| Motion sickness is induced by unusual acceleration, mainly resulting in signs and symptoms of the autonomic nervous system(e.g., nausea, vomiting, dizziness). The most dramatic form of motion sickness is seasickness. Although seasickness don't result in life-threatening, it makes people feel uncomfortable, increases physical exertion, causes fatigue, decreases people,s ability to perform job tasks, and also is an important reason for the accident at sea and is one of the main factors of non-battle casualties. Therefore, the mechanisms of motion sickness have attracted much attention, especially army. Though, Many investigators have studied on motion sickness, the precise etiology of it remains under investigation. Many theories (e.g.,the sensory conflict and neural mismatch theory; the"toxin detector"hypothesis; the neurotransmitter hypothesis; the referred visceral discomfort hypothesis) were used to explain the development of motion sickness, however no one can elucidate fully.The changes of serum metabolites can be induced by stress. Studies have shown that a stress response exists in the deveplopment of motion sickness, however the changes of metabolites in motion sickness is not clear. Blood metabolite levels can reflect molecular physiological changes and may be a useful tool for clarifying the underlying molecular mechanisms of physiological changes and the pathophysiology of diseases.ObjectiveBased on the analysis of changes of both metabolites and stress hormones involved in metabolic regulation, the animal experiment was adopted to observe whether above metabolic and hormonal changes could affect the tolerance of human body to acceleration providing new insights that elucidate the etiology and pathogenesis of motion sickness that are induced by repetitive acceleration.Methods1. Metabolite changes in serum before and after exposure to repetitive acceleration between the MS and NMS groups1.1 Subjects and Ethics Statement60 healthy male volunteers were recruited from students at the Second Military Medical University. Each subject provided informed consent, and the study protocol was approved by the Ethics Committee of the Medical Faculty of Second Military Medical University, China. The subjects were informed not to eat high purine foods or drink carbonated beverages from 24 hours before the trial until the experiment was finished. Subjects were asked not to consume any food from 18:00 h before (after dinner the night before the trial) until after the study was completed. The subjects were also instructed to sleep at 21:00 h.1.2 Exposure to AccelerationAfter resting blood pressure, heart rate, height, weight and forehead temperature were measured, the subjects were seated in chairs fixed in a 6-degree-of-freedom ship motion simulator (length: 3 m, width: 4 m, height: 2.5 m). The subjects were divided into 4 batches and were exposed to repetitive vertical acceleration for 15 min after adaptation for 5 min (the acceleration was 0.27g using a sine function with a frequency of 0.26 Hz ). The experiment was performed from 9:00 h until 10:30 h. The room temperature was 15.2°C, and the cabin temperature was 18.0°C. After the end of the acceleration exposure, subjects who developed nausea and vomiting were classified as the motion sickness (MS) group, and the others who did not develop nausea and vomiting were classified as the no motion sickness (NMS) group.1.3 Sample Preparation and Spectral AcquisitionBlood samples were collected using BD Vacutainer tubes (Becton Dickinson Medical Devices, Shanghai) immediately before and after exposure to repetitive acceleration. The plasma was separated by centrifugation at 3000 g for 20 min, and the supernatant was collected and stored at -80°C. Internal standards (10μL of L-2-chlorophenylalanine in water, 0.3 mg/mL; 10μL of heptadecanoic acid in methanol, 1 mg/mL) were added to each 100μL serum sample. After shaking the serum, 300μL of a methanol and ethyl chloroform mixture (methanol: ethyl chloroform, 1:3 v/v) was added to the mixed solution to precipitate protein. All samples were shaken and stored at -20°C for 10 min, then centrifuged and 10,000 g for 10 min. The 300μL supernatant was injected into a glass sampling vial to evaporate to dryness under vacuum at room temperature. After 80μL methoxyamine at a concentration