Effect And Mechanism Of Echinacoside On Simulation Plateau Hypobaric Hypoxia-induced Cognitive Impairment In Mice

Background:China has the largest and the highest altitude plateau area,and the largest population in the world.Its border line is as long as 4,000 kilometers,and the military and strategic status of which is very important.The plateau has a special natural environment.As the altitude increases,the air pressure decreases and the oxygen partial pressure in the air also decreases.Human body's ability to adapt to low oxygen environment is comparatively weak,and easily suffers from plateau disease,which threatens the health and life safety of officers and soldiers entering the plateau from the plain.To develop the medicines for prevention and treatment of plateau diseases is an important requirement to ensure the combat effectiveness of plateau troops,as well as an urgent need to maintain border stability and national security.After entering the plateau area,hypobaric and hypoxia are the first environmental factors perceived by the body and will cause a series of physiological dysfunction and pathological changes,a series of non-specific clinical syndromes(including dyspnea,headache,insomnia,decreased attention,etc.),and acute mountain sick(AMS),including the life-threatening high altitude pulmonary edema(HAPE)and high altitude cerebral edema(HACE)as well as emotional and neurocognitive impairments will occur.As the core organ of the human body.The brain consumes a large amount of oxygen(equivalent to 20% of the total oxygen consumption of the human body).Hypoxia leads to abnormal metabolism of nerve cells and imbalance of cerebral vascular regulation,which directly damages neurons and thus triggers a series of neurological changes,such as insomnia,memory loss,cognitive decline,sensitivity to external reactions and behavioral abnormalities.In severe cases,it can lead to sleep respiratory dysfunction,even appear a symptom like agitated,convulsion,coma or ataxia.How to prevent and treat the brain damage caused by high altitude hypobaric hypoxia and its influence on mental activity has attracted many researchers' attention and become the frontier and hotspot of high altitude medical research.Especially considering the needs of military training,military decision-making,military operations and future wars,the prevention and protection of cognitive impairment of officers and soldiers in plateau special working environment is particularly important.At present,the prevention and treatment of brain cognitive impairment in high altitude hypobaric and hypoxic environment is mainly based on hypoxic acclimatization and clinical intervention of drugs(galantamine,Isradipine,Chinese herb extracts,etc.).However,at present,there are still lack of special drugs to improve the cognitive dysfunction at high altitude in China.Based on the above facts.It is urgent to seek new drug targets and develop related control drugs.Echinacoside(ECH)is a natural phenylethanoid glycosides compound separated from cistanche.Studies have shown that ECH can improve antioxidant level,and has anti-apoptosis effect on nerve cells,anti-inflammation,anti-tumor,bone protection and learning and memory improvement ability,as well as immune regulation,etc.ECH also has the effect of promoting the recovery of cholinergic neurotransmitter level in rat brain,improving the function of central cholinergic system,promoting protein and nucleic acid synthesis,and regulating immune status and improving learning and memory.The in-depth study on the brain protection effect and its mechanism in the special region of the plateau is expected to provide experimental basis for the search of new drugs with the ability to improve the plateau cognitive dysfunction from traditional Chinese medicine and natural products.It will provide a new target for the prevention and treatment of hypoxic brain injury at high altitude and a new idea for clinical treatment.Objectives:In this study,a low-pressure and hypoxic mouse model was made by using a low-pressure oxygen chamber to observe the protective effect of ECH on improving the cognitive function of mice.To investigate the effects and mechanisms of ECH on improving cognitive function in mice in hypoxia condition through the perspectives of behavior,brain tissue structure,oxidative stress and Keap1/Nrf2/ARE signaling pathway.At the same time,in order to provide experimental basis for the new clinical use of ECH,we need to further explore whether ECH pretreatment can alleviate oxidative stress injury in HT22 cells under hypoxia and whether Keap1/Nrf2/ARE signaling pathway mediates its protective effect through cytological study in vitro.Methods:1.Protection of ECH on hypobaric hypoxia-induced cognitive function impairment in C57 mice1.1 Morris water maze navigation training was conducted on C57 mice,the time to the platform(escape latency),route trajectory and average swimming speed were recorded.All groups except the Control group were placed in the low-pressure oxygen chamber for 7d.After the low-pressure and hypoxic experiment,the platform was removed to test the memory ability of each group of mice in space exploration,and the number of times the mice crossed the platform,the percentage of time they stayed in the target quadrant and the percentage of time they stayed in the target quadrant were observed and recorded.1.2 Animal behavior tests: recorded the number of horizontal and vertical activities of the mice within 120 s.After the hypobaric hypoxia experiment,the horizontal and vertical activities of mice in each group within 120 s were observed and recorded again.1.3 HE staining was used to observe the morphology of pyramidal cells in the hippocampal CA1 region of C57 mice in each group and to calculate and analyze the number of normal pyramidal cells.1.4 Hippocampal cell mitochondria in C57 mice of each group was observed and analyzed by use transmission electron microscopy and image collection.2.Mechanism of ECH on hypobaric hypoxia-induced cognitive function impairment in C57 mice2.1 The hippocampal tissue of mice was removed.The contents of SOD,CAT,GSH-Px and MDA were determined by taking appropriate amount of tissue homogenate.2.2 RT-PCR was used to detect the expression of Nrf2,HO-1,NQO1 and ?