Cellular senescence is a permanent state of cell cycle arrest that occurs in proliferating cells under various stresses causing age-related disorders. This study investigated the role of D-galactose in inducing premature senescence of neural stem cells (NSCs) and the genes involved in this process. After NSC isolation and proliferation, senescence was induced with 10, 20, or 30µM concentrations of D-galactose for 24h. Cell viability was tested using the MTT assay, and senescent cells were identified based on increased lysosomal β-galactosidase activity. In addition, levels of NO and malondialdehyde (MDA) oxidative biomarkers but also total thiols (T-SH) and total antioxidant FRAP, as well as inflammatory cytokines, were investigated. Besides, RNA-Seq was performed on the various groups, the gene network was mapped, and genes with the most significant changes were examined. Treatment of NSCs with 20 or 30µM concentrations of D-galactose caused a significant decrease in cell survival, a number of neurospheres, and a number of neurosphere-derived cells, compared to the control group. In addition, the number of BrdU + cells significantly decreased after induction of NSC senescence with 10 or 20μM D-galactose, whereas aging-related β-galactosidase (SA-β-gal) increased significantly. Moreover, treatment with 10 or 20μM D-galactose showed a significant increase in NO production, but not malondialdehyde (MDA). However, the levels of total thiol (T-SH) and antioxidant FRAP levels decreased significantly. Furthermore, TNF-α and IL-6 cytokines significantly increased in NSCs treated with 20μM, but not 10μM, D-galactose. Finally, a gene network consisting of 860 gene orthologs was mapped using RNA-Seq. Protein interactions were obtained in 11 hub genes which were classified using gene ontology based on molecular function, biological processes, or cellular processes. Genes with the most significant changes in aging included Fn1, Itga2, Itga3, Itga6, Itga8, Ptk2b, Grin2b, Cacna2d3, Pde4d, Shisa6, and Stac3. This study showed that D-galactose reduces NSC proliferation and antioxidant activity while increasing oxidative stress and inflammatory cytokines. A survey of the genes involved in premature aging may be used as therapeutic candidates for aging-related disorders.
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