Autophagy Regulates Notch Signal Expression In Neural Stem Cells And The Effect Of Astragaloside Ⅳ

ObjectiveIn order to further study the mechanism of Astragaloside IV regulating the proliferation and differentiation of neural stem cells C17.2 injured by Aβ,we seek the possible target of Astragaloside IV in the treatment of Alzheimer’s disease through network pharmacology;detect the effect of Astragaloside IV on the expression of Notch signal pathway proteins and autophagy-related proteins in neural stem cells,and explore the relationship between Astragaloside IV and their regulatory mechanisms.Methods:The neural stem cell C17.2 cells were cultured,and the effect of Astragaloside IV on the proliferation of C17.2 cells was determined by CCK-8 method.and then effect of Astragaloside IV on the differentiation of C17.2 cells was determined by immunofluorescence.The target genes of drugs are obtained by database,and the target genes of diseases are obtained by Gene Cards and OMIM.The Wayne diagram of drug target genes and disease target genes is drawn,and then the target genes are imported into STRING database to draw PPI network.GO and KEGG are enriched and analyzed by R language to predict the mechanism of action.Western blot method was used to detect the effect of Astragaloside IV on the expression of Notch1 and NICD proteins in Notch signal pathway and the expression of autophagy-related proteins LC3 and p62,ATG16L1 and Beclin1.Results:The results of CCK-8 detection showed that 10^-6mol/L Astragaloside IV showed strong proliferation-promoting effect at all time points,and the proliferation-promoting effect of 10^-6mol/L Astragaloside IV at 24 h was significantly higher than that of 10^-5mol/L group at the same time（p<0.05）.In the immunofluorescence experiment,C17.2 cells cultured with 10^-5mol/L（high dose group）and 10^-6mol/L（low dose group）changed from polygonal to fusiform and showed the characteristics of differentiation after 48 hours,while the morphology of C17.2 cells did not change significantly when the concentration of Astragaloside IV IV was 10^-7mol/L and 10^-8mol/L.Immunofluorescence detection was carried out by using Nestin antibody,a marker of neural stem cells,and DCX⁺,a marker of neuronal differentiation.The results showed that the DCX⁺ratios of high-dose group and low-dose group were 34.2±1.9%and 26.0±3.10%,respectively.However,there were almost no DCX⁺antibody positive cells in control group,10^-7mol/L group and10^-8mol/L group.Network pharmacological analysis found that there were 168 AS-IV drug targets related to AD and 19 highly correlated signal pathways were found by KEGG results.Among them,Notch signal pathway is an important signal pathway of Astragaloside IV acting on AD.Western blot of C17.2 cells injured by Aβwas treated with high dose Astragaloside IV.The results showed that the Notch1 protein and intracellular NICD of C17.2 cells were significantly decreased after Aβinjury for 24 h and 48 h,but Astragaloside IV could alleviate this decrease.Compared with the control group,the ratio of LC3II/I in Aβinjury group and injury plus drug group increased,and the result of P62 was consistent with that of LC3II/I,indicating that AS-IV can promote the Notch signal pathway and induce the activity of autophagy.After DAPT inhibited the activity ofγ-secretase,the expression of NICD decreased.AS-IV could increase the expression of Notch1 but not NICD,and AS-IV could still increase the ratio of LC3II/I,indicating that the stimulation of AS-IV on autophagy activity of C17.2 cells did not depend on Notch signal pathway.ATG16L1si RNA was used to knock down the ATG16L1 of C17.2 cells.The expression of Notch1 protein in C17.2 cells was significantly increased after knock down.After the addition of AS-IV,the amount of Notch1 and NICD were further increased.Cells were treated by hydroxychloroquine to inhibit autophagy,Notch1 and LC3II/I were found accumulated,and after the addition of Astragaloside IV,the levels of Notch1,NICD and LC3II/I increased.Further mechanism of promoting autophagy by Astragaloside IV was studied and found that Astragaloside IV could induce the expression of Beclin1.Conclusion:Astragaloside IV can promote the proliferation and differentiation of cultured neural stem cells C17.2.The expression of Notch1 protein decreased and the level of autophagy increased after Aβ_1-42injury for 24 h,and the autophagy function was damaged by Aβinjury for a long time（48 h）.Astragaloside IV can reduce the damage of Aβto autophagy and increase the expression of Notch signal pathway.After the inhibitor of Notch1 signal pathway inhibited the production of NICD,AS-IV could still promote the increase of autophagy,while after the inhibition of early and late autophagy,the levels of Notch1 and NICD increased,and the expression of Notch1and NICD further increased after the addition of drugs.In the early stage of autophagy,Notch1 protein can be encapsulated by phagocytic vesicles and finally degraded by autophagy.Astragaloside IV is independent of autophagy in the activation of Notch1signal.Astragaloside IV could also induce the increase of Beclin1 protein expression.It is concluded that Astragaloside IV can promote the expression of Notch1 protein in C17.2 cells and enhance autophagy by promoting the expression of autophagy regulatory protein Beclin1.