The Initial Studies About The Neuroprotective Function And Mechanism Of Neuroglobin

Stroke is one of the diseases which seriously threaten human health and affect the quality of life. Up to date, No neuroprotective agents have been confirmed with striking therapeutic effects in phase III clinical trials. The pathogeneses of stroke include NO injury, acidosis, toxicity excitatory amino acids, injury of free radical. Ca²⁺ antagon, excitatory amino acids antagon and free radical antagon are usually been used in clinical treatments of stroke. However, their clinical efficacies are not widely accepted.By reviewing the utilization of oxygen, it was found that HGB (Haemoglobin, HGB) and MGB (Myoglobin, MGB) play an important role in the transport, store and utilization of oxygen. However, brain consumes oxygen in a different way. Although the weight of human brain accounts for 2% of the body, the oxygen consumption of NGB accounts for 20% of total oxygen consumption of the body even in the resting state.How the nervous system utilizing oxygen remain unclear. The discovery of neuroglobin (NGB) provides us an enlightenment about the oxygen utilization in the brain. NGB is a member of the globin family. NGB shares little amino-acid sequence similarity with vertebrate MGB (< 21% identity) and HGB (< 25% identity). Like other globins, NGB can reversely bind oxygen and nitric oxide (NO). NGB oxygen affinity is higher than that of HGB, but lower than that of MGB. NGB makes it easy to transport O2 through BBB (blood-brain barrier), and up-regulate the O2-comsumption of brain. Hypoxia and ischemia can up-regulate the expression of NGB. Over-expression of NGB by transfection can protect neurons from hypoxia injury, while the neuroprotective mechanism of NGB is still unclear.We have finished several initial studies about the neuroprotective function and mechanism of NGB in many ways. First, we constructed and identified the siRNA eukaryotic expression vector (pSL-NGB) targeting NGB. After transfection with eukaryotic expression vector pSL-NGB, PC12 were incubated with 300μM CoCl₂, the MTT results showed that endogenous NGB can protect PC12 cells from CoCl₂-induced cell injury. Second, we constructed and identified another eukaryotic expression vector pCMV-myc-NGB. After transfection with pCMV-myc-NGB, PC12 cells were lysised or incubated with 300μM CoCl₂, the expression of NGB were identified by anti-myc antibody using Western blot technique, and the cellular viability was measured by MTT assay. The results of Western blot showed that the reconstruction plasmid expressed NGB protein. The MTT results showed over-expression of NGB also can protect PC12 cells from CoCl₂-induced cellular injury. The neuroprotective function of NGB has been confirmed, and enables us to study the neuroprotective mechanism of NGB. As a member of globin, NGB can also bind NO. After study, we found over-expression of NGB can protect PC12 cells from NO donor, SNP (sodium nitroprusside)-induced cells injury by up-regulating the expression of bcl-2 gene, but didn't change either extracellular or intracellular concentration of NO. Because down-regulation of bcl-2 expression is one of the pathogeneses of SNP-induced cell injury, NGB may affect anti-apoptosis gene bcl-2 by an unclear way, and up-regulate the expression of bcl-2 gene to take neuroprotective effect.Energy exhaustion is one of the most important pathogenesis in ischemia and hypoxia injury. To better understand the molecular mechanism of neuroprotective effects of NGB, our lab used yeast two-hybrid technique to screen for proteins interacting with NGB from a human fetal brain cDNA library using NGB as bait. A candidate clone coding the C-terminal portion of Na⁺, K⁺-ATPaseβ2 subunit (ATP1b2) was identified. The interaction of NGB and ATP1b2 was confirmed by GST-pull down and co-immunoprecipitation assay. As Na⁺, K⁺-ATPase is an important enzyme for energy consumption, it consumes 40⁵0% of ATP even in physical condition. According to these, we hypothesized that down-regulation of Na⁺, K⁺-ATPase activity inhibited by NGB could possibly provide a unique neuroprotective mechanism for the neuronal cells by down-regulating the metabolism level. After research, our lab also demonstrated that recombinant NGB compared with GST could inhibit the Na⁺, K⁺-ATPase activity in a cell-free system. Over-expression of NGB in NG108-15 can also inhibit the Na⁺, K⁺-ATPase activity, and the inhibition was in a dose dependent and time-dependent manner. To identify this possible neuroprotective mechanism of NGB, we used ouabain to inhibit the activity of Na⁺, K⁺-ATPase directly, and the results showed low-dose ouabain can protect PC12 cells from CoCl₂-induced hypoxia injury. Results suggested that inhibition of Na⁺, K⁺-ATPase activity may be one of the NGB neuroprotective mechanisms.Further more, we used rhNGB (recombinant human neuroglobin) to study the function of NGB. Results showed that low-dose rhNGB can also protect neurons from CoCl₂-induced hypoxia injury. After preparing the AP4-NGB fusion protein, we incubated AP4-NGB with primary cultured neurons. Results showed that there were potential NGB-binding proteins in the surface of cultured primary neurons.In summary, the neuroprotective mechanism and function of NGB were explored in this study. We identified the neuroprotective mechanism of endogenous NGB and over-expressed NGB. NGB can protect PC12 cells from SNP-induced cytotoxicity, probably due to up-regulation of the anti-apoptosis bcl-2 gene. Regarding on the meaning of the interaction between NGB and NKA1b2, we found that NGB could inhibit the enzymatic activity of Na⁺, K⁺-ATPase via itsβ2 subunit, which is a potential survival strategy for neuronal cells in early stage of hypoxia. We also found rhNGB has neuroprotective function, and AP4-NGB can bind the surface of neuron. All the results are important to understand the function of NGB, and provide more clues for the clinical application of NGB.