Neuroprotective Mechanism Of Tat-NGB Under Hypoxia And Application Of A Novel Hypoxia Model

Normal oxygen partial pressure of human tissue is 2-9%(14-65 mm Hg), whereas the oxygen partial pressure of intake air is 21%(160 mm Hg). Tissue hypoxia appeared when the outside air pressure was low or oxygen transport and use were blocked. Hypoxia is common stress for human and animals. A large number of studies have shown that hypoxia can cause diseases of cardiovascular system, immune system, respiratory system, digestive system, the motor system and many other physical disorders even life-threatening pulmonary edema or cerebral edema, such as acute and chronic high altitude disease caused by high altitude, neonatal hypoxia induced seizures, cerebral infarction caused by the local blood supply disorder. These common diseases are closely related with tissue or cells hypoxia. Therefore, the pathogenesis and interventions of hypoxia related diseases are hotspots of research. Recently, protective additives of hypoxia injury are mainly focused on polysaccharide antioxidant or natural extracts based on the free radical scavenging effects. Seaweed polysaccharides and a number of other anti-oxidation substances have achieved cell repair and the protective effects in vitro, however, because the disease such as stroke arose quickly with a blood brain barrier hindering, there were little clinical drugs applied to hypoxia injury. Neuroglobin(NGB) has been highly concerned since found in 2000. It mainly distributes in the neurons and retina cells. As the third kind of globins, NGB has the ability of carrying oxygen similar to hemoglobin and myoglobin. A large body of researches showed that NGB expression increased significantly in neurons under hypoxic conditions. Other studies demonstrated that NGB has the ability of scavenging superoxide free radical. In the current study, NGB was chosen to be a potential protein drug for prevention and treatment of hypoxic diseases. And the recombinant NGB was attempted to express in a eukaryotic expression system, therefore, the expressed proteins could be of high fidelity(not yet reported at present). Furthermore, the recombinant NGB fused with a membrane penetrating peptide tag HIV-1 Tat to overcome the blood-brain barrier obstacles. Neural protective functions were also verified in the study.In the hypoxic research, hypoxia dynamic is an important link. The current physical methods as hypoxic incubator had deficiencies as equipment dependence, high costs, complicated operations. Whereas, the chemical hypoxic inducing agents as Co Cl2, Na2S2O4 had heavy metal toxicity or instability. In the current paper, Na2SO3 was introduced to be a hypoxia inducing agents without the above shortcomings, relied on investigation and preliminary work. Furthermore, the mechanisms of hypoxic regulation in E. coli were preliminarily studied based on the Na2SO3 application.The brain occupies an important position in the human and animal body. Brain mass accounts for only 2% of body weight but use 20% of the energy. Traditional theory holds that the brain is selfish in the use of energy, its energy storage is few and obtains from peripheral nerve tissue for need, even to damage other organs. The finding of NGB further supported selfishness of the brain, for its high oxygen carrying capacity could guarantee the energy supply of brain. In the current study, special status of brain under nutrition extremely lack would be discussed on water regulation and autophagy.The main results and conclusions are as follows:1. Eukaryotic expression and purification of recombinant NGB and its protective effect on hypoxia injuryThe Ngb genes were amplificated by RT-PCR using previously constructed human neuroglobin prokaryotic expression plasmid p ET28b-Ngb as templates. Ngb genes and p PIC8 k vectors were digested by Eco RI、Not I and connected to complete the construction of p PIC8 k. Then the recombinant vectors were linearized respectively by SalⅠ、BglⅡ and electrically transformed into Pichia pastoris GS115. MD plates were used to screen positive clones. The both MD and MM plates were introduced to screen out methylotrophic strains. Followed, strains with high plasmid copy were selected by geneticin G418 and colony PCR. The selected yeast strains were introduced to do expression test and chose the strains with high output by SDS-PAGE. The above steps had successfully realized recombinant NGB expression in eukaryotic system. Exogenous recombinant proteins by eukaryotic modification had more fidelity with its original function. In addition, recombinant NGB could be directly secreted into the culture to simplify collection of target proteins and avoid invalid expressions