| Objective:To explore the regulatory role of dihydromyricetin(DHM)and voluntary wheel-running exercise inducd autophagy in brain aging of senescence accelerated mouse prone/8 mice(SAMP8).Method:1.Animal experiments: Totally 20 6-month-old male SAMP8 mice with body weight of 280 ± 20 g were randomly divided into four groups including aging model group(Con),low-dose DHM(D1),high-dose DHM(D2),and high-dose DHM combined with chloroquine(D2Q)groups,with 5 mice in each group.In addition,20 SAMR1 mice were also divided into four same groups.After adaptive feeding for 1 week,the mice from Con group was naturally reared for 2 months without any interventions.The mice from D1 and D2 groups were administrated with DHM at the doses of 15 and 30 mg/ml/d by gravage,respectively.The mice from D2 Q group were administered with DHM at the dose of 30 mg/ml/d by gravage coupled with intraperitoneal injection of CQ at the dose of 5 mg/ml/d.After 2-month interventitons,all mice were sarficed by cervical dislocation after eyeball blood collection.Hippocampal and cerebral cortex tissues were harvested in ice and then sequentially subjected to nitrogen frozen and storage at-80 ℃ refrigerator immediately.The apoptosis in hippocampal tissue was evaluated by TUNEL staining.The neurons in hippocampal tissue were examined by nissl staining.Autophagosomes in hippocampal tissues were observed by TEM.The expression of AD-like pathological proteins in hippocampal tissues was evaluated by Western blot for validating the degree of brain aging in SAMP8 mice.And Western blot was performed to detect the expression of senescence related proteins in the hippocampus to verify the degree of senescence.And the changes of autophagy and apoptosis related proteins were also detected by Western blotting to evaluate the levels of autophagy and apoptosis.2.Cell experiments: Neuroblastoma cells(SH-SY5Y)were cultured and inoculated into 6-well plate when cell population reached up to 70%.The cells were divided into control group(Con),D-gal-induced aging group(D-gal),DHM treatment group(D-gal+DHM),SIRT1 inhibitor EX527(5 mM)group(D-gal+EX527),DHM combined with EX527(5 mM)group(D-gal+DHM+EX527),DHM combined with EX527(2 mM))group(D-gal+DHM+EX527).The cells in Con group were cultured normally.D-gal+EX527(5 mM)group,D-gal+DHM+EX527(5 mM)group and D-gal+DHM+EX527(2 mM)group were treated with 2mL 5mM and 2mM EX527 for 1h.D-gal+DHM group,D-gal+DHM+EX527(5 mM)group and D-gal+DHM+EX527(2mM)group were treated with 2mL 12.5mg/ml DHM for 2 hrs.D-gal group,D-gal + DHM group,D-gal+EX527(5 mM)group,D-gal+DHM+EX527(5 mM)group and D-gal+DHM+EX527(2 mM)group were cultured with 40mg/ml D-gal culture medium.Cell samples were collected after 24 hrs.Western blot was used to detect the expression level of SIRT1 to verify whether it passed through SIRT1 pathway and changes in autophagy related proteins and aging-related proteins were detected to evaluate the levels of autophagy and aging.Results:1.Animal experiments : 1)TUNEL staining showed that the apoptosis rate of the hippocampal tissue in the two dosage of DHM group was significantly reduced,compared with the model group(p<0.001),while that was significantly increased in the combined chloroquine group(p<0.001).2)Nissl staining showed that the neurons in the DHM combined with chloroquine group were loose in arrangement and most of the nuclei were deeply stained,compared with the model group.The high dose group had more orderly arrangement and better effect.3)The results of transmission electron microscopy showed that the number of autophagosomes in hippocampus increased in high dose group compared with model group.4)Western blotting results showed that the expression levels of AD pathology-like protein APP,p-Tau /Tau and BACE1 in the hippocampus of mice in the model group were higher due to brain senescence,and the expression levels were significantly decreased after the treatment of different doses of DHM.On the contrary,the expressions of the above pathology-like protein were increased after the treatment of chloroquine combined with DHM.Both dosage of DHM inhibited the expression of senescence-related proteins such as Ac-p53(p<0.01,p<0.01),P16(p<0.01,p<0.001),and the expression level of Ac-p53 increased after the