Relationship between neural stem cells aging and nuclear factor erythroid 2 related factor 2/antioxidant response element signaling pathway

doi:10.16098/j.issn.0529.1356-2020.06.002

Abstract

Abstract:

Objective To establish aging model of mouse neural stem cells (NSCs) in vitro and study the relationship between D-galactose (D-gal)-induced NSCs aging and nuclear factor erythroid 2 related factor 2 (Nrf2)/antioxidant response elemen (ARE) signaling pathway. Methods The whole brain of newborn C57BL/6J mouse was obtained. NSCs were isolated and identified, and cultured the cells to the third generation. The NSCs were randomly divided into control group and aging group. The NSCs of control group were cultured in NSCs medium for 48 hours, and the NSCs of aging group were added with D-galactose (D-gal,final concentration of 10 g/L) on the basis of the control group. Cell counting kit-8(CCK-8) was used to detect the activity of NSCs; Senescence-associated β-galactosidase (SA-β-gal) staining was used to detect the percentage of senescence-positive neurospheres; Trypan blue staining was used to detect cell viability; Enzyme-labeled colorimetry assay was used for detection of the activity of superoxide dismutases (SOD), catalase (CAT) and malondialdehyde (MDA) in the cell culture supernatants; Western blotting was used to detect the expression of Nrf2, heme-oxygenase-1 (HO-1) protein; Real-time PCR was applied to detect the expression level of GCLC, GCLM genes. Results Compared with the control group, the proliferative activity of NSCs in the aging group decreased significantly, the percentage of SA-β-gal staining positive neurons increased obviously, the activity of trypan blue stained NSCs decreased explicitly, SOD activity and CAT activity decreased markedly, and MDA content increased significantly. The expression levels of Nrf2/ARE signaling pathway-related proteins Nrf2 and HO-1 were significantly down-regulated in cells, the expression of pathway-associated GCLC and GCLM genes was down-regulated. Conclusion The addition of D-gal to the culture system can construct an in vitro aging model of NSCs. The possible mechanism is closely related to the inhibition of the antioxidant activity of Nrf2/ARE signaling pathway.

Key words: D-galactose, Neural stem cell, Aging, Nuclear erythroid related factor 2/antioxidant response elemen signaling pathway, Real-time PCR, Mouse

CLC Number:

R338.1

WU Qi WANG Shun-he CHENG Xiao XIANG Yue CHEN Lin-bo WANG Zi-ling XIAO Ha-xian-zhi JIANG Rong WANG Lu WANG Ya-ping. Relationship between neural stem cells aging and nuclear factor erythroid 2 related factor 2/antioxidant response element signaling pathway[J]. Acta Anatomica Sinica, 2020, 51(6): 815-820.

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