| In recent years, along with the accelerated aging of the population, aging and degeneration of the brain has become widespread and serious diseases affecting the elderly ability of living. The major topic of aging research is still limited to some preliminary experiments and theories, of which oxygen free radicals and apoptosis is the greatest impact. The free radical theory of aging consider that apoptosis caused by oxygen free radicals is the major reason affecting the aging process, the mechanism of the injury remains to be explored.Hippocampus is a key component of learning and memory, and is extremely susceptible to aging, oxygen free radical damage to neurons in the hippocampus was important reason for the decrease in memory lead. PMA as a cancer inducer can product reactive oxygen species by stimulating cell and be used to induce apoptosis. But there is no reports about hippocampal neurons induced apoptosis so far. A large body of research shows phosphatidylserine (PS) plays an important role in the enhancement of learning and memory, improve brain function, maintenance and repair of nerve cells. However, previous investigatator has researched physiological functions of the PS in vivo, they did not study at the cells level.Therefore, we extracted PS from fresh cattle in the brain, using PMA-induced apoptosis in rat hippocampal neurons. We studied the antioxidant and anti-apoptotic role of PS on hippocampal neurons for the first time.Objetive: To study the antioxidant effect of different concentrations of PS on primary cultured rat hippocampal neurons and compare the antioxidation of two kinds PS from self extracts and Sigma.Methods: We extracted PS from fresh cattle in the brain, and also determined the nature and the amounts by thin-layer chromatographic (TLC) and by thin-layer chromatographic scanner. We cultured primary neonatal rat hippocampal neurons, PMA with different concentrations at different times can produce reactive oxygen species (ROS) which can induce apoptosis by stimulated cells. The apoptosis rat of cells after acridine orange (AO) staining was detected by Confocal, inverted fluorescence microscope was used to detect apoptotic cells period. Early and late apoptotic cells can be detected by experimental PS valgus. NFDS-1 was used to detect the H2O2 content of cells after stimulating,and tested the protective effect of PS on hippocampal neurons.Results: It was simple and had high extraction rate and high sample recovery in this method by thin-layer chromatographic. PMA-induced apoptosis results showed that it is suitable for the establishment of neuronal apoptosis model in the concentration of 10-2g/L and the time for 24h. Both PS in the concentration of 1.5mg/L and 15mg/L all had obvious anti-apoptotic effects, the differences was significant compared with 10-2g/L PMA grope, but the grope of 15mg/L was more effective; However, the concentration of 150mg/L had no apparent protective effect on cells; In the same concentration, PS which was extracted by ourselves was somewhat better in the inhibition of apoptosis than the Sigma′s. The result using inverted fluorescence microscope also testified that the grope of 15mg/L PS can effectively inhibit neuronal apoptosis caused by PMA. In the NFDS-1 experiment, control group did not show fluorescent phenomenon after adding the fluorescent probe; The group injuried 24 hours by PMA, whereas, showed strong fluorescent; and the group was protected by 15mg/L PS before PMA injury had stronger fluorescent than control group, but obviously weaker than PMA injuried. Annexin V-FITC and PI experiment can detected early and late apoptosis of cells clearly.Conclusion:1) PMA at a certain concentration can produce ROS free radical oxidation and injury rat hippocampal neurons by stimulating nerve cells. In this study, it is reliable to establish PMA–induced apoptosis model in hippocampal neurons.2) PS at a certain concentration can increase the ability of antioxidant in hippocampal neurons. It can be concluded that PS can protect the nerve cells in the cell level; Further it is affirmed that the PS will promote learning, enhance memory and improve the role of age-related diseases.3) In the same concentration, the PS which was extracted by ourselves is better in the inhibition of apoptosis than the Sigma′s. This experiment proves that the method is feasible and reliable to extract and separat PS. Innovative:1) We provided a practical separation parameters of brain phospholipid by TLC.2) We adopted PMA-induced apoptosis in hippocampal neurons for the first time and acquired the most appropriate drug concentration and time.3) We have studied the antioxidant and anti-apoptotic of phosphatidylserine on hippocampal neurons in the cells level and achieved the desired results, which can provided an effective drug for treating and preventing the aging and brain related diseases... |
PDF Full Text Request