| ObjectiveBrain aging has become a key research topic in the field of neuroscience and aging.Studies have shown that brain aging causes a number of central nervous system degenerative diseases,such as Parkinson's disease and alzheimer's disease.Ginseng is one of the clinical "tonic drugs" in Chinese medicine.The main pharmacophase is ginsenoside,but there are dozens of components.Our previous research proved that ginsenoside Rg1 is an anti-aging monomer component in ginseng,It can effectively antagonistic the attenuant,delay the mouse brain aging,improving the ability of learning and memory in aging mice,and the relationship between delayed brain aging and neural stem cells(NSCs)was preliminarily discussed,but it was not clear that Rg1 regulated NSCs aging mechanism.Recent studies have shown that the excessive activation of Wnt/?-catenin signaling pathway is closely related to stem cell senescence,and whether Rg1 delays the aging of NSCs is related to the regulation of the signaling pathway.This research adopts the D-galactose(D-gal)build nestin protein-green fluorescent protein(nestin-GFP)transgenic mice aging model,discusses Rg1 ginsenosides protection or postpone hippocampus of aging mice and the regulation of NSCs aging relationship,to interpret whether Rg1 can regulate NSCs senescence by inhibiting the over-activation of Wnt/?-catenin signaling pathway,aging research to find new ways to slow brain aging to provide theoretical and experimental basis.Methods1.Aging mouse model construction and administration: 40 male Nestin-GFP transgenic mice were randomly divided into 4 groups.Aging group: mice were injected subcutaneously with D-gal(200mg/kg qd×42d);Rg1 aging group: D-gal was injected according to the aging group method,and ginsenoside Rg1(40mg/kg qd×27d)was injected from the 16 th day;Rg1 Control group: mice were injected subcutaneously with normal saline(200 mg/kg qd x 42d),ginsenosides were injected on the 16 th day(the dose and time were the same as those of the Rg1 aging group),and the control group: mice were injected equal volume and isochronous saline.The aging model was established and the related experiments were performed on the 2nd day after administration.(1)Dynamic observation of mouse in each group.(2)Effect of Rg1 on learning and memory ability of mice: the Morris water maze experiment was conducted on the second day of modeling,and the spatial learning and memory capacity of mice was detected.(3)Effect of Rg1 on relative quantity of NSCs: the mice head was extracted,and the fresh hippocampal tissues of the four groups were fully collected and frozen sections were taken for each group to analyze the fluorescence intensity of NSCs in the hippocampus region of each group.(4)Effect of Rg1 on senescence of NSCs: analysis of the positive percentage of NSCs of senescence in the hippocampal tissues and the staining of NSCs;(5)Effect of Rg1 on oxidative and antioxidant indices of hippocampal tissue: to prepare the homogenate of the hippocampal tissue,and to detect the content of SOD,T-AOC and MDA in homogenate.(6)Effect of Rg1 on inflammation factor of hippocampal tissue: the detection of Il-1,Il-6 and TNF-? in the homogenate of the hippocampal tissue;(7)Effect of Rg1 on aging of hippocampal tissue: the expression of P53 and P21 in hippocampal tissue was detected by Western blotting.2.The whole brain of Nestin-GFP transgenic newborn rat was extracted,and NSCs were extracted and cultured to P3 generation.The following indicators were detected:(1)NSCs has been trained to identify the NSCs with their green fluorescence,marking Sox2 to red fluorescence(2)Effect of Li Cl on the proliferation and survival of NSCs: the P3 generation NSCs were cultured in different concentrations(5mmol/l,10 mmol/l,20 mmol/l,40 mmol/l)of the LiCl medium(Wnt/?-catenin signal agonist)for 1d,2d,3d,4d,5d,6d and 7d,and the cell proliferation and survival detection of each group were detected by CCK-8 method.(3)Effect of ginsenoside Rg1 on proliferation and survival of NSCs: the P3 generation NSCs were cultured in different concentrations(5?g/ml,10 ?g/ml,20?g/ml,40?g/ml)of the Rg1 medium(Wnt/?-catenin signal agonist)for 1d,2d,3d,and 4d,The OD value of each group was detected by CCK-8 method.(4)The effects of ginsenoside Rg1 and LiCl(Wnt/?-catenin signal agonist)on proliferation and survival rate of NSCs:the NSCs were randomly divided into four groups.LiCl group:P3 NSCs were cultured in the final concentration of 20mmol/l of LiCl medium for 48h;Rg1+LiCl group: P3 generation NSCs were cultured in LiCl medium(dose with LiCl group)for 24 h,and the Rg1 of ginsenoside Rg1(final concentration of 20 ?g/ml)was added for 24 h.Rg1 group:P3 generation NSCs were cultured for 24 h in the conventional culture medium,plus ginsenoside Rg1(dose with Rg1+LiCl group)to continue to be cultured for 24h;Control group:P3 NSCs were cultured in conventional medium for 48 h.Check the following indicators:1)The growth of NSCs in each group was observed by optical microscope.2)Flow detection of cell cycle of each group;3)Brdu test the proliferation ability of each group;4)Fluorescence microscope observed the fluorescence intensity of Nestin and Sox2 of NSCs in each group;5)The SA-?