| Objective:The biology of aging and anti-aging is the common concern of social science and natural science major research issues,especialby brain aging is one of the important research contents of aging. Our previous studies our, brain aging is closely related to neural stem cell senescence.We have shown that "Qi" pathway can delay brain aging and NSCs aging in traditional Chinese medicine,so looking for a new way to delay the brain aging and prevent neurodegenerative disease in natural medicine is worth of pay high attention.Angelica is "supplement and quicken the blood "in clinical traditional Chinese medicine,has thousands of years history of clinical application.Modern medicine has proofed that ASP is the important potency constituent of Angelica.It has the effect of antioxidation,radioprotection,antineoplastic, enhance immune,promoting hemopoiesis and decelerating stem cells aging.So far,whether ASP could delay aging especial brain aging,which hasn’t been reported.Senescence belongs vacuity detriment category in Chinese medicine theory,as blood stasis due to qi deficiency, deficiency of kidney and blood stasis, two spleen and kidney deficiency with blood stasis syndrome. We presume that it can delay aging and antagonize aging damage factor by Chinese medicine "supplement and quicken the blood"or"supplement the blood and benefiting vital energy". We combine traditional Chinese medicine theory and modern medical aging theory and use modern biology research method to investigate the relationship of ASP on delaying brain aging and regulating NSCs aging by duplicating brain aging model and NSCs vitro aging model,aim to elucidate the senescene mechanism delaying brain aging and regulating NSCs aging by"supplement the blood and benefiting vital energy", in order to provide the theory and new thread of preventing and curing neurodegenerative disease.Methods:1.Forty SD rats at 3 months of age were divided into four groups in random. Aging model group were treated with D-gal(120mg/kg/day) for 42 days by subcutaneous injection. ASP aging group were also injected with D-gal with the same dose and time as aging model group, and from the 14th day on, rats were given ASP (100 mg/kg) by intraperitoneal injection for 28 days. Normal control group were received saline with the same volume for 42 days. ASP control group were given saline with the same volume for 14 days, and received ASP (100mg/kg) by intraperitoneal injection for 28 days. One the day of 41 treatment, BrdU (50 mg/kg in saline) was administered intraperitoneally three times with an interval of 4 hours. The Morris water maze test was carried out to test spatial learning and memory ability of rats after modeling. SA-β-Gal staining was detected brain aging. The anti-oxidant ability in hippocampus were quantified by chromatometry. The capability of hippocampus to secrete IL-1β, IL-6 and TNF-a were assayed with ELISA. The telomere activities was assayed by Southern blotting.The telomere length was assayed by TRAP-PCRThe BrdU> GFAP、β-III tubulin were stained by immunofluorescence.2.1solate cells from fetal SD rats for primary culture. Identify cultured cells were NSCs by flow cytometry and immunofluorescence.The third generation of NSCs were divided into control group(conventional culture), aging group (add 10 mg/ml D-gal), ASP group (add 100 ug/ml ASP after handling as same as control group),ASP anti-aging group (add 100 ng/ml ASP after handling as same as aging group),each group was cultured for 48 hours.SA-P-Gal staining display the aging neurospheres, calculating the percentage of positive neurospheres.The amount of MDA were measured by chromatometry and the level of ROS was assayed with flow cytometry. The expressions of NSCs senescence associated p19、p21、p53 mRNA in each group were examined by RT-PCR.Result:1. In ASP aging group,the capacities of learning and memory of rats were remarkablely ameliorated,the ROD of SA-β-Gal staining in brain sections were markedly decreased,the SOD and GSH-Px activities and the consumption of GSH in hippocampus were significantly increased,while the amount of MDA was diminshed. The secretory capability of IL-1β, IL-6, TNF-a were observabry reduced,both the telomere length and telomere activities were significantly increased.The number of β-tubulin Ⅲ+ and BrdU+ were remarkablely increased,the number of GFAP+was significantly decreased compared with aging group.2.Isokte and identify NSCs successfully,nestin of cells was positive by immunofluorescence staining. Compared to aging group, the numbers of reforming neurospheres were observably increased in ASP aging group,the percentage of SA-β-Gal positive neurospheres was remarked ly decreased,the amount of MDA and the level of ROS was significantly decreased, the expressions of senescence associated p19、p21、p53 mRNA were striking reduced.Conclusions:1.Injection of D-gal can duplicate aging rats model and construct brain aging model.2. ASP has the anti-aging effect of brain aging induced by D-gal3.ASP maybe decrease the expression level of inflammation factor and elevated the antioxidant capacity by regulating telomere systemof cells in brain tissue to antagonize the effect of brain reduced by aging agent4.D-gal can reduce NSCs aging in vitro,ASP antagonize NSCs aging reduced by aging agent.Objective To investigate the effect of Angelica Sinensis polysaccharide(ASP) on the spleen structure and function of aging model rats and its relative mechanism, in order to provide theoretical and experimental evidences and find the effective ingredient from traditional Chinese medicine to delay immune system aging. Methods 40 SD rats were randomly divided into normal control group, ASP control group, aging model group and ASP aging model group. Aging model group were treated with D-galactose (120mg/kg/day) for 42 days by subcutaneous injection. ASP aging group were also injected with D-galactose with the same dose and time as aging model group, and from the 14th day on, rats were given ASP (100 mg/kg) by intraperitoneal injection for 28 days. Normal control group were received saline with the same volume for 42 days. ASP control group were given saline with the same volume for 14 days, and received ASP (100mg/kg) by intraperitoneal injection for 28 days. After 2 days of finishing injections the spleen index were measured, paraffin section were made to observe spleen microscopic structure. Senescence-accociated β-galactosidase (SA-β-Gal) staining was detected aging sptenocytes. The proliferative capacity of splenocytes with stimulating by Concanavalin A (Con A) was measured by CCK-8. The cell cycles of splenocytes were assayed with flow cytometry. The capability of splenocyte to secrete TNF-α,GM-CSF and reactive oxygen species(ROS), malondialdehyde(MDA), superoxide dismutase (SOD)were assayed with ELISA. The change of cell aging related protein P53、P21、 RB were detected by Western blotting analysis. Results comparing the aging rats induced by D-galactose with normal control group,the biological feature of spleens in aging model group as follow:spleen index, splenic white pulp area proportion, the proliferative capacity of splenocytes stimulated by Con A, and S-phase rate in cell cycle, the secretory capability of TNF-α and GM-CSF, the active content of SOD were obviously decreased;The percentage of SA-β-Gal positive cells, ratio of G1 and G2/M stages, the production of ROS and MDA in splenocytes were significantly increased, and the expression of P53、P21、RB were also up-regulated, comparing ASP aging group with aging model group, ASP could remarkably increase spleen index, splenic white pulp area proportion, the proliferative capacity of splenocytes stimulated by Con A, S-phase ratio, the secretory capability of TNF-a and GM-CSF, the active content of SOD. ASP also could obviously increase the percentage of SA-β-Gal positive cells, ratio of Gl and G2/M stages, the production of ROS and MDA in splenocytes, and down-regulate the expression of P53、P21、RB. Conclusion The spleen structure and function were obviously damaged of D-galactose-induced aging model rats.ASP has definitely protective effects on its injury. |
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