabinoid system. PLoS One 5 (5), e10752. Cluny, N.L., Naylor, R.J., et al., 2008. The effects of cannabidiol and tetrahydrocannabinol on motion-induced emesis in Suncus murinus. Basic Clin. Pharmacol. Toxicol. 103 (2), 150–156. De Petrocellis, L., Di Marzo, V., 2009. An introduction to the endocannabinoid system: from the early to the latest concepts. Best Pract. Res. Clin. Endocrinol. Metab. 23 (1), 1–15. Derbenev, A.V., Stuart, T.C., et al., 2004. Cannabinoids suppress synaptic input to neurones of the rat dorsal motor nucleus of the vagus nerve. J. Physiol. 559 (Pt 3), 923–938. Evanson, N.K., Tasker, J.G., et al., 2010. Fast feedback inhibition of the HPA axis by glucocorticoids is mediated by endocannabinoid signaling. Endocrinology 151 (10), 4811–4819. Fujii, Y., Nakayama, M., 2007. Efficacy of dexamethasone for reducing postoperative nausea and vomiting and analgesic requirements after thyroidectomy. Otolaryngol. Head Neck Surg. 136 (2), 274–277. Harris, A.L., 1982. Cytotoxic-therapy-induced vomiting is mediated via enkephalin pathways. Lancet 1 (8274), 714–716. Hesketh, P.J., 2008. Chemotherapy-induced nausea and vomiting. N. Engl. J. Med. 358 (23), 2482–2494. Hill, M.N., Ho, W.S., et al., 2005. Chronic corticosterone treatment increases the endocannabinoid 2-arachidonylglycerol in the rat amygdala. Eur. J. Pharmacol. 528 (1–3), 99–102. Hill, M.N., Patel, S., et al., 2005. Downregulation of endocannabinoid signaling in the hippocampus following chronic unpredictable stress. Neuropsychopharmacology 30 (3), 508–515. Hu, D.L., Zhu, G., et al., 2007. Staphylococcal enterotoxin induces emesis through increasing serotonin release in intestine and it is downregulated by cannabinoid receptor 1. Cell Microbiol. 9 (9), 2267–2277. Ko, J.Y., Wu, R.W., et al., 2012. Cannabinoid receptor 1 mediates glucocorticoidinduced bone loss in rats by perturbing bone mineral acquisition and marrow adipogenesis. Arthritis Rheum. 64 (4), 1204–1214. Kohl, R.L., 1986. Dexamethasone mimicks the antimotion sickness effects of amphetamine and scopolamine. Acta Astronaut. 13 (9), 565–571. Li, X., Jiang, Z.L., et al., 2005. Plasma vasopressin, an etiologic factor of motion sickness in rat and human? Neuroendocrinology 81 (6), 351–359. Malik, N.M., Liu, Y.L., et al., 2007. Differential effects of dexamethasone, ondansetron and a tachykinin NK1 receptor antagonist (GR205171) on cisplatin-induced changes in behaviour, food intake, pica and gastric function in rats. Eur. J. Pharmacol. 555 (2–3), 164–173. Mo, F.F., Qin, H.H., et al., 2012. Acute hyperglycemia is related to gastrointestinal symptoms in motion sickness: an experimental study. Physiol. Behav. 105 (2), 394–401. Money, K.E., Lackner, J.R., Cheung, R.S.K., 1996. The autonomic nervous system and motion sickness. In: Yates, B.J., Miller, A.D. (Eds.), Vestibular Autonomic Regulation. CRC Press, Boca Raton, FL, pp. 147–173 Money, K.E., 1970. Motion sickness. Physiol. Rev. 50 (1), 1–39. Oosterveld, W.J., 1995. Motion sickness. J. Travel Med. 2 (3), 182–185. Otto, B., Riepl, R.L., et al., 2006. Endocrine correlates of acute nausea and vomiting. Auton Neurosci. 129 (1–2), 17–21.
105
Pagano, C., Rossato, M., et al., 2008. Endocannabinoids, adipose tissue and lipid metabolism. J. Neuroendocrinol. 20 (Suppl 1), S124–S129. Parker, L.A., Limebeer, C.L., et al., 2009. The FAAH inhibitor URB-597 interferes with cisplatin- and nicotine-induced vomiting in the Suncus murinus (house musk shrew). Physiol. Behav. 97 (1), 121–124. Ray, A.P., Griggs, L., et al., 2009. Delta 9-tetrahydrocannabinol suppresses vomiting behavior and Fos expression in both acute and delayed phases of cisplatininduced emesis in the least shrew. Behav. Brain Res. 196 (1), 30–36. Reason, J.T, 1978. Motion sickness adaptation: a neural mismatch model. J. R. Soc . Med. 71 (11), 819–829. Rich, W.M., Abdulhayoglu, G., et al., 1980. Methylprednisolone as an antiemetic during cancer chemotherapy—a pilot study. Gynecol .Oncol. 9 (2), 193–198. Rossi, C., Pini, L.A., et al., 2008. Endocannabinoids in platelets of chronic migraine patients and medication-overuse headache patients: relation with serotonin levels. Eur. J. Clin. Pharmacol. 64 (1), 1–8. Roux, J., Wanaverbecq, N., et al., 2009. Depolarization-induced release of endocannabinoids by murine dorsal motor nucleus of the vagus nerve neurons differentially regulates inhibitory and excitatory neurotransmission. Neuropharmacology 56 (8), 1106–1115. Sam, T.S., Chan, S.W., et al., 2001. Action of glucocorticoids to antagonise cisplatininduced acute and delayed emesis in the ferret. Eur. J. Pharmacol. 417 (3), 231–237. Storr, M., Sibaev, A., et al., 2004. Cannabinoid receptor type 1 modulates excitatory and inhibitory neurotransmission in mouse colon. Am. J. Physiol. Gastrointest. Liver Physiol. 286 (1), G110–G117. Storr, M.A., Sharkey, K.A., 2007. The endocannabinoid system and gut-brain signalling. Curr.Opin .Pharmacol. 7 (6), 575–582. Strewe, C., Feuerecker, M., et al., 2012. Effects of parabolic flight and spaceflight on the endocannabinoid system in humans. Rev. Neurosci. 