| Objective: Qinghai-Tibet Plateau,an average elevation of more than 4000 meters,known as the "roof of the world ".However,due to the thin air,low atmosphere,strong ultraviolet light and other factors,the natural environment in most plateau areas is very harsh.Acute exposure to high altitude hypoxic environment,there is a risk of disease.A series of physiological and pathological changes will occur in the human body under the influence of environmental factors such as high altitude acute hypoxia,among which acute hypoxic environment is the most important factor leading to these changes.The brain is one of the most important oxygen-consuming organs in the body,and it has high sensitivity to hypoxia damage in acute hypoxia state,especially the damage of hippocampus structure and function is the most obvious.Therefore,how to prevent this pathological injury through drugs is a hot topic in brain science of high altitude medicine.Methods:1.To establish Model: male SD rats,from the altitude of 2250 m through 5 hours of rapid advance to the altitude of 4200 m site to establish a rat high altitude acute hypoxia hippocampal injury model.Through Morris water maze experiment to detect learning and memory ability;through HE staining,Nissl staining to observe the changes of hippocampal histopathology;transmission electron microscope to observe the ultrastructure changes of hippocampal neurons;to detect the expression of SOD、GSH-Px and MDA in the brain horse;TUNEL method to detect the apoptosis rate of hippocampal neurons in rats.2.The protective mechanism and optimal dose screening of rat hippocampus injury model with high altitude acute hypoxia through SIRT1/PGC-1α pathway regulation: male SD rats were randomly divided into five groups: low altitude control group(2250 m),acute high altitude hypoxia group(4200 m),low dose acute +hypoxia group(25mg/kg/d),medium dose +acute hypoxia group(50 mg/kg/d)and high dose +acute hypoxia group(100 mg/kg/d).Crocin was injected intramuscularly once a day in crocin group,and daily intramuscular injection of the same volume of 0.9% Nacl in the control group.After 3 days of continuous administration,rats were transported to high altitude environment(Gande County,Guoluo Prefecture,Qinghai Province,4200 m)for 72 hours.Morris water maze test to detect learning and memory ability;HE staining,Nissl staining and transmission electron microscopy to observe hippocampal histopathological changes;immunohistochemistry,RT-PCR、Western blot detection of hippocampal SIRT1、PGC-1α、TFAM、caspases-3、Bcl-2、Bax expression;detection of mental horse SOD、GSH-Px and MDA expression and detection of neuronal apoptosis in rats.3.To verify the protective effect of crocin(100mg/kg/d)pretreatment on the model rats in high altitude acute hypoxia for 1,3,5 and 7 days and the related molecular mechanism: establish hypoxia model group and crocetin group(100mg/kg/d),Morris water maze test was used to detect the learning and memory ability of high altitude hypoxia for 1,3,5,7 days;He staining,Nissl staining and transmission electron microscopy to observe the pathological changes of hippocampus;the expression of SIRT1,PGC-1α,TFAM,caspases-3,Bcl-2 and Bax were detected by immunohistochemistry,RT-PCR and Western blot;the levels of SOD,GSHPx and MDA in hippocampus of each group at 1,3,5,7 days of high altitude hypoxia were detected by kit,and the apoptosis level of neurons in hippocampus of each group at 1,3,5,7 days of high altitude hypoxia was detected by TUNEL method.4.To verify the protective effect of crocin on HT22 cells under hypoxia and the molecular mechanism through SIRT1/PGC-1α pathway:(1)to establish(1%O2)hypoxic HT22 cell injury model;(2)CCK-8 method was used to detect the effect of crocin(0.001,0.1,0.1,1,10,100μmol/L)on HT22 cells in 12 and 24 hours of hypoxia(1% O2);,and the optimal dose range was determined.