-Gal staining of each group of neurospheres was used to analyze the positive rate of aging and staining.;6)Observation of ?-catenin distribution in NSCs by Laser Confocal Scanning.7)Western Blotting was used to detect changes in the protein content of Wnt/?-catenin pathway in each group.Results1.Internal results1)Aging group mouse hair color gradually dull,listless and lethargic,diet decreased,body mass increased slowly,show natural aging signs.There was no significant correlation between the control group,Rg1 group and Rg1 aging group.2)The results of Morris water maze experiment showed that the spatial learning and memory ability of mice in the aging group was significantly reduced,while the spatial learning and memory ability of Rg1 group was significantly improved.3)Immunofluorescence observation showed that the green fluorescence intensity of Nestin-GFP in the hippocampal DG region of the aging group decreased significantly,while the green fluorescence intensity of Nestin-GFP in the hippocampus of the Rg1 group was increased.4)The SA-?-gal staining results showed a significant increase in the number of SA-?-gal staining positive particles in the hippocampal CA3 area of the aging group,while the number of SA-?-gal staining positive particles in the hippocampal CA3 area of Rg1 group and Rg1 group was significantly reduced.5)Oxidative and antioxidant capacity tests showed that compared with the normal control group,the activity of SOD and T-AOC in the homogenate of hippocampus in aging mice decreased,but the content of MDA increased.However,the activity of SOD and T-AOC in hippocampal homogenate of Rg1 senescence group was higher than that in aging group,and the content of MDA was decreased.6)The results of ELISA showed that the contents of IL-1?,IL-6 and TNF-? in the hippocampal homogenate of the aging group were significantly increased,while the levels of IL-1?,IL-6 and TNF-? in the hippocampus region of the Rg1 group were significantly reduced.7)Western Blotting test showed that the contents of P53 and P21 protein in the hippocampal region of the aging group were significantly increased,while the contents of P53 and P21 in the hippocampal area of Rg1 aging group were significantly reduced.2.In vitro results1)Observation of the growth of neural stem cells in each group showed that the number of neurons in the LiCl group was different and the number decreased,while the number of neurons in the Rg1+LiCl group was significantly increased,and the size rule;2)Flow testing each cell cycle show that: LiCl group of NSCs S and G2 cell percentage decrease,G1 phase cell percentage increase,Rg1 + LiCl NSCs S and G2 cell percentage increase in the LiCl group,G1 phase cell percentage decrease.3)Brdu test showed that the proliferation ability of NSCs in the LiCl group decreased,and the proliferation capacity of NSCs in Rg1+LiCl group was enhanced.4)Fluorescence microscopy observed the fluorescence intensity of Nestin and Sox2 in each group showed no significant difference in fluorescence intensity of NSCs.5)Each nerve ball SA-?-gal aging staining positive rate: the positive rate of aging of the neural bulb in the LiCl group was increased,and the positive rate of the aging of the neural stem cells in the Rg1+LiCl group was decreased.6)The ?-catenin distribution of each group of NSCs: the Li Cl group NSCs cytoplasm ?-catenin is less and the number of cells is small.It was found that that Rg1+LiCl group had a large ?-catenin of NSCs and a large number of cell;7)Western Blottiing was used to detect the changes in the related protein content of Wnt/?-catenin pathway in each group cells,indicating that the activation correlation protein content of the Wnt/?-catenin pathway in the LiCl group was increased,the activation of Wnt/?-catenin pathway-associated protein was decreased in Rg1+Li Cl group.Conclusion1.D-gal can replicate the brain aging model.Ginsenoside Rg1 can antagonize the aging of mice brain caused by D-Gal,and its possible mechanism is related to the oxidative stress damage of hippocampal NSCs induced by Rg1 antagonist anti-attenuate agents,and regulate the downstream P53-P21 signaling pathway.2.LiCl can activate the Wnt/?-catenin signaling pathway of NSCs,ginsenoside Rg1 can antagonize the senescence of NSCs caused by activation of Wnt/?-catenin signaling pathway and delay brain aging. |
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