of 15 mg/mL in pyridine was added to the residue, the solution was placed at 37°C for 90 min. 80μL BSTFA ( 1% TMCS) was added to the solution, which was maintained at 70°C for 60 min for analysis. Samples (injection volume: 1μL) were injected into an Agilent 6890N gas chromatograph coupled to a Pegasus HT time-of-flight mass spectrometer (GC-TOF/MS) (Leco Corporation, St. Joseph, MI, USA). The GC-TOF/MS was operated at a constant flow rate of 1.0 ml/min using helium as the carrier gas in full scan mode (m/z 30-600) with the injection, transfer, interface temperatures the ion source temperature set at 270°C, 260°C and 200°C, respectively. The temperature of the GC oven was initially held at 80°C for 2 min, raised to 180°C at 10°C /min, raised to 230°C at a constant rate of 6°C /min, raised at 40°C /min to 295°C and, finally, maintained for 9 min.1.4 Data Reduction and Pattern RecognitionRaw data were converted to the NETCDF format by Databridge (Perkin-Elmer Inc., U.S.A.) and processed by MATLAB (MathWorks, Inc.). Baseline corrections, peak discrimination and alignment, internal standard exclusion, and normalization to the total sum of the chromatogram were executed before the three-dimensional matrix including the arbitrary compound index (paired retention time-m/z), sample names (observations), and normalized peak areas (variables) were imported to SIMCA-P 12.0 Software (Umetrics, Ume?, Sweden). Principle component analysis (PCA), an unsupervised projection multivariate method, was used to overview serum GC-TOF/MS data from the subjects pre- and post-exposure. Orthogonal partial least-squares-discriminant analysis (OPLS-DA) was subsequently used to determine the most significant metabolites between subjects pre- and post-exposure using the VIP value (VIP>1). The VIP value is a computation of the influence of every x term in the model on the y variable. Larger VIP values suggest a greater influence of the x term on the y variable. Differential metabolites from the OPLS-DA model were also validated with univariate analysis using both a parametric test (paired sample t-test) and a nonparametric test (Wilcoxon signed rank test) with a significance level of 0.05. A paired sample t-test or the Wilcoxon signed rank test was employed to determine whether the levels of metabolites in the MS and NMS groups respectively obtained from the OPLS-DA model were different between subjects pre- and post-exposure. An independent samples t-test or the Wilcoxon rank sum test test with a significance level of 0.05 was adopted as the cutoff to determine different metabolites derived from the OPLS-DA model between MS group and NMS group, during the pre- and post-exposure phases respectively. 2. Comparisons of blood stress hormones of metabolic regulation between MS and NMS groups2.1 Measurements of serum insulin, glucagon and cortisol concentrations using RIA Serum insulin, glucagon and cortisol concentrations were measured using radioimmunoassay kits for each hormone (Beijing, North Institute of Biological Technology Co).2.2 Measurement of serum epinephrine concentrations using ELISA Epinephrine levels were analyzed by immunoenzyme assay (commercial kits epinephrine-ELISA, ZYMO RESEARCH, China).3. Effect of blood glucose levels change on the severity of motion sickness in rats3.1 AnimalsMale Sprague-Dawley (SD) rats (n=36) weighing 250-300 g were purchased from Sino-British SIPPR/BK Lab Animal Ltd (Shanghai, China). All animals were housed individually under controlled conditions (lighting: 8:00-20:00, and temperature: 22±2°C) and fed with standard rat chow and tap water ad libitum. The animal experiment was approved by the Animal Use and Care Committee for Research and Education of Second Military Medical University (Shanghai, China).100 rats were exposed to acceleration, and the motion sickness index (MSI) of each rat was recorded. The rats that were sensitive to motion sickness ( MSI was greater than 9) were selected for experimental use. After deadaption for 2 weeks, the selected rats were randomly divided into three groups as follows: the acceleration model group (n=14), which were exposed to acceleration; the insulin group (n=14), which were injected insulin intraperitoneally (1 unit/kg) 30 min before exposure to acceleration; and the control group (n=8).3.2 Exposure to