as inclusion.The above selected yeast strains with high NGB expression were used to produce recombinant NGB and achieved protein enrichment and purification by Ni-NTA affinity chromatography. Then the purified proteins Tat-NGB and NGB were respectively cocultured with PC12 cells. The recombinant NGB were detected by Western Blot after 12 h coculture. The results demonstrated that there was no NGB signal detected in the total proteins of PC12 cells with His-NGB, whereas, in the Tat-NGB group NGB bands were clear, demonstrating that the fusion of HIV-1 Tat mediated the recombinant NGB for transmembrane. The recombinant NGB in the cells could be accumulated to a stable level within 1.5 h. The entering speed could meet the time window of therapeutic intervention in hypoxic cells. CCK-8 kit was introduced to monitor cell viability under hypoxia. Results showed that, PC12 cells with Tat-NGB had a significant higher viability than the control cells and cells with NGB. The results indicated that the recombinant Tat-NGB could not only transmembrane but also play a protection role in hypoxia injury repair. To sum up, this part of the study firstly realized the expression and purification of recombinant NGB in eukaryotic expression system of Pichia pastoris. The obtained recombinant NGB not only fidelitied the original function of NGB to protect PC12 cells from hypoxic damage but also realized the function of transmembrane by HIV-1 Tat, which provided basis for its potential applications as neuroprotective drugs.2. Hypoxic inducing effect of Na2SO3 on microorganisms and study of hypoxia regulation mechanismFirstly, we used growth curve to detect effects of sodium sulfite on different E. coli strains(BL21(DE3), HB101, DH5α, OP50). The results showed that sodium sulfite had obvious and wide growth inhibition on E. coli. The dissolved oxygen in the medium was determined at different point during the growth and had confirmed that the inhibition on E. coli was due to hypoxia in the medium. The anaerobic state could be stably maintained for up to 8 h. By means of TEM(transmission electron microscopy), bacterial cells were observed and there were no obvious cell rupture or injury. In conclusion, sodium sulfite could deprive oxygen in the medium for a stable period without damage on bacterial cells. The hypoxia effect was similar to physical methods. Therefore, sodium sulfite could be used as an ideal hypoxia inducing agent for anaerobic microbial culture and hypoxia study.Considering 99% of animal intestinal bacteria were anaerobic, study of Na2SO3 was extended to animal. The solutions were injected into mice gastrointestinal tract, and the effects of Na2SO3 on animal body were discussed on oxidative stress. The results showed that body weight of animal was not significantly affected, MDA concentration and SOD activity were both increased. The increased MDA inhibited a chronic damage on mice cells, therefore, Na2SO3 of high concentration was not suitable for in vivo studies. For rich flora in the gut, Na2SO3 of suitable concentration may be helpful for the growth, so this point will be the key in further research.The traditional hypoxia agent Co Cl2 has serious heavy metal toxicity. The follow-up study was focused on the toxicity of Co2+ and other heavy metal ions. Based on the growth curves of E. coli, we found that heavy metal ions had significant inhibiting effects on the microbial growth, but there were differences of the action mode for different metals.autophagy 3. Self regulation of mice brain under extreme nutrition lack through AQPs andThe mice were divided into groups with water deprived, food deprived and double deprived. During the experiments, the brain weight remained stable while the body weight and blood glucose sharply decreased. Further,HE staining shows that the cerebral cells held normal morphology and there were not obvious damages. Therefore, the brain had abilities for self protection maintaining physiological functions and morphology during extreme nutritional condition. The normal water contents of brain and stable Aqp-1, Aqp-4 expression demonstrated that the brain could maintain water content by regulations of AQPs. To determine the autophagy level in cerebral cells, LC3 Proteins were detected by Western Blot method. Results showed that autophagy levels of the experimental groups were all increased, especially the group without both water and food. So in addition to competing energy from surrounding organs, the brain also had its own self rescue behavior based on its own materials through autophagy. The study had expanded the selfish brain theory by water content regulation and supplemented the theory by autophagy from a non selfish perspective.