The Protection Of Several Polyphenolson On Memory Of Rats Under Hypobaric Hypoxia And The Underying Molecular Mechanism

Background and aims: On the plateau, air pressure and oxygen partial pressure decrease as altitude became higher and higher. Hypobaric hypoxia could have deleterious effects on cognitive functions, such as short-term memory, attention span and the ability of thinking and judgement of human beings and consequently harm normal life, working and studying. As the economy and society in plateau region develop, the cognitive detrimentis more and more prominent. From a military perspective, rapidly movement and trans-regional strategic projection is more and more regular, but advancing forces have no time to take adaptive training on plateau area and consequent decrease of information work ability and serious altitude stress would weaken the battle effectiveness. Therefore, it is extremely significant to develop the medicine and provide biomedical measure that could alleviate cognition impairment and promote cognitive adaptation for military medicine in the new era. Memory is important for cognition. Although the underlying mechanism of memory injury under hypobaric hypoxia is a puzzle, mitochondrial homeostasis disorderand synapse damage are the important element. Therefore, how to ameliorate mitochondrial homeostasis disorder and synapse damage may be a effective measure for alleviating memory injury under hypobaric hypoxia. Phytochemicals, which are derived from plants, have various kinds of biological functions. Among various phytochemicals, polyphenols have aroused broad concern and research and been proved to have anti-inflammatory, anti-microbial, anti-oxidant, and anti-carcinogenic as well as hepatoprotective effects. Some studies demonstrated polyphenols could improve some kinds of diseases and promote a quick recovery, including protecting neurons from hypoxia-ischemia brain injury or neurodegenerative disorders. Polyphenols might alleviate hypobaric hypoxia induced neuron injury through protecting mitochondrial quantity, structure and function in neurons.We speculated that polyphenols might protect hypobaric hypoxia-induced memory impairment through exerting biological functions. Therefore, the present study aim to investigate polyphenols that could protect memory ability from hypobaric hypoxia and discuss the effects and underlying mechanism of polyphenols on higher brain functions of memory through regulating mitochondrial homeostasis and synapse in rats.Methods: In present study, the rats were exposed continuously to HH equivalent to 5000 m for 1, 3, 7 and 14 days in a decompression chamber. Spatial memory was tested by Morris water maze. We applied the exposure time-7 days to create the rat model of memory impairment under hypobaric hypoxia to conduct the further experiments. Next, rats received resveratrol(RSV), quercetin(QUE), dihydromyricetin(DHM) and myricetin(MYR) daily(50, 75 or 100mg/kg.bw/d) via gavage during the period of exposure for hypobaric hypoxia, respectively. Cognitive performance was assessed by the Morris water maze test and indicated QUE and DHM could alleviate memory impairment induced by HH. Then we investigated the biological mechanism of QUE and DHM on memory ability under hypobaric hypoxia in rats. With transmission electron microscope, we observed the protective effect of QUE and DHM on mitochondria and synaptic structure of neurons; Furthermore, we used quantitative real-time PCR(Qrt-PCR), enzyme linked immunosorbent assay and western blot(WB) to investigate the effect of QUE and DHM on mitochondrial quantity, the activity of mitochondrial respiratory chain complexes and the expression of proteins related with mitochondrial biogenesis, dynamics and synapse; With HT-22 cell model of hypoxia by exposure to a mixture of ultra-high purity gases(5 % CO2, 92%N2) in an automatic multi-gas incubator, we investigated the effect of DHM on mitochondrial homeostasis and synaptic plasticity via SIRT3 signaling.Results: 1. After 7 and 14 days of exposure to the simulated high altitude of 5000 m, Morris water maze test indicated the spatial memory of rats was significantly impaired compared to the rats of normoxia group. And there is no significant difference in the decrease of spatial memory of rats between 7 days group and 14 days group. 