| Objective:To explore the regulation effect of the voluntary wheel-running exercise on miR-130 amediated autophagy in the process of natural brain aging.Methods:In animal experiment: Totally 30 male Sprague-Dawley(SD)rats of SPF grade with body weight of 160±20g.Among them,20 rats aged 10 weeks were randomly divided into old control group(OC)and old voluntary wheel running group(OVR)with 10 rats in each group.Before the experiment,all rats were given adaptive feeding for 2 weeks.Then,other rats were continuously housed to the age of 21 months old to build a natural aging model.The rats from of OVR group were housed for 8 weeks to 23 months in a cage with a running wheel and and the rats in group OC were naturally fed to 23 months of age without treatment.At the end of all experiments,another 10 3-month-old rats were taken as the young control group(YC),and all rats were drawn blood from eyeball and were decapitated.After 8 weeks voluntary wheel running,the hippocampus and cerebral cortex were separated on the ice and stored at-80? freezer immediately for further analysis.The changes of miRNAs in hippocampus were detected by Real-time-PCR.Tunel staining was used to evaluate the level of apoptosis in hippocampus,the changes of autophagosome in hippocampus were observed by TEM.The expression of aging-related proteins in the hippocampus of the Western blot test to verify the degree of aging,to detect the changes of autophagy and apoptosis related protein to evaluate the level of autophagy and apoptosis.In cell experiments: Cultured neuroblastoma cells(SH-SY5Y)cells were seeded in 6-well plates in about 70% confluence.The cells were divided into control group,D-gal induction group,miR-130 a transfected group(D-gal+miR-130 a mimics)and transfected control group(miR-130 a mimics).The cells in Con group were cultured normally.The D-gal+miR-130 a mimics group and miR-130 a mimics group were transfected with miR-130 a mimics at a final concentration of 20 n M.The cells in Con group were cultured normally.The D-gal+miR-130 a mimics group and the miR-130 a mimics group were transfected with miR-130 a mimics at a final concentration of 20 n M.After 4 to 6 hours of transfection,D-gal group and D-gal+miR-130 a mimics group were cultured with D-gal medium,and the miR-130 a mimics group was changed to normal culture medium.Cell samples were collected after 48 h,Western blot detection of aging-related proteins to verify the degree of aging,autophagy-related protein changes detected to assess the level of autophagy.Result:In animal experimen: 1)Real-time-PCR results showed that compared with young control rats,miR-130 a was significantly decreased(p<0.001)in hippocampus of rats in old control group and exercise could reverse this condition(p<0.001).2)TUNEL staining showed that compared with the young control group,the apoptotic rate of hippocampus in old control group was significantly increased(p<0.001),and then decreased significantly after the voluntary wheel-running exercise(p<0.001).3)The results of TEM showed that the number of autophagosome in the in old control group decreased significantly compared with the young control group,while the number of autophagosome in the hippocampus of the old voluntary wheel running group increased significantly more than that of that in the old control model group.4)Western blot results showed that voluntary wheel-running exercise can inhibit the expression of aging-related protein Ac-p53 and p21(p<0.001),and in the apoptosis,the expression of pro-apoptotic proteins Bax and caspase3 were increased(p<0.01,p<0.05)during natural aging in rats,and decreased after the voluntary wheel-running exercise.The autophagy-related proteins showed that the expression of Beclin 1,ATG7 and LC3 in old control group was significantly increased,(p<0.05,p<0.001,p<0.001),and there was a significant improvement after free runner exercise(p<0.001).While the negativerelated protein p62 significantly increased(p<0.01)and was significantly inhibited after voluntary wheel-running exercise(p<0.01),suggesting that motor activation of autophagy,increased autophagic lysosomal degradation.In addition,the expression of autophagy pathway related signal protein Sirt1 and p-AMPK(phosphorylation AMPK)were inhibited in the natural aging process of rats(p<0.001),whereas exercise could significantly up-regulate the phosphorylation of AMPK(p<0.001).In cell experiment: Western blot results showed that D-gal could induce the expression of aging-related p53(p<0.001),while p21 also increased.LC3 was also inhibited(p<0.001),and p62 p62 appeared to be ascending.However,overexpression of miR-130 a mimic in the aging cell model inhibited the acetylation of p53 and inhibited the expression of p21(p<0.001).At the same time,the expression of LC3 was also up-regulated(p<0.001)and the degradation of p62 was promoted(p<0.001).ConclusionsDuring the process of natural aging and cell senescence,miR-130 a regulates the process of brain aging by regulating apoptosis and autophagy.In addition,voluntary wheel-running exercise improves cell apoptosis by activating miR-130a-induced autophagy,protect neurons and ultimately delay brain aging process. |
PDF Full Text Request