Effects Of Autophagy Stimulant Rapamycin On Hippocampal Neurogenesis And Learning/Memory In Middle-aged Mice

Objective:To investigate the effect of promoting the autophagy on hippocampal neurogenesis in middle-aged mice,and then to find some effective methods to activate hippocampal neurogenesis in vivo,that may provide a new idea for the prevention and treatment of neurodegenerative diseases.Methods:1.The total hippocampal tissue protein was isolated from the newborn and middle-aged mice(6 months old),and the autophagy-related protein,such as LC3? in hippocampus was detected by Western blotting,to evaluate the effect of age on autophagy.2.The autophagy activator,Rapamycin(4mg/kg)was injected intraperitoneally in the middle-aged mice group by once every other day,and the protein in hippocampus was extracted after continuously administrated for 4 weeks.The phosphorylation levels of rapamycin targeted protein,mTOR and its downstream target protein ULK1ser757 were detected,and the autophagy-related proteins,LC3 ? and p62 in hippocampus were measured to observe the changes of autophagy.3.The quantitative changes of Ki67 and DCX in hippocampal SGZ region of rapamycin group and DMSO control group were detected by immunofluorescence staining,and the effect of autophagy on hippocampal neurogenesis was observed.4.After were treated by rapamycin for 4 weeks,the learning and spatial memory abilities of middle-aged mice were tested by a new objective recognition and Y-maze.To assess the effect of autophagy on learning and memory ability of mice.Results:1.The results of Western blot showed that the expression of LC3 II in the hippocampus of middle-aged mice group was decreased by 24.66 ±6.26%,compared with that of newborn mice group,suggesting that the level of hippocampal autophagy decreased significantly in middle-aged mice.2.After an intraperitoneal injection of rapamycin in middle-aged mice for four weeks,the results of Western blot detection showed that the phosphorylation levels of mTOR and ULK1 ser757 in hippocampus of middle-aged mice were 28.16 ±0.05 and 24.30 ±0.05% lower than those of DMSO control group,respectively.The LC3 II expression in hippocampus of rapamycin group was significantly higher than that of DMSO control group,while the expression of p62 protein was decreased.The results suggest that intraperitoneal injection of rapamycin can effectively inhibit hippocampal mTOR of middle-aged mice,and then to activate ULK1,to promote the hippocampal autophagy in middle-aged mice.3.The results of immunofluorescence staining showed that after an intraperitoneal injection of rapamycin,the number of Ki67+ and DCX+ cells in hippocampal SGZ region of middle-aged mice were increased by 55.38 ± 13.06%(P<0.05)and 39.32 ± 12.01%(P<0.05)times,respectively compared with DMSO control group,and the length and number of DCX+ cells in rapamycin group increased significantly.The results of new objective recognition experiment showed that its index of rapamycin group was 67.50 ±3.40%,which was significantly higher than that of the DMSO control group 57.45%± 2.65%(P<0.05);The results of Y-maze spontaneous alternating test suggested that there was no significant difference in the number of entering the arm between rapamycin group and DMSO control group,but the correct alternating response rate of rapamycin group was 10.52 ±2.57% higher than that of DMSO control group(P < 0.001).It is suggested that rapamycin can effectively promote hippocampal neurogenesis and enhance learning and memory in middle-aged mice.Conclusion:The level of hippocampal autophagy in middle-aged mice decreased significantly,and intraperitoneal injection of rapamycin could effectively up-regulated the level of hippocampal autophagy,to promote hippocampal neurogenesis and enhance learning and memory in middle-aged mice.