2. Morris water maze test showed both QUE100(100mg/kg.bw/d) and DHM100(100mg/kg.bw/d) could significantly improve the spatial memory of rats under HH. Inaddition, we did not observed resveratrol and myricetin significantly influence the spatial memory of rats under HH. 3. The observation of transmission electron microscope showed QUE100 and DHM100 could significantly ameliorated hippocampus mitochondrial and synaptic lesions induced by hypobaric hypoxia. Qrt-PCR results showed that QUE100 and DHM00 could markedly increase hippocampus mitochondrial DNA copy number in rats. In addition, activity of oxidative respiratory chain complexes were detected, and we found complex II, complex IV and complex V activity was protected following administration of QUE100, and complexⅠ, complex II, complex IV and complex V activity was protected following administration of DHM100. Furthermore, WB detections indicated QUE100 could up-regulate the expression of SIRT1, PGC-1α, the proteins related with mitochondrial biogenesis(Nrf1 and TFAM) and the proteins related with mitochondrial fusion(Mfn1 and Mfn2), but down-regulate the expression of the proteins related with mitochondrial fission(Fis1 and Drp1). Moreover, QUE100 increased expression of fibronectin type III domain-containing protein 5(FNDC5) and brain-derived neurotrophic factor(BDNF), showing the PGC-1/FNDC5/BNDF pathways might be involved in neuronal adaptation exerted by QUE100. Meanwhile, WB results showed DHM100 could up-regulate the expression of PGC-1α and TFAM, which are involved in mitochondrial biogenesis, and synaptic markers PSD95 and SYP protein in rats. Furthermore, we found DHM100 reduced MDA level in hippocampal neurons. Moreover, IP analysis showed DHM could increase SIRT3 expression in a dose-dependent manner and synchronously reduce the level of FOXO3 acetylation. 4. To investigate the role of SIRT3 in the change of FOXO3 acetylation, we applied SIRT3 si RNA on HT-22 cells. As a result, the level of FOXO3 acetylation was significantly increased upon SIRT3 knockdown, suggesting that DHM-induced FOXO3 deacetylation occurs via SIRT3. Upon SIRT3 or FOXO3 knockdown, mitochondrial reactive oxygen species production reduced by DHM was significantly increased, indicating that mitochondrial reactive oxygen species production was inhibited via SIRT3 deacetylating FOXO3. This was confirmed by measuring intracellular reactive oxygen species levels with DCFH-DA. DHM exerted similar effects on the levels of MDA, a marker of lipid peroxidation. An ELISA-based kinase activity assay results showed that DHM couldimprove the activity of complexes I, IIin HT-22 cells, but the effect was reversed upon SIRT3 or FOXO3 knock-down. Similar results were observed for ATP level, indicating that the effects of DM on mitochondrial function are exerted via SIRT3 deacetylating FOXO3. A WB analysis of HT-22 cell lysate revealed that SIRT3 or FOXO3 knockdown suppressed DHM-increasing PGC-1α and TFAM levels and the relative mt DNA copy number under hypoxic conditions. Furthermore, upon SIRT3 knockdown, the increase in SYP and PSD95 expression induced by DHM was suppressed. Taken together, these results indicate that DHM could protect hippocampal neurons against HH-induced neurodegeneration and memory impairment by promoting mitochondrial biogenesis and synaptic function via SIRT3/FOXO3 signaling.Conclusion: 1.The present study create the rat model of memory impairment under hypobaric hypoxia successfully and found the spatial memory of rats was markedly impaired after 7 days’ exposure to simulated high altitude of 5000 m. 2. To studythe effects of four common polyphenols on rat model of memory impairment under hypobaric hypoxia, we found both QUE and DHM could obviously reduce memory injury of rats under hypobaric hypoxia. However, RSV and MYR could not markedly reduce memory injury of rats under hypobaric hypoxia. 3. Both QUE and DHM can effectively alleviate mitochondrial homeostasis disorder and hippocampal neuronal synapse damage. 4. QUE could clearly regulate mitochondrial dynamics and increase BDNF expression in the hippocampus.SIRT1/PGC-1α/Nrf1/Tfam pathway are involve in the regulation of QUE on mitochondrial homeostasis and PGC-1α/FNDC5/BDNF pathway plays a role in the protection of QUE on hippocampal neuronal synapse damage. 5. DHM treatment could induce SIRT3 to deacetylate FOXO3 to alleviate mitochondrial homeostasis disorder, including enhancing mitochondrial biogenesis and restoration of mitochondrial function, diminish oxidative stress by reducing reactive oxygen species generation in hippocampal neurons, ameliorate synaptic structure andultimately protect neuronal function. In summury, the present study investigated the effects of four common polyphenols on rat model of memory impairment under hypobaric hypoxia and the underlying mechanism.Our findings suggest new idea for understanding the mechanism of memory impairment after exposure to hypoxia and provide new way for the research on effective biomedical measure for altitude sickness.