ABSTRACT
AIM:To investigate the effect of nuclear factor E2-related factor 2 (Nrf2) activation by 18α-gly-cyrrhetinic acid (18α-GA) on the proliferation and self-renewal of adult neural stem cells (aNSCs), and to explore an ef-fective way of maintaining the viability of aNSCs .METHODS:NSCs were dissociated from subventricular zone of the mice at postnatal days 0, 60, and 300.The expression levels of Nrf2 in the NSCs at various ages were compared .After treatment with 18α-GA, the expression of Nrf2 was examined by real-time PCR and Western blot.shRNA lentiviral vector (LV) car-rying green fluorescent protein (GFP) gene was constructed to knock down Nrf2 expression.The knockdown efficiency in the aNSCs was detected by real-time PCR and Western blot .Subsequently , the aNSCs were divided into DMSO group , 18α-GA group, LV-GFP group and LV-Nrf2-shRNA group.BrdU incorporation assay , Tuj1 staining, CCK-8 assay, Hoechst 33342/PI staining and detection of reactive oxygen species ( ROS) were performed to analyze the proliferation , dif-ferentiation, viability, apoptosis and oxidative stress levels of the NSCs .RESULTS:The mRNA expression level of Nrf2 in adult and aged NSCs was significantly lower than that in newborn NSCs (P<0.01), while the ROS level of aNSCs was significantly higher (P<0.05).After treatment with 18α-GA, the expression level of Nrf2 in the aNSCs was significantly up-regulated as compared with DMSO group ( P<0.01).Increased number of BrdU +and Tuj1 +cells was observed in 18α-GA group, indicating that 18α-GA-treated cells had higher viability (P<0.05).Meanwhile, there were fewer apop-totic cells and lower ROS level in 18α-GA group than those in DMSO group (P<0.05).After knockdown of Nrf2 in aNSCs and then treated with 18α-GA, there were less BrdU +and Tuj1 +cells, as well as the aNSCs with lower viability in LV-Nrf2-shRNA group (P<0.05).Moreover, the ROS level was increased in LV-Nrf2-shRNA group as compared with LV-GFP group (P<0.05).CONCLUSION:Activation of Nrf2 by 18α-GA elevates the antioxidant capacity of aNSCs , thus ameliorating the cell proliferation and differentiation potentials .
ABSTRACT
AIM:To investigate the effect of nuclear factor E2-related factor 2 (Nrf2) activation by 18α-gly-cyrrhetinic acid (18α-GA) on the proliferation and self-renewal of adult neural stem cells (aNSCs), and to explore an ef-fective way of maintaining the viability of aNSCs .METHODS:NSCs were dissociated from subventricular zone of the mice at postnatal days 0, 60, and 300.The expression levels of Nrf2 in the NSCs at various ages were compared .After treatment with 18α-GA, the expression of Nrf2 was examined by real-time PCR and Western blot.shRNA lentiviral vector (LV) car-rying green fluorescent protein (GFP) gene was constructed to knock down Nrf2 expression.The knockdown efficiency in the aNSCs was detected by real-time PCR and Western blot .Subsequently , the aNSCs were divided into DMSO group , 18α-GA group, LV-GFP group and LV-Nrf2-shRNA group.BrdU incorporation assay , Tuj1 staining, CCK-8 assay, Hoechst 33342/PI staining and detection of reactive oxygen species ( ROS) were performed to analyze the proliferation , dif-ferentiation, viability, apoptosis and oxidative stress levels of the NSCs .RESULTS:The mRNA expression level of Nrf2 in adult and aged NSCs was significantly lower than that in newborn NSCs (P<0.01), while the ROS level of aNSCs was significantly higher (P<0.05).After treatment with 18α-GA, the expression level of Nrf2 in the aNSCs was significantly up-regulated as compared with DMSO group ( P<0.01).Increased number of BrdU +and Tuj1 +cells was observed in 18α-GA group, indicating that 18α-GA-treated cells had higher viability (P<0.05).Meanwhile, there were fewer apop-totic cells and lower ROS level in 18α-GA group than those in DMSO group (P<0.05).After knockdown of Nrf2 in aNSCs and then treated with 18α-GA, there were less BrdU +and Tuj1 +cells, as well as the aNSCs with lower viability in LV-Nrf2-shRNA group (P<0.05).Moreover, the ROS level was increased in LV-Nrf2-shRNA group as compared with LV-GFP group (P<0.05).CONCLUSION:Activation of Nrf2 by 18α-GA elevates the antioxidant capacity of aNSCs , thus ameliorating the cell proliferation and differentiation potentials .
ABSTRACT
BACKGROUND: Adult neural stem cells have the potential for self-renewal and differentiation into multiple cell lineages via symmetric or asymmetric cell division. Preso1 is a recently identified protein involved in the formation of dendritic spines and the promotion of axonal growth in developing neurons. Preso1 can also bind to cell polarity proteins, suggesting a potential role for Preso1 in asymmetric cell division. METHODS: To investigate the distribution of Preso1, we performed immunohistochemistry with adult mouse brain slice. Also, polarized distribution of Preso1 was assessed by immunocytochemistry in cultured neural stem cells. RESULTS: Immunoreactivity for Preso1 (Preso1-IR) was strong in the rostral migratory stream and subventricular zone, where proliferating transit-amplifying cells and neuroblasts are prevalent. In cultured neural stem cells, Preso1-IR was unequally distributed in the cell cytosol. We also observed the distribution of Preso1 in the subgranular zone of the hippocampal dentate gyrus, another neurogenic region in the adult brain. Interestingly, Preso1-IR was transiently observed in the nuclei of doublecortin-expressing neuroblasts immediately after asymmetric cell division. CONCLUSION: Our study demonstrated that Preso1 is asymmetrically distributed in the cytosol and nuclei of neural stem/progenitor cells in the adult brain, and may play a significant role in cell differentiation via association with cell polarity machinery.