| Spaceflight special environments such as microgravity and chronical sleep deprivation can cause muscle atrophy, osteoporosis, immune, and learning and memory ability decline. To complete a variety of fine complex manipulation tasks, the astronaut needs to keep a good cognitive state during space flight. Thus, the effects of spaceflight special circumstances on learning and memory ability are increasingly arousing people’s attention. But so far, the underlying mechanisms of the effects of spaceflight special environments on learning and memory ability are still largely unknown.In addition, effective countermeasures are also very lacking. Ginseng is a precious traditional Chinese medicine. A number of studies have demonstrated that ginsenosides, the main active ingredients of ginseng, have significant learning and memory ability-improving effects. Metabonomics is a new systems biology research method, the comprehension, sensitivity, and unbiased nature make it contributes to clarifying the mechanism of spaceflight special environments on learning and memory ability and the intervention targets of ginseng.The main contents and results of this thesis are shown as follows:1. The simulated microgravity rats model was established by hindlimb unloading and intervened by ginseng total saponins (GTS). During the 7-days tail suspension, the food and water intake, as well as the body weight of each rat were monitored daily. At the end of tail suspension, the learning and memory ability of the rats was evaluated by the shuttle box behavioral test. To determine corticosterone (CORT) level, compute the organ index, and perform toluidine blue-based Nissl body staining, plasma, the cortex, thymus, adrenal gland, spleen, and brain were collected. The results showed that, compared with the control group, hindlimb unloading resulted in significantly reduced body weight gain, significantly decreased learning and ability, markedly increased plasma CORT level, obvious thymus and spleen atrophy and adrenal hypertrophy, notably damaged hippocampus CA1 pyramidal neuron. Oral administration of GTS (100 mg/kg body weight) could significantly improve the learning and memory ability, reduce the elevated plasma corticosterone levels, relieve the hypertrophy of thymus and spleen and atrophy of the adrenal cortex, and protect hippocampus pyramidal neurons.2. An ultra performance liquid chromatography-quadrupole-time of flight mass spectrometry (UPLC-QTOF-MS) method for the determination of rat plasma, urine, and hippocampus was established. Firstly, the preparation methods of urine, plasma, and hippocampus were optimized. Secondly, the elution gradient program, injection volume, column temperature, capillary voltage, ion source temperature, cone voltage, temperature and flow of desolvation gas, precision, repeatability of the analytical method, and stability of the samples were optimized, and the optimal parameters of the UPLC-QTOF-MS method for the analysis of urine, plasma, and hippocampus were obtained.3. An UPLC-QTOF-MS based untargeted metabolomics was performed to study the effects of simulated MG on the metabolome of rats urine, plasma, and hippocampus. The obtained LC-MS raw data sets were firstly converted to.CDF file format. The converted data sets were then processed using R programming language package XCMS (version 1.38.0) and CAMERA (version,1.20.0) for peaks extraction, nonlinear retention time correction and metabolite peaks annotation. Then the resultant data sets were further performed multivariate statistical analyses including principal component analysis (PCA) and partial least-squares discriminant analysis (PLS-DA) using SIMCA-P software (version 11.5, umetrics, Sweden). The identified potential biomarkers by PLS-DA were then analyzed by ANOVA or Student’s t-test to identify more reliable biomarkers. The chemical structures of the resultant biomarkers were identified by online MS databases and MS/MS information of the raw data. Finally, the biological significances of the identified biomarkers were comprehensively elucidated. The results show that after being exposed to 7-days simulated microgravity, the metabolic profiles of the urine, plasma, and hippocampus were significantly changed. GTS treatment can significantly ameliorate these changes. Eleven, twelve, and seventeen biomarkers were identified in urine, plasma, and hippocampus, respectively. Important biomarkers include taurine, adenine, kynurenic acid, valine, choline, tryptophan, sphingosine. creatine, ornithine, hypoxanthine, and guanine, and involved metabolic pathways include amino