Anti-hypoxia Effect And The Molecular Mechanism Of The Free Radical Scavenger Tempol

Objective:1. Establish the BALB/c mouse normbaric hypoxia model to evaluate the anti-hypoxia effect of Tempol. Preliminary study of the antihypoxic mechanism of Tempol.2. Establish PC12cells and H9C2cells hypoxia models observe the injury in cells of the hypoxic environment, screening the optimal concentration of Tempol in hypoxic cells,study the protective molecular mechanism of Tempol in cells by the hypoxic injury.3. Observe the influence of Tempol on hypoxic H9c2cells ROS.MIF-1α. anti-oxidation system and apoptosis. further investigate the possible mechanisms of Tempol.Method:1.The BALB/c mouse models of normbaric hypoxia and decompression hypoxia were used to evaluate the survival time of mice in hypoxia after given different doses of Tempol, evaluate changes of LD contents LDH activity、SOD activity and MDA content,Preliminary study of the antihypoxic mechanism of Tempol.2. Cultivate PC12cells and H9C2cells in anoxic environment, screening the best concentration of Tempol in hypoxic cells, observe the cell morphology, determin the cell viability before and after hypoxia. assay ROS content, SOD activity, LDH content,MDA content and T-AOC ability.use Real-time PCR analysis HIF-1α,VEGF, Caspase-3mRNA expression levels, extract total protein and use Western-blot detect HIF-1α,VEGF proteins expression.3. Use YC-1blocked HIF-1α. observe the change of ROS content. HIF-1α,SOD activity.CAT activity and apoptosis in anoxic H9c2cells of Tempol.Result:1.Compared to the model group, three dose of Tempol enhanced the survival time of mice in both normbaric hypoxia models.and extension of time than acetazolamide.The mice were exposed continuously to a simulated high altitude.Coinpared with the model group,Tempole could decrease the content of LD.LDH.MDA and increase the activity of SOD in heart and brain.2, Hypoxic environment significantly inhibited the PC12cells and H9C2cells activity, compared with normal control group, cultured PC12cells and H9C2cells in hypoxic environment for48h. the cells activity significantly inhibited. The Tempool final concentration in1×10-6mol·L-1could significantly improve the anoxic cells activity.Tempol concentration in1×10-6mol·L-1could effectively reduce the content of ROS,LDH,MDA. increase the activity of SOD.T-AOC in hypoxic cells.At the same time,Tempol could decrease CK activity in H9C2cells.Compared with hypoxia model group, Tempol could increase hypoxic PC12cells HIF-1α.VEGF mRNA and protein expression, cultured PC12cells in hypoxia inveronment for36h,48h.on the other hand, Tempol could significantly decrease Caspase-3mRNA expression after exposed to the hypoxic24hours.Compared with hypoxia model group,Tempol could increase hypoxic H9C2cells HIF-1HIF-1α.VEGF mRNA and protein expression, cultured H9C2cells in hypoxia inveronment for24h,36h and48h, reduced the expression levels of Caspase-3mRNA in the hypoxic environment.3. The YC-1concentration in4×10-5mol·L-1could effectively block the expression levels of HIF-la in H9C2cells after hypoxia for24h, resulting in hypoxia H9C2cells VEGF. SOD, CAT expression has also been reduce, Tempol can be increased the expression levels of HIF-la after YC-1blocking HIF-lamRNA and protein, at the same,increased the expression levels of VEGF, SOD, CAT mRNA and protein.reduced the expression levels of caspase-3, reduced apoptosis in H9C2cells.Conclusion:1. Tempol has significant anti-hypoxia effect on hypoxic mice, the possible mechanism is related to increase SOD activity, decrease LD content,LDH activity,MDA content in heart and brain.protect the integrity of the cell membrane, affecting the cell cycle, anti-apoptosis, increased the gene expression levels of HIF-1α in hypoxic PC12cells.2. Tempol has significant anti-hypoxia effect on PC12cells and H9C2cells, the possible mechanism correlated to improve cells activity, clear intracellular excessive ROS, increase intracellular antioxidant capacity, and reduce the accumulation of lipid peroxidation products, protect the integrity of cell embranes, anti-apoptosis and increase the gene expression levels of HIF-1α、VEGF and decrease the gene and protein expression levels of Caspase-3in hypoxic cells. 3. In the absence of oxygen. Tempol protection mechanisms H9c2cells may be elevated HIF-la expression activated VEGF. SOD. CAT’s high expression increased antioxidant capacity, remove excess free radicals, reduce apoptosisto achieve. The specific mechanism is in need of further study.