The Studies On Mechanisms Of Dexamethasone’s Anti-motion Sickness And Its Application

Motion sickness is a malady characterized by a combination of signs and symptoms,like pale, cold sweat and emesis, that accompany movement or perceived environmentalmovement, and can be triggered by many different circumstances such as traveling inautomobiles, aircraft, boats and exposure to moving visual scenes. Nowadays, althoughvarious pharmacological and non-pharmacological countermeasures have been used for theprevention and treatment of motion sickness, most of them have adverse effects onpsychomotor performance, like drowsiness, impairement of memory abiliy, inattention andlow work-efficiency, thus limiting their wider applications. DEX has been to treat variousinflammatory and autoimmune diseases, as well as used in cancer patients emsis,postopetavie nausea and vomiting and other treatment-related adverse effects. Clinicaltrials have found that DEX has a relatively antiemetic broad-spectrum. Since1986, Kohl etal has proposed the use of DEX to modulate motion sickness due to its long acting andslow tolerance properties and fewer adverse effects as compared with amphetamine andscopolamine, but its mechanism has not been demonstrated clearly.A parabolic flight experiment showed that motion sickness was accompanied by asignificant lowering of peripheral ECS reactivity. A study on metabolic differencesbetween nausea/vomiting subjects and non-nausea/vomiting ones caused by accelerationexposure showed that arachidonic acid as the downstream metabolite of endocannabinoidswas increased significantly in nausea/vomiting subjects, suggesting that motion sicknessmay be associated with impairment to endocannabinoid activity. There are bidirectionaland functional correlations between glucocorticoids and the ECS. Glucocorticoids recruitthe ECS to exert rapid negative feedback control of the hypothalamic-pituitary-adrenal(HPA) axis during stress. The ECS in turn plays an essential role in the down-regulationand habituation of HPA axis activity in response to repeated stress. It is therefore believedas an important regulator of the interaction under both physiologic conditions and stress.In former animal experiments, we found that Dexamethasone Acetate has anti-motionsickness effects on rats while has the side effect on weight increasing, while gensenosidescould decrease this side effects. Researchers found that gensenoisde Rg1is the functionalligand of the glucocorticoid that has the glucocorticoid-like effect,and it could enhance thelearning and memory ability of rats. Gensenoisde Re could decrease the intake of kaolininduced by cisplatin which is used for chemotherapy, it may imply that Gensenoisde Recould decrease the emesis caused by chemotherapy. ObjectiveThe aim of the present study was to determine whether DEX had an anti-motionsickness effect in rats and explore the mechanism of this action. And to study the effect ofDexamethasone Acetate combined with gensenoside Rg1,Re for anti-motion sickness,anti-fatigue and the ability of learning and study of the rats in accelerating condition.Methods1. The mechanisms of DEX`s effects on anti-motion sicknsson rats1.1AnimalsThirty-three male Sprague–Dawley (SD) rats aged6weeks and weighing190-240gwere purchased from Sino-British SIPPR/BK Lab Animal Ltd (Shanghai, China).Animals were housed in Plexiglas box cages in the colony room at an ambienttemperature of22±2℃with a12h light/12h dark schedule (lights on at8a.m.) andmaintained on an ad libitum schedule of food and water.Rats were randomly assigned to four groups: control group (n=8), where the animalsjust watched the acceleration apparatus and experienced the noise of the apparatus withoutexperiencing actual acceleration; acceleration model group (n=8), where the animalsreceived intragastric administration of water30min before acceleration exposure; DEXgroup (n=9), where the animals received intragastric administration of0.05mg/kg DEX30min before acceleration exposure, ensuring that the drug was completely absorbedaccording to the criteria defined by the National Institute for the Control of Pharmaceuticaland Biological Products (Beijing，China); AM-251/DEX group (n=8), where the animalsreceived intragastric administration of5mg/kg AM-251(Tocris, Ballwin, MO)15minbefore intragastric administration of0.05mg/kg DEX30min before acceleration exposure.Selection of DEX dosage and administration was based on our previous experiment.During the30-min rotation, rats were given no food or tap water.1.2Accelerating exposure and tissue preparation.1.2.1AnimalsRats in the acceleration model group, DEX group and AM-251/DEX group weresubjected to rotation. Each rat was enclosed in a cuboid plexiglass box which was suspended on a metal frame revolving around an axis parallel to the floor. It began to rotateat a constant angular acceleration of16°/s2in clockwise direction until the angular velocityreached120°/s2, then the box started to slow down at a constant angular deceleration of48°/s2.1s pause later, the box continued to rotate in counter-clockwise direction in thesame manner. The rotating procedure last30min. After the rotation, motion sicknesssymptoms were observed and recorded.1.2.2Observation of motion sickness symptomsBecause rats had no emetic reaction, motion sickness symptoms were observed afterrotation and recorded according to the motion sickness index (MSI) that reflects theseverity of gastrointestinal symptoms caused by motion stimulation (Yu, Cai et al.2007;Wei, Wang et al.2011). Evaluation criteria of MSI as follows: each fecal granule scored1and none scored0; urination scored1.2and none scored0; severe piloerection scored1.2,slight piloerection scored0.6, and none scored0; tremor scored1.2and none scored0(Yu,Cai et al.2007). MSI was calculated by the sum of all these scores.1.2.3Tissue and blood preparationAnimals in each group were anesthetized with10%0.3g/kg chloral hydrateintraperitoneally (i.p.) and blood was drawn by cardiac puncture, followed by transcardialperfusion of60ml chilled saline.5ml blood was collected by cardiac puncture, using aBD-Falcon vacutainer. Blood samples were centrifuged at3000rpm for20min at4℃, andthe plasma layer was collected and stored at80℃. The brain was immediately dissectedand cooled in iced saline for1min to remove the dorsal vagal complex (DVC) andstomach carefully. The dissected tissues were frozen in liquid nitrogen immediately andstored at80℃for further use.1.3Measurement of AEA and2-AG levels in plasmaAnalysis the plasma levels of AEA,2-AG in control group, acceleration model groupand dexamethasone group was performed on the LC/MS/MS system.1.4Determination of NAPE-PLD、DAGL-a、FAAH、MAGLmRNA levels in DVC and stomachReal-time quantitative PCR was performed using IQ5Real-Time PCR DetectionSystem to measure the expression levels of NAPE-PLD、DAGL-a、FAAH、MAGL mRNAin DVC and stomach. 1.5Determination of cannabinoid-1receptor（CB1R）mRNAand protein levels in in DVC and stomachReal-time quantitative PCR was performed using IQ5Real-Time PCR DetectionSystem to measure the expression levels of CB1R mRNA expression in DVC and stomach.CB1R protein levels in DVC and stomach were determined by Western blot.2. Studies on Dexamethasone Acetate combined withgensenoside Rg1, Re for strengthening the learning, memoryand the anti-fatigue ability of the rat in motion sickness2.1AnimalsFifty-four male Sprague–Dawley (SD) rats weighing250-300g were purchased fromSino-British SIPPR/BK Lab Animal Ltd (Shanghai, China). Animals were housed inPlexiglas box cages in the colony room at an ambient temperature of22±2℃with a12hlight/12h dark schedule (lights on at8a.m.) and maintained on an ad libitum schedule offood and water. The rats were randomly divided into8groups, control group (intragastricadministration of water1ml/100g), scoplamine group(intragastric administration ofscoplamine1mg/100g), acceleratione group (intragastric administration of water1ml/100g), Rg1,Re group(intragastric administration of Rg12mg/kg,Re0.5mg/kg),Dexamethasone Acetate group (intragastric administration of Dexamethasone Acetate0.05mg/kg), Dexamethasone Acetate with low, middle, high dosage of Rg1,Re group(lowdosage group, intragastric administration of Dexamethasone Acetate0.05mg/kg with Rg11mg/kg, Re0.2mg/kg; middle dosage group, intragastric administration of DexamethasoneAcetate0.05mg/kg with Rg12mg/kg,Re0.5mg/kg; high dosage group, intragastricadministration of Dexamethasone Acetate0.05mg/kg with Rg15mg/kg, Re1mg/kg).2.2Acceleration exposure and motion sickness index2.2.1AnimalsAcceleration group, scoplamine group, Rg1,Re group, Dexamethasone Acetate group,Dexamethasone Acetate with low, middle, high dosage of Rg1,Re rats were were subjectedto rotation. Each rat was enclosed in a cuboid plexiglass box which was suspended on ametal frame revolving around an axis parallel to the floor. It began to rotate at a constant angular acceleration of16°/s2in clockwise direction until the angular velocity reached120°/s2, then the box started to slow down at a constant angular deceleration of48°/s2.1spause later, the box continued to rotate in counter-clockwise direction in the same manner.The rotating procedure last30min. After the rotation, motion sickness symptoms wereobserved and recorded.2.2.2Observation of motion sickness symptomsThis part is the same as1.2.2.2.3Recording the body weights of ratsRemark rats`body weight for three days on9o`clock.2.4Rolling stick experimentAfter the acceleration exposure, rats were immediately placed on the rolling stickdevice and recorded the during-time on the rolling stick.2.5Observation of spontaneous locomotionAfter the acceleration exposure, rats were immediately placed to the spontaneouslocomotion device to record the the total distance and exploratory activity of spontaneouslocomotion for3minutes.3. Statistical analysesThe results are expressed as means±SD. Statistical analysis was carried out by usingSPSS16.0statistical software. One-way analysis of variance (ANOVA) and nonparametrictest were performed for the analysis of the biochemical indices. Differences wereconsidered significantly at P<0.05level.Results1. The mechanisms of dexamethasone`s effects onanti-motion sicknss on rats1.1. Comparison of motion sickness severity between the fourgroupsAfter acceleration exposure, MSI in acceleration group (MSI=12±3.31) was higherthan control group (MSI=3.2±1.0)(P<0.001). There was no significant difference in MSI between control group (MSI=3.2±1.0) and DEX group (MSI=4.4±1.36)(P>0.05).MSI inAM-251/DEX group (MSI=8.53±1.18) was lower than that in acceleration model group(MSI=12±3.31)(P<0.05), and higher than that in control group (MSI=3.2±1.0)(P<0.001)and DEX group (MSI=4.4±1.36)(P<0.001).1.2. Changes in plasma AEA and2-AG+1-AG concentrations inratsAfter acceleration exposure, AEA levels were similar in three groups, control group(7.60±1.07ng/ml), acceleration model group (6.19±0.98ng/ml) and dexamethasone group(7.39±1.92ng/ml), respectively. While plasma2-AG+1-AG level was decreased inacceleration model group (32.39±10.91ng/ml) as compared with control group(67±19.84ng/ml) and DEX group(54.28±20.28ng/ml)(P<0.05).1.3. Expressions of endocannabinoid enzymes (NAPE-PLD,FAAH, DGL-a and MAGL) responsible for biosynthesis anddegradation in the rat DVC and stomachIn DVC, NEPE-PLD mRNA expression was decreased in dexamethasone group (P<0.01), compared with control group. FAAH mRNA was increased in dexamethasone group,comparing with control group (P<0.01) and acceleration group (P<0.05). DGL-a mRNAexpression was decreased in acceleration group, comparing with control group (P<0.001)and dexamethasone group (P<0.01). MAGL mRNA expressions in acceleration group anddexamethasone group was decreased, comparing with control group (P<0.01; P<0.01).In stomach, NAPE-PLD mRNA showed no significant differences in three groups(P>0.05). FAAH mRNA expression in acceleration group was higher than control group(P<0.01) and dexamethasone group (P<0.05). DGL-a mRNA expressions in accelerationgroup and dexamethasone group were higher than control group (P<0.05; P<0.05). MAGLmRNA expression in acceleration group was higher than control group (P<0.01) anddexamethasone group (P<0.05).1.4. The effect of acceleration exposure and DEX on theexpression of CB1R message RNA and protein in the rat DVC andstomach The transcription of CB1R message in the DVC was decreased in acceleration modelgroup compared with control group (P<0.05), but it remained normal in DEX group.CB1R mRNA expression in the stomach was decreased in acceleration model groupcompared with control group (P<0.001), but DEX remained CB1R mRNA expression innormal. CB1R expression was further investigated by Western blot. The changes of proteinlevel of CB1R in the DVC and stomach were the same as the changes in mRNA levels.2. Studies on Dexamethasone Acetate combined withgensenoside Rg1, Re for strengthening the learning, memoryand the anti-fatigue ability of the rat in motion sickness2.1Motion sickness indexAfter acceleration exposure, the MSI acceleration model group was higher thancontrol group (P<0.001). Scopolamine, Dexamethasone Acetate, Dexamethasone Acetatecombined with low, middle and high dosage of gensenoside Rg1,Re decreased significantly,comparing with the acceleration group(P<0.01, P<0.01, P<0.01, P<0.01, P <0.05).2.2The effect on rats`s body weightBefore the day of experiments, on the day of experiments, after the day ofexperiments, after2days of experiments, the body weight of rats changed significantly.Dexamethasone Acetate influenced the body weight increasing, while DexamethasoneAcetate with middle dosage Rg1and Re lessened the side effect of sigle usingDexamethasone Acetate.2.3The duration on rolling stickAfter acceleration exposure, the duration on rolling stick in scopolamine group waslower than control and acceleration group (P<0.05, P<0.01). Dexamethasone Acetate withmiddle dosage Rg1and Re group was higher than control group, acceleration group andscopolamine group (P<0.001, P<0.01, P<0.001). Dexamethasone Acetate with high dosageRg1and Re group was higher than control group, acceleration group and scopolaminegroup (P<0.01, P<0.05, P<0.001).2.4The effects on spontaneous locomotionAfter the acceleration exposure, the total distance of spontaneous locomotion inacceleration group, Rg1and Re group, scopolamine group and Dexamethasone Acetate group were shorter than control group (P<0.001, P<0.01, P<0.001, P<0.05); the totaldistance of spontaneous locomotion in Rg1and Re group, Dexamethasone Acetate group,Dexamethasone Acetate with low, middle, high dosage Rg1and Re group was higher thanacceleration group(P<0.01, P<0.01, P<0.001, P<0.001, P<0.001) and scopolaminegroup(P<0.01, P<0.01, P<0.001, P<0.001, P<0.001). Comparing with control group,Dexamethasone Acetate with low, middle, high dosage Rg1and Re group had nosignificant difference(P>0.05).After the acceleration exposure, the exploratory activity of spontaneous locomotion inacceleration group, scopolamine group, Rg1and Re group were lower than in controlgroup(P<0.001, P<0.05, P<0.001); the exploratory activity of spontaneous locomotion inDexamethasone Acetate group, Dexamethasone Acetate with low, middle, high dosage Rg1and Re group was higher than acceleration group(P<0.001, P<0.001, P<0.01, P<0.01).Conclusion1. DEX exerted its anti motion sickness effect partly through modulating CB1receptor expression in the DVC and stomach, and regulating2-AG level in blood.2. Dexamethasone Acetate with middle dosage of Rg1and Re has the effect ofanti-motion sickness, anti-fatigue, strengthening ability of learning and memory andlessening the adverse effect of Dexamethasone Acetate on rats`normal increase of bodyweight.