| Objective Due to the accelerating aging of the world’s population,Alzheimer’s disease(AD),Parkinson’s syndrome(PD)and other neurodegenerative diseases(DDNS)have become important diseases that endanger the life and health of the elderly,so accelerate the promotion of the nervous system Senescence biology and related disease prevention and treatment are of great significance.The preliminary work of the research group has proved that the occurrence and development of neurodegenerative diseases are closely related to the aging and functional degradation of neural stem cells(NSCs).By interfering with the process of cellular oxidative stress injury,NSCs aging can be significantly delayed,and the corresponding clinical symptoms of neurodegenerative diseases can be reduced,but the mechanism is not yet clear.Ginseng,known as the "King of Herbs",has long been regarded as an essential medicine for "qi" in the field of traditional medicine in the motherland,and Rg1,which is one of its saponin components,plays an anti-aging function in ginseng.Previous research by the research group has shown that ginsenoside Rg1 has a significant effect of resisting the aging of NSCs and can improve neurodegenerative symptoms of brain aging animals.The mechanism remains to be discussed in depth.The latest theory believes that the Nrf2 / ARE signaling pathway plays a key role in stem cells against oxidative stress damage.Nuclear factor 2(Nrf2)can enhance the expression of antioxidant genes by interacting with antioxidant response elements(ARE),thereby regulating cells Anti-oxidative stress and redox reactions.The latest research proves that the aging of NSCs in the elderly may be related to the block or partial block of this pathway.To date,there has been no report of the relationship between Rg1 delaying NSCs aging and regulating Nrf2 / ARE signaling pathway.If we can find the target of Rg1 regulating NSCs aging,it will have important theoretical significance and clinical application value.In this experiment,the senescence agent D-galactose(D-gal)was used to construct the NSCs aging model in vitro to study the relationship between the ginsenoside Rg1 delaying NSCs aging and the Nrf2 / ARE signaling pathway.The purpose was to provide a theory for finding NSCs aging targets.Basic and experimental basis.Methods By stripping whole brains of newborn mice and cultivating NSCs to P3 generation,a cell aging model can be constructed,and Rg1 intervention is added to detect various corresponding indicators.1.Peel off the whole brain of the suckling rat within 1 day of birth,cut the whole brain on the ice,then add trypsin and gently pipette through the pasteurized tube for 5min,centrifuge at 1000 r / min for 5min and discard the supernatant.Under dark conditions,add 3m L of NSCs complete culture medium to each cell culture flask,incubate for 2 days in an incubator at 37 ° C,5% CO2 concentration,collect the NSCs that aggregated into balls,and digest them into single cells with trypsin and then continue to cultivate.This process needs to be repeated many times.When NSCs are bred to the P3 generation,follow-up experiments are conducted.2.Inoculate NSCs into cell culture flasks at 5 × 105 / m L and divide them into four groups.D-gal group(aging model group)added D-gal with a final concentration of 10 g / L to jointly cultivate for 48h;Rg1 group was first cultivated for 24 h,then added Rg1 with a final concentration of 20 ug / m L to jointly cultivate for 24h;blank control group,directly routine 48 hours incubation;D-gal + Rg1 group(Rg1anti-aging model group)first added D-gal with a final concentration of10 g / L to incubate for 24 h,and then added Rg1 with a final concentration of 20 ug / m L to incubate for 24 h.After the construction of the experimental model is completed,the corresponding subsequent experimental design can be carried out on the 2d.3.3.Immunofluorescence was used to detect nestin expression,and immunofluorescence was used to identify the percentage of Nestin expressed by NSCs in each group.4.The CCK-8 method was used to detect the proliferation of NSCs,and the enzyme-labeled optical density was used to measure the proliferation of neurospheres.5.SA-β-gal staining was used to detect NSCs,and cell senescence was evaluated based on the number and percentage of positive cells stained.Evaluation of cell viability.6.Trypan blue staining to detect NSCs,according to the number and percentage of positive staining cells7.Enzyme-labeled colorimetric method was used to detect the content of oxidative stress-related indexes SOD,MDA,CAT,4-HNE and8-OHd G in the culture supernatant of NSCs,and to evaluate cell aging.8.Fluorescence quantitative PCR was used to detect the expression levels of Nrf2 / ARE signaling pathway related genes GCLC,GCLM,NQO1 and GSTM-1 in NSCs,and to evaluate the relationship between NSCs aging and Nrf2 / ARE pathway activation or block.9.Western-blot detects the expression levels of Nrf2 / ARE signaling pathway-related proteins Nrf2,HO-1 and Keap1 in NSCs,and evaluates the key targets of NSCs aging and Nrf2 / ARE pathway.Result1.Observe the growth status of NSCs: Compared with the blank control group,the Rg1 group formed neurospheres more regularly and the total amount increased significantly.The total size of the neurospheres formed in the D-gal group was different,and the total amount of neurospheres decreased.Compared with the D-gal group,the size of the neurosphere formed in the D-gal + Rg1 group was more regular,and the total amount increased.2.Nestin immunofluorescence identification proved that the four groups of neurospheres all showed positive expression of nestin.3.CCK-8 method was used to detect the proliferation of NSCs.The experimental results showed that the proliferation of the D-gal group was significantly inhibited compared to the blank control group,while the proliferation of the Rg1 group was significantly improved compared to the D-gal + Rg1 group.4.SA-β-gal staining of neurospheres in each group proved that,compared with the blank control group,the positive percentage of neurospheres in the D-gal group increased significantly,and the positive percentage of neural stem cells in the Rg1 group decreased significantly compared to the D-gal + Rg1 group.5.Trypan blue staining test for NSCs in each group proved that the survival rate of NSCs decreased when the D-gal co-culture group was added to the culture system compared with the blank control group.Compared with the corresponding control group,the survival rate of NSCs increased significantly when the Rg1 co-culture group and the D-gal + Rg1 group were added in vitro.6.The oxidative stress index detection of NSCs in each group proved that the activity of SOD and CAT in the supernatant of the D-gal group was significantly lower than that of the blank control group;the contents of MDA,4-HNE and 8-OHd G increased significantly.Compared with the corresponding control group,the Rg1 co-cultivation group and the D-gal + Rg1 group were significantly reduced in the culture supernatant.7.Fluorescence quantitative PCR was used to detect the expression of Nrf2 / ARE pathway-related genes in NSCs of each group.The results showed that the expression level of antioxidant genes related to this pathway in NSCs was reduced in the D-gal co-culture group compared to the blank control group.Compared with the corresponding control group,the Rg1 co-culture group and D-gal + Rg1 group in vitro increased the expression of Nrf2 / ARE pathway-related genes in NSCs.8.Western-blot detected the content of Nrf2 / ARE pathway-related proteins in NSCs of each group.The results showed that the content of Nrf2 / ARE pathway-related proteins in NSCs decreased when the D-gal co-culture group was added to the blank control group.Compared with the corresponding control group,the Rg1 co-culture group and the D-gal+ Rg1 group in vitro increased the content of Nrf2 / ARE pathway-related proteins in NSCs.Conclusion1.Using the aging agent D-gal can successfully build NSCs aging model in vitro.2.Ginsenoside Rg1 can antagonize the effect of D-gal on the decay of NSCs.3.Ginsenoside Rg1 resists the decay effect of D-gal on NSCs and is related to the reduction of cellular oxidative stress damage.4.The possible mechanism of ginsenoside Rg1 antagonizing the aging of NSCs caused by D-gal is related to the regulation of key targets of Nrf2 / ARE pathway. |
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