AccelerationAcceleration model and insulin groups were put into the rotation device. The detailed rotation methods were as described as Yi-Ling Cai's report. Each rat was enclosed in a cuboid plexiglass box which was suspended on a metal frame revolving around an axis parallel to the ?oor. It began to rotate at a constant angular acceleration of 16°/s~2 in clockwise direction until the angular velocity reached 120°/s~2, then the box started to slow down at a constant angular deceleration of 48°/s~2. A 1 s pause later, the box continued to rotate in counter-clockwise direction in the same manner. The rotating procedure last 30 min.The control group were put into the device but not rotated. After the rotation, motion sickness symptoms were observed and recorded. All rats were sacrificed by decapitation immediately. The plasma was separated by centrifugation at 3000 g for 20 min at -4°C, and the supernatant was collected and stored at -80°C.3.3 Motion sickness symptoms measurementEvaluation criteria for motion sickness index was followed: per fecal granule was scored 1, none was scored 0; urination was scored 1.2, none was scored 0; severe piloerection was scored 1.2, light piloerection was scored 0.6, none was scored 0; tremor was scored 1.2, none was scored 0. Motion sickness index was calculated by the sum of all scores.3.4 Blood glucose levels DeterminationThe levels of blood glucose were measured using full-featured blood glucose meter (ACCU. Check, Roche, German).3.5 Hormone DeterminationThe methods of measurement of animal insulin, glucagon, corticosterone and epinephrine were the same as for human.4. Statistical AnalysisChanges in hormone concentrations of human before and after exposure to repetitive acceleration were analyzed by using the paired samples t-test or the Wilcoxon signed rank test. Differences in hormone concentrations of human between the two groups during pre- and post-exposure phases were analyzed using an independent samples t-test or the Wilcoxon rank sum test. And differences in hormone, glucose concentrations and motion sickness index of rats were analyzed among three groups using one-way analyses of variance (ANOVA) of SPSS statistical program, followed by LSD post hoc tests. The critical p-value was set at 0.05. Results1. Metabolite changes in serum after exposure to repetitive acceleration between the MS and NMS groups1.1 The incidence of motion sicknessAfter exposure, 30 Subjects who developed nausea and vomiting were classified as the MS group, and the others who did not develop nausea and vomiting were classified as the NMS group.1.2 Metabolic variations induced by repetitive accelerationA total of 35 key metabolites were significantly perturbed as a result of repetitive acceleration. The levels of Inosine, Hypoxanthine, Urea, Glycolic acid, L-Alanine, L-Histidine, L-Isoleucine, L-Lysine, L-Methionine, L-Ornithine, L-Phenylalanine, L-Proline, L-Serine, L-Threonine, L-Tryptophan, L-Tyrosine, L-Valine, Creatinine, 3-Hydroxyisobutanoic acid, 4-Hydroxy-L-proline, Pyroglutamic acid and Carbodiimide, Fumaric acid, L-2-Aminobutyric acid decreased wheras the levels of n-Dodecanoic acid, n-Tetradecanoic acid, Oleic acid, Linoleic acid, Palmitic acid, Elaidic acid, Stearic acid,β-Hydroxybutyric acid, Arachidonic acid, Glucose and Pyruvic acid increased.1.3 Metabolite changes in serum after exposure to repetitive acceleration between the MS and NMS groupsTo search for potential physiological or physiopathological differences between the two groups, we compared the directions of alteration (increased or decreased concentration) of the 35 metabolites identified using the OPLS-DA model. There were 26 metabolites including glucose and pyruvic acid, with the same alteration tendency in both groups. Interestingly, 9 metabolites did not exhibit the same direction of alteration between the two groups. Serum urea concentrations decreased in the NMS group but did not change in the MS group. The concentrations of L-Histidine, L-Ornithine, L-Serine, L-Tyrosine, Pyroglutamic acid and Fumaric acid decreased in the MS group but did not change in the NMS group. Serum concentrations of n-Dodecanoic acid and n-Tetradecanoic acid were elevated in the MS group but did not change in the NMS group. 