| With the development and progress in the field of science and technology, manned spaceflight, challenge for astronauts to spend long time is big problem. The special environment in which astronauts stay, including microgravity, noise, change of circadian rhythms and narrow space, will have influence on the physiology and psychology of astronauts, thus affecting their work ability. To make sure the healthy safe and efficient work of astronauts in long period spaceflight, relative research has been made hotspot and difficulties in the astromedicine. At present, effects of complex aerospace environment to the body and brain function of astronauts are relatively lagged. For the abovementioned question and need of future development of aerospace, research on the complex aerospace environment to the brain function of astronauts are very meaningful and important.This study was designed to investigated the two basic research aspects in space. On one way, simulated microgravity rats model was designed to monitor the physiology and biochemistry parameters of their brain using 18 O labeled quantitative proteomics technology to identify the diffierentially expressed protein between the model group and control group.The effect of complex environment such as microgravity, noise, change of circadian rhythms and narrow space was induced to estimate the changes in hippocampus of brain cognition and emotion function. Furthermore, 18 O labeled and unlabeled “SWATH quantitation” technology, was used to investigate the effect of the complex environment at protein level in rat hippocampus. Following are the main achievement in our study,1. A medium and long term simulated microgravity rats model was successfully built and assessed. A rat tail suspension model(30o) was used to explore the effects of 21 days of prolonged simulated microgravity(SM). Compared with the control, the food-intake of SM group decreased 0.51% and the body weight of SM group increased 0.75%,which can simulate the real microgravity effectively and be considered as a successful model. To determine whether the SM animals show the major depression symptom anhedonia, the sucrose consumption levels in both the groups were measured. There are no significantly difference between the two groups. We apply the open field test to examine the motivation level and the anxiety. Compared with the control group, SM groups showed a significant decrease in locomotion and rears of open field activity. The central stop has the same tends. While, SM groups showed significantly increased in grooming and fecal pellet. These data suggested that SM groups have reduced interest in moving and show anxiety. To determine whether the SM animals show the major depression symptom despair, the immobility time in both the groups were measured. There are no significantly difference between the two groups.Furthermore,SM groups showed significant increase in the lipid peroxidation product(malondialdehyde,MDA) and H2O2 compared to the control group. Superoxide dismutase is significantly decreased compared to the control group, which shows oxidative stress of medium and long term simulated microgravity rat’s hippocampus.2. From the level of protein, we confirmed that medium and long term simulated microgravity caused oxidative stress damage and make disordered metabolism. A comparative 18O-labelled quantitative proteomic strategy was used to analyze the differential expression of rats hipocampus proteins. A total of 679 proteins were identified as having a spectrum score greater 15 and an FDR of 1% at the protein level. Forty-two proteins exhibited significant changes; 21 were up-regulated, and 21 were down-regulated. These differentially expressed protein are mostly enriched in(1) damage of oxidative phosphorylation(mitochondrial complex I、III、IV downregulated)(2) damage of TCA cycle( isocitric acid dehydrogenase and malic acid dehydrogenase downregulated)(3)oxidation disorder of fatty acid(long chain fatty acid CoA associated subunit 6 upregulated、long chain acetyl- CoA dehydrogenase 、acetyl- CoA synthetase family protein 2 and acylamide hydrolase downregulated). At the same time, antioxidation protein DJ-1 and peroxiredoxin 6 are upregulated, which may be correlated with cellular compensatory. Western blot analysis of proteins DJ-1 and COX 5A confirmed the mass spectrometry results.3 SM took effects on the neurotransmitter release and elimination of rats hippocampus. Excitatory neurotransmitter glutamate is increased and inhibitory neurotransmitter is decreased, which maybe toxic to neurons. A comparative 18O-labelled quantitative proteomics strategy was applied to detect the differential expression of rats hippocampus cellular membrane protein. A total of 873 proteins were identified as having a spectrum score greater 15 and an FDR of 1% at the protein level. Fifty-three proteins exhibited significant changes; 31 were up-regulated, and 22 were down-regulated. These differentially expressed protein are mostly enriched in(1) interference of SNARE formation(esicle-associated membrane protein 3(VAMPA3) and syntaxin-1A downregulated)(2) affection of synaptic vesicle recycling(syntaxin-binding protein 5(tomosyn), rab3 A downregulated and its effector rim2 upregulated)(3)affection of elimination and release of glutamate in synaptic cleft(Glial