0 (0), 1–8. Van Sickle, M.D., Oland, L.D., et al., 2001. Cannabinoids inhibit emesis through CB1 receptors in the brainstem of the ferret. Gastroenterology 121 (4), 767–774. Van Sickle, M.D., Oland, L.D., et al., 2003. Delta9-tetrahydrocannabinol selectively acts on CB1 receptors in specific regions of dorsal vagal complex to inhibit emesis in ferrets. Am. J. Physiol. Gastrointest. Liver Physiol. 285 (3), G566–G576. Vane, J.R., Botting, R.M., 1996. Mechanism of action of anti-inflammatory drugs. Scand. J. Rheumatol. Suppl. 102, 9–21. Wang, S., Lim, G., et al., 2007. Central glucocorticoid receptors regulate the upregulation of spinal cannabinoid-1 receptors after peripheral nerve injury in rats. Pain 131 (1–2), 96–105. Wei, X., Wang, Z.B., et al., 2011. Verification of motion sickness index in mice. CNS Neurosci .Ther. 17 (6), 790–792. Wu, R.W., Lin, T.P., et al., 2011. Cannabinoid receptor 1 regulates ERK and GSK3beta-dependent glucocorticoid inhibition of osteoblast differentiation in murine MC3T3-E1 cells. Bone 49 (6), 1255–1263. Yates, B.J., Miller, A.D., et al., 1998. Physiological basis and pharmacology of motion sickness: an update. Brain Res. Bull. 47 (5), 395–406. Yu, X.H., Cai, G.J., et al., 2007. A novel animal model for motion sickness and its first application in rodents. Physiol. Behav. 92 (4), 702–707. Zoerner, A.A., Gutzki, F.M., et al., 2011. Quantification of endocannabinoids in biological systems by chromatography and mass spectrometry: a comprehensive review from an analytical and biological perspective. Biochim. Biophys. Acta 1811 (11), 706–723.
Contents lists available at ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Pulmonary, gastrointestinal and urogenital pharmacology
Dexamethasone alleviates motion sickness in rats in part by enhancing the endocannabinoid system Yan Zheng a,b,1, Xiao-Li Wang a,1, Feng-Feng Mo a, Min Li a,n a b
Department of Military Hygiene, Faculty of Naval Medicine, Second Military Medical University, 800 Xiang Yin Road, Shanghai, China Department of Nutrition, Tong Ren Hospital Affiliated to Shanghai JiaoTong University School of Medicine, 1111 Xian Xia Road, Shanghai, China
art ic l e i nf o
a b s t r a c t
Article history: Received 3 May 2013 Received in revised form 22 January 2014 Accepted 24 January 2014 Available online 4 February 2014
Low-dose dexamethasone has been widely used for the prevention of nausea and vomiting after chemotherapy and surgical procedures and to treat motion sickness due to its minimal adverse effects, but the mechanisms underlying its anti-motion sickness effects are poorly understood. Previous studies have demonstrated that the endocannabinoid system is suppressed by motion sickness but stimulated by dexamethasone. The aim of the present study was to determine whether dexamethasone has an antimotion sickness effect in rats and to elucidate the mechanism of this action. We used HPLC–MS/MS to measure the plasma concentrations of anandamide and 2-arachidonoylglycerolþ1-arachidonoylglycerol, and we employed real-time quantitative PCR (qRT-PCR) and/or Western blot analysis to assay the expression of N-acylphosphatidyl-ethanolamine hydrolyzing phospholipase D, sn-1-selective diacylglycerol lipase, fatty acid hydrolase, monoacylglycerol lipase and endocannabinoid CB1 receptor in the dorsal vagal complex and stomach of rats exposed to a motion sickness protocol. The results showed that dexamethasone lowered the motion sickness index and restored the levels of endogenous cannabinoids and the expression of the endocannabinoid CB1 receptor, which declined after the induction of motion sickness, in the dorsal vagal complex and stomach. & 2014 Elsevier B.V. All rights reserved.
Keywords: AEA 2-AG Glucocorticoid Nausea and vomiting
1. Introduction Motion sickness is a malady characterized by a combination of signs and symptoms, such as gasping, drowsiness, general inactivity and excessive vomiting, that accompany movement or perceived environmental movement (Oosterveld, 1995; Yates et al., 1998). Motion sickness can be triggered by many different stimuli, including traveling in automobiles, aircraft, spacecraft or boats and exposure to moving visual scenes (Money, 1970; Reason, 1978). A parabolic flight experiment showed that motion sickness was Abbreviations: AEA, anandamide; ACTH, adrenocorticotropin; ACN, acetonitrile; 2-AG, 2-arachidonylglycerol; AP, area postrema; AVP, Arginine vasopressin; CIV, chemotherapy induced vomiting; CNS, central neural system; CRH, corticotropin releasing hormone; DAGL-a, sn-1-selective diacylglycerol lipase; DMNV, dorsal motor nucleus of the vagus; FAAH, fatty acid hydrolase; GAPDH, glyceraldehyde phosphate dehydrogenase; PONV, postoperative nausea and vomiting; PVN, paraventricular nucleus; TRPV1, transient receptor potential vanilloid-1; MAGL, monoacylglycerol lipase; NAPE-PLD, N-acylphosphatidyl-ethanolamine hydrolyzing phospholipase D; NTS, nucleus tractus solitarius; NR, nucleus of raphe; HPA, hypothalamic-pituitary-adrenal; Δ9-THC, delta-9-tetrahydrocannabinol n Corresponding author. Tel.: þ 86 21 81871120. E-mail addresses: [email protected] (Y. Zheng), [email protected] (X.-L. Wang), [email protected] (F.-F. Mo), [email protected] (M. Li). 1 Yan Zheng and Xiao-Li Wang contributed equally to this work http://dx.doi.org/10.1016/j.ejphar.2014.01.047 0014-2999 & 2014 Elsevier B.V. All rights reserved.
