| Background and purpose:Nowadays,people often celebrate,make friends and tribute with the wine.Wine,whose main composition is ethanol,is a kind of small molecular neurotropic substance and it can pass through the blood brain barrier quickly,resulting in the damage of central nervous system directly.Hippocampal neurons are one of the most complicated cells in structure and function and also the most special cells of central nervous system.They are much sensitive to ethanol toxic effects,and the exposure to ethanol for a long time can cause changes in the structure and function of hippocampal neurons,depressing the ability of cognition and memory.This research built models of rat hippocampal cells with the different concentration of ethanol and procyanidins in grape seeds firstly,and then observed the changes of cell morphology at different time points by the laser confocal microscope,and used enzyme standard instrument to determine the content changes of SOD,MDA and LDH.The results of this research might provide theory basis for the further exploration in mechanism of damages that ethanol caused on the central nervous system and how to reduce the damages.Methods:1.Isolated hippocampus neural cells from SD rat infants that were clean level(SPF level)and cultured cells to the seventh day in vitro.Used NSE immunocytochemical staining to identify the purity of hippocampus neural cells and selected the hippocampus neural cells with good condition and higher purity.Divided the cultured hippocampal neurons into low dose group,middle dose group and high dose group,and added ethanol in the solution making the final concentrations of ethanol 0.5g\L,1.5g\L and 3.0g\L.Added equal PBS solution into the control group.Cultured neural cells in the CO2 incubator for 1h,4h,8h,12 h and 24 h.Collected supernatant for application and replaced nutrient solution after washing 3 times.Added low,middle and high dose of grape seed procyanidins respectively,making its final concentration 1.0mg\L,2.5mg\L and 5.0mg\L.Added equal PBS into control group.Cultured neural cells in the CO2 incubator for 8h and 24 h.Collected supernatant for application.2.Used WST-1 method,TBA method and Lactate dehydrogenase Kit(micro ELISA method)to measure the activity of SOD and the concentrations of MDA and LDH in supernatant at different time points and different concentrations of ethanol and grape seed procyanidins.3.Used laser confocal microscope to observe the changes of hippocampus neural cells morphology at different time points and different concentrations of ethanol and grape seed procyanidins.4.Statistical analysis methods: The obtained data were expressed in s.Used multiple factor analysis of variance and LSD method for comparing between two groups of SPSS22.0 statistical software to analyze data with test level=0.05.Results:1.Compared with control group,the activity of SOD and the concentrations of MDA and LDH in supernatant under different concentrations and different time points were statistically significant(P<0.05).Under the low dose of ethanol,the changes in content of SOD,MDA and LDH were not obvious.Under the middle and high dose of ethanol,the activity of SOD at different time points decreased significantly.However,the content of MDA and LDH increased significantly.After 1h and 4h,the activity of SOD decreased gradually and the content of MDA and LDH increased gradually.Under high dose of ethanol for 8h,the activity of SOD was only 10.67±0.393U\ml which was the minimum value.Compared with the low dose group,the activity of SOD in the high dose group of GSP at 8h increased significantly by1.09±0.987 U/ml and the content of MDA is floating biggest,reduced the 0.192 ±0.002 nmol/mL.Compared with the high dose group of ethanol at the two time points,in the high dose group of ethanol and the low dose group of GSP the activity of SOD increased slightly and the content of MDA decreased slightly.Compared with the low dose group,the content of LDH in the low dose group of ethanol and the high dose group of GSP for 8h decreased significantly.Compared with the middle dose group of ethanol,the content difference of LDH in the low,middle,high dose of GSP had statistical significances.Compared with the high dose group of ethanol,the content of LDH in the high dose group of GSP changed significantly,decreased the 189.78±1.68 U/L。2.After exposure to different concentrations of ethanol for different times,used immunofluorescence staining to observe the cells.The body of cells in control group showed fusiform,trilateral and oval form.The nucleus located in the center of the cell with full shape and a clear boundary.It stretched out one or more coarser filamentous protuberances,interconnecting and forming the neutral network.Dying was relatively uniform.After exposure to ethanol which concentration was 0.5g\L,the cell form didn’t change obviously at any time.After exposure to ethanol which concentration was 1.5g\L for 4h,intercellular wire start decreased and after 8h,the damaged cell started releasing NCAM which prompted some cells to gather.As the exposure time increasing,the density of cells decreased,and satellite phenomenon appeared.The intercellular wire fracture and some even disappear.After exposure to 3.0g\L concentration of ethanol for 1h,cells started gather and the intercellular wire became slender even fracture.As the time goes on,neurogliocyte increased and almost all of the hippocampal cells gathered with small nucleus and deep staining.The cells were short and the connections between them were hardly visible.The neural network structure had been completely destroyed and some cells even died.After exposure to GSP,the boundaries of some cells were clear and the intercellular wire became coarsen obviously and its dying was deep.The connection between cells increased.Some bodies were normal approximately and connections recovered.The phenomenon that cells gathered into a cluster was significantly reduced and sparse neural network were visible.Conclusions:1.After exposure to ethanol,the activity of SOD in supernatant decreased significantly and the content of MDA and LDH increased significantly,which were both related to exposure time,showing a trend of dose dependent.This proved that ethanol could induce oxidative stress and produce toxic effect on hippocampal cells.GSP could increase significantly the activity of SOD and decreased the content of MDA and LDH in supernatant of hippocampal cells which were damaged by ethanol,showing a trend of dose dependent.It reminded that GSP had the effect of antioxidant and protected hippocampal cells.2.After exposure to ethanol,the density of hippocampal cells reduced.Cell body was damaged,and boundaries were fuzzy.The intercellular wire became shorter and slender and some even fractured and disappeared.The structure of neural network was destroyed.All concentrations of GSP had protective effect on hippocampal cells which could repair the damaged hippocampal cells.The high dose of GSP could maintain the normal morphology of cell body which was damaged by ethanol.The intercellular wire increased and became coarse,forming sparse neural network structure. |
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