| ObjectiveThe world has entered an aging society,and the nervous system is one of the systems with the earliest aging and the greatest damage.Studies have found that regulating the senescence of neural stem cells(NSCs)can significantly delay brain aging,but the mechanism remains unclear.According to reports,both the physiological and pathological brain aging process is accompanied by a decrease in autophagy flux in the brain,which shows that autophagy is closely related to brain aging.Ginseng is a traditional Chinese medicine for ‘bu qi',and ginseng saponin is its main active ingredient.Our research group confirmed that ginsenoside Rg1 was an important monomer component in ginseng and had anti-aging effects.It can regulate the senescence of NSCs through anti-oxidation and antagonism of inflammatory cytokine production,thereby delaying the effect of brain aging,but whether ginsenoside Rg1 regulates the autophagic flow of NSCs to delay brain aging has not been reported.This study was to investigate the mechanism by which ginsenoside Rg1 regulates the autophagy activity of NSCs to delay brain aging.Methods1.Aging C57BL/6J mouse model construction and administration: Two months old male or female mice were purchased from Cyagen Biosciences Inc.(Animal Certificate of Conformity: SYXK Guangdong 2008-0090).After adapting to the environment at the Animal Experimental Center of Chongqing Medical University,mice(n = 60)were randomly divided into normal group,Rg1 group,D-gal +Rg1 group and D-gal group.The D-gal group was subcutaneously injected with D-galactose(200mg/kg),qd×42.The D-gal + Rg1 group was intraperitoneally injected with Rg1(40mg/kg)since the 16 th day of the replication in the brain aging model,qd×27.The Rg1 group was subcutaneously injected with the same amount of saline,qd×15,and following intraperitoneally injected with Rg1(40mg/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.(1)Diet and body weight of mice in each group were monitored after daily injection of the drug.(2)Morris Water Maze Performances: The morris water maze is a 1.2m diameter and 0.5m high bucket,which is divided into four quadrants.The water temperature is controlled at 22 to 25 °C.A 10 cm diameter platform was fixed in a quadrant of the platform 1 cm below the horizontal plane.The drug maze test was started on the 43 rd day after the completion of the drug intervention experiment.The positioning navigation test lasted for 6 days,4 times every day in the morning and afternoon.The mouse was placed in the water from the 4 water inlet points to the pool wall,and the time to find the platform within 2 minutes(escape latency time)was recorded.Space exploration experiment: On day 7,the platform was removed.The mouse is optionally placed in the water,and the number of times the mouse crossed the original platform position and percentage of occupancy time in the original platform quadrant within 120 s is measured.During the entire cognitive function test process,keep the indoor environment such as lighting and items in the laboratory consistently to eliminate environmental interference.2.Neural stem cells were isolated and extracted from the brain tissue of mice,cultured and purified to the third generation,and the corresponding indicators were detected after drug intervention.(1)NSCs separation and extraction: Neonatal mouse(within 1 day after birth)dissected the whole brain,shredded brain tissue and digested with the enzyme for 5 minutes.The supernatant was removed by centrifugation and the cells were grown in suspension in NSC medium.Two days later,the neurospheres were collected and digested with enzymes into single cells.This process is repeated continuously so that NSCs can be naturally purified until experimental use.The cells were cultured to the 3P and an experiment was conducted.(2)NSCs were seeded in culture flasks which were divided into four group.The D-gal group was cultured with D-gal(10mg/mL)for 48 h.D-gal + Rg1 group was first cultured with D-gal(10mg/mL)for 24 h,following by adding Rg1(20?g/mL)with medium containing Rg1 and D-gal for 24 h.The Rg1 group was routinely cultured for 24 h,and then added Rg1(20?g/mL)for 24 h.The normal group was routinely cultured for 48 h.(3)NSCs of each group were identified by immunofluorescence.(4)NSCs senescence was detected by SA--gal senescence staining.(5)NSCs cycle was detected by flow cytometry.(6)NSCs proliferation was detected by EDU test.(7)The autophagosome morphology of NSCs was observed by electron microscopy.(8)The expression of autophagy protein of NSCs was detected by immunofluorescence and western-blot assay.(9)Confocal microscope was performed to observe the difference of the number of autophagosomes and autolysosomes of NSCs.Results1.In the D-gal group,mice gradually showed neurodegenerative manifestations,such as lethargy,listlessness,dullness of reaction,lethargy,and somnolence,with dull hair color,reduced diet and water intake,decreased immunity,and other aging phenomena.Senescence signs were not obvious in Normal group,Rg1 group and D-gal +Rg1 group.2.Morris water maze behavior test results: The cognitive and learning and memory abilities of the D-gal group were significantly decreased.The neurological function of the mice was significantly improved after the injection of Rg1.3.Growth of each group of NSCs: The growth of neural stem cells in each group: The size of the neurospheres in the D-gal group was irregular,and the total number of neurospheres was reduced.After the intervention of Rg1,the size of the neurospheres in the D-gal+Rg1 group was uniform,the density distribution was uniform,and the total number of neurospheres increased.4.Nestin protein immunofluorescence test results: After Rg1 intervention(D-gal+Rg1 group),the expression of Nestin protein in NSCs increased,and the number of positive cells also increased.Neurosphere identification showed no significant change in the strength of Nestin protein.5.SA-?