accompanied by a significant decrease in the reactivity of the peripheral endocannabinoid system (Chouker et al., 2010). A study of metabolic differences between subjects who experience nausea/ vomiting upon acceleration and those who do not revealed that the level of arachidonic acid, the downstream metabolite of endocannabinoids, was significantly increased in nausea/vomiting subjects (Mo et al., 2012), suggesting that motion sickness may be associated with an impairment of endocannabinoid activity (Chouker et al., 2010). Dexamethasone is often administered to cancer patients to counteract chemotherapy-induced vomiting, postoperative nausea/vomiting and analgesic requirements after thyroidectomy (Fujii and Nakayama, 2007), but its mechanism of action has not been clearly demonstrated (Malik et al., 2007; Hesketh, 2008). In 1986, Kohl proposed the use of dexamethasone to modulate motion sickness due to its long-acting, slow tolerance and fewer adverse effects compared with amphetamine and scopolamine (Kohl, 1986). The endocannabinoid system comprises (i) cannabinoid receptors (cannabinoid type 1 receptor (CB1R), cannabinoid type 2 receptor and transient receptor potential vanilloid-1); (ii) their specific endogenous ligands (endocannabinoids), which include N-arachidonylethanolamine (anandamide, AEA), 2-arachidonoylglycerol (2-AG), noladin ether and virodhamine; and (iii) a number of biosynthetic and
100
Y. Zheng et al. / European Journal of Pharmacology 727 (2014) 99–105
degradation enzymes (De Petrocellis and Di Marzo, 2009). NAcylphosphatidyl-ethanolamine hydrolyzing phospholipase D (NAPEPLD) and sn-1-selective diacylglycerol lipase (DAGL-a) are responsible for the synthesis of AEA and 2-AG, respectively, whereas fatty acid hydrolase (FAAH) is responsible for the degradation of AEA, and monoacylglycerol lipase (MAGL) is responsible for the degradation of 2-AG (Pagano et al., 2008). There are bidirectional and functional correlations between glucocorticoids and the endocannabinoid system. Glucocorticoids recruit the endocannabinoid system to exert rapid negative feedback control of the hypothalamic–pituitary–adrenal (HPA) axis during stress conditions (Evanson et al., 2010). AEA and 2-AG, as endogenous endocannabinoids at the terminals of vagal afferents in the gastrointestinal tract, have been demonstrated to participate in the complex regulation of food intake as well as emesis induced by toxins (Hu et al., 2007). Recently, researchers have found that AEA and 2-AG play important roles in the pathophysiology of nausea and vomiting induced by conditions such as migraine and cancer chemotherapy drugs and the conditioned gaping response elicited by a lithium-paired context (Rossi et al., 2008; Parker et al., 2009). To understand the protective effect of dexamethasone against motion sickness in rats and determine whether this effect is mediated by stimulating the endocannabinoid system, we measured the levels of two major endocannabinoids AEA and 2-AG in plasma and their turnover enzymes in the dorsal vagal complex (DVC) and stomach. We analyzed the mRNA and protein levels of CB1R in these tissues to further elucidate the role of endocannabinoid signaling in the course of motion sickness.
administration method were selected based on our previous experiments. During the 30 min rotation, the rats were given no food or tap water and were enclosed in a cuboidal Plexiglas box suspended on a metal frame, which revolved around an axis parallel to the floor as described by Cai et al. (2010). It has been demonstrated in our laboratory that this magnitude of stimulation can induce motion sickness in rats (Cai et al., 2010; Mo et al., 2012). Dexamethasone acetate was dissolved in distilled water. AM251 was dissolved in a vehicle composed of 2% DMSO plus 1% Tween-80 (Sigma-Aldrich, St. Louis, MO). Preliminary experiments showed that the motion sickness index induced by acceleration stimulation in gastric lavage-treated animals was not significantly different (P 40.05) from the response of animals in the control group. 2.2. Observation of motion sickness symptoms Because the rats had no emetic reaction, motion sickness symptoms were observed after rotation and recorded according to the motion sickness index, which reflects the severity of gastrointestinal symptoms caused by motion stimulation (Yu et al., 2007; Wei et al., 2011). The evaluation criteria of the motion sickness index were as follows: 1 point was given for each fecal granule, urination scored 1.2 points, severe piloerection scored 1.2 points, slight piloerection scored 0.6, and tremor scored 1.2 (Yu et al., 2007). For each criterion, 0 points were allotted if the symptom was not present. The motion sickness index was calculated by summing of all of these scores. 2.3. Tissue and blood preparation
