Activation Of Proteasome Activity Promotes Neural Stem Cell Viability Through Insulin-like Growth Factor-1/Nuclear Factor Erythroid 2-related Factor 2 Signaling

Objective:To investigate whether insulin-like growth factor 1(IGF-1)up-regulates proteasomal activity of neural stem cells(NSCs)through activation of the proteasome-associated nuclear transcription factor Nrf2(NF-E2-related factor 2),and that to promote the proliferation and differentiation of NSCs.The aim is to seek effective targets for maintaining the self-renewal vitality of NSCs,and to provide new ideas for the treatment of neurodegenerative diseases to delay aging.Methods:1.After isolated and cultured of neonatal(Postnatal day 0,P0)mouse hippocampal NSCs,IGF-1 was treated for 24 h,and the proteasome activity of NSCs and the mRNA expression of its subunits PSMB1,PSMB2,PSMB5,and PSMA3 in different concentrations of IGF-1 were analyzed.2.BrdU incorporation and Tuj1 immunofluorescence staining were used to detect the effect of IGF-1 on the proliferation and differentiation potential of NSCs.3.Nrf2 immunofluorescence staining detects the expression of proteasome-associated nuclear transcription factor Nrf2 after IGF-1 interferes with NSCs.4.Design and synthesis of siRNA targeting Nrf2 and its negative control(NC)sequence,electrotransfection of NSCs,from the mRNA and protein levels to verify the knockout efficiency.IGF-1(100 ng/mL)was used to treat NSCs in siRNA group and NC group.Proteasome activity was detected by fluorescence microplate reader,BrdU and Tuj1 immunofluorescence staining,SA-β-gal staining,and CCK8 experiment to analyze whether IGF-1 affected proteasomal activity of NSCs through activation of Nrf2,and then regulate NSCs activity.Results:1.The proteasomal activity of NSCs in the 50 ng/m L and 100 ng/m L IGF-1 groups was 1.75±0.26(P<0.01)times and 2.02±0.19 times(P<0.05)in the PBS control group,respectively,with increasing IGF-1 concentration.After 24 hours of IGF-1 treatment,total RNA of NSCs was extracted,and the expression of proteasome subunits was detected by Real-time PCR.20,50,and 100 ng/m L IGF-1 could increase mRNA expression of PSMA3 in NSCs compared with PBS control group by 1.11±0.02 times(P<0.05),1.16±0.03 times(P<0.01),and 1.17±0.03 times(P<0.01).In addition,100 ng/m L IGF-1 also increased the mRNA expression of NSCs PSMB5 by 1.18±0.04 times(P<0.05).2.BrdU immunofluorescence staining showed that IGF-1 could increase the proliferation of NSCs for 24 h.The positive rates of BrdU in the 50 ng/mL group and 100 ng/m L group were 37.83%±2.71% and 39.36%±3.28%,respectively,which was significantly higher than the PBS control group 30.08%±2.42%(P<0.05).The results of Tuj1 immunofluorescence staining also showed that the number of Tuj1 positive newborn neurons was 34.34% ± 34% and 47.27% ± 3.37%,respectively,when NSCs were treated with 50 ng/mL and 100 ng/m L IGF-1 for 3 days,which was significantly higher than that of PBS control group 20.22%±1.92%(P<0.05),suggesting that IGF-1 can promote the differentiation of NSCs into neurons.3.IGF-1 interfered with NSCs for 24 h.Nrf2 immunofluorescence staining showed that the nuclear fluorescence signal of IGF-1 group was enhanced.MetaMorph analysis software showed that as the concentration of IGF-1 increased,the nuclear/cytoplasmic ratio(nuclear/cytoplasmic ratio)of Nrf2 fluorescence signal was gradually increased.The 20 ng/mL,50 ng/m L,and 100 ng/mL IGF-1 groups were 1.32±0.12,1.41±0.11,and 1.52±0.21 times higher than the PBS control group,respectively(P<0.05).It is suggested that IGF-1 can promote the transcriptional activation of proteasome-associated nuclear transcription factor Nrf2.4.NSCs were electroporated with siRNA targeting Nrf2.Western blot results showed that siRNA interference efficiency was 34.28%±7.28%(P<0.01).100 ng/m L IGF-1 was used to act on the siRNA group and the NC control group.The proteasome activity of the siRNA group was 0.37±0.04 lower than that of the NC group(P<0.001).In addition,the proliferation and Tuj1 positive rate of NSCs in siRNA group were also decreased by 6.24%±0.83%(P<0.05)and 6.34%±1.05%(P<0.05),and cell viability was decreased by 0.83±0.07(P<0.001),the positive rate of senescent cells increased by 12.39%±1.37%(P<0.05).It is suggested that IGF-1 may up-regulate the proteasome activity of NSCs by activating Nrf2,thereby increasing cell viability.Conclusion:1.IGF-1 can increase the proteasome activity of NSCs,promote proliferation and differentiation of NSCs,and maintain cell self-renewal.2.IGF-1 may up-regulate the proteasome activity of NSCs by activating Nrf2,thereby increasing cell viability.