acid metabolism, neurotransmitter metabolism, sphingosine metabolism, phospholipid metabolism, taurine and hypotaurine metabolism, purine and pyrimidine metabolism etc.4. Thirty-three metabolites originated from the metabolic pathways identified by untargeted metabolomics,8 hippocampus neurotransmitters (acetylcholine (Ach). homovanillic acid (HVA), epinephrine (E) and norepinephrine (NE), dopamine (DA), serotonin (5-HT), glutamic acid (Glu), and γ-aminobutyric acid (GABA)) were quantitatively determined by an ultra fast liquid chromatography tandem mass spectrometry (UFLC-MS/MS) method. Sixteen plasma amino acids were determined by a high performance liquid chromatography fluorescence detection (HPLC-FLD) method. According to the correlation matrix analysis, hierarchical cluster analysis, heatmap analysis, metabolic pathway analysis, metabolic enrichment analysis, ANOVA, and receiver operating characteristic curve (ROC) analysis, the results were interpreted and elucidated. The results showed that in the targeted validation studies,16,10, and 17 biomarkers in urine, plasma and hippocampus were identified. Of these biomarkers, tryptophan. kynurenine, lysine, and cystathionine were identified in both urine and hippocampus; phenylalanine, aspartic acid, and asparagine were identified both in plasma and hippocampus, and tyrosine and threonine were identified simultaneously in the 3 biological samples. GTS can restore most of the identified biomarkers to the normal direction. The results of ROC analysis showed that the simulated MG induced decline of the learning and memory ability was mainly mediated by the imbalance of the level of Tyr, Trp, Asn, kynurenic acid, Phe, Asp, choline, Val, and 3-hydroxykynurenine; The learning and memory ability-improving effects of GTS was mainly mediated by restoring the levels of Tyr, Trp, Asn, kynurenic acid. The results of neurotransmitters determination showed that, compared with the normal control group, the levels of 7 neurotransmitters except NE were significantly affected by simulated MG. The levels of Glu and 5-HT were significantly elevated, while the levels of other five neurotransmitters were markedly decreased. GTS can restore the levels of most neurotransmitter to the normal direction, especially Glu, Ach, and HVA. The results of amino acid determination show that, compared with normal control group, the levels of 10 amino acids (Thr, Ala, Met, Val, Leu, His, Iso, Asp, Phe, and Glu) were significantly changed. Of the 10 changed amino acids,9 were decreased except Glu. GTS can restore the levels of most amino acid to the normal direction, especially Phe.5. The molecular mechanisms of the effects of chronic sleep disturbance (CSI) on learning and memory ability of mice were studied using above established UPLC-QTOF-MS-based untargeted metabolomics method. This study combines the novel object preferences and temporal order judgment behavioral test, serum corticosterone (CORT), brain tissue malondialdehyde (MDA), superoxide dismutase (SOD) and catalase (CAT) level determination. The results showed that CSI could significantly reduce the discrimination index of mice in the new object recognition and temporal judgment test, increase serum CORT and MDA levels, and reduce the level of SOD and CAT. Multiple serum biomarkers were identified which is closely related to the decreased learning and memory ability, such as choline, valine, uric acid and allantoic acid, visual steroids, carnitine. Compared with the normal control group, significantly elevated levels of uric acid, allantoic acid and carnitine derivatives, and significantly decreased levels of visual steroidal compounds, choline, and valine were detected in the CSI mice.Firstly, this study using a combination of untargeted and targeted metabonomics methods, from the perspective of endogenous metabolites, systematically studied the mechansims of simulated microgravity on learning memory ability and the regulation targets of GTS. The results showed that the simulated microgravity could lead to a variety of endogenous metabolism pathways imbalance and learning and memory ability decline. GTS mainly through regulating the metabolism of amino acids, kynurenine, neurotransmitters and phospholipids to ameliorate the decreased learning and memory ability. This study helps to deepen the understanding of the underlying mechanisms of spaceflight special circumstances induced learning and memory ability damage, and provides scientific evidence for choosing ginseng as an effective countermeasure. |
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