2. Materials and methods 2.1. Animals and acceleration exposure Thirty-three six-week-old male Sprague-Dawley (SD) rats weighing 190–240 g were purchased from Sino-British SIPPR/BK Lab Animal Ltd. (Shanghai, China). The animal protocols and procedures were approved by the Animal Use and Care Committee for Research and Education of the Second Military Medical University (Shanghai, China), and experiments were carried out in accordance with the guidelines published by the National Institutes of Health (USA) regarding the care and use of animals for experimental procedures. The animals were housed in Plexiglas box cages in the colony room at an ambient temperature of 2272 1C with a 12 h light/12 h dark schedule (lights on at 8 a.m.) and were maintained on an ad libitum schedule of food and water. The rats were randomly assigned to four groups: the control group (n ¼8), which received an intra-gastric administration of water and, 30 min later, watched the acceleration apparatus and experienced the noise of the apparatus without experiencing actual acceleration; the acceleration model group (n¼ 8), which received an intra-gastric administration of water 30 min before acceleration exposure; the DEX group (n ¼9), which received an intra-gastric administration of 0.05 mg/kg dexamethasone acetate 30 min before acceleration exposure, ensuring that the drug was completely absorbed according to the criteria defined by the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China); and the AM-251/DEX group (n¼ 8), in which the animals were injected intraperitoneally with 5 mg/kg N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4methyl-1H-pyrazole-3-carboxamide (AM-251) (Tocris, Ballwin, MO) 15 min before the intra-gastric administration of 0.05 mg/kg dexamethasone acetate 30 min before acceleration exposure. Rats in the acceleration model, the DEX and AM-251/DEX groups were subjected to rotation. The dexamethasone acetate dosage and
Animals in each group were anesthetized by an intraperitoneal (i.p.) injection of 10% 0.3 g/kg chloral hydrate, and blood was drawn by cardiac puncture followed by a trans-cardiac perfusion of 60 ml chilled saline. A blood sample (5 ml) was collected by cardiac puncture using a BD-Falcon vacutainer. Blood samples were centrifuged at 3000 rpm for 20 min at 4 1C, and the plasma layer was collected and stored at 80 1C. The brain was immediately dissected and cooled in iced saline for 1 min before the dorsal vagal complex was carefully removed. The stomach was also removed for analysis. The dissected tissues were immediately frozen in liquid nitrogen and stored at 80 1C for further use. 2.4. HPLC–MS/MS This analysis was performed on an LC/MS/MS system consisting of a Triple Quad™ 5500 (Applied Biosystems, Concord, ON, Canada), a SIL-HTc gradient pump (Shimadzu, Kyoto, Japan), two LC-20AD transfer pumps (Shimadzu, Kyoto, Japan), a DGU-20A3 vacuum pump (Shimadzu, Kyoto, Japan), a Gemini C18 chromatographic column (2.0 50 mm, 5 μm, Phenomenex, USA) and a C18 guard column (4.0 3.0 mm, 5 μm, Phenomenex, USA). Data acquisition and analysis were performed using Analyst software (version 1.5.1; Applied Biosystems/MDS Sciex). AEA, 2-AG, AEA d8 and 2-AG d8 were purchased from Cayman Chemicals (Nottingham, UK). Stock solutions were prepared at 0.1 mg/ml in acetonitrile for 2-AG, 2.5 μg/ml in acetonitrile for 2-AG d8, 0.5 mg/ml in methanol for AEA and 10 μg/ml in methanol for AEA d8. Stock and working solutions were stored at 80 1C or 20 1C. During sample preparation, all steps were carried out at 4 1C to prevent the isomerization or degradation of the compounds. After spiking with the internal standard, 300 ml of the plasma sample/calibration sample was treated by the liquid–liquid extraction method with 1 ml hexane/ ethyl acetate (1:1, v/v) by vortex shaking (approximately 1500 moves/min) for 10 min followed by centrifugation at 15,000 rpm
Y. Zheng et al. / European Journal of Pharmacology 727 (2014) 99–105
for 5 min at 4 1C. The upper organic phase was evaporated to dryness at room temperature, reconstituted with 50 ml of a 40% acetonitrile/water mixture, centrifuged at 15,000 rpm for 3 min at 4 1C and injected (5 ml) into the LC/MS/MS system. Multiple reactions monitoring of the product ions from the precursor ions of the analyte of interest was carried out, and the results were quantified. Details of the ions monitored (m/z) are as follows: 379.3287.4 amu for 2-AG and 1-AG, 387.4-294.4 amu for 2-AG-d8 and 1-AG-d8, 348.3-62.1 amu for AEA and 356.3-62.1 amu for AEAd8. Calibration curves (r2 ¼ 0.9988 for AEA and r2 ¼0.9980 for 2-AG) were constructed for both 2-AG and AEA. Because 1-AG and 2-AG undergo rapid isomerization, it is difficult to report with certainty the quantities of each species originating from tissues; thus, the results are reported as the sum of the individual peaks of 2-AGþ1AG (Zoerner et al., 2011).
101
peroxidase-labeled secondary antibodies (1:2000, anti-rabbit IgG, Jackson Immunoresearch, West Grove, PA, USA) at room temperature. The bands were then visualized with an ECL system (Millipore Corp, Bedford, MA, USA). The density of the bands was analyzed with a Gel Doc image system (Bio-Rad, San Diego, CA, USA). Signal intensities were normalized against the internal control (GAPDH). 2.7. Data analysis Statistical analysis was performed using the SPSS v16.0 statistical program. Data were tested with a one-way analysis of variance (ANOVA) followed by least significant difference (LSD) pair-wise comparison tests. All data are presented as mean values 7S.E.M. P values less than 0.05 were considered significant in all tests.
