| Objective:To investigate the inhibition effect of Lychee seed total saponins on neuronal apoptosis of hippocampal in rats with Alzheimer's disease induced by A?,and to explore its mechanism.Methods:The normal learning and memory male Sprague-Dawley(SD)rats were selected with Morris water maze,which were randomly divided into sham operation group and operation group.The operation group were injected 10?g(2?g/1?l)state of aggregation A?25+35 in intracerebroventricular to prepare the model of rats with AD,three weeks after the operation,The model rats were selected with Morris water maze,which were randomly divided into model group?0.42mg·kg-1 donepezil positive group?0.12g·kg-1,0.24g·kg-1 and 0.47g·kg-1 lychee seed total saponins treatment group;The treatment group intragastric administration,one time every day for one month.Morris water maze examination was used to detect learning and memory ability of the AD rats;Brain of rats were poured into 4%paraformaldehyde for fix,and the brain was made by paraffin embedding,coronal microtome section.TdT-mediated dUTP nick end labeling(TUNEL)and the immunohistochemical staining(SP)were employed to observe the degree of neuronal apoptosis and the positive expression of Akt and GSK-3? in hippocampus,the positive rate of apoptotic cells was calculated by GD-10.0 image analysis system and the average optical density was analysised by Image pro plus 6.0 software of expression of Akt and GSK-3?;Caspase-3 gene expression in rat hippocampus were detected by RT-PCR method,and the average gray value of caspase-3 gene expression were calculated by gel quantitative analysis software Gel-Pro analyzer 4.0.Results:(1)Learning and memory capacity:Compared with the sham operation group,the escape latency of rats in model group was significantly increased(P<0.01),the numbers of rats crossing platform and the time spent in target quadrant were decreased significantly(P<0.01);Compared with the model group,each dose lychee seed total sapopnins can shorten the escape latency of rats(P<0.05,P<0.01)and can increase the numbers of rats crossing platform(P<0.01)?0.47g·kg-1 lychee seed total saponins treatment group can increase the time spent in target quadrant(P<0.05);Compared with the treatment groups,there are significant differences in the escape latency of rats,in the fourth and fifth day of the place navigation exercise(P<0.05),and the numbers of rats crossing platform was significantly different between the low dose and high dose group(P<0.05).(2)Observed under the microscope:?Neuronal apoptosis:a few brown cell and which nucleus were stained in brown in hippocampal CA1 area in the sham operation group and a number of brown cell and which nucleus with brown were seen in hippocampal CA1 area in the model group,which the apoptotic index of cells increased obviously(P<0.01);Each dose lychee seed total sapopnins,the number of brown cell and which nucleus were stained in brown in hippocampal CA1 area decreased significantly,so the same as the apoptotic index of cells(P<0.01),and there was significantly different between the Litchi total saponins of three dose groups(P<0.05,P<0.01).?The expression of Akt:Many brown cell?deeply brown stained cytoplasm,and have a positive expression of the brown particles in nuclei in hippocampal CA1 area in the sham operation group,a few brown cell?light staining cytoplasmic and rare nuclei positive expression of the brown particles in hippocampal CA1 area in model group was significantly decreased,compared with the model group,each dose lychee seed total sapopnins can increase brown cell with deeply stained brown cytoplasm and have a positive expression of the brown particles in nuclei of hippocampal CA1 area,and more obvious with the increase in the dose effect.The average optical density of positive staining of Akt expression,compare with the sham operation group,model group was significantly lower(P<0.01);Compared with model group,each dose lychee seed total sapopnins can increase the average optical density(P<0.01);Compared with the treatment groups,there are significant differences between low dose and high dose group(P<0.05).?The expression of GSK-3?:Very small number of brown cells with regularly arranged and round shape in hippocampal CA1 area in the sham operation group,disarranged brown cells increased significantly in hippocampal CA1 area in model group was significantly increased,each dose lychee seed total sapopnins can reduce brown cells in hippocampal CA1 area,and more obvious with the increase in the dose effect.The average optical density of positive staining of GSK-3?expression,compare with the sham operation group,model group was significantly higher(P<0.01);Compared with model group,each dose lychee seed total sapopnins can shorten the average optical density(P<0.01);Compared with the treatment groups,there are significant differences between low dose and high dose group(P<0.05).(3)Expression of Caspase-3 mRNA:In every group,there was obvious amplified strap of ?-actin mRNA in the 445bp,and there was amplified strap of Caspase-3 mRNA which is different intensity but sharply demarcated in the 218bp.Compared with sham operation group,the intensity of Caspase-3 mRNA amplified segmental are more obvious,also the relative gray value of Caspase-3 mRNA was increased significantly in the model group(P<0.05);Compared with model group,the intensity of Caspase-3 mRNA amplified segmental are attenuated,and the relative gray value of Caspase-3 mRNA was decreased significantly in the pretreatment group(P<0.05,0.01);Compared with the treatment groups,there are significant differences between low dose and high dose group(P<0.05).Conclusion:1.The model of rats with AD were induced successfully by intracerebroventricular injection of trace state of aggregation A?25-35.2.Lychee seed total sapopnins can improve learning and memory of rats with AD,and there is a dose-dependent.3.The possible mechanism include:(1)The lychee seed total saponins activating Akt of the signaling transduction pathway P13-K/Akt to reduce the secretion of GSK-3?,thus,to reduce the expression of Caspase-3 and weaken the apoptosis cascade reaction triggerred by Caspase-3.(2)The lychee seed total saponins activating Akt of the signaling transduction pathway PI3-K/Akt to inhibit the expression of Caspase-3 directly,thus,to weaken the apoptosis cascade reaction triggerred by Caspase-3. |
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