| Objectives:Chronic hypoperfusion cerebral ischemia causes brain tissue damage and brain atrophy,leading to vascular cognitive impairment(VCI).This study explores the effects of aerobic exercise on the spatial learning and memory function of rats with chronic cerebral ischemia,as well as the changes in the structure and function of hippocampal synaptic plasticity,and reveals its differentially expressed micro RNAs(miRNAs)through hippocampal gene high-throughput sequencing,providing experimental basis for aerobic exercise to interfere with VCI.Methods:(1)36 SPF healthy male Sprague-Dawley(SD)rats were divided into sham operation group(n=12)and operation group(n=24)by random number table.The bilateral common carotid artery occulusion(2-VO)was duplicated to establish a rat model of chronic cerebral ischemia.In the sham operation group,only the blood vessels were separated without occlusion.After the operation,small animal magnetic resonance imaging 3D time-of-fight(3D-TOF)angiography was used to detect whether the bilateral common carotid artery occlusion is successful.(2)The rats in the operation group were randomly divided into 2 groups according to the random number table,the model group and the exercise group,with 12 rats in each group.Then,aerobic exercise training was carried out,using a treadmill device,the slope was set to0°,the treadmill speed was set to 15 m/min,1 hour a day,5 days a week,and lasted for 4weeks.The sham operation group and the model group only grasped under the same conditions without intervention.(3)After aerobic treadmill,Morris water maze was performed to test the spatial learning and memory function of rats in each group;Golgi staining was used to observe the number and morphological changes of hippocampal synapses and dendritic spines in rats,and electrophysiological was used to detect long-term potentiation(LTP)in hippocampus of rats;Western blotting(WB)was used to detect the expression of glutamate receptor protein and immediately early genes(IEGs);miRNA high-throughput sequencing was used to detect the differential expression of miRNAs,then target gene prediction and signal transduction pathway enrichment analysis were performed,Quantitative Real-time PCR(q RT-PCR)was used to verify the expression level of miRNAs,WB was used to detect the level of protein related to downstream signaling pathways.Results:(1)The effect of aerobic treadmill exercise on the spatial learning and memory function of chronic cerebral ischemia model rats: In the learning phase,compared with the sham operation group,the escape latency of the model group was significantly increased(P<0.001),compared with the model group,the escape latency in the exercise group was significantly reduced(P<0.05,P<0.01,P<0.001).In the test phase,compared with the model group,the number of crossing platform and the percentage of target quadrant duration in the exercise group were significantly increased(P<0.001),and there was no significant difference in average swimming speed among the three groups.(2)The effect of aerobic treadmill exercise on the dendritic spine density and LTP of hippocampus in rats with chronic cerebral ischemia: Golgi staining results showed that compared with the sham operation group,the dendritic spine density in the hippocampus was significantly reduced in the model group(P<0.001).Compared with the model group,the dendritic spine density increased significantly in the exercise group(P<0.001).The results of electrophysiological showed that compared with the sham operation group,the field excitatory postsynaptic potential(f EPSP)slope of hippocampal in the model group was decreased significantly after 100 Hz single pulse stimulation(P<0.001).Compared with the model group,the f EPSP slope of hippocampus was significantly increased in the exercise group(P<0.001).(3)The effects of aerobic treadmill exercise training on synaptic plasticity-related proteins and immediate-early gene proteins related to learning and memory in chronic cerebral ischemia model rats: WB results showed that the expression levels of N-methyl-D-aspartate receptor 1(N-methyl-D-aspartate receptor 1,NMDAR1)and subtypes of NMDAR2 A and NMDAR2 B in the model group were higher than those of the sham operation group(P<0.05,P<0.01),and the expression level ofα-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor 1(AMPAR1)had a downward trend(P=0.104).The phosphorylation levels of NMDAR2 A,NMDAR2B and AMPAR1 were all markedly reduced(P<0.05);compared with the model group,the hippocampal expression levels of NMDAR1,NMDAR2 A,NMDAR2B,and AMPAR1 in the exercise group increased significantly(P<0.05),the phosphorylation levels of NMDAR2 A,NMDAR2B,and AMPAR1 also increased significantly(P<0.05,P<0.01).In addition,the expression level of c-Fos,early growth response factor 1(Egr1)and activity-regulated cytoskeletal(Arc)in the hippocampus of model group was significantly lower than that of the sham operation group(P<0.05,P<0.01,P<0.001).And compared with the model group,the expression of c-Fos,Egr1 and Arc in the hippocampus of the exercise group was significant increased(P<0.01).(4)The effect of aerobic treadmill exercise on the miRNAomics of chronic cerebral ischemia model rats: The results of hippocampus miRNA high-throughput sequencing analysis of the three groups showed that compared with the sham operation group,there were15 miRNAs with differential expression ≥1.5 times in the hippocampus of the model group(P<0.05),among which 11 were up-regulated and 4 were down-regulated;compared with the model group,there were 12 miRNAs with differential expression ≥ 1.5 times in the hippocampus of the model group(P<0.05),of which 7 were up-regulated and 5 were down-regulated.Then q RT-PCR was used to verify the sequencing results of miRNAs,and the results showed that the expression levels of miR-144-3p,miR-144-5p,miR-1188-5p,miR-130b-3p,miR-155-5p,miR-34b-5p,miR-15b-3p and miR-27b-5p differed significantly.Among them,the expression levels of miR-144-3p,miR-144-5p,miR-155-5p,miR-130b-3p and miR-15b-3p were significantly different in the model group and exercise group.(5)The effect of aerobic treadmill exercise on the signal pathway of chronic cerebral ischemia model rats: The target gene prediction and signal transduction pathway analysis of the miRNAs differentially expressed in the hippocampus of the three groups revealed that the mammalian target of rapamycin(mTOR)signal pathway(P<0.05)was closely related to the synaptic plasticity.Through online website analysis and literature review,this study found that the target genes of miR-144-3p,miR-155-5p and miR-130b-3p were related to the Phosphatase and tensin homolog deleted on chromosome ten(PTEN)/Phosphoinositide 3-kinase(PI3K)/ Protein kinase B(PKB/AKT)/ mTOR signaling pathway.(6)The effect of aerobic treadmill exercise on the mTOR signaling pathway of chronic cerebral ischemia model rats: WB results showed that compared with the sham operation group,the expression level of PTEN in the hippocampus increased significantly in the model group(P<0.05),the expression level of PI3Kα had a downward trend(P=0.239),and the expression levels of PI3Kβ,p-AKT/AKT and p-mTOR/mTOR had a significant decrease(P<0.05).Compared with the model group,the expression level of PTEN in the hippocampus was significantly reduced in the exercise group(P<0.05),the level of PI3Kαhad a rising trend(P=0.337),the expression levels of PI3Kβ,p-AKT/AKT and p-mTOR/mTOR were significantly increased(P<0.05,P<0.01).Conclusion:This study suggests that aerobic treadmill exercise may affect the expression levels of hippocampal miR-144-3p,miR-155-5p,and miR-130b-3p,target and regulate PTEN expression levels,activate downstream PTEN/PI3K/AKT/mTOR signaling pathway,and enhance synaptic plasticity,thus improving spatial learning and memory function. |
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