Thioredoxin Antioxidant Stress Research Based On The Level Of Nitric Oxide

Objective: Nitric oxide (NO) is widely distributed in various tissues in vivo,especially nervous tissue. It is a new biological signal molecule, and was selected asthe star moleculesthe by Science magazine in United States in1992. NO is a veryunstable biological free radicals, small molecules, simple structure, gas under normaltemperature, slightly soluble in water, fat-soluble, and can quickly spread through thebiofilm, biological half-life is only3-5s, generate depend on nitrogen oxide synthase(nitric oxide synthase, NOS).It has a very important biological role the regulation ofheart, cerebral blood vessels, nerves and immune.Therefore, it has been taken theuniversal attention. L-arginine (L-Arg) is one of the essential amino acids in the body,which can be converted into L-citrulline and NO under nitric oxide synthase (NOS).However, due to the recent academic controversial about the cytoprotection of NO–theexistence of NO is a "double-edged sword". Because NO can affect the activity of Trxand its own influence on apoptosis is extremely complex. We can indirectly control thecontent of NO in the cells by controlling the amount of NO donor(L-arginine) to,thereby establishing NO cell toxicity model, and then to explore the role of NO inoxidative stress injury and the relationship between the TRX and NO.Thioredoxin (TRX) is a new member of anti-redox protein as we all know. Itplays an important role in the anti-oxidation system. Thioredoxin is a small moleculeprotein (about12kDa). It as a hydrogen donor in many of the reduction reaction, andcould scavenge oxygen free radicals generated in cells due to oxidative damage in acertain extent. TRX has been proven to have a protective effect for the body’s nervecells, but the mechanism is unclear. The oxygen free radicals are widely involved in avariety of neurological diseases, and could been the basis of the incidence. In thisexperiment, we use Neuro-2A as the research object. Neuro-2A cells is mice neuralcrest parent tumor cells which has the same morphology and physiological andbiochemical characteristics with normal nerve cell and retains a series of normal nerve cell function, has a wide range of representative role for the experimental study of thenervous system.This study was designed to investigate the protective effect of thioredoxin innerve cells oxidative stress, the relationship between TRX and NO and the influenceof TRX in NO signaling pathway. Thus further complete research data about the TRXspecific protective effect on the nervous system, providing a new way for clinicaltreatment of nervous system diseases.Methods: With the Neuro-2A cell as the experiment object, using the hypoxicbox to simulate the environment within the body of hypoxia and adding the L-Arg(concentration to1mmol/L) to imitate the production pathway of NO in humanbody,we can establish a NO cell toxicity model of the Neuro-2A cell. After4hours ofhypoxia/reoxygenation, Observe the shape, the viability, the apoptosis and the contentof malondialdehyde (MDA) and NO and the vitality of superoxide dismutase(SOD) inboth normal cells and TRX gene transfection ones.Results:1. the hypoxia cell group gets a greater damage than the normal one,which looks sparse, poor adherence, protuberance back, shrinking and round obviously.however, the normal one looks regular,well adherence, and only a bit of them shrinkingand round.2. Detecting the cell viability by MTT assay:the viability of two groups of cellsreduced after making the hypoxia and adding L-Arg after. But the TRX genetransfection cells are more viable than normal ones in every experiment group（P<0.05or P<0.01）.3. The result of MDA and SOD in cells: comparing with the normal cells, the hypoxicones get a gradual raise of MDA and a reduce of vitality of SOD（P<0.05）. Comparingwith the hypoxic cells, L-Arg hypoxic ones get a gradual raise of MDA and a reduce ofvitality of SOD（P<0.05）. Comparing with the normal Neuro-2A cells, the TRX genetransfection Neuro-2A ones get a reduce of MDA and a raise of vitality of SOD（P<0.05）.4. The result of apoptosis rate: comparing with the normal cells, the hypoxic ones get agradual reduce of the survival rate and a raise of apoptosis rate（P<0.05）. Comparingwith the hypoxic cells, L-Arg hypoxic ones get a gradual reduce of the survival rate anda raise of apoptosis rate（P<0.05）. Comparing with the normal Neuro-2A cells, the TRX gene transfection Neuro-2A ones get a reduce of the apoptosis rate and a raise ofsurvival rate in every experiment group（P<0.05）.5. The result of NO in cells: comparing with the bland group, the NO in the addingL-Arg only is similar. Comparing with the normal cells, the hypoxic ones get a gradualraise of the content of NO（P<0.05）. Comparing with the hypoxic cells, L-Arg hypoxicones get a gradual raise of the content of NO（P<0.05）. Comparing with the normalNeuro-2A cells, the TRX gene transfection Neuro-2A ones get a reduce of the contentof NO in every experiment group（P<0.05）.Conclusion:1. TRX gene expression of thioredoxin protein has the role ofanti-oxidative damage, protecting the Neuro-2A cells suffered oxidative stress to someextent.2. TRX protect Neuro-2A cells may be by combined with NO, reducing the NOcontent, scavenging oxygen free radicals, and enhancing antioxidant capacity tomaintain the level of intracellular SOD.