The Mechanism Of Ginkgo Biloba Extract And Creatine Phosphate Sodium's Protection From Apoptosis Of PC-12 Cells Induced By A β25-35

Objective: This experiment uses amyloidβ(Aβ) 25-35 to induce apoptosis in PC12 cells, establishing the cell type of Alzheimer's disease, and then provides Ginkgo biloba extract and creatine phosphate sodium to intervene. To explore whether the two play protective roles in neuronal apoptosis induced by Aβ25-35.Materials and methods: Choosing the PC12 cells on the logarithmic growth phase, after cell count , inoculating thems in 96-well plates on the density of 104/mL. Culturing cells for 24 hours, exchange a half of nutrient medium,then add different concentrations of Aβ25-35(A:0μmol/L (control group),B:10μmol/L,C:20μmol/L,D:30μmol/L,E:40μmol/L). Culturing cells for 24 hours, obtain the cell survival percentage,carry on the statistical analysis by the SPSS software, choosing variance analytic method. Discoverying the damage effect on PC-12 cells obvious when Aβ25-35 in the concentration of 30μmol/L. Culture the cells 24 hours according to the same condition and then add Aβ25-35 to them on the density of 30μmol/L, 12 hours later, add the gingko leaf extraction and the creatine phosphoric sodium according to the following grouping method : the Ginkgo biloba extract group A:Aβ0umol/L (control group), B:Aβ30umol/L,C:Aβ30umol/L + EGB 100mg/L , D: Aβ30u mol /L+ EGB200mg/L, E: Aβ30umol/L + EGB400mg/L); the creatine phosphate sodium group:(A: Aβ0umol/L (control group),B:Aβ30umol/L.C:Aβ30umol/L+PCr10mmol/L,D:Aβ30umol/L+PCr20mmol/L,E:Aβ30umol/L+PCr30mmol/L), culturing for 12 hours,and then use CCK-8 to measure the cell survival percentage,after statistical analysis, we draw a conclude: the Ginkgo biloba extract(200mg/L) and the creatine phosphate sodium group (20mmol/L) play obvious protective role in the damage. According to the above methods, we use Aβ25-35 (30μmol/L) to damage the PC12 cells, 12 hours later, add the Ginkgo biloba extract (200mg/L), the creatine phosphate sodium(20mmol/L) to protect cells,after raising 12 hours, mark the PC12 cell with line plastochondria membrane potential special fluorescent probe JC-1. Start the laser confocal microscopy, Choose the 488nm argon ion laser and the 543nm helium neon laser as the stimulation light, 530±15 nm and 615±30 nm channel as the reception channel. Use Lasersharp 2000 software to scan the picture, and carry on finally the image in the Laserpix software processing. Simultaneously choose the cell, in the concentration of 104/mL , to fling the piece at the speed of 600r/min, detect Apoptosis by TUNEL the reagent box of Apoptosis, Calculate the apoptosis rate, and collect the image,and then use SPSS statistical software for statistical analysis. Results: When Aβ25-35 in the concentration of 30μmol/L, effects of injury to PC12 cells are obvious. It has significant differences from groups: 0μmol/L, 10μmol/L, 20μmol/L, (p<0.01) and similar to group of 40μmol/L (P>0.01). After intervention in different concentrations of Ginkgo biloba extract and sodium creatine phosphate,measure OD value,use the analysis of variance method to obtain that the group of EGB 200mg/L has significant differences with the group of Aβ30μmol/L and EGB 100mg/L (P<0.01) and has no significant differences with the group of EGB 400mg/L and Aβ0μmol/L (P>0.01) .The group of PCr 20mmol/L has obvious differences with the group of Aβ30μmol/L and PCr 10 mmol/L (P<0.01)and has no significant differences with the group of PCr 30 mmol/L and Aβ0μmol/L (P>0.01). Add Aβto PC12 cells in concentrations of 30umol/L(referred to as Group Aβ, the same below), after 24 hours, Mitochondrial membrane potential shows that red fluorescence intensity decreased significantly and green fluorescence intensity enhanced , which means that MMP lower. After dealing with PC12 cells according to the group of Aβ30umol/L+EGB 200mg/L (referred to as ginkgo group, the same below) and the group of Aβ30umol/L+ PCr20mmol / L (referred to as sodium creatine phosphate group, the same below), red fluorescence intensity markedly improved, and green fluorescence intensity decreased significantly, which means MMP increases. Group Aβ(30 umol/L Aβ), EGB (Aβ30 umol/L+EGB200mg/L) and PCr (Aβ30umol/L+PCr20mmol/L) have TUNEL-positive cells, that are, apoptotic cells,POD- marked green fluorescent cells significantly, DAB color,its nucleus is stained brown. The apoptosis rate of group Aβ(63%) is higher than that ginkgo group (21%) and creatine phosphate group (22%) (p <0.01).Conclusion: 1.Aβ25-35 in the concentration of 30 umol/L can induce apoptosis in PC12 cells, establishing the cell type of Alzheimer's disease. 2.Ginkgo biloba extract in the concen- tration of 200mg/L,creatine phosphate sodium in the concent- ration of 20 mmol/L can significantly increase the survival rate of cells by raising the level of mitochondrial membrane potential and the inhibition of apoptosis,and so play the role in protecting PC-12 cells injuried by Aβ25-35.