-GCS in hippocampus.2.3 Western blot was used to detect the protein expressions of Keap1,Nrf2,HO-1,NQO1 and ?-GCS in the hippocampus.2.4 Immunofluorescence(IF)was used to detect the ROS content and expression in the hippocampal tissue.The integral optical density(IOD)was calculated and analyzed.3.Protection of ECH on hypoxia-induced hypoxia impairment in HT22 cells3.1 The proliferation of mice hippocampal neuron cell line HT22 cells was detected after anoxic culture for 6h,and the number and morphology of cells were observed.3.2 The supernatant was collected after HT22 cells were lysed uniformly,and the activity of SOD,CAT,GSH-Px antioxidant enzyme and MDA content were detected.3.3 Western blot was used to detect the protein expressions of Keap1 and Nrf2 in HT22 cells.Results:1.Protection of ECH on hypobaric hypoxia-induced cognitive function impairment in C57 mice1.1 Morris water maze data showed that the results of positioning navigation training increased with the extension of training time.The swimming path and average swimming speed improved significantly.Hypobaric hypoxia significantly reduced the percentage of target quadrant stop time,the percentage of target quadrant stop path and the times of cross-platform of mice,while ECH and GBE significantly increased the percentage of quadrant stop time,the percentage of target quadrant stop path and the times of cross-platform in mice,and the effect was proportional to the concentration of ECH.1.2 The results of the first animal behavior test showed that there was no significant difference in horizontal and vertical activities among the groups,indicating the consistency of mouse behavior.The second animal behavior test showed that hypobaric hypoxia significantly increased the horizontal activity and decreased the vertical activity of mice.Whereas ECH and GBE significantly reduced the horizontal and increased the vertical activities of mice,and this effect was proportional to the ECH concentration.1.3 Results of HE staining confirmed that hypobaric hypoxia can result in mice hippocampal CA1 region pyramidal cells arranged loosely,and most of them were contracted and deeply stained.The number of pyramidal cells in the hippocampal CA1 region of each ECH group was abundant and closely arranged,and a very small number of pyramidal cells were contracted and hyperchromatic,and this protective effect was in direct proportion to the ECH concentration.1.4 Results of transmission electron microscopy showed that mitochondrial morphology in hippocampal cells of normal mice are mainly tubular with structure no change.Hypobaric hypoxia can result in several mitochondria swollen and dilated,showing loss of internal cristae structure.In ECH groups,there were several large but intact mitochondria with normal structure.The morphological analysis of the mitochondria showed that,compared with the mice exposed to low pressure and low oxygen,the higher the dose of ECH,the better the mitochondrial structure of mice.2.Mechanism of ECH on hypobaric hypoxia-induced cognitive function impairment in C57 mice2.1 Hypobaric hypoxia can result in the activity of SOD,CAT and GSH-Px in the hippocampal tissue of the mice significantly decreased,and the content of MDA significantly increased.In ECH groups,the activities of SOD,CAT and GSH-Px in the hippocampal tissues of the mice significantly increased,while the contents of MDA significantly decreased.2.2 RT-PCR results showed that hypobaric hypoxia can result in Nrf2 of the hippocampal tissue of mice increased and HO-1,NQO1,and ?-GCS decreased.In ECH groups,Nrf2,HO-1,NQO1,and ?-GCS significantly increased,and this protective effect was in direct proportion to the ECH concentration.2.3 Western blot results showed that hypobaric hypoxia can result in Nrf2 of the hippocampal tissue of mice increased and HO-1,NQO1,and ?-GCS decreased.In ECH groups,Nrf2,HO-1,NQO1,and ?-GCS significantly increased,and this protective effect was in direct proportion to the ECH concentration.2.4 The results of immunofluorescence showed that hypobaric hypoxia can result in the production of ROS in the hippocampus CA1 region,while ECH and GBE significantly inhibited the accumulation of ROS in the hippocampus CA1 region,with the increase in ECH concentration,this inhibition of ROS production in the CA1 region of hippocampal cells increases.3.Protection of ECH on hypoxia-induced hypoxia impairment in HT22 cells3.1 Through the proliferation of HT22 cells,it was found that hypoxic induction could reduce the activity of cells,cause obvious changes in cell morphology,and cause the cells to shrink,become round and leave the culture plate.In hypoxic culture,pretreated HT22 cells with ECH can inhibit the decrease of cell activity and the change of cell morphology.3.2 Hypoxic induction could reduce the activity of SOD,CAT and GSH-Px in HT22 cells and increase the content of MDA.The activities of SOD,CAT and GSH-Px in HT22 cells significantly increased and the contents of MDA significantly decreased in ECH groups,.3.3 Western blot results showed that hypoxic can result in Keap1 of HT22 cells significantly increased and Nrf2 slightly increased.In ECH groups,Keap1 of HT22 cells significantly decreased and Nrf2 significantly increased,and the ECH-H group has the most obvious effect.Conclusions:1.Ethological experiments showed that hypobaric hypoxia induced spatial cognitive impairment in C57 mice,and ECH has a protective effect on spatial cognitive impairment under hypobaric hypoxia.2.Morphological experiments showed that ECH has a good protective effect on mitochondria in hippocampal cells of C57 mice.3.Low oxygen space can cause damage on pyramidal cells in hippocampal of C57 mice,and ECH has certain protective effect on the injury.The possible mechanism may be related to it stimulating the Keap1/Nrf2/ARE pathway and at the same time improving the SOD,CAT and GSH-Px activity in hippocampus,reducing the content of MDA.4.ECH pretreatment is one of the effective strategies for alleviating the brain injury of the mice under hypobaric and hypoxia,which can provide a new idea for the discovery of innovative drugs for the prevention and treatment of cognitive impairment under hypobaric and hypoxia.