combination of chloroquine(p<0.01).In terms of cell apoptosis,in the hippocampus of SAMP8 mice with rapid senescence,the expression of pro-apoptotic proteins decreased after the treatment of two dosage of DHM(p<0.001,p<0.001),the expression level increased again after the combination of chloroquine(p<0.001),the expression level increased in the high-dose group of anti-apoptotic protein Bcl2(p<0.01),and decreased after the combination of chloroquine(p<0.05).Autophagy related protein showed that the expression of autophagy positively related proteins Beclin1 and Atg7 increased after the treatment of two dosage of DHM(p<0.001,p<0.01),(p<0.01,p<0.01),while decreased after chloroquine was added(p<0.001).Compared with model group,autophagy negatively related protein P62 significantly decreased(p<0.001,p<0.05)in the two DHM group,suggesting that DHM activated cell autophagy and increased the degradation of autophagy-lysosome.In addition,the expression of SIRT1,a signal protein involved in the autophagy pathway,increased significantly after DHM treatment(p<0.001,p<0.0001),while the expression of Sirt1 decreased significantly after chloroquine treatment(p<0.001).Voluntary wheel-running exercise: Western blot results showed that the expression levels of AD-like proteins APP,p-Tau/Tau and BACE1 in hippocampus of mice in group V were significantly lower than those in group Con(p< 0.01).On the contrary,the expression of AD-like proteins such as LC3II/LCⅠ in model group was increased by chloroquine combined with free running exercise.The ratio of 3I,the expression of Atg7 and Beclin1 decreased,the expression of autophagic substrate P62 increased,while the expression of autophagy-related protein was significantly increased and the expression of P62 protein was down-regulated by free-wheel exercise,while the expression of apoptosis-related protein Bax was up-regulated in VQ group,while the expression of Bcl-2 was down-regulated in model group,while the expression of autophagy-related protein Bax was up-regulated in V group and on group.Compared with the control group,the apoptosis-related protein Bax in group V was significantly decreased(p < 0.01)and the protein level of Bcl-2 was significantly increased(p < 0.01),but the result was contrary after intraperitoneal injection of chloroquine.2.Cell experiments: Western blotting results showed that D-gal induced the expression of Ac-p53 /P53,P21 and P62(p<0.001).When DHM was added into the senescent cells model,it inhibited the expression of P53,acetylation(p<0.0001),increased the expression of LC3 Ⅱ/Ⅰ and promoted the degradation of P62,increased the expression of SIRT1(p<0.01)at the same time.When SIRT1 inhibitors EX527 was added into senescent cells model,the expression of SIRT1 was significantly inhibited(p<0.01),the expression of P62 was increased,the expression of LC3 Ⅱ/Ⅰ was decreased,the expression of P21 and Ac-p53(p<0.001)were increased,compared with D-gal + DHM group.Compared with D-gal + DHM group,the expression level of SIRT1 in D-gal + DHM + EX527(2 mM)group decreased after inhibitor were added(p<0.05),the expression level of P62 in D-gal + DHM + EX527(5 mM)group and D-gal + DHM + EX527(2 mM)group increased(p<0.05,p<0.05),the expression level of LC3 Ⅱ/Ⅰ in D-gal + DHM + EX527(5 mM)group decreased(p<0.05),the expression level of acetylated P53 increased in the D-gal +DHM+EX527(5 mM)group and the D-gal +DHM+EX527(2 mM)group(p<0.01,p<0.01),while the expression of P21 increased in the D-gal +DHM+EX527(5 mM)group(p<0.05),indicating that autophagy was inhibited.Conclusion:During the rapid senescence process of SAMP8 mice and cell senescence induced by D-gal,DHM can execute the prevention of neurons and delay brain aging process through inducing SIRT1-mediated autophagy,which is validated by down-regulated expression of AD-like proteins,up-regulated expression autophagy-related proteins and suppressed apoptosis as well as less damaged nurons in hippocampal tissue of SAMP8 mice upon DHM intervention.Voluntary wheel-running exercis may reduce the expression of AD pathology-like protein by activating autophagy and reducing apoptosis to achieve the protection of neurons and ultimately delay the process of brain aging. |
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