| Objective: Taking 16-month-old C57BL/6J naturally aging mice as the research object,long-term low-intensity aerobic exercise intervention was carried out to detect the effect of aerobic exercise on learning and memory ability and hippocampal neuron structure of aging mice,and the regulatory effect of exercise on DAXX-ASK1-JNK pathway and its effect on hippocampal neuron apoptosis were studied through transcriptome sequencing and molecular biology experiments.Methods:(1)After 1 week of adaptive feeding,12 male C57BL/6J mice at the age of 4 months were included in the youth group(group C,12 mice/group),and 24 male C57BL/6J mice at the age of 16 months were randomly divided into two groups,namely,aging group(group A,12 mice/group)and aging exercise group(group E,12mice/group).(2)Group E mice were given aerobic exercise intervention at a speed of12m/min for 60 min,lasting for 8 weeks,60min/day,5 times/week.Mice in group C and group A were naturally fed for 8 weeks.(3)After 8 weeks,behavioral tests were carried out on three groups of mice,and samples were taken 24 hours after the end of behavioral tests.Three mice in each group were perfused and fixed with 4%paraformaldehyde for Nessler’s staining;Three mice in each group were perfused and fixed with 3% glutaraldehyde for transmission electron microscopy;The hippocampus of 3 mice in each group was taken for RNA-seq;The hippocampal tissue of 3 mice in each group was detected by Western blot.Results:(1)The data of the open field experiment showed that the total distance of the three groups of mice in the open field was not significantly different at the same time,suggesting that the autonomous activity ability of the mice in each group was similar.The time of activity in the central region of the three groups of mice was different.Compared with group C,the time of activity in the central region of group A mice was significantly shorter(P<0.05).Compared with group A mice,the time of activity in the central region of group E mice was longer,but there was no significant difference(P>0.05).The Barnes maze experiment was divided into learning stage and testing stage.The data showed that during the first day of learning,the latency of group C mice was the shortest,and the number of errors before finding the dark box was the least.Compared with group C,the latency and number of errors of group A and E mice were significantly increased(P<0.05),while the latency and number of errors of group A and E mice were not significantly different(P>0.05).In the second and third day of learning,the latency of mice in each group was shortened,and the number of errors was reduced.The latency of mice in Group C was the shortest.Compared with Group C,the latency of mice in Group A was significantly increased(P<0.05),and the latency of mice in Group E was significantly shortened(P<0.05).On the fourth day of learning,mice in each group could quickly find the dark box,and there was no significant difference between the latency and the number of errors between the groups(P>0.05).After 4 days of study,in the 90 s Barnes maze test on the fifth day,the average latency of mice in Group C was the shortest.Compared with Group C,the average latency of mice in Group A was significantly longer(P<0.01).Compared with Group A,the average latency of mice in Group E was significantly shorter(P<0.05).There was no significant difference in the number of errors of mice in each group before finding the dark box(P>0.05).(2)Nissl staining results: The neurons in the CA1 area of the hippocampus of mice in Group C and Group E have complete morphology and structure,uniform distribution,dense arrangement,rich Nessler body and deep staining.In group A,the structure of neurons in CA1 area of mice was not clear,the outline was fuzzy,the arrangement was scattered,loose,irregular,the staining was shallow,and the number of neurons was reduced.(3)The results of transmission electron microscope observation showed that the neuronal organelles in hippocampus CA1 area of group C mice were abundant,the mitochondria were oval,the mitochondrial cristae were clear,and the nuclear membrane was intact.In group A mice,apoptotic bodies were observed,with atrophy of the nucleus of neurons,swelling and vacuolation of mitochondria,blurred and broken mitochondrial cristae,and dissolution of peripheral nerve fibers.No apoptotic bodies were found in the hippocampal neurons of group E mice.Compared with group A,the morphology and structure of mitochondria were good,no vacuoles and swelling were found in mitochondria,and the organelles were abundant.(4)Transcriptome sequencing results: compared with Group A,9 genes in Group E were up-regulated and 6 genes were down-regulated.Compared with Group C,28 genes in Group A were up-regulated and 82 genes were down-regulated.The key genes of DAXX are closely related to the regulation of cell apoptosis.(5)Western blot detection of DAXX-ASK1-JNK pathway protein and downstream protein results: The expression of DAXX,ASK1 and JNK protein in hippocampus of group A mice was significantly higher than that of group C and E(P<0.05).The expression level of Csapase3 and Bax protein in group C and E was significantly lower than that in group A(P<0.05)and the expression level of Bcl-2 protein and the ratio of Bcl-2/Bax were significantly higher than those in group A(P<0.05).Conclusions:(1)Aging can lead to the decline of learning and memory ability of mice,the damage of neuron structure in hippocampus,and the increase of cell apoptosis.Exercise can improve the decline of learning and memory ability and neuron damage caused by aging,and reduce cell apoptosis.(2)Through transcriptome sequencing,it was found that the differential gene DAXX between Group A and Group E is closely related to cell apoptosis.Exercise may inhibit the DAXX-ASK1-JNK pathway and thereby reduce cell apoptosis by downregulating DAXX. |
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