2.5. Real-time quantitative PCR 3. Results Tissue samples were homogenized in TRIzol reagent (Invitrogen, USA), and total RNA was extracted. Complementary DNA (cDNA) was synthesized using oligo dT primers and the PrimeScript RT Reagent Kit (Takara, Japan) according to the manufacture’s protocol. Reverse transcription of total RNA was performed at 95 1C for 5 min. Real-time PCR reactions were carried out in a Rotor-Gene PCR machine (RG-3000A, Corbett Research) as follows: an initial denaturation step at 95 1C for 5 s followed by 40 cycles of 95 1C for 5 s, annealing at 60 1C for 20 s and extension at 72 1C for 30 s. The amount of cDNA per sample was determined using a SYBR Premix Ex Taq™ kit (Takara, Japan). The progression of the PCR reaction was assessed by changes in the amount of SYBR Green dye attached to double-stranded DNA. The target genes were normalized to the endogenous control gene, glyceraldehyde phosphate dehydrogenase (Gapdh). The primers used for real-time PCR were as follows: CB1R: sense primer 5'-TCAAAGCTGACGCCCTGAC-3', antisense primer 5'-GAACATAACGATGCGATACACG-3'; NAPE-PLD: sense primer 5'-TGATGGTGGAAATGGACGAG-3', antisense primer 5'-AATGTTGGGAGGGCGTGA-3'; FAAH: sense primer 5'-ATTGCCCAGTGGAAAGCG-3', antisense primer 5'-GGATGAACGACCCTCACGAT-3'; MAGL: sense primer 5'-AGAGGATGGTGGTATCGGACTT-3', antisense primer 5'-GCAGATGAGTGGGTCGGAGT-3'; DAGL-α: sense primer 5'-TGGAACCGTGGCAGAATG-30 , antisense primer 5'-AAGGAGACAGGGCTTTGGAT-3'; and GAPDH: sense primer 5'-GGCTCTCTGCTCCTCCCTGTTCTA-3', antisense primer 5'CGTCCGATACGGCCAAATCCGT-3'. 2.6. Western blot analysis Tissue samples were lysed in 1 ml tissue and cell lysis solution plus protease and phosphatase inhibitors (Keygen Biotech, Nanjing, China) and then centrifuged at 10,000g for 5 min at 4 1C. The supernatant was collected as the total protein extract, and the protein concentration was determined spectrophotometrically according to the bicinchoninic acid (BCA) method. Equal amounts of protein (60 μg per lane) from each sample were loaded on a 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). Following electrophoresis, the proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Biotech, Nanjing, China) using a Mini-Trans-Blot Electrophoresis Transfer Cell (Bio-Rad, Hercules, CA, USA). Nonspecific protein binding of antibodies was blocked by treating membranes in 5% nonfat milk in Tris-buffered saline containing Tween 20 (TBST; 10 mM Tris–HCl, 150 mM NaCl, 0.05% Tween-20). The membrane was incubated overnight at 4 1C with the CB1 rabbit polyclonal antibody (1:200; Abcam Ltd., Hong Kong) or anti-GAPDH (1:1000; Sigma-Aldrich, St. Louis, MO, USA) in TBST with 1% bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO, USA). The membrane was then washed with TBST buffer and incubated for 1 h with
3.1. Comparison of motion sickness severity among the four groups. The motion sickness index of the DEX group was significantly lower than that of the acceleration model group (4.4 71.36 vs. 12 73.31, P o0.001), whereas there was no significant difference between the control and DEX groups (3.2 71.0 vs. 4.4 71.36). AM251 (5 mg/kg) partially abolished the effect of DEX (P o0.001). The motion sickness index of the AM-251/DEX group was lower than that of the acceleration model group (8.53 71.18 vs. 12 73.31, Po 0.05) and higher than that of the control (P o0.001) and DEX groups (P o0.001) (Fig. 1). 3.2. Changes in plasma AEA and 2-AG þ 1-AG concentrations in rats AEA levels were similar across all groups (7.60 71.07 ng/ml in the control group vs. 6.19 70.98 ng/ml in the acceleration model group and 7.39 71.92 ng/ml in the DEX group) (Fig. 2A), whereas plasma 2-AG þ 1-AG levels were lower in the acceleration model group (32.39 710.91 ng/ml) than in the control and DEX groups (67 719.84 ng/ml and 54.28 720.28 ng/ml, respectively; P o0.05) (Fig. 2B). 3.3. Expression of endocannabinoid biosynthesis and degradation enzymes (NAPE-PLD, FAAH, DGL-a and MAGL) in the rat DVC and stomach Compared with the levels in the control group, the mRNA levels of NAPE-PLD and FAAH were decreased and increased, respectively, in the DVC after acceleration stimulation. These changes
Fig. 1. Comparison of motion sickness index in control group, acceleration model group, DEX group, and AM-251/DEX group. nStatistically significant when compared with control group (Po 0.001); #Statistically significant when compared with acceleration model group (P o0.05); Statistically significant when compared with DEX group(Po 0.001); and one-way ANOVA followed by LSD pairwise comparison tests, n¼ 8–9.
102
Y. Zheng et al. / European Journal of Pharmacology 727 (2014) 99–105
were significantly enhanced by DEX treatment. Compared with the levels in the control group, the mRNA levels of both DGL-a and MAGL were decreased and DGL-a was enhanced after motion stimulation. MAGL mRNA expression was markedly reduced by DEX administration (Fig. 3). Motion stimulation up-regulated FAAH, DGL-a and MAGL mRNA expression in the stomach relative to the levels in the control group, whereas DEX down-regulated the FAAH and MAGL levels compared with the those in the acceleration model group (Fig. 4).
3.4. Effect of acceleration exposure and DEX on the expression of endocannabinoid CB1 receptor mRNA and protein in the rat DVC and stomach mRNA levels of endocannabinoid CB1 receptor in the DVC were decreased in the acceleration model group compared with the control group (0.98 70.21 vs. 1.68 70.12) but were unaffected in the DEX group (1.78 70.55) (Fig. 5A). The levels of endocannabinoid CB1 receptor mRNA in the stomach were similarly decreased in the acceleration model group compared with the control group (1.04 70.20 vs. 1.87 70.17) and were unchanged in the DEX group (1.80 70.21) (Fig. 5B). Endocannabinoid CB1 receptor expression was further investigated by Western blotting (Fig. 6). To quantify protein expression levels, the optical density of the Western blot data was calculated and normalized to the GAPDH control. The protein levels of endocannabinoid CB1 receptor in the DVC were 0.046 70.015, 0.02 70.001 and 0.045 70.003 in the control, acceleration model and DEX groups, respectively (Fig. 6A,B), and 1.17 70.27, 0.84 70.18 and 1.16 70.20, respectively, in the stomach (Fig. 6C,D).
4. Discussion
Fig. 2. Comparison of plasma concentrations of endocannabinoid anandamide (A) and 2-AG (B) among control group, acceleration model group and DEX group. nn Indicates significantly lower 2-AG plasma concerntrations comparing with the control group, Po 0.01. One-way ANOVA followed by LSD pairwise comparison tests, n ¼8–9.
