The Effects Of Notoginsenoside R₁ On A?_25-35-induced Neuronal Apoptosis And Learning And Memory Impairment Of AD Model Mice Through JAK2/STAT3 Pathway

Objective:The Effects of Notoginsenoside R1 on A?25-35-induced Neuronal Apoptosis and Learning and Memory impairment of AD Model Mice through JAK2/STAT3 pathwayMethods:1.In vitro-the rat neuroma cell PC12 cells were taken as the research objects,and A?_25-35 fragments were used to establish the AD cell model of PC12 cells,and AG490 and NGR1 were used to intervene.The cell viability was detected by CCK-8.Apoptosis was detected by Annexin V-FITC/PI staining FCM.The expressions of JAK2,STAT3 genes were detected by real-time PCR method.Western blot were used to detect the phosphorylation levels of JAK2 and STAT3.2.In vivo-Kunming mice were randomly divided into 7 group:control group,A?_25-35 group,A?_25-35+AG490 group,high dose A?_25-35+NGR₁ group,low dose A?_25-35+NGR₁ group,high dose A?_25-35+AG490+NGR₁ group and low dose A?_25-35+AG490+NGR₁ group.The model group and each treatment group were injected with A?_25-35 fragment by lateral ventricle injection method.The control group was injected with the same dose of saline.AG490 group and high dose A?_25-35+AG490+NGR₁group are given JAK inhibitor AG490 at the dose of 1mg/kg intraperitoneal injection every other day before model establishment for 12