Effects Of Soybean Phospholipid On Learning And Memory Related Genes And Abilities Of Antioxidation

Phospholipid is a natural and nutritional health product, which is mainly in animal and plant cells and the main component of biological membranes. Soybean phospholipid can maintain the normal structure and function of biofilms, and keep closely related with cells growth, proliferation, differentiation and cancer, what's more, its degradation products are also involved in cell signal transduction and metabolic regulation, improving memory, relaxing the consenescence, regulating blood fat, anti-tumor, caring of the brain, protceting the liver, and other physiological functions. In this study, we concentrate on the effects of soybean phospholipid on learning and memories related genes and antioxidant capacity, and explore its machanism and provide experimental basis to the application of soybean phospholipid and conservation to nerver cells.Methods:1. Modeling of D-galactose induced senile rats. According to weight, randomly divided 50 SD male rats into 5 groups: nomal control group, model control group, low dose of soybean phospholipids group (0.5 g/kg/d), middle dose of soybean phospholipid group (1.0 g/kg/d) and high dose of soybean phospholipids group (1.5 g/kg/d). All of the model control group and the groups of soybean phospholipid were injected with D-galactose(50 mg/kg/d), while the normal control group was injected with physiologic saline. The rats were gavaged and injected regular daily. The learning and the memory abilities were tested on the 30th day, then the rats were killed on the 45th day, and contents of MDA, SOD and CAT activity in tissues were determined.2. Modeling D-galactose induced senile rats, then rats were killed on the 45th day, to extract the total RNA of hippocampus and cerebral cortex, using reverse transcription polymerase chain reactin (RT-PCR) method, quantitative analysis of the expression of Bax, p53, Bcl-2, c-fos, NCAM genes in hippocampus and cerebral cortex.3. Cultured nerve cells of rats. Take the newborn rats within 24h, use 75% alcohol disinfection, open the brain of the rats, removing the meninges and blood vessels, cutting into pieces of 1mm3, digesting with 0.125% trypsin on 37℃for 20 min, then 1000 r/min centrifugal 5 min, take precipitation, made into cell suspension. Adjusted the densities to 2 X 106cells/mL, seeded into culture plates by 0.1% L-polylysine coated off, cultured under the conditions of 37℃,5% CO2 and saturated humidity. The whole medium was changed after 24 h,3 d after inoculation, the whole medium was changed again, adding different concentrations of soybean phospholipids containing complete medium and continue to develop. Do the next experiment after 6 days'cell culture. Cultured brain cells of rats with the same way.6 d after cell cultured, the cells were collected by MTT and trypan blue staining method to detect cell viability, and determined the content of total protein, MDA and the activity of CAT.4. Cell morphology was observed under inverted microscope, using the method of MTT to assay the viability of cells, AO/EB fluorescentstained, Hoechst 33258 fluorescentstained, single cell gel electrophoresis to detect apoptosis of cells, observing the cell surface ultrastructure byscanning electron microscope.Results:1. Compared with normal control group, the learning and memory abilities of model control group were greatly declined (P<0.05), the contents of MDA were obvious increased, while the abilities of SOD and CAT were decrease (P<0.05) in all tissues. The learning and memory abilities of soybean phospholipid groups were greatly improved (P<0.05), and the contents of MDA were lower, the activities of SOD and CAT were also greatly increased (P<0.05). All the results showed that we had built up the model of D-galactose induced senile rats, and soybean phospholipid can strengthen memory ability and antioxidant capacity.2. Compared with the control group, in the model control group, both the ratio of Bax/β-actin and p53/β-actin increased, the difference were significant (P<0.01); both the ratio Bcl-2/β-actin and NCAM/β-actin decreased, the difference were significant (P<0.01); c-fos/β-actin increased (P>0.05). Compared with the model control group, in the groups of soybean phosphlipid, the ratio of Bax/β-actin was significantly decreased (P<0.05); the ratio of p53/β-actin tended to decrease (P>0.05); both the ratio of Bcl-2/β-actin and NCAM/β-actin were increased, the difference were significantly (P<0.01); the ratio of c-fos/β-actin was decreased significantly (P<0.01). All the results showed that soybean phosphlipid can decrease the expression of Bax, p53, c-fos and enhance the expression of Bcl-2, NCAM in Hippocampus and Cortex tissues of induced senile rats.3. The results of MTT showed that, compared with the nomal group, when treated with soybean phosphlipid from 10-7 mg/mL to 10-5 mg/mL, the activity of nerve cells increased while the concentration increased, and there showed significant difference when the concentration increased to 10-5 mg/mL (P<0.05). However, when the treated soybean phosphlipid increased from 10-5 mg/mL to 10-3 mg/mL, the activity of nerve cells decreased, and the activity of nerve cells of 10-3 mg/mL treated group even lower than the control group. These results show that only in a certain range of concentration, soybean phospholipid can increase the activity of nerve cells. Trypan blue staining showed that the survival rate of cells between the treated groups and the control group were not significant (P>0.05). The contents of total protein and MDA in nerve cells were not significantly different (P> 0.05), and the activity of CAT was increased (P>0.05). All the results showed that soybean phosphlipid can enhance the antioxidant capacity of nerve cells to some extent.4. Under the inverted microscope, the cells began to adhere after inoculated 4h, distribut uniformly, cells appeared circle and transparent shapes; cultured 24 h, most cells were spindle-shaped, cells were in a three-dimensional bulge, the surface smooth, most cells appeared a unipolar apophysises, and a small number appeared bipolar cell apophysises. After 3 days'culturing, the cells were mostly spindle-shaped morphology, and cells' bodies were full, cell apophysis increased and longer, bipolar and multipolar apophysises increased significantly, lighting links were obviously, among the cells began to form a complete neural network architecture. Cultured after 6 days, cells were spindle-shaped, triangle-shaped and other shapes, cells'bodies increased significantly, the apophysises become larger, longer and more. The normal control group and the soybean phospholipid groups showed shades of structural features in different colour of fluorescence by AO/EB fluorescent staining. Cell morphology was normal and nuclear chromatin was green. Hoechst 33258 fulorescent staining showed that the nuclear morphology of all groups were normal and showing a uniform weak blue fluorescence. In the single cell gel electrophoresis, all the groups were no smearing. And both Hoechst 33258 fluorescence staining and single cell gel electrophoresis results had shown that soybean phospholipid can increase the density of cells. Under the scanning electron microscopy, neurons of soybean phospholipid groups were in rich of surface folds, more apophysises, bipolar and multipolar differentiated clearly and showed net structures. All the results showed that a certain concentration of soybean phosphlipid can maintain the nomal structure of nerve cells, enhance the sensity of nerve cells and enhance the differentiation and growth of nerve cells.Conclusion:1. Soybean phospholipid can strengthen memory ability and may have something to do with cells decrepitude and relax the consenescence caused by D-gallstone.2. Soybean phospholipid can delay cell senescence by down-regulated Bax, c-fos and p53 and upregulated Bcl-2and NCAM, in order to improve the learning and memory ability of D-galactose induced senile rats.3. At a certain concentration range, soybean phospholipid can increase the activity of cells and increase the effects of antioxidant.in nerve cells of rats and help the removal system clear and maintain the radical to nomal degree.4. Soybean phospholipid can promote the growth of nerve cells and maintain the normal structure of nerve cells and so on.