The motion sickness index is regarded as an appropriate measure for evaluating the severity of motion sickness in rodents (Yuet al., 2007; Wei et al., 2011; Mo et al., 2012). Motion sickness index scores were increased after motion stimulation in the control and acceleration model groups, whereas dexamethasone protected rats against motion sickness, confirming the previous finding that dexamethasone has an anti-motion sickness effect (Kohl, 1986). The ability of dexamethasone to modulate motion sickness-induced gastrointestinal symptoms was partially reversed by treatment with the selective endocannabinoid CB1 receptor antagonist AM-251. The incomplete reversal was most likely due to dexamethasone exerting its antiemesis effect via multiple mechanisms. Researchers have postulated that DEX may suppress the production of inflammatory autacoids, prostaglandins, thromboxane, leukotrienes and cytokines, thus decreasing histamine production and/or release from mast cells involved in mediating the emetic response (Rich et al., 1980; Harris,
Fig. 3. NAPE-PLD (A), FAAH (B), DGL-a (C), and MAGL (D) mRNA expression in rats dorsal vagal complex. nIndicates significant difference comparing with the control group, Po 0.01. One-way ANOVA followed by LSD pairwise comparison tests, n¼ 8–9.
Y. Zheng et al. / European Journal of Pharmacology 727 (2014) 99–105
103
Fig. 4. NAPE-PLD (A), FAAH (B), DGL-a (C), and MAGL (D) mRNA expression in rats stomach. nIndicates significantly higher mRNA expression comparing with the control group, P o 0.05. One-way ANOVA followed by LSD pairwise comparison tests, n¼ 8–9.
Fig. 5. Comparision of CB1R messenger RNA relative quantity in the dorsal vagal complex (DVC) (A) and stomach (B) among control group (solid bar), acceleration model group (open bar) and dexamethasone group (gray group). nSignificant difference (P o0.05) of acceleration model group from control group. nn Significant difference (P o 0.001) compares with control group. One-way ANOVA followed by LSD pairwise comparison tests, n¼ 4–5.
1982; Vane and Botting, 1996; Sam et al., 2001; Becker, 2010). In 1986, Kohl proposed that dexamethasone may exert its anti-motion sickness effect through genetic induction mechanisms requiring new protein synthesis (Kohl, 1986). We observed that plasma levels of 2-AG and AEA decreased during motion sickness and that this decrease was abrogated by dexamethasone treatment. We also evaluated the expression of endocannabinoid enzymes in the DVC and stomach. In the central nervous system, motion sickness stimulation primarily decreased
the levels of the enzymes that synthesize 2-AG, indicating that 2AG levels may decrease in the central neural system in response to motion stimulation. Dexamethasone treatment blocked this effect. In contrast, in the periphery, motion sickness stimulation primarily enhanced the expression of 2-AG and AEA degradation enzymes, thereby decreasing blood 2-AG and AEA levels. Dexamethasone treatment restored 2-AG and AEA levels in response to motion stimulation. To some extent, these findings suggest that changes in the peripheral expression of endocannabinoid enzymes coincide with changes in AEA and 2-AG plasma levels. We believe that 2-AG plays an important role in suppressing nausea and vomiting in the CNS, whereas in the periphery, both 2-AG and AEA appear to inhibit motion sickness-induced emesis. Plasma endocannabinoid levels may reflect, to some extent, a spill-over of endocannabinoids from peripheral tissues involved in nausea and vomiting (Caraceni et al., 2010). The reduction in plasma 2-AG observed in the acceleration model group suggests that the motion sickness stimulation may be accompanied by reductions in 2-AG. This observation is in accordance with the findings of Chouker et al. (2010). Motion-induced nausea/vomiting is known to be linked to a pronounced activation of the glucocorticoid- and sympatico-adrenergic stress response systems (Otto et al., 2006), and 2-AG may regulate the corticosterone response to stress (Storr and Sharkey, 2007). Our results show that dexamethasone treatment restored plasma endocannabinoid levels under acceleration stimulation. We thus postulate that motion sickness stimulation may influence AEA and 2-AG levels in the DVC and stomach, respectively. However, dexamethasone may exert its effect by increasing 2-AG levels in the DVC and stomach as well as AEA levels in the stomach. One limitation of this study is that we did not measure AEA and 2-AG levels in the DVC or stomach. Further study is needed to determine whether dexamethasone influences AEA and 2-AG levels in the DVC and stomach. We also present evidence that motion sickness down-regulated the expression of the endocannabinoid CB1 receptor. A recent study reported similar findings, namely, that endocannabinoid CB1 receptor expression levels differed between individuals with and without motion sickness (Chouker et al., 2010). We found that motion sickness decreased the expression of the endocannabinoid CB1 receptor in the DVC and stomach. Previous studies have
104
Y. Zheng et al. / European Journal of Pharmacology 727 (2014) 99–105
Fig. 6. Western-blot detection showed the expression levels of CB1R protein expression in dorsal vagal complex (A) and stomach (C). Quantitative analysis of western-blot corresponding to CB1 receptor in dorsal vagal complex (B) and stomach (D), respectively. nPo 0.05; nnPo 0.01, one-way ANOVA followed LSD pairwise comparison tests, n¼ 4–5.
