Effect Of Hypoxia On CXCR4 Expression In Neural Stem Cells And Its Mechanisms

Hypoxia is one of the important pathological stimuli that are often associated with a variety of clinical diseases and military work in special environment. The prospect of manipulating endogenous neural stem cells to replace damaged tissue and correct functional deficits offers a potential therapeutic strategy for hypoxia injury in CNS. After hypoxia injury, NSCs migrate to harmed tissue and differentiate into progeny, which then participate in tissue repair and function rebuilding. The elucidation of the molecular mechanisms underlying the effect of hypoxia on NSCs biologic characteristic may provide the crucial information toward the blockade of hypoxia injury and the therapeutic intervention for hypoxia-related clinical diseases and hypoxia injury in military work. Effects of hypoxia on NSCs result from not only direct impact on cells, but also indirect impact via remodeled cell mini-environment. In this study, we established primary cultured NSCs model in vitro. To understand the regulation of hypoxia on NSCs functions, we observed the effect of hypoxia on the expression of CXCR4 in NSCs. Furthermore, since it is reported that hypoxia up-regulated endocannabinoids in CNS, we also observed the effect of endocannabinoid 2-AG on NSCs differentiation and the involvement of STAT3 signaling pathway. Results favor the understanding on the indirect regulation of hypoxia on NSCs differentiation through endocannabinoids. Our data provide not only the evidence on the effects of hypoxia on NSCs biologic characteristic and relative molecular mechanisms, but also the cue for hypoxia-related diseases therapy.1.Effect of hypoxia on CXCR4 expressionObjective:To observe the expression changes of CXCR4 in NSCs in different hypoxia conditions and explore the molecular mechanism of the effect of hypoxia on NSCs functions. Methods:(1)Primary cultured NSCs were divided into 5 groups: Control, hypoxia (1%) for 4 h, hypoxia for 16 h, hypoxia for 4 h and reoxygenation for 8 h, hypoxia for 16 h and reoxyenation for 8 h. CXCR4 mRNA and protein expression levels in NSCs were determined by RT-PCR and Western-blot, respectively.(2)NSCs were incubated with the supernatant of 16 h hypoxia-treated N9 cells for 4 h or 16 h. CXCR4 mRNA and protein expression levels in NSCs were determined by RT-PCR and Western-blot, respectively.(3)N9 cells were employed and treated by hypoxia for 4 h or 16 h. CXCR4 mRNA and protein expression levels in NSCs were determined by RT-PCR and Western-blot, respectively. Cell migration capability was measured by transwell assay.Results:(1)No significant changes of CXCR4 mRNA and protein expression in NSCs were observed after 4 h or 16 h hypoxia exposure. In addition, no significant changes of CXCR4 expression in NSCs were observed after 4 h hypoxia/8 h reoxygenation or 16 h hypoxia/8 h reoxygenation.(2)No significant changes of CXCR4 mRNA and protein expression in NSCs were observed after 4 h or 16 h incubation with the supernatant of 16 h hypoxia-treated N9 cells.(3)CXCR4 mRNA and prtein expression levels in N9 cells were up-regulated by 4 h or 16 h hypoxia treatment that resulted in the significant enhance of cell migration capability.Conclusions:CXCR4 expression in NSCs cannot be impacted by hypoxia stimulus directly or incubation with the supernatant of 16 h hypoxia-treated N9 cells. However, enhanced CXCR4 expression in N9 cells was observed under same hypoxia condition. Regulation of NSCs CXCR4 expression may not be one of the potential mechanisms underlying the effect of hypoxia on NSCs functions.2. The role of STAT3 signaling pathway in endocannabinoid 2-AG-induced NSCs astroglial differentiationObjective:To observe the effect of 2-AG on NSCs differentiation and the role of STAT3 signaling pathway in 2-AG-induced NSCs astroglial differentiation. Methods:(1)CB1 and CB2 mRNA expression levels were determined by RT-PCR. Expression levels of CB1 and CB2 proteins were measured by cytoimmunochemistry.(2)The ratio of astroglia to neuron after 2 d or 6 d treatment by 2-AG (1μM or 10μM) was determined by cytoimmunochemistry.(3)STAT3 phosphorylation levels after 15 min, 30 min or 60 min treatment by 2-AG (1μM or 10μM) were determined by Western-blot. A STAT3 specific inhibitor, Stattic, as well as CB1 and CB2 antagonists, AM251 and AM630, was used to confirm the effects of 2-AG on STAT3 phosphorylation by pretreatment for 1 h.(4)The nuclear translocation and DNA-binding of STAT3 after 10μM 2-AG treatment for 1 h were determined by EMSA. GFAP mRNA expression levels after 1μM or 10μM 2-AG treatment for 1 h were measured by RT-PCR.(5)JAK1-3 and Src phosphorylation after 30 min treatment by 2-AG (1μM or 10μM) were determined by Western-blot.(6)The effect of STAT3 inhibitor, Stattic, on 2-AG-induced NSCs astroglial differentiation was observed by cytoimmunochemistry.Results:(1)Both CB1 and CB2 mRNA were detected in NSCs. Furthermore, both CB1 and CB2 protein were co-expressed with Nestin.(2)The ratios of astroglia were increased significantly in NSCs differentiation induced by both two doses of 2-AG treatment.(3)STAT3 phosphorylation levels were increased significantly induced by both two doses of 2-AG treatment. This effect was blocked by STAT3 inhibitor and CB1 antagonist, but not CB2 antagonist.(4)Enhanced STAT3 DNA-binding and STAT3 downstream gene GFAP mRNA expression were showed and these effects were blocked by STAT3 inhibitor and CB1 antagonist.(5)No significant changes on JAK1-3 phosphorylation levels in NSCs were observed after both two doses of 2-AG treatment. However, 2AG-induced Src phosphorylation level was significantly increased which can be blocked by CB1 astagonist.(6)2-AG-induce NSCs astroglial differentiation was blocked by STAT3 inhibitor incompletely. Conclusions:(1)NSCs astroglial differentiation is promoted by endocannabinoid. It may be one of the indirect ways by which NSCs differentiation was impact by hypoxia via endocannabinoid.(2)Src/STAT3 signaling pathway is involved in 2-AG-induced NSCs differentiation. Our data provide crucial information for the molecular mechanism elucidation of the indirect effects of hypoxia on NSCs differentiation through endocannabinoid.