Mechanism Of Mitochondrial DNA Oxidative Damage In Alzheimer Disease Cell Model And Ginsenosides Efficacy

Background and objectsAlzheimer disease(AD) is a neurodegenerative disease which developswith age, characterized by evident learning and memory impairment anddementia.Recent research suggested that increase of oxygen free radicalsproduction and reduction of cleanability play an important role in cell apoptosisof AD.Oxygen free radicals are must produced in mitochondria and effect onmitochondria's respiration fuction in turn.The abnormal structure and functionof mitochondria could contribute to the progress of AD.Oxygen free radicals inthe cell body are must produced by the oxidative phosphorylation ofmitochondria.So the mitochondria are must easily damaged by oxygen freeradicals.Energy metabolism decreased when the brain cells suffered fromAD,but so far it is not reported whether the decrease is related to themitochondria oxidative damaged. Ginsenosides,a kind of Chinese herb,canimprove the ability of learning and reach longevity.Modern medical sciencesbelieve Ginsenosides can prevent oxidative damage .However,it is not reportedhow it fuctions on the damage.This study explored the relation between AD andmitochondria oxidative damaged and the mechanism of Ginsenosides onmitochondria oxidative damaged,through observing the structure and fuctionaldamage of AD model cells and Ginsenosides'effect on them,which laystheoretical foundation for further elucidating its mechanism and treatments forAD.Methods The cells were devided into control group,model group and Ginsenosidesgroup. Control group cells were incubated with normal cuture ,AD model groupcells were made by adding β-amyloid protein protein 25~30(Aβ25~30) intothe cuture. In Ginsenosides group,cells disposed to Aβwere treated withGinsenosides. Cell activity ,apopotisis rate of cell was detected by MTT andPI stain. Cell ultralstructure was observed under electronic microscope.SODactivity ,MDA content and COX activity were measured byUV-spectrophotometer. Fluidity of mitochondrial membrane was detected byflurescenece spectrophotometer. Adenosine level were measured with highperformance liquid chromatography. Mitochondrial respiration fuction werecaculated by oxygen electrode.The mitochondrial DNA COXⅠ, COXⅡandCOXⅢgene were amplified by PCR and the were processed to sequenceanalysis.Results1. The cell activity in model group was significantly more than those in the control group(P < 0.01) and the Ginsenosides group(P < 0.01). The apoptosis rate in model group was significantly lower than those in the control group(P<0.01) and the Ginsenosides group(P<0.01).2. The ultrastructure damage in model group was more severious than the control and Ginsenosides groups.3. SOD activity in model group was lower than the other 2 groups(P<0.01)。 MDA level was much higher in model group than the other 2 groups (P<0.01)。 Mitocho -ndrial membrane fluidity in model group was worse than the other 2 groups(P<0.01)。4. Cell activity in model group was positively related to SOD activity and with MDA negatively.Apoptosis rate was significantly related to SOD activity negatively and with MDA positively.The viscous coefficient of mitochondria membrane was significantly related to SOD activity negatively and with MDA positively.5. COX activity in model group was significantly higher than in the other groups (P<0.05); In model group ,COX Ⅰprotein expression was higher than in the other two groups(P<0.01);EC was higher than in the other two groups (P<0.05); RCR and OPR were lower than in the other two groups (P<0.01).6. COXⅠprotein expression was positively related to COX activity.EC was positively related to COX activity, RCR,OPR and cell activity. COX activity was negatively related to MDA level.7. COXⅠA gene sequence analysis :More gene mutation was found in model group.In control group, we found 3 base replacements(1 samesense),no gene insertion or absense. In model group, 11 base replacements(4 samesense), 5 absense,4 insertion were found.In Ginsenosides group ,there were 3 base replacements(1 samesense) and 1 base absense.COX I B gene sequence analysis : In control group, we found 2 base replacements(both samesense),no gene insertion or absense. In model group, 34 base replacements(2 samesense), 4 absense were found.In Ginsenosides group ,there were 2 base replacements(1 samesense), no gene insertion or absense.8. COXⅡgene sequence analysis : In control group, we found 2 base replacements(1 samesense),no gene insertion or absense. In model group, 21 base replacements(7 samesense), 7 gene insertion and 1 absense were found.In Ginsenosides group ,there were 4 base replacements(2 samesense) and 1 gene insertion.9. COXⅢgene sequence analysis : In control group, we found 3 base replacements(2 samesense),no gene insertion or absense. In model group, 14 base replacements(3 samesense), 7 gene insertion and 1 absense were found.In Ginsenosides group ,there were 4 base replacements(3 samesense) , no gene insertion or absense.10. In model group,COX mRNA expression was lower than the control(P<0. 01) and Ginsenosides groups(P<0. 05). COXⅠ,COXⅡmRNA expression were positively related to COX activity and EC.Conclusion1. The AD model constructed by disposing PC12 cells with Aβwas reliable . The model stimulated AD's typical change apoptosis at the cell ,protein and gene level.2. Ginsenosides can increase cell activity, alleviate the ultralstructure damage,decrease apoptosis rate.3. In model group, SOD activity was decreaed,MDA level increased and...