Swimming Attenuates D-galactose-induced Brain Aging Via MiR-34a-mediated Autophagy

Objective:To explore the regulatory role of miR-34 a in D-gal-induced brain aging during exercise intervention.Methods:1.Totally 30 male Sprague-Dawley(SD)rats with the age of 8 weeks old were randomly divided into three groups including normal control group(Control),D-gal-induced aging model groups(D-gal),and D-gal treatment combined with swimming intervention group(D-gal + EXE)with 10 rats in each group.After adapting to new environment for 1 week,the rats from D-gal + EXE group were subjected to 10 min swimming training without any loading.Meanwhile,a programmed increase in swimming time of 10 min was provided during each-training period.Once the swimming training reached up 60 min,the rats will provided with 60 min swimming training once a day for 6 consecutive weeks.The rats from C group were administrated with distilled water,and the rats from other groups were subjected to subcutaneous injection of D-gal at the daily dose of 200 mg/kg for 6 consecutive weeks.After swimming training for 6 consecutive weeks,Morris water maze was used to evaluate hippocampus-dependent spatial learning and memory capacity of D-gal-induced aging rats.All rats were sacrificed under anesthesia after the last MWM for 24 h.Blood samples from the rats were collected in coagulation tubes,and serum was separated by centrifugation at 1400 ×g at 4 °C for 10 min.Hippocampal tissues were collected and isolated immediately and frozen in liquid nitrogen for future analysis.The content of MDA in hippocampal tissue was determined using thiobarbituric acid method.The activity of SOD in serum was also assayed using SOD assay kit.The autophagosomes and mitochondria were detected by transmission electron microscopy,and the expression of miR-34 a was determined by Real-time-PCR,and the autophagy-related proteins were evaluated by Western blot.2.SH-SY5 Y cells were rinsed with pre-warmed phosphate-buffered saline(PBS)before dissociation by 0.25% trypsin-EDTA solution.When the cell confluence reached up to approximately 50-70%,the cells were transfected with miR-34 a inhibitor at the final concentration of 10,20 and 40 nM using Lipofectamine 2000 according to the manufacturer's protocol.The cells were harvested after another 48 h cultivation.The expression of miR-34 a was examined by Real-time-PCR,and the autophagy-related proteins were evaluated by Western blot.Results:1.The number of crossing platform position in D-gal-induced aging group revealed a significant decrease when compared with the normal control group,indicating the impairment of learning and memory capacity.As we expected,swimming intervention significantly increased the number of crossing platform position when compared with the D-gal-induced aging group and normal control group.On the fifth day of swimming training,the mean swimming speed of D-gal-induced aging rats was significantly decreased when compared with that in the normal control group,suggesting the presence of aging behavior of D-gal-induced rats.D-gal treatment caused a significant increase in MDA level in hippocampal tissue,and SOD activity is significant increase in D-gal + EXE group.The expression level of miR-34 a was significantly up-regulated in hippocampal tissue of D-gal-induced aging rats when compared with the normal control group,swimming training markedly down-regulated the expression of miR-34 a.On the other hand,the autophagy markers such as LC3,Beclin1 and Atg7,and the degradation of p62 were decreased in the presence of D-gal administration,while swimming training markedly increased the expression of LC3,Beclin1 and Atg7,and accelerated the degradation of p62.At the same time,D-gal treatment resulted in a significant increase in expression levels of Drp1 and Mfn2 proteins,and swimming training significantly up-regulated PGC-1?.Compared with the normal control group,swimming training significantly decreased the expression of Drp1.2.The inhibition of miR-34 a was confirmed by Real-time-PCR analysis,and the expression level of miR-34 a was markedly lower in miR-34 a inhibitor-transfected SH-SY5 Y cells than the mock-transfected SH-SY5 Y cells both in the presence and absence of D-gal treatment.The reduced expression of miR-34 a was accompanied by the increased expression of autophagy-related proteins including LC3II/I,Beclin1 and Atg7 and the accelerated degradation of p62.D-gal treatment markedly increased the expression of Drp1 and Mfn2.On the contrary,the protein expression levels of Drp1 and Mfn2 were reversed by miR-34 a inhibitor.Conclusions: MiR-34 a may be involved in the pathogenesis of brain aging by regulating the expression of autophagy-related proteins and mitochondrial dynamics in D-gal-induced aging rat and cell models.