-gal senescence staining test results: Ginsenoside Rg1 atten uates D?gal induced NSCs senescence.The percentage of senescence nerospheres in the D-gal group was significantly increased as compar ed to the Normal group.The D-gal + Rg1 group decreased the perce ntage of senescence nerospheres.6.Flow cytometry results in detecting cell cycle of NSCs: Ginsenoside Rg1 promotes cell division.after D-gal treatment of mouse neural stem cells,the proportion of cells in G2 phase and S phase decreased,and the proportion of G0-G1 phase increased,which was significantly different from Normal group,but after Rg1 intervention,compared with D-gal group,the proportion of cells in the G2 phase and the S phase increased,and the proportion of the G0-G1 phase decreased in the D-gal+Rg1 group.7.EDU detection of proliferation results of NSCs in each group: Ginsenoside Rg1 promoted NSCs proliferation.The number of EDU+ cells in the D-gal group was less than that in the D-gal+Rg1 group8.Transmission electron microscope observation results: Transmission electron microscopy observation of autophagosomes in NSCs.the autophagosomes wrapped in the bilayer membrane structure of the normal group were rare.Compared with the normal group,the number of autophagosomes in the D-gal group and the D-gal+Rg1 group increased significantly.In the D-gal group,autophagosomes with bilayer membrane structure were observed,but after Rg1 intervention,the autolysosomes of the monolayer membrane structure of D-gal+Rg1 group increased,and autophagosomes with bilayer membrane structure was also observed.It is indicated that Rg1 can promote the increase of the number of autolysosomes in aging neural stem cells induced by D-gal.9.Results of expression of autophagy-related proteins in NSCs: Ginseng Rg1 regulates autophagy-related protein expression.Immunofluorescence and western-blot assay indected that Rg1 increased the expression of LC3-II protein,decreased the expression of P62 protein in aging NSCs.10.Laser confocal microscopy results: Ginseng Rg1 increased the autophagic flux.the results showed that the total number of spots in single cells of D-gal group were more than that in normal group,yellow spots significantly increased compared with the normal group,red spots were rare,indicating autophagic flux diminished;The average red spot per cell in the D-gal+Rg1 group was more than that of the D-gal group,and the yellow spot was less than the D-gal group,indicating that Rg1 promotes autophagosome degradation and the autophagic flux increased.Conclusion1.D-gal can successfully replicate the brain aging model and NSCs in vitro aging model.2.Ginsenoside Rg1 can delay D-gal-induced brain aging in mice and antagonize D-gal inducing senescence of NSCs in vitro.3.Both aging and degenerative diseases of the nervous system may be associated with abnormal autophagy in NSCs.Brain aging is closely related to senescence and autophagy of NSCs.4.The protective effect of ginsenoside Rg1 on D-gal-induced NSCs senescence to delay brain aging may be related to promote cell proliferation,increase the number of autophagosomes,regulate autophagy proteins,and increase autophagy flux of NSCs.The incidence of neurodegenerative diseases is severely increasing with the aging.It has been proposed that NSCs(neural stem cells)help to control aging,but the mechanisms responsible remain unclear.Angelica polysaccharide is an active ingredient of Angelica sinensis in traditional Chinese medicine,which possesses versatile pharmacological activities including anti-oxidative and anti-aging effects.In this study,D-gal(D-galactose)was used to construct an aging model of Nestin-GFP transgenic mice brain tissues and NSCs.Mouse model was subcutaneously injected with D-gal,as we observed that mice consistently displayed acceleration of aging-like behavior change.Conversely,aging retardation was achieved in Nestin-GFP mice Induced by D-gal that was locally injected with ASP(Angelica polysaccharide).Mechanistically,we isolated and cultured NSCs in vitro.ASP protected NSCs by increasing the cell proliferation;decreasing the number of SA-?-gal stained neurons;increasing the activity of SOD(superoxide dismutase)and T-AOC(total antioxidant capacity),decreasing the content of MDA(malondialdehyde);decreasing the levels of IL-1b,IL-6,TNF-a;and down-regulated the expression of cellular senescence associated genes p53,p21 in the aging NSCs.In conclusion,ASP can delay aging speed by protecting NSCs and promote neurogenesis by enhancing the antioxidant and anti-inflammatory capacity,up-regulation of p53/p21 signaling pathway.As to provide theoretical basis for treatment for brain aging related diseases,add new scientific connotation for "qi and blood theory" and "supplement blood and delay aging" of Traditional Chinese Medicine. |
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