| Objective The incidence of neurodegenerative diseases are severely increasing with the aging.The development and progression of neurodegenerative diseases is linked to the senescence of neural stem cells.Unfortunately,there has been no effective way to prevent neurodegenerative diseases.Medical practice indicated that the aging could be delay through the "replenishing qi and blood".Angelica is one of the Chinese medicine with the effect of "replenishing qi and blood,anti-aging,repair damage" and so on.To investigate the effect of angelica sinensis polysaccharide(ASP)on the brain senescence of Nestin-GFP mice induced by D-galactose and its possible mechanism we constructed the model of neural stem cells(NSCs)in vitro and in vivo.Methods 1.Male Nestin-GFP transgenic mice(n = 60)aging from 6 to 8 weeks old were randomly divided into D-gal group,ASP+D-gal group,normal group and ASP group.The D-gal group was subcutaneously injected with D-galactose(200mg/kg),qd×42.The ASP+D-gal group was intraperitoneally injected with ASP(140mg/kg)since the 16 th day of the replication in the brain aging model,qd×27.The ASP group was subcutaneously injected with the same amount of saline,qd×15,and following intraperitoneally injected with ASP(140mg/kg)qd×27.The normal group was subcutaneously injected with an equal volume of saline within the same time.Perform the related experiment on the second day after finishing copying the model.Learning and memory abilities were measured by Morris water maze.Frozen sections were made to observe the hippocampus fluorescence intensity.The percentage of senescent cells in DG of hippocampus was detected by SA-?-Gal staining.The activities of SOD,T-AOC and contents of MDA in hippocampus were quantified by enzyme colorimetric method.The activity of Na+-K+-ATPase in hippocampus was quantified by chromatometry.The level of IL-1?,IL-6 and TNF-a proinflammatory cytokines in hippocampus were detected by methods of ELISA.Western Blot was used to detect the changes of P53 and P21 contents in hippocampus.2.NSCs were isolated from the brain of neonatal Nestin-GFP transgenic mice and primary cultured to P3 generation.The CCK8 assay was used after the P3 generation of NSCs were treated with different concentration of ASP(10,20,40,80,160?g/ml)respectively for 24,48,72h.3.The P3 generation of NSCs was randomly divided into four groups: normal group,D-gal group(treated with 20mg/ml D-gal for 24h),ASP group(treated with 100?g/ml ASP for 48h),ASP+D-gal group(co-treated with 20mg/ml D-gal and 100?g/ml ASP for 48h).Then the Nestin immunofluorescence was used to identify NSCs.The number of senescent positive neurospheres was detected by SA-?-Gal staining.The activities of SOD,T-AOC and contents of MDA in NSCs were quantified by chromatometry.The level of IL-1?,IL-6 and TNF-a proinflammatory cytokines in NSCs were detected by methods ELISA.The content of ROS in NSCs was detected by flow cytometry.Western Blot was used to detect the changes of P53 and P21 contents in NSCs.Results 1.In vivo,compared with the normal group,in D-gal group,the spatial learning and memory capacities were weaken,the fluorescence intensity decreased in the dentate gyrus(DG)of hippocampus,SA-?-Gal positive granules increased in section of brain tissue,the activity of SOD,T-AOC decreased in hippocampus while the contents of MDA increased in hippocampus.The activity of Na+-K+-ATPase in hippocampus was decreased.The level of IL-1?,IL-6 and TNF-a increased in hippocampus.The contents of P53 and P21 in hippocampus were increased.Compared with the D-gal group,in ASP+D-gal group,the spatial learning and memory capacities were enhanced,the fluorescence intensity increased in the DG area of hippocampus,SA-?-Gal positive granules decreased in section of brain tissue,the activity of SOD,T-AOC increased in hippocampus while the contents of MDA decreased in hippocampus.The activity of Na+-K+-ATPase in hippocampus was increased.The level of IL-1?,IL-6 and TNF-a in hippocampus decreased.The contents of P53 and P21 in hippocampus were decreased.2.In vitro,the culture of the neurospheres showed Nestin green fluorescence positive reaction.ASP can increase the ability of NSCs to proliferate,which is positively correlated with the concentration of ASP.3.In D-gal group,the number of SA-?-gal-positive neurospheres were increased,the activity of SOD and T-AOC decreased and the content of MDA increased.The activity of in IL-1?,IL-6 and TNF-? were increased,the level of ROS was increased.The contents of P53 and P21 protein were increased.Compared with the aging model group,in ASP+D-gal group,the number of SA-?-gal stained neurons in the ASP+D-gal group was decreased,the activity of SOD and T-AOC was increased,the content of MDA was decreased,the contents of IL-1?,IL-6 and TNF-? were decreased,the level of ROS was decreased,the contents of P53,P21 protein were decreased.Conclusion 1.In this study,the model of D-gal replication was used as a model of brain senescence.2.ASP can antagonize brain aging induced by D-galactose in mice.In addition,improvement of antioxidant ability,down regulation the level of proinflammatory cytokines and maintaining the number of neural stem cells in hippocampus may be the underlying anti-aging mechanism of ASP.3.ASP can antagonize D-gal-induced NSCs senescence in vitro,and its mechanism may be related to the oxidative and antioxidant capacity and senescence-related genes of NSCs. |
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