Role of Wnt/β-catenin signaling pathway in proliferation of neural stem cells of neonatal rats in vitro after hyperbaric oxygen therapy

Abstract

Objective To investigate the role of Wnt/β-catenin signaling pathway in hyperbaric oxygen (HBO)therapy for hypoxic-ischemic brain damage(HIBD). Methods The neural stem cells (NSCs) of neonatal rats were randomly divided into control group and 8 treatment groups, then the treatment groups were cultured in the supernatant of brain homogenate to simulate micro-environment of HIBD and normal brain respectively.Through electroporation β-catenin siRNA and negative control plasmid was transfected into NSCs respectively, then HBO therapy was performed.Immunocytochemical staining was performed simultaneously in the 9 groups of NSCs on precoated chamber slides to detect the differentiation of NSCs.Quantitative reverse transcriptase(RT)-PCR was used to detect the relative content of Ngn1 mRNA and bone morphogenetic protein(BMP4) mRNA in the of NSCs.Western blot was used to detect the relative content of Ngn1 protein and BMP4 protein in the NSCs. Results In vitro, HBO alone promoted NSCs infected with negative control siRNA (ncNSCs) to differentiate into neurons and oligodendrocytes and depressed astrogliosis; HIBD or normal brain tissue extract cultures promoted ncNSCs to differentiate into neurons and oligodendrocytes but depressed astrogliosis, and the effect of HIBD brain extract cultures was superior to the latter and could be further increased by HBO; β-catenin siRNA decreased the NSE-positive neurons and increased glial fibrillary acidic protein-po-sitive astrocytes in the siNSCs (NSCs infected with β-catenin siRNA) in vitro, the effect could not be inversed by HBO, but could be alleviated; HBO increased the level of Ngn1 mRNA and Ngn1 protein, decreased BMP4 mRNA and BMP4 protein of ncNSCs, transfection of β-catenin siRNA could down-regulate the expression of Ngn1 mRNA and up-regulate BMP4 mRNA of NSCs in vitro respectively. Conclusions HBO can promote NSCs cultured with HIBD brain extract cultures to differentiate into neuronal or Oligodendrocyte, and inhibit them to differentiate into astrocytes.HBO therapy promotes the proliferation of NSCs in vitro, an effect which is correlated with β-catenin protein.HBO therapy can promote neurogenesis by β-catenin-induced activated Ngn1, and repress astrocytogenesis by β-catenin-induced down-regulated BMP4.There are potential cooperative actions of BMP4 and Ngn1 on differentiating rat NSCs in cerebral ischemic brain.The ability of Ngn1 to promote neurogenesis may allow Ngn1 to act as a potent neuronal commitment factor. Key words: β-catenin; RNA interference; Hyperbaric oxygen therapy; Hypoxic-ischemic brain damage; Electroporation