(3)the protective molecular mechanism of crocin on HT22 cells in hypoxia condition: normal oxygen control group,hypoxia(1% O2)group,SIRT1 inhibitor(EX-527,100μmol/L)+ hypoxia(1% O2)group,crocin 25μmol/L + hypoxia(1% O2)group,crocin 50μmol/L + hypoxia(1% O2)group,crocin100μmol/L + hypoxia(1%O2)group,the ultrastructural changes of HT22 cells were observed by transmission electron microscope;SIRT1,PGC-1 α,TFAM,Caspases-3,Bcl-2 and Bax expressions were detected by Western blot and RT-PCR;SOD,GSH-Px and MDA expressions were detected by kit and flow cytometry was used to detect the apoptosis rate.Results:1.The model of high altitude acute hypoxia hippocampal injury model was established successfully.Morris water maze experiment showed that the latency and distance of the model group increased significantly(P < 0.05),while the percentage of target quadrant decreased(P < 0.05);the results of histopathology showed that the neurons in the hippocampus of the model group were disorganized,the nucleus was shrunk,there were local eosinophilic neurons,and the IOD value of Nissl staining of neurons in the CA1 area of the hippocampus was significantly reduced(P < 0.05);The results of transmission electron microscopy showed that the structure of neurons in CA1 area of hippocampus in the model group was not clear,the number of mitochondria decreased significantly,the mitochondria were highly swollen and the cristae were damaged seriously;the activity of GSHPx and SOD decreased significantly in the model group(P <0.05),and the content of MDA increased significantly(P <0.05);TUNEL staining showed that the number of TUNEL apoptosis positive cells in CA1 area of hippocampus in acute high altitude hypoxia group was significantly higher than that in control group(P < 0.05).2.Crocin(25mg/kg/d,50mg/kg/d,100mg/kg/d)pretreated by SIRT1/PGC-1α pathway had a dose-dependent protective effect on rats in the model group.Morris water maze experiment showed that the latency and distance of saffron pretreatment group decreased significantly(P < 0.05),while the percentage of target quadrant increased(P<0.05),showing a dose-dependent effect(P <0.05);the results of histopathology showed that he staining showed that crocetin pretreatment group could significantly reduce the pathological damage of hippocampal neurons in rats with acute high altitude hypoxia for 72 hours,Nissl staining showed that the IOD value of hippocampal CA1 neurons in rats increased significantly(P <0.05),transmission electron microscopy showed that crocin pretreatment group returned to normal nuclear structure of hippocampal CA1 neurons,mitochondrial swelling The results showed that the mitochondrial ridge structure tended to be normal in a dose-dependent manner;Crocin pretreatment could significantly increase the expression of GSHPx and SOD,and decrease the content of MDA(P < 0.05).Meanwhile,crocin pretreatment could significantly reduce the number of apoptosis positive cells(P <0.05),showing a dose-dependent effect(P < 0.05);Crocin pretreatment could significantly up regulate the expression of SIRT1,PGC-1 α,TFAM,bcl-2 m RNA and protein,down regulate the expression of Bax and caspase-3(P < 0.05),showing a dose-dependent effect(P < 0.05);the results showed that crocetin(100mg/kg/d)had the best effect.3.The protective effect of crocin(100mg/kg/d)on hippocampal neurons of rats exposed to high altitude acute hypoxia for 1,3,5 days was significant,which was related to the molecular protective mechanism of SIRT1/PGC-1α pathway.Morris test showed that compared with hypoxia model group,the learning and memory ability of crocin pretreatment group was significantly improved on the 1,3 and 5 day of acute high altitude hypoxia(P < 0.05);through morphological observation,it was found that the acute high altitude hypoxia environment led to the disorder of nerve cell arrangement in the hippocampus of the rat brain,with local soluble necrosis,mitochondrial edema and structural damage,especially on the 1,3 and 5 days,and the intervention of crocin also significantly reduced the