glutamate transporter 1(GLT1) downregulated, vesicular glutamate transporters(VGluTs), VGluT1 and VGluT2,upregulated)(4) affection of synthesis of GABA(Glutamate decarboxylase 2(GAD 65) and glutamate decarboxylase 1(GAD 67), downregulated). Proteomics results showed that elimination of glutamate and synthesis of GABA are influenced, while overexpressed excitatory transmitter glutamate and overweak inhibitory transmitter both caused the neuron damage. Thus, through multiple reaction monitoring(MRM), we analyzed the level of neurotransmitter in rats brain, and we found that the concentration of Glu was 101.60 μg/mg wet tissue in the rat hippocampus under SM, compared to 85.29 μg/mg in the control, indicating remarkable enhancement, while the concentration of GABA was 27.69 μg/mg wet tissue in the rat hippocampus under SM, compared to 37.64 μg/mg in the control, a dramatic drop. The results are same with change of glutamate transporter and glutamate decarboxylase and these changes all indicated SM effects on the neurotransmitter release and elimination of rats hippocampus. Western blot analysis of protein PSD-95 confirmed the mass spectrometry results.3 A simulated spaceflight complex environment rats model(SSCE)was successfully built and assessed. We employed the tail suspension model with noise(80 db), circadian rhythms(1.5 h) and limited space to simulate spaceflight complex environment. Compared with the control, the food-intake of SSCE group decreased 4.2% and the body weight of SSCE group increased 5.9%. Forced swim test(FST), sucrose consumption test and open field test(OFT) were applied to determine wheteher the SSCE animals show the major depression symptom. Compared with the control groups, SSCE groups showed significantly decrease in sucrose consumption levels. Moreover, SSCE groups showed a significant decrease in locomotion and rears of open field activity. The central stop has the same tends. While, SSCE groups showed significantly increased in grooming and fecal pellet. In addition, SSCE groups showed significantly increased in immobility time compared with the control groups.5.Under the simulated spaceflight complex environment, rats hippocampus mitochondrial energetic metabolism showed disorder. A comparative 18O-labelled quantitative proteomics strategy was applied to detect differential expression of the mitochondrial proteins in simulated spaceflight complex environment rats model. A total of 515 proteins were identified as having a spectrum score greater 15 and an FDR of 1% at the protein level. Eighty-nine proteins exhibited significant changes; 12 were up-regulated, and 77 were down-regulated. These differentially expressed protein are mostly enriched in(1) oxidative phosphorylation(mitochondrial complex I、III、IV downregulated) and(2) the TCA cycle(pyruvate dehydrogenase complex and aconitic acid synthase downregulated).6. Simulated spaceflight complex environment took effects on the process of endoplasmic reticulum protein and transportation of synaptic vesicle. Excitatory neruo transmitter glutamate is increased and inhibitory neurotransmitter is decreased, which maybe toxic to neurons. Using unlabeled “SWATH quantition” technology, a total of 4250 proteins were identified as having an FDR of 1% at the protein level. 1112 proteins exhibited significant changes, 812 were up-regulated and 300 were down-regulated. Sec 61 and sec 62 are involved in the process of endoplasmic reticulum protein. AP180、AP2 kinase、dynamin-1 and clathrin-light chain are involved in the transportation of synaptic vesicle. PSD-95、neurexin 2 and neuroligin-1 are involved in the synaptic transmission. Through multiple reaction monitoring(MRM), we found that the concentration of Glu was 154.38 μg/mg wet tissue in the rat hippocampus under simulated spaceflight complex environment, compared to 85.68 μg/mg in the control, indicating remarkable enhancement, while the concentration of GABA was 15.42 μg/mg wet tissue in the rat hippocampus under simulated spaceflight complex environment, compared to 36.44 μg/mg in the control, a dramatic drop. These changes all indicated simulated spaceflight complex environment did harm to rats hippocampus. Western blot analysis of protein GSK-3 β confirmed the mass spectrometry results.7. Simulated spaceflight complex environment had more significant effect on rats brain nervous system than SM. This conclusion resulted from the behavior、change of hippocampus mitochondrial protein、cellular membrane protein and neurotransmitters.In all, our research focused on the future need of development of manned spaceflight. In order to enhance the adaptability and decision ability of astronauts, using two level simulated conditions, respectively solo-simulated gravity and simulated spaceflight complex environment, we built rats models to simulate influence of spaceflight complex environment on mental behavior. And in combination with proteomics technology, we explored the molecular mechanisms of the spaceflight complex environment on mental behavior. This research will help in construction of future design of spaceflight complex environment and support the research on the protective measures to astronauts. This research is also meaningful to the pathogenesis of mental disorder on earth. |
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