| As the age increases,the organs,tissues and cells of the aging body will gradually become irreversible,comprehensive structural and functional decline,natural aging is not a disease,but it is closely linked with many elderly diseases.In the process of brain aging,due to the progressive decline of brain structure and function,will eventually lead to a variety of neurodegenerative diseases.Neurodegenerative disease is caused by a variety of causes of chronic progressive neuronal degeneration,loss and death of patients with behavioral disorders and dysfunction of neurological diseases.Modern studies have found that neural stem cells(NSCs)exist in specific parts of the brain,such as dentate gyrus of the hippocampus,which maintain and control the homeostasis of the nervous system structure and function through its proliferation and differentiation.The stress and damage state of the aging body are maintained and the steady-state ability of recovery is reduced,which is closely related to the decrease in the number of stem cells and the decline of the function.At present,the occurrence and development of neurodegenerative diseases are closely related to the aging of neural stem cells.As the process of population aging was accelerated,the increasingly serious of neurodegenerative diseases has become a major disease harm to human health.Unfortunately,there has been no effective way to prevent neurodegenerative disease.Ginseng is the main drug active ingredient of ginseng,it has a wide range of pharmacological effects,has been widely used in clinical treatment of multiple diseases or adjuvant therapy.In recent years,ginsenoside Rg1 is an important monomer component of ginsenoside,with a clear anti-aging effect,but its mechanism is not clear,it is worth in-depth study.The previous research has proved that: Ginsenoside Rg1 could antagonize the brain aging induced by D-galactose in rats,and significantly improved the behavior of brain aging rats and promoted the neurogenesis of NSCs in SVZ region.(2)The vitro experiments also proved that Rg1 can delay the senescence of NSCs and improve the proliferation and differentiation of aging NSCs.However,the relationship between ginsenoside Rg1 delaying brain senescence and regulating the senescence of NSCs and its mechanism is not clear.To date,there is no report from the perspective of neural stem cells to explore the mechanism of delayed aging of ginseng.In this study,the aging model of Nestin-GFP transgenic mice and the in vitro model of NSCs were established.The effects of Rg1 on the proteome of aging neural stem cells were analyzed by iTRAQ and bioinformatics.The in-depth mechanism of ginsenoside Rg1 on brain aging and NSCs were explored.The new ideas for the prevention and treatment of senile diseases of the nervous system with the ginsenoside Rg1 were provided.1.Materials and Methods1.1 C57BL/6 mice,6–8 weeks old,were randomly divided into 4 groups(control,D-gal-treatment,Rg1,and D-gal plus Rg1 treatment).In the D-gal-treatment group,D-gal(120 mg/kg/d)was injected subcutaneously daily into mice for 42 days.In the D-gal plus Rg1 treatment group,ginsenoside Rg1(20 mg/kg/d)was concomitantly injected intraperitoneally daily for 28 days from day 15 of D-gal injection.In the Rg1 group,the mice were treated with saline at the same volume of D-gal injection for 42 days and combined with Rg1(20 mg/kg/d)intraperitoneally for 28 days from day 15 of saline injection.All control animals were subcutaneously and intraperitoneally injected with the same volume of saline.And the following aspects of detection were operated:1)Morris water maze was used to evaluate the spatial learning and memory ability of each group of mice.2)Immunofluorescence was used to detect the number of neural stem cells in hippocampus of mice.3)Senescence-related ?-galactosidase(SA-?-Gal)staining to detect the level of hippocampal tissue senescence in each group.4)The changes of hippocampus tissue oxidative parameters and inflammatory factors were detected in each group.5)The changes in senescence-related signaling pathways were explored in the hippocampus of mice in each group.1.2 The NSCs were isolated from the hippocampus of C57 BL / 6 Nestin-GFP transgenic mice.The third generation NSCs were divided into 4 groups and the corresponding treatments were performed: In the D-gal-treatment group,the NSCs were treated with D-gal(10mg/ml)for 48 h.In the D-gal plus Rg1 treatment group,ginsenoside Rg1(20 ?g/ml)was concomitantly administrated of D-gal treatment for 48 h.In the Rg1 group,the NSCs were treated with PBS at the same volumn of D-gal and combined with Rg1(20?g/ml)for 48 h.Control group was processed with the same volume of PBS for 48 h.And the following aspects of detection were operated:1)CCK-8 was used to detect the proliferation of neural stem cells in each group.2)Senescence associated ?