damage of neurons and mitochondria;Compared with the control group,crocin pretreatment group significantly increased the relative expression of SIRT1,PGC-1 α,TFAM,Bcl-2 in m RNA and protein level(P < 0.05),decreased the expression of Bax and caspase-3(P < 0.05);at the same time,crocin pretreatment can significantly increase GSH-Px and SOD activity and reduce MDA content in hippocampus of rats in 1,3,5 days of hypoxia group;crocin pretreatment significantly reduced the apoptosis of hippocampal neurons in rats at 1,3,5 days of acute high altitude hypoxia(P < 0.05).4.The protective effect of crocin pretreatment on HT22 cells under hypoxia is related to SIRT1/PGC-1α pathway molecular protection mechanism.The transmission electron microscope showed that the cytoplasmic structure of HT22 cells in O2% hypoxia model group was loose at 12 and 24 hours,the nuclear membrane was shrunk,the mitochondria swelling was obvious,the mitochondrial ridge disappeared,and the damage was more serious at 24 hours than at 12 hours,and the damage was more obvious in SIRT1 inhibitor + hypoxia group;In the crocin + hypoxia group,the nuclear structure of HT22 gradually returned to normal,the swelling of mitochondria and endoplasmic reticulum decreased,and the structure of mitochondria ridge tended to normal,showing a dose-dependent effect;Compared with the control group,the expression of SIRT1,PGC-1α,TFAM,bcl-2 protein and m RNA decreased significantly in the hypoxia group and SIRT1 inhibitor + hypoxia group at 12 and 24 hours(P <0.05),while the expression of Bax,caspase-3 protein and m RNA increased significantly(P <0.05),and the inhibition effect of inhibitor + hypoxia group was more significant than that of hypoxia group(P <0.05);the expression of SIRT1,PGC-1α,TFAM,Bcl-2 protein and m RNA in hypoxic group decreased significantly,while Bax,caspase-3 protein and m RNA decreased significantly(P < 0.05),in a dose-dependent manner(P < 0.05);compared with the control group,SOD,GSHPx activity decreased,MDA expression increased(P<0.05),and SOD,GSHPx activity decreased at 12 and 24 hours in hypoxic group and inhibitor+ hypoxicgroup(P <0.05)The inhibition effect of inhibitor + hypoxia group was more obvious than that of hypoxia group(P<0.05),while the activity of SOD and GSHPx in each crocin group(25,50,100 μmol/L)+ hypoxia group was significantly increased,MDA was decreased,and the expression was dose-dependent(P <0.05);compared with the control group,the apoptosis rate of hypoxia group and inhibitor + hypoxia(1% O2)group was significantly increased in 12 and 24 hours(P <0.05),and the apoptosis rate of inhibitor + hypoxia group was significantly higher than that of hypoxia group(P <0.05),but the apoptosis rate of crocin+hypoxia group was significantly decreased(P<0.05),which was dose dependent Conclusion:1.The model of hippocampal injury in high altitude acute hypoxic rats was successfully established.2.Crocin(25mg/kg/d,50mg/kg/d,100mg/kg/d)pretreatment can significantly improve the cognitive dysfunction and pathological damage of hippocampal neurons in rats with high altitude acute hypoxic brain damage.Through regulating the expression of SIRT1/PGC-1α pathway related factors,crocin can play a role in anti mitochondrial damage,anti oxidative stress and anti apoptosis,showing a dose-dependent effect.3.Crocin(100mg/kg/d)pretreatment significantly improved the learning and memory ability of rats on the 1,3 and 5 day of high altitude acute hypoxia,significantly reduced the pathological damage of hippocampal neurons,and also played the role of anti mitochondrial damage,anti oxidative stress and anti withering by regulating SIRT1/PGC-1α pathway.4.Crocin can reduce the mitochondrial structure damage of HT22 cells in 12 and 24 hours under the condition of 1%O2 by regulating the expression of SIRT1/PGC-1α pathway related factors,and play the role of anti oxidative stress and anti apoptosis. |
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