| Objective: Oxidative stress is the main cause of brain aging,which affects the cognitive function.Astaxanthin(AST)and Sirt1/PGC-1α pathway has strong antioxidant effect,and is efficient in removing ROS,Therefore,the purpose of this study is to investigate whether astaxanthin can improve the cognitive function of Aβ25-35 mediated in mice through Sirt1/PGC-1α pathway,and whether astaxanthin can realize its protective effect on neurons of hippocampal structure.The results of this study have important scientific significance for proving the regulatory mechanism of astaxanthin on oxidative stress in hippocampus,and will lay a reliable theoretical foundation for the prevention and treatment of brain aging and Alzheimer’s disease,as well as the further development and clinical application of astaxanthin.Methods: 1.Animal model and grouping: Sixty 6-week-old ICR mice were randomly divided into four groups: Control group(n = 15),Aβ group(n = 15),Aβ+AST group(n =15),and Aβ+AST+NAM group(n = 15).For Aβ group,Aβ25-35(10μM,5 μL)was injected into the right lateral ventricle continuously 7 days to establish the aging mouse model: For Aβ+AST group,the mice were also fed with AST(10 μM,10 mg/kg/d)for30 days.For Aβ+AST+NAM group,nicotinamide(NAM,500 μM,500mg/kg/d)was intraperitoneally injected additionally for 7 days in form of intragastric gavage.2.Morris water maze test was used to examine the spatial learning and memory of mice in each group.3.The protein expression levels of BDNF and SYN in three subregions(CA1,CA3,DG)of hippocampal formation was examined by immunofluorescence method.The expression levels of Sirt1 and PGC-1α pathway proteins and Bcl-2 and Bax in three subregions of hippocampus were observed.4.Western blotting was used to detect the protein expression levels of BDNF and SYN as well as Sirt1 and PGC-1α pathway proteins and Bcl-2,Bax in the hippocampus of mice in each group.5.RT-PCR was used to detect the mRNA expression of BDNF,SYN,Sirt1,PGC-1α,Bcl-2 and Bax in the hippocampus of mice in each group.6.The changes of ROS,SOD,MDA and GSH-Px in the hippocampus of mice in each group were tested by the oxidative stress index detection method.The changes of ROS,SOD,MDA in the in each group of PC12 cells were tested.7.CCK8 method,Annexin V/PI staining,and senescence related β-gal staining were used to detect the apoptosis and senescence of PC12 cells in each group.8.Statistical analysis method: experimental data were expressed as mean ± standard deviation.Statistical analysis was performed by using Graph Pad Prism5.0.Comparison between two groups was analyzed with Tukey test,P<0.05 indicates that the difference is statistically significant.Results: 1.Morris water maze behavioral test results showed that Aβ25-35 injection through lateral ventricle could decrease the learning and memory ability of mice,and in vitro astaxanthin supplement could improve the learning and memory ability of mice.In addition,astaxanthin could improve the cognitive function of Aβ 25-35 mediated mice through Sirt1/PGC-1α pathway.2.Immunofluorescence staining results showed that astaxanthin can up-regulate the expressions of Sirt1 and PGC-1α in mouse hippocampus,and astaxanthin can up-regulate the expressions of SYN,BDNF,Bcl-2 and down-regulate the expression of Bax in mouse hippocampus through Sirt1/PGC-1α pathway.3.Western blotting results confirmed that astaxanthin-treated mice increased the expression levels of Sirt1,PGC-1α,SYN,BDNF and Bcl-2 in the hippocampus,while decreased the expression levels of Bax.4.Real-time PCR results confirmed that the expression levels of Sirt1 mRNA,PGC-1αmRNA,SYN mRNA,BDNF mRNA and Bcl-2 mRNA in the hippocampus of mice were increased after astaxanthin-treated,while the expression level of Bax mRNA was decreased.The protein expression trend of RT-PCR was consistent with Western blotting results.5.The detection results of oxidative stress related indexes showed that astaxanthin could reduce the contents of ROS and MDA,and increase the contents of SOD and GSH-Px in the hippocampus of aging model mice.In addition,astaxanthin could decrease the ROS and MDA content,while increase the SOD content in PC12 cells of Aβ group.6.CCK8 assay and Annexin-V/PI double staining assay confirmed that the viability of PC12 cells decreased with the Aβ25-35 concentration.Astaxanthin can reduce the apoptosis rate of PC12 cells.7.The results of β-galactosidase staining showed that Aβ25-35 increased the number of positive stained PC12 cells,while astaxanthin decreased the number of positive stained PC12 cells.Conclusion: 1.This study demonstrated that astaxanthin can up-regulate the expressions of BDNF,SYN,Sirt1 and PGC-1α in the hippocampus of mice,and improve the cognitive dysfunction of Aβ25-35 mediated mice through Sirt1/PGC-1α pathway.2.The results of this study confirmed that astaxanthinin can up-regulate the expression of Bcl-2 and down-regulate the expression of Bax,and further exert the protective effect on the apoptosis of hippocampal neurons induced by Aβ25-35 through the Sirt1/PGC-1αpathway.3.The results of this study indicated that astaxanthin can enhance the activity of PC12 cells and protect against Aβ25-35 mediated PC12 cell senescence and apoptosis through Sirt1/PGC-1α pathway. |
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