-galactosidase(SA-?-Gal)staining was used to evaluate the aging of neural stem cells in each group;3)The levels of reactive oxygen species and antioxidant enzymes in neural stem cells were investigated by flow cytometry.4)Western blot was used to explore the changes of senescence-related Akt / mTOR signaling pathway in neural stem cells.1.3 Changes in the proteome of neural stem cells in D-gal and Rg1 + D-gal groups were detected by iTRAQ and bioinformatics analysis.2 Results2.1 In vivo results1)The results of morris water maze test showed that the spatial memory capacity was significantly weaker than the other groups after the treatment of D-gal,and the spatial memory capacity of the aging mice with treatment of Rg1 has been significantly improved.2)Immunofluorescence assay suggest that the number of Nestin and SOX-2 positive cells of the aging group was significantly decreased.The number of Nestin and SOX-2 positive cells in the Rg1 + D-gal group was significantly higher than that in the D-gal group.3)The results of SA-?-Gal indicated that the positive rate of staining in Rg1 + D-gal group was lower than that in D-gal group.4)The results showed that the levels of MDA in the hippocampus were significantly increased and the activities of SOD and GSH-px were significantly decreased in the D-gal group,while the levels of SOD and GSH-px were increased in the Rg1 + D-gal group.The levels of IL-1?,IL-6 and TNF-? in the hippocampus of the D-gal group were significantly higher than those in the Rg1 + D-gal group,while the levels of IL-1?,IL-6 and TNF-? in the Rg1 + D-gal group were decreased.5)The levels of p19 and p21 mRNA in hippocampus were increased,the levels of p53 and Rb were increased,and the levels of p-Rb were decreased in the D-gal.In the Rg1 + D-gal group,p19 and p21 mRNA levels and p53 and Rb protein levels were lower in the D-gal group and p-Rb levels were elevated.The levels of phosphorylated I?B(p-I?B)and NF-?B in the D-gal group were significantly higher.The levels of phosphorylated I?B(p-I?B)and NF-?B in Rg1 + D-gal group were lower than those in D-gal group2.2 In vitro results1)Ginsenoside Rg1 can increase the proliferation of aging NSCs,and at 20?g / ml and below the concentration of NSCs increased the ability to increase the value of Rg1 was proportional to the concentrations.2)The percentage of positive neurons in the D-gal group was significantly higher than that in the other three groups,while the percentage of positive neurospheres in the Rg1 + D-gal group was lower.3)The level of ROS in D-gal group was significantly higher than that in D-gal group,but the ROS level in Rg1 + D-gal group was significantly lower than that in D-gal group.Ginsenoside Rg1 can increase the activity of intracellular SOD and GSH-px antioxidant enzymes.4)Ginsenoside Rg1 can downregulate the AKT / mTOR signaling pathway.5)Bioinformatics analysis of proteome in D-gal and Rg1 + D-gal group showed that the expression of some enzymes related to activity was changed,such as oxidoreductase activity,Hydroxymethylglutaryl-Co A reductase kinase activity and acetyl-Co A carboxylase kinase activity.The mTOR signaling pathway,the important pathway for regulating cell senescence,is inhibited after Rg1 treatment.In addition,we also found that Regulation of autophagy-related signal pathway has changed.3 Conclusions3.1 Ginsenoside Rg1 can delay D-gal-induced mouse brain aging.3.2 Rg1 delay brain aging may be by reducing the oxidative stress and then down-regulation of the relevant signal pathway in the hippocampus.3.3 Ginsenoside Rg1 can delay the aging of NSCs,but also can enhance the proliferation of aging NSCs,suggesting that it has anti-aging effect and treatment of aging,and the mechanism may be related to down-regulation of aging-related AKT / mTOR signaling pathway.3.4 Ginsenoside Rg1 regulates the changes of oxidoreductase activity,hydroxymethylglutaryl-Co A reductase kinase activity and acetyl-Co A kinase-associated protein expression,and changes in mTOR signaling pathways and autophagy-related signaling pathways in aging NSCs. |
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