The Protective Effects Of Soybean Isoflavone Cooperated With Folic Acid On The Neural Tube Defects Of Rat Induced By Cyclophosphamide

Objective:To study the mechanism of soybean isoflavone cooperated withfolic acid 's effects on the neural tube defects of rat induced by cyclophosphamide on the basis of formal research. The mechanism to study included: 1 .The influence of soybean cooperated with folic acid on the per-oxidation status of pregnant rats induced by cyclophosphamide ; the best combination group and the relationship of the two factors on the orthogonal experimental design; 2.The apoptosis-suppressing effect of genistein cooperated with folic acid on theapoptosis of fetus's neuron induced by cyclophosphamide. Methods:l.vivistudy: put the female rat and the male rat in the same cage by the ratio of 1:1 overnight, the female rat that were detected with the vaginal plug the next morning were recognized as the pregnant rat ,and the day was the 0 day of gestation. Pregnant rats were randomly divided into the control group, model group, combination experimental group and the isoflavone solo-experimental group according to their body weight. Each group had 7 animals except that the combination experiment were repeated 4 times. The pregnant rats of the experimental group were oral administrated with isoflavone and folic acid according to the prescribed doses from the 0th day to the 20th day of gestation. In the combination group, the two factors, soybean isoflavone and folic acid were designed as three levels, (SI: 40,80, 160mg/kg.bw, FA: 0.35, 0.7, 1.4mg/kg.bw), the experiment were strictly carried on according to the L9(34)orthogonal experimental table. The doses of the SI solo-experimental group were based on the dose and the twice dose of the soybean isoflavone in the best combination experimental group. On the 13th day of gestation , the pregnant rats of the model and the experimental group were given cyclophosphamide 12.5mg/kg via intraperitoneal adminstration; the animals of the control group were given 0.3ml N.S in the same way. On the 14th day of gestation, two rats of each group were executed and the brains of the embrys were fixed in the neutral formalin solution as the specimen of the immuohistochemmistry .The other pregnant rats were executed on the 20th day of gestation. The 1001 whole blood of the jugular was used as the mensuration of whole blood selenium level; the other blood of the jugular was centrifuged to obtain the serum for the examination of the levels of glutathiom glutathion-peroxidase, selenium, mangnase, iron; the embryos were carefully checked and the malformation (meningoencephalocele) rate were recorded. 2.vitro study: (1) the observation on early apoptosis of neuron by flow cytometry: Neuronal preparations at 48 hours were randomly divided into control group, model group and seven experimental groups added with different concertrations of genistein or (and) folic acid. The cells of control group were directly collected at 54 hours of cultivation. For the model group, neuronal preparations at 48 hours were added with cyclophosphamide at the concertration of 8g/ml and collected after 6 hours. For the experimental group , genistein and folic acid were added into neuronal preparations at a range of concertration 30miniutes before the CP was added .(2) The observation on the changes of memberane of early apoptosis neuron by scanning electron microscope(SEM): thetreatment was as same as the groups' described above. Results:1. The effects onthe per-oxidation status of pregnant rats: The administration of CP made a obvious decline of the levels of glutathionx glutathion-peroxidase and the related mineral elements seleniuiru mangnase, iron compared with the control group. After the adminstration with isoflavone and folic acid , the results showed that: (1) The interaction effects of the two factors have significantly compact on these indices .The coadministration of the high dose of isoflavone(160mg/kg.bw) and the middle dose of folic acid(0.7mg/kg.bw) could have the best effect .(2) When the indices related with the antioxidation reached the highest levels, they may maintain at the close...