Protective Effect Of Oyster Extract On Apoptosis Of Nerve Cells Induced By Hyperthermia

Objective:Aristogenesis and well education and the elevation of population diathesis are great plan which related to our country wealthy and strong. The coming years,the work of aristogenesis has gained significant achievrment,but there are many problems to be solved.for instance,because of the severely environmental pollution accompanied by the development of the industry, the incidence rate of congenital malformation was so high.This serious burden afflict society as well as the affected families.Neural tube defects(NTD) was one of the most common and pernicious congenital malformation in these birth defects and it is the major reason to effect aristogenesis rate in our country. Hyperthermia is a common physico-teratogen.It induced multiple congenital abnomaly ,and it was the major environmental agent to lead to NTD.It was hard for pregnant women to ward off its affect. Epidemiologic survey showing, 10 percent of NTD were induced by hyperthermia.At present,we didn't get useful drag to rivalry NTD,therefore,it possesses important significance for reducing congenital malformation to search approach of preventing and therapy NTD. Oyster is oceanshell animal,it is rich in several nutritional ingredient that human body requires.The medicine usage of oyster shell and meat were awared in our country in the past ,and it was used in clinic constantly.It was recorded in Chinese medicine that oyster played a role in preventing miscarriage.There is an opinion that eating oyster after pregnancy,the moon calf would not be born. Modern medicine investigation indicatethat oyster has multi-bioactivity. Taurine and zinc is abundant in its extract.Taurine is a conditional essential amino acid,there are abundant of taurine in cells.It is a potent antioxidant and has an effect of removing free radical in the body.Moreover,it inhibited cell apoptosis in ischemic myocardial cells and protects injuried cells.It is reported that taurine and zinc could promote early embryo development and oppose teratogenic effect. It has been proved by a lot of animal experiments that hyperthermia could promote cell apoptosis in the neuroepithelial and mesenchyme cells of embryo,make viable count to decrease,reduce neural tube closure delayed or not closure.Thereby,neural tube defect,such as anencephalia, exencephalia, meningocele and so on were formed.Taurine inhibited cell apoptosis in ischemic myocardial cells, could promote early embryo development.If it has protective effect on apoptosis of NTD induced by hyperthermia,it will rivalry NTD.Therefore ,we proposed hypothesis that oyster extract rivalry NDT induced by hyperthermia.We have establisted an animal model of neural tube defects induced by hyperthermia in golden hamster.Furthermore we did some works about the developmental mechanism of NTD on this animal model. In this experiment we want to observe protective effect of oyster extract on apoptosis in neural tube defect induced by hyperthermia in vivo. To observe protective effect of oyster extract on apoptosis of neural stem cells induced by hyperthermia in vitro. Therefore, to observe and discuss antagonism of oyster extract on NTD induced by hyperthermia and to explain conceivable mechanism of action. Guiding people to utilize natural food to reduce birth defect, at the same time, promoting development and utilization of oyster food. Method:1.Experiment in vivo: Adult nulliparity female golden hamsters were offered by beijing experimental animal investigation center. Every night female hamsters were mated with mature male golden hamsters. Pregnant golden hamsters were divided randomly into 4 groups:control group treated by hyperthermia and oyster treated group I, II, III. Pregnant golden hamsters of control group were bathed in water(42 °C) 20 min on day 8 after pregnancy. All pregnant golden hamsters of oyster treatment were used oyster extract respectively from 4 to 8 day after pregnancy ,then treated as control group.All of the pregnant golden hamsters were killed at 8 hours, 16 hours after bathed , the uteri were excised,then some of them were fixed with 4% paraformaldehyde,the rest were fixed with improved bouins fixation fluid, then dehydrated ,embedded in wax, finally the specimens were sliced into section of 5um in thickness. The nuclear fast red-gentian violet staining method was used to observe the histological and quantitative alteration of apoptosis cells during development of neural tube of all groups embryo. Immunohistochemistry method and image analysis were employed to explore the expression of Caspase-3 in the neuroepithelial and mesenchyme cells of each mouse embryo group.2. Experiment in vitro: The mature female mice were mated with mature male mice.The pregnant mice were kill on the day 13 of pregnancy, cerebral cortex of embryo were excised,neural stem cells(NSCs) were cultivanted and identified by fluorescence microscope. Neural stem cells which were passaged 3 times were divided randomly into 4 groups xontrol group I and oyster treated group I,II,III, three groups were treated by oyster extract with different concentration. .At the same time,control group II only containing culture fluid and control groupIIIcontaining culture fluid and oyster extract were designed. All oyster treated groups and control groups were treated by hyperthermia. The vitality of neural stem cells was detected by MTT assay. Western-blotting was employed to explore the expression of p53 in cerebral neural stem cells of each group. Result:1.Nuclear fast red-gentian violet staining: at 8 and 16 hours after hyperthermia,the disposition of neuroepithelial cells were not regulate in control group,the number of apoptosis cells on neuroepithelial and mesenchymal cells was more. Compared with the control groups,apoptosis index of neuroepithelial and mesenchymal cells in the oyster-treated groups decreased.2. Caspase-3 immunohistochemistry staining: At 8 and 16 hours after hyperthermia,the Caspase-3 positive cells were found in neuroepithelial and mesenchymal cells randomly.The brown-yellow granules were found in intracytoplasm and cell nucleus. At 8 and 16 hours after hyperthermia,expression of Caspase-3 in the neuroepithelial cells was weakly positive or negative in the oyster-treated that was weaker than control groups.3. Neural stem cells: After cultivation 24 hours,the neurospheres with inequality of size were formed and there were 4-20 cells in each neurosphere.The size of neuropheres was increasing with days,smooth and glossy,not with obvious prominence.The cells in the neurospheres were uniformity in the size,and refraction of cells was strong.Cells in the neurospheres proliferated quickly after passage.Immumofluorescence staining shows that most cells in the neurospheres were positive of nestin.4. MTT: Compared with control group I ,the size of neuraospheres in the oyster-treated group II,III was bigger,the number of neurospheres was more. Cell boundary in the neurosphere was distinct and the cell has a better refraction.The numerical value of MTT in the oyster-treated group II ,III was higher than control group I.5. Western-blotting: Compared with control group I ,the expression of p53 in the oyster-treated group II ,III was weaker than in control group I.Conclusion:1.In vivo,oyster extract had an effect to reduce apoptotic cells in neural tube defects induced by hyperthermia ,and it could weaken the expression of apoptosis-associated protein Caspase-3.So we think that oyster extract had a protective effect on apoptotic cells in neural tube defects induced by hyperthermia.2.In vitro, oyster extract could enhanced proliferation activity of neural stem cells that were treated by hyperthermia, and it could weaken the expression of apoptosis-associated protein p53.So we think that oyster extract had a protective effect on apoptosis of NSCs treated by hyperthermia.Significance:The result of this study revealed that oyster extract had a protective effect on apoptotic cells in neural tube defect induced by hyperthermia,it had a protective effect on apoptosis of NSCs treated by hyperthermia.This is an evidence that oyster extract has an antagonistic action on NTD induced by hyperthermia to some degree.Therefore,it provides theory and experiment proof for precaution and cure of NTD induced by hyperthermia,guiding people to utilize natural food to reduce birth defect,at the same time,for promoting development and utilization of oyster food.