Transcriptional Regulation Mechanisms Of Hypoxia-Induced Neuroglobin Gene Expression And Identifying Compounds That Upregulate Neuroglobin For Endogenous Neuroprotection

Part Ⅰ Transcriptional Regulation Mechanisms of Hypoxia-Induced Neuroglobin Gene ExpressionNeuroglobin (Ngb) has been identified as a novel endogenous neuroprotectant. However, little is known about the regulation mechanisms of Ngb expression, especially under hypoxia conditions. In this study, we located the core proximal promoter of mouse Ngb gene to a554bp segment, which harbors putative conserved NFκB, Egr1binding sites. Over-expression and knock-down of transcription factors p65, p50, Egr1or Sp1increased and decreased Ngb expression, respectively. Experimental assessments with transfections of mutational Ngb gene promoter constructs, as well as EMS A and ChIP assays demonstrated that NFκB family members (p65, p50, cRel), Egr1, and Sp1bound in vitro and in vivo to the proximal promoter region of Ngb gene. Moreover, a κB3site was found as a pivotal cis-element responsible for hypoxia-induced Ngb promoter activity. NFκB (p65) and Sp1were also responsible for hypoxia-induced upregulation of Ngb expression. Although there are no conserved HREs (hypoxia-response elements) in the promoter of mouse Ngb gene, our results suggested that HIF1α is also involved in hypoxia-induced Ngb upregulation. In conclusion, we identified that NFκB, Egr1, and Sp1played important roles in regulation of basal Ngb expression via specific interactions with the mouse Ngb promoter. NFκB, Sp1and HIF1α contributed to the upregulation of mouse Ngb gene expression under hypoxic conditions. Part ⅡIdentifying Compounds That Upregulate Neuroglobin for Endogenous NeuroprotectionStroke is one of the leading causes of death worldwide. Previous studies indicate that Neuroglobin is a novel neuroprotective protein that protected against hypoxic/ischemic neuron injury, oxidative injury, neurodegenerated disease. Thus, up-regulating Ngb may be a novel therapeutic approach for the intervention and treatment of stroke and related disorders. To identify natural compounds that increase Ngb expression, we developed both mouse and human reporter system that stably expressed a luciferase reporter gene directed by around2000bp of the mouse and human Ngb promoter, respectively. Preliminary validation of both reporter system using a small pool of natural compounds identified several Ngb activators:Polydatin, Icariin, Genipin, Genistein, Daidzein, Biochanin A, Formononetin for increasing mouse Ngb promoter activity; Polydatin, Genistein, Daidzein, Biochanin A, Formononetin for increasing human Ngb promoter activity. Those potential activators of the Ngb promoter were further evaluated by secondary analyses. RT-PCR and Western blotting further confirmed that Polydatin, Genistein, Formononetin were able to significantly increase Ngb mRNA levels and protein levels. Moreover, they also significantly decreased OGD-induced the rate of death of mouse primary cortical neuron. Our results suggested that Polydatin, Fonnononetin are potential neuroprotective drugs and our mouse and human reporter system could be used for high-throughput screening.