demonstrated that the functional endocannabinoid CB1 receptor, distributed in the DVC and the gastro-enteric tract, participates in intestinal motility, gastrointestinal transit, gastric secretion and colonic propulsion and mediates anti-emetic responses (Van Sickle et al., 2001; Casu et al., 2003; Derbenev et al., 2004; Storr et al., 2004; Roux et al., 2009). Other studies have found that delta-9tetrahydrocannabinol (Δ9-THC) and cannabinoids, both synthetic endocannabinoid CB1 receptor agonists, have anti-emesis effects (Van Sickle et al., 2001; Ray et al., 2009). Δ9-THC has been demonstrated to have an endocannabinoid CB1 receptor-dependent anti-emetic effect on motion-induced emesis in Suncus murinus (Cluny et al., 2008). In contrast, dexamethasone may lower the motion sickness index by enhancing the expression of endocannabinoid CB1 receptor. Both in vitro and in vivo studies have found that glucocorticoid can up-regulate the expression of endocannabinoid CB1 receptor via glucocorticoid receptor-dependent transcriptional and translational regulation (Wang et al., 2007; Wu et al., 2011; Ko et al., 2012). However, it should be pointed out that the glucocorticoid-mediated regulation of endocannabinoid CB1 receptor expression is not ubiquitous throughout the CNS. Hill et al. reported that corticosteroids did not affect endocannabinoid CB1 receptor density in the amygdala (Hill et al., 2005) and that glucocorticoids are responsible for the down-regulation of hippocampal endocannabinoid CB1 receptor expression (Hill et al., 2005). The endocannabinoid CB1 receptor may reduce emesis by acting on terminals of NTS neurons that project to the output neurons of the DMNV and by inhibiting transmitter release from interneurons of the NTS (Van Sickle et al., 2003). We thus conclude that dexamethasone may decrease the motion sickness index through both central and peripheral mechanisms, potentially by modulating the endocannabinoid system. Money et al. proposed that motion sickness responses could be divided into two categories: those related to stomach emptying and those related to “stress,” which may be secondary to stomach emptying (Money et al., 1996). Motion sickness-provoking conditions can thus be considered stressful stimulation. Resistant subjects present greater increases in stress-related hormones than susceptible subjects during stimulation (Li et al., 2005; Strewe
et al., 2012). Chouke et al. hypothesized that low levels of endocannabinoids accompanied by a low expression of the endocannabinoid CB1 receptor may indicate a failure to up-regulate endocannabinoid signaling during stress exposure in individuals with motion sickness (Chouker et al., 2010).
5. Conclusions Enhanced expression of the endocannabinoid CB1 receptor in the DVC and stomach and increased 2-AG levels suggest that dexamethasone may lower the motion sickness index by activating glucocorticoid receptors and influencing endocannabinoid levels, thereby stimulating endocannabinoid CB1 receptors to counteract stress and attenuate nausea and vomiting symptoms. Further studies are required to fully understand the role of the endocannabinoid system in the course of motion sickness and to identify other mechanisms underlying the anti-motion sickness effect of dexamethasone.
Acknowledgments This work was supported by (i) the Youth Grant of Second Millitary Medical University, China(2011QN03), (ii) the Military Special Research Project of Traditional Chinese Medicine (10ZYZ130), and (iii) the Shanghai Public Health funding for Construction of Key Disciplines.
References Becker, D.E., 2010. Nausea, vomiting, and hiccups: a review of mechanisms and treatment. Anesth. Prog. 57 (4), 150–156. Cai, Y.L., Wang, J.Q., et al., 2010. Decreased Fos protein expression in rat caudal vestibular nucleus is associated with motion sickness habituation. Neurosci. Lett. 480 (1), 87–91. Caraceni, P., Viola, A., et al., 2010. Circulating and hepatic endocannabinoids and endocannabinoid-related molecules in patients with cirrhosis. Liver Int. 30 (6), 816–825.
Y. Zheng et al. / European Journal of Pharmacology 727 (2014) 99–105
Casu, M.A., Porcella, A., et al., 2003. Differential distribution of functional cannabinoid CB1 receptors in the mouse gastroenteric tract. Eur. J. Pharmacol. 459 (1), 97–105. Chouker, A., Kaufmann, I., et al., 2010. Motion sickness, stress and the endocannabinoid system. PLoS One 5 (5), e10752. Cluny, N.L., Naylor, R.J., et al., 2008. The effects of cannabidiol and tetrahydrocannabinol on motion-induced emesis in Suncus murinus. Basic Clin. Pharmacol. Toxicol. 103 (2), 150–156. De Petrocellis, L., Di Marzo, V., 2009. An introduction to the endocannabinoid system: from the early to the latest concepts. Best Pract. Res. Clin. Endocrinol. Metab. 23 (1), 1–15. Derbenev, A.V., Stuart, T.C., et al., 2004. Cannabinoids suppress synaptic input to neurones of the rat dorsal motor nucleus of the vagus nerve. J. Physiol. 559 (Pt 3), 923–938. Evanson, N.K., Tasker, J.G., et al., 2010. Fast feedback inhibition of the HPA axis by glucocorticoids is mediated by endocannabinoid signaling. Endocrinology 151 (10), 4811–4819. Fujii, Y., Nakayama, M., 2007. Efficacy of dexamethasone for reducing postoperative nausea and vomiting and analgesic requirements after thyroidectomy. Otolaryngol. Head Neck Surg. 136 (2), 274–277. Harris, A.L., 1982. Cytotoxic-therapy-induced vomiting is mediated via enkephalin pathways. Lancet 1 (8274), 714–716. Hesketh, P.J., 2008. Chemotherapy-induced nausea and vomiting. N. Engl. J. Med. 358 (23), 2482–2494. Hill, M.N., Ho, W.S., et al., 2005. Chronic corticosterone treatment increases the endocannabinoid 2-arachidonylglycerol in the rat amygdala. Eur. J. Pharmacol. 528 (1–3), 99–102. Hill, M.N., Patel, S., et al., 2005. Downregulation of endocannabinoid signaling in the hippocampus following chronic unpredictable stress. Neuropsychopharmacology 30 (3), 508–515. Hu, D.L., Zhu, G., et al., 2007. Staphylococcal enterotoxin induces emesis through increasing serotonin release in intestine and it is downregulated by cannabinoid receptor 1. Cell Microbiol. 9 (9), 2267–2277. Ko, J.Y., Wu, R.W., et al., 2012. Cannabinoid receptor 1 mediates glucocorticoidinduced bone loss in rats by perturbing bone mineral acquisition and marrow adipogenesis. Arthritis Rheum. 64 (4), 1204–1214. Kohl, R.L., 1986. Dexamethasone mimicks the antimotion sickness effects of amphetamine and scopolamine. Acta Astronaut. 13 (9), 565–571. Li, X., Jiang, Z.L., et al., 2005. Plasma vasopressin, an etiologic factor of motion sickness in rat and human? Neuroendocrinology 81 (6), 351–359. Malik, N.M., Liu, Y.L., et al., 2007. Differential effects of dexamethasone, ondansetron and a tachykinin NK1 receptor antagonist (GR205171) on cisplatin-induced changes in behaviour, food intake, pica and gastric function in rats. Eur. J. Pharmacol. 555 (2–3), 164–173. Mo, F.F., Qin, H.H., et al., 2012. Acute hyperglycemia is related to gastrointestinal symptoms in motion sickness: an experimental study. Physiol. Behav. 105 (2), 394–401. Money, K.E., Lackner, J.R., Cheung, R.S.K., 1996. The autonomic nervous system and motion sickness. In: Yates, B.J., Miller, A.D. (Eds.), Vestibular Autonomic Regulation. CRC Press, Boca Raton, FL, pp. 147–173 Money, K.E., 1970. Motion sickness. Physiol. Rev. 50 (1), 1–39. Oosterveld, W.J., 1995. Motion sickness. J. Travel Med. 2 (3), 182–185. Otto, B., Riepl, R.L., et al., 2006. Endocrine correlates of acute nausea and vomiting. Auton Neurosci. 129 (1–2), 17–21.
105
Pagano, C., Rossato, M., et al., 2008. Endocannabinoids, adipose tissue and lipid metabolism. J. Neuroendocrinol. 20 (Suppl 1), S124–S129. Parker, L.A., Limebeer, C.L., et al., 2009. The FAAH inhibitor URB-597 interferes with cisplatin- and nicotine-induced vomiting in the Suncus murinus (house musk shrew). Physiol. Behav. 97 (1), 121–124. Ray, A.P., Griggs, L., et al., 2009. Delta 9-tetrahydrocannabinol suppresses vomiting behavior and Fos expression in both acute and delayed phases of cisplatininduced emesis in the least shrew. Behav. Brain Res. 196 (1), 30–36. Reason, J.T, 1978. Motion sickness adaptation: a neural mismatch model. J. R. Soc . Med. 71 (11), 819–829. Rich, W.M., Abdulhayoglu, G., et al., 1980. Methylprednisolone as an antiemetic during cancer chemotherapy—a pilot study. Gynecol .Oncol. 9 (2), 193–198. Rossi, C., Pini, L.A., et al., 2008. Endocannabinoids in platelets of chronic migraine patients and medication-overuse headache patients: relation with serotonin levels. Eur. J. Clin. Pharmacol. 64 (1), 1–8. Roux, J., Wanaverbecq, N., et al., 2009. Depolarization-induced release of endocannabinoids by murine dorsal motor nucleus of the vagus nerve neurons differentially regulates inhibitory and excitatory neurotransmission. Neuropharmacology 56 (8), 1106–1115. Sam, T.S., Chan, S.W., et al., 2001. Action of glucocorticoids to antagonise cisplatininduced acute and delayed emesis in the ferret. Eur. J. Pharmacol. 417 (3), 231–237. Storr, M., Sibaev, A., et al., 2004. Cannabinoid receptor type 1 modulates excitatory and inhibitory neurotransmission in mouse colon. Am. J. Physiol. Gastrointest. Liver Physiol. 286 (1), G110–G117. Storr, M.A., Sharkey, K.A., 2007. The endocannabinoid system and gut-brain signalling. Curr.Opin .Pharmacol. 7 (6), 575–582. Strewe, C., Feuerecker, M., et al., 2012. Effects of parabolic flight and spaceflight on the endocannabinoid system in humans. Rev. Neurosci. 0 (0), 1–8. Van Sickle, M.D., Oland, L.D., et al., 2001. Cannabinoids inhibit emesis through CB1 receptors in the brainstem of the ferret. Gastroenterology 121 (4), 767–774. Van Sickle, M.D., Oland, L.D., et al., 2003. Delta9-tetrahydrocannabinol selectively acts on CB1 receptors in specific regions of dorsal vagal complex to inhibit emesis in ferrets. Am. J. Physiol. Gastrointest. Liver Physiol. 285 (3), G566–G576. Vane, J.R., Botting, R.M., 1996. Mechanism of action of anti-inflammatory drugs. Scand. J. Rheumatol. Suppl. 102, 9–21. Wang, S., Lim, G., et al., 2007. Central glucocorticoid receptors regulate the upregulation of spinal cannabinoid-1 receptors after peripheral nerve injury in rats. Pain 131 (1–2), 96–105. Wei, X., Wang, Z.B., et al., 2011. Verification of motion sickness index in mice. CNS Neurosci .Ther. 17 (6), 790–792. Wu, R.W., Lin, T.P., et al., 2011. Cannabinoid receptor 1 regulates ERK and GSK3beta-dependent glucocorticoid inhibition of osteoblast differentiation in murine MC3T3-E1 cells. Bone 49 (6), 1255–1263. Yates, B.J., Miller, A.D., et al., 1998. Physiological basis and pharmacology of motion sickness: an update. Brain Res. Bull. 47 (5), 395–406. Yu, X.H., Cai, G.J., et al., 2007. A novel animal model for motion sickness and its first application in rodents. Physiol. Behav. 92 (4), 702–707. Zoerner, A.A., Gutzki, F.M., et al., 2011. Quantification of endocannabinoids in biological systems by chromatography and mass spectrometry: a comprehensive review from an analytical and biological perspective. Biochim. Biophys. Acta 1811 (11), 706–723.
