The Study Of Protective Effect Of Schisandrone On Hippocampal Neurons Damage Induced By Amyloid-beta Protein

This study is compound with cell and animal experiments. On the cell experiment, the hippocampal neurons damaged by amyloid-beta protein and protected by Schisandrone are cultured through primary culture technic and additionally feeding with amyloid-beta protein or Schisandrone. The reactive oxygen species(ROS) and intracellular calcium concentration([Ca~2+]i) are measured and compared with each other. On the animal experiment, the animal damaged by amyloid-beta protein and proteced by Schisandrone are treated through injected with amyloid-beta protein into lateral cerebral ventricle and continuous fed with Schisandrone dissolving in corn oil. The capability of learn and memory of every rat is tested by Y maze experiment. The unit of resisting reactive oxygen species of hippocampus is measured and calculated, and the morphology is observed by Nissl and Congo Red staining.The experiment techniques in this study are concerned with primary culture of hippocampal neuron, Coomassie brilliant blue protein quantitation, spectroscopical chromatometry, mensuration and detection of intracellular reactive oxygen species and free calcium concentration, intragastric administration, lateral cerebral ventricle injection, Y maze experiment, Nissl and Congo Red staining.The main methods and results of this study are as follows: 1. Cell Experiment1.1 Effect of Schisandrone on intracellular ROS induced by amyloid-beta protein in cultured hippocampal neuronsThe hippocampal neurons of one-day-old male Sprague-Dawley(SD) rats cultured for seven days are divided randomly to A, B and C groups. The group A is cultured with 1μmol/L Aβ25-35- The group B is cultured with lumol/L Aβ25-35 and additionally with 100μmol/L Schisandrone. The group C , the blank control group is cultured only with B27. Then all the groups are cultured for another two days. On the nineth day the concentration of ROS in every group is measured and calculated. Theconcentration of ROS in every group is 99.8778 ± 17.7379U/ml of A group, 65.9318 ±24.3944U/ml of group B, and 48.9732 + 28.2315U/ml of group C. The data is significantly different with each other(P<0.05). These results indicate that amyloid-beta protein can induce the increase of the intracellular ROS, aggravating the oxidative stress, which can be partly prevented by Schisandrone.1.2 Effect of Schisandrone on [Ca ~2+]i induced by amyloid-beta protein in cultured hippocampal neuronsThe hippocampal neurons damaged by Ap2s-35 and protected by Schisandrone are cultured and divided through the same way. On the nineth day, every dish with cultured cells is rinsed with Kreb's-HEPES for three times and fed with lml Kreb's-HEPES dissolving lOul pluronic F-127 and 3ul Fura-2/Am, the final concentration is 0.04% and 4umol/L. All dishs are layed into incubator with 37°C and 5% carbon dioxide for twenty to thirty minutes. Then the dish is rinsed with Kreb's-HEPES for another three times. The well marked and dispersing cells are selected for dectection and analyses by Metafiour soft ware. The ratio of 340nm to 380nm is represented the real concentration of intracellular calcium. The ratio of group A is 0.5951±0.0311, group B is 0.4168±0.0468 and group C is 0.3617±0.0197, which is significantly difference with each othoer. These results demonstrate that the increace of intracellular calcium can be induced by amyloid-beta protein and be partly blocked by Schisandrone.2. Animal Experiment2.1 Effect of Schisandrone on the influence of amyloid-beta protein on learn and memory of ratsThirty SD rats are divided randomly to A, B and C group. Every group with ten rats are raised in standard environment for seven days. Schisandrone dissolving in 2ml corn oil concentration lmmol/L is fed by intragastric administration to the rats of group B, and 2ml pure corn oil is fed to the rats of group A and B one day at a time. On the eighth day, amyloid-beta protein dissolving in 5ul saline with concentration lOmmol/L is injected by stereotaxis injection into lateral cerebral ventricle of group Aand B, and 5ul saline is injected into lateral cerebral ventricle of group C. All rats are raised for another seven days in the same way. On the sixteen day all the rats are tested by Y maze experiment. The data of average ability of learning are as follows(times): 27.9±13.13 of group A, 10.9± 11.50 of group B, 7.7± 10.46 of group C; the average ability of memory are(times): 20.4±13.66 of group, 6.3±11.34 of group B, 4.7±6.09 of group Co The data of A and B, A and C is significantly difference(p<0.05), the data of B and C is no difference at 0.05. These data imply that amyloid-beta protein is harm to the ability of learn and memory of rats and the Schisandrone is beneficial to improve the ability.2.2 Effect of Schisandrone on the influence of amyloid-beta protein on unit resisting ROS of rat's hippocampusThe SD rats have been passed Y maze experiment are decapitated, and the hippocampus of every rat are dissociated under the anatomical microscope and then are homogenated by hand. The clear supernatant liquid of homogenate centrifugalization is diluted with saline to 1%. The protein quantity is measured through Coomassie brilliant blue quantitation, and the unit of resisting ROS is measured by spectroscopical chromatometry. The concentration of unit resisting ROS of group A is 16.081 ±5.418U/mg, the group B is 32.893 ±3.776U/mg and the group C is 61.014+14.390U/mg, which is significantly difference with each other. These results demonstrate that the decreace of the concentration of unit resisting ROS can be induced by amyloid-beta protein and be partly blocked by Schisandrone.2.3 Nissl and Congo Red stainingThe rats are decapitated, and brain are dissociated and be fixed, cut to sections. The section is stained by Nissl and Congo Red staining to observe the loss of neurons and the deposition of amyloid-beta protein. The results of Nissl staining reveal that morphous of neurons are decreased and deflected in group A. The deposition of amyloid-beta protein are not observed obviously through Congo Red staining in three group section. These observation indicated that amyloid-beta protein can lead to the loss of neurons and the effect maybe indirect pathway besides the direct action.