| Research purposes:Alzheimer’s disease（Alzheimer’s disease,AD）is a recessive,age-dependent,cognitive impairment characterized by progressive neurodegenerative disease,there is no cure at present.Although there are more and more studies on the etiology and pathogenesis of AD,the etiology of AD is still inconclusive.at present,the main pathogenesis of AD are β-amyloid deposition,Tau protein phosphorylation,neuroinflammation,oxidative stress,mitochondrial dysfunction,autophagy disorder and other mechanisms have been proposed to promote the occurrence and development of AD.In the field of autophagy,a large number of studies have shown that adenylate-activated protein kinase（Adenosi A 5’-monophosphate（AMP）-activated protein kinase,AMPK）is a key molecule in the regulation of biological energy metabolism.It is expressed in a variety of metabolism-related organs and can be activated by various stimuli of the body,including cellular pressure,exercise,many hormones and substances that can affect cell metabolism.It can also affect the process of autophagy through multiple intracellular pathways.Activation of AMPK expression directly inhibits the production of rapamycin target protein（mammalian target of rapamycin,mTOR）in mammals,which in turn activates Unc-51-like kinase 1（Unc-51 li KE autophagy activating kinase-1 ～ ULK1）to form AMPK/mTOR/ULK1 pathway,which induces autophagy and accelerates A β clearance to alleviate AD symptoms.At present,the existing research is mainly through drugs to treat AD,and developed several new drugs to treat AD,but their therapeutic effect on AD patients is very limited,the best strategy for AD is to prevent and delay the development.A large number of studies have shown that exercise can improve brain function,improve cognitive ability and delay the occurrence and development of AD,and some studies have shown that exercise may enhance autophagy by activating AMPK signal to accelerate the clearance of A β,improve learning and memory ability,and alleviate AD disease,but the specific mechanism is not clear,and compared with drug research,the research of exercise therapy is still less.Therefore,this study established AD model rats by intraperitoneal injection of D-galactose,tried to use treadmill exercise to intervene AD model rats through AMPK/mTOR/ULK1 signal pathway,and explored the effect of treadmill exercise intervention on learning and memory ability of AD model rats,as well as the regulation and molecular mechanism of AMPK/mTOR/ULK1 signal pathway in AD,hoping to provide a new target for the treatment of AD.Research method: In this study,48 SD male healthy rats were randomly divided into4 groups with 12 rats in each group: normal control group（NC group）,normal exercise group（NCE group）AD model group（NE group）and AD exercise group（KE group）.The NE and KE groups were injected intraperitoneally with D-galactose at the dose of 300mg/（kg ·d）every morning,while the NC and NCE groups were injected with the same dose of normal saline as the model exercise group.Exercise intervention for the NCE group and KE group,and treadmill training for the NCE group and KE group on the afternoon of each training day（4:00-6:00）.Every Monday,Tuesday,Wednesday,Friday,and Saturday are training days.Four and Sunday were rest periods for a total of 8 weeks.The rats in the NC and NE groups were placed on the stationary treadmill for the same time,and the slope was zero.Morris water maze experiment was carried out after eight weeks of exercise intervention,mainly five-day positioning navigation experiment and one-day space exploration experiment to test the learning and memory ability and space exploration ability of rats.48 rats were cut off from water and food after the end of Morris water maze experiment.24 hours later,the rats were weighed,anesthetized and then sampled.Six of the twelve rats in each group were perfused and fixed,dehydrated,transparent,embedded and sectioned,and stained with HE for histological examination to observe the structure of hippocampal neurons.The other 6 rats were decapitated and the hippocampus was stored in cryopreservation at-80 ℃ for molecular biological detection.The protein contents of AMPK,ULK1,mTOR and LC3 were detected by Western-blot,and the activity of superoxide dismutase（SOD）and the content of malondialdehyde（MDA）were detected by enzyme labeling method.Research results:1)the results of the five-day positioning navigation experiment showed that with the progress of the positioning navigation experiment,the time of finding the platform was gradually shortened and the escape latent ability increased,especially in the NCE group,and the other three groups also shortened in varying degrees.It can be seen from the table that the second day,the third day and the fourth day of the positioning navigation experiment were shortened obviously,and the shortening was not obvious on the fifth day.The overall results of the navigation experiment were as follows: NCE group < NC group < KE group < NE group.2)the number of times of crossing the virtual platform in the spatial exploration experiment of the four groups was NCE group > NC group > KE group > NE group,and the result of target quadrant stay time was NCE group > NC group > KE group > NE group.3)histological results showed that the neurons in hippocampal CA1 region of NC group and NCE group had high cell density,regular arrangement,similar size and morphology,and complete structure.Compared with NC group,neurons in hippocampal CA1 region of NE group had nuclear condensation and pyknosis,a large number of dead cells,disordered arrangement of neurons,decrease in number and incomplete structure,increased gap between neurons and inconsistency in size.In KE group,the nuclei of neurons in hippocampal CA1 region were slightly condensed and pyknotic,with a small number of dead cells,a relatively chaotic arrangement of neurons,a small number of cells and a relatively complete structure,and a slight increase in the gap between neurons.There was no difference in neurons in hippocampal CA1 region between NCE group and NC group.Compared with NE group,the number of dead cells in KE group decreased,the structure of neurons arranged neatly,the morphology and structure of neurons were relatively complete,and the gap between cells decreased.4)the expression of AMPK,mTOR and ULK1 protein in hippocampus of rats was detected by Western Blot assay.The results showed that compared with NC group,the expression of AMPK protein in NE group decreased significantly,while the expression of AMPK protein increased in NCE group.There was no significant difference in AMPK protein expression between KE group and NC group.The expression of ULK1 protein in NE group was significantly lower than that in NCE group,while the expression of ULK1 protein in KE group was not significantly different from that in NC group,while mTOR protein was significantly increased in NE group,decreased in NCE group and decreased in KE group.Compared with NE group,the expression of AMPK protein in KE group increased significantly,the expression of ULK1 protein increased significantly in KE group,and the expression of mTOR protein decreased significantly in KE group.5)the results of SOD,MDA and GSH-PX showed that the activity of SOD in NCE group was the highest,followed by NC group and KE group,and the lowest in NE group.The activity of MDA was opposite to that of SOD.The results of SOD activity:compared with NC group,SOD activity decreased significantly in NE group,SOD activity decreased in KE group,SOD activity increased in NCE group,and it increased significantly in KE group compared with NE group.MDA concentration results: compared with NC group,MDA concentration in NE group and KE group increased significantly,MDA concentration decreased in NCE group,but the difference was not statistically significant（P > 0.05）,and MDA concentration in KE group was significantly lower than that in NE group.Research conclusions:1)8-week moderate-intensity treadmill exercise intervention can effectively improve the learning ability and memory ability of AD model rats,and delay the morphological changes of hippocampal neurons.2)8-week moderate-intensity treadmill exercise can activate AMPK/mTOR/ULK1 pathway,induce hippocampal neuron autophagy and improve autophagy dysfunction in AD rats.3)8-week moderate-intensity treadmill exercise can increase the activity of SOD and decrease the concentration of MDA in AD model rats,reduce the oxidative stress of AD model rats and improve the pathological symptoms of AD. |