1.4 Variations of metabolites in serum between the MS and NMS groups during the pre-exposure phaseAmong the 35 metabolites identified using the OPLS-DA model, we found 19 that were differentially expressed between the MS and NMS groups prior to exposure. Among these differentially expressed metabolites, higher levels of L-Tryptophan, L-Isoleucine, L-Lysine, L-Methionine, L-Valine, L-Threonine, L-Histidine, L-Ornithine, L-Phenylalanine, L-Serine, L-Tyrosine and Pyroglutamic acid were observed in the MS group, whereas the levels of Elaidic acid, Linoleic acid, Hypoxanthine, Creatinine,Carbodiimide, 3-Hydroxyisobutanoic acid and Pyruvic acid were higher in the MS group than in the NMS group.1.5 Variations of metabolites in serum between the MS and NMS groups during the post-exposure phaseDuring the post-exposure phase, elevated levels of L-Isoleucine, L-Methionine, L-Ornithine, Creatinine, Hypoxanthine, Pyruvic acid, 4-Hydroxy-L-proline, Glucose, Oleic acid and Urea were observed in the MS group relative to the NMS group.Interestingly, the levels of serum 4-hydroxy-L-proline, glucose, oleic acid and urea were higher in the NAV group than in the NNV group after exposure but not prior to exposure, however only the elevation of glucose levels in the MS group was higher than that in the NMS group.2. Comparisons of blood stress hormones of metabolic regulation between MS and NMS groupsSerum cortisol and epinephrine levels increased (respectively) by 30% (P<0.001) and 24% (P<0.001) in the MS group and by 26% (P<0.001) and 18% (P<0.001) in the NMS group. The insulin level decreased by 33% (P<0.01) in the MS group and by 17% (P<0.01) in the NMS group. Prior to exposure, the level of insulin in the MS group was lower than in the NMS group (P<0.05). The glucagon/insulin ratio was elevated after exposure to acceleration in both groups. Both the glucagon concentration and the glucagon/insulin ratio were significantly higher in the MS group than in the NMS group before (P<0.01) and after (P<0.05) exposure.3. Effect of blood glucose levels change on the severity of motion sickness in ratsSerum insulin levels in the insulin group was 3.88 times higher than in the acceleration model group (P<0.001), while 2.57 times higher than in the control group (P<0.001). Serum glucose levels in the insulin group was lower significantly than in both the acceleration model (P<0.001) and the control groups (P<0.001). Motion sickness index in the insulin group was also lower markedly than in the acceleration model group (P<0.05). After exposure, serum corticosterone and epinephrine levels in the insulin group was respectively 1.49 (P<0.01) and 5.21 (P<0.001) times higher than in the control group.Glucagon levels were not significantly different among three groups. However, the glucagon/insulin ratio was significantly lower in the insulin group than in both acceleration model (P<0.001) and control groups (P<0.001)Conclusions1. A total of 35 key metabolites invovled in purine metabolism, amino acid metabolism, fatty acid metabolism and glucose metabolism were significantly perturbed as a result of repetitive acceleration.2. 9 metabolites which included 4 glycogenic amino acids (L-histidine, L-ornithine, L-serine and L-tyrosine ), 2 fatty acids (n-dodecanoic acid and n-tetradecanoic acid), urea, pyroglutamic acid and fumaric acid,didn't exhibit the same direction of alteration between the two groups.3. Serum glucose level increased more in the MS group than that in the NMS group. Acute hyperglycemia during the acceleration may be related to gastrointestinal symptoms in motion sickness.4. After exposure, cortisol, epinephrine levels and the rate of glucagon/insulin increased, wheras insulin concentrations decreased in both two groups;Prior to exposure, insulin levels in the NMS group were higher than that in the MS group, wheras the rate of glucagon/insulin in the MS group was higher than that in the NMS group.5. It suggested increased insulin levels in rats prior to exposure release the elevation of blood glucose levels during the exposure, and relieve gastrointestinal symptoms in motion sickness .Above all, it hints higher insulin levels in the NMS group before exposure could suppress the elevation of serum glucose, relieving gastrointestinal symptoms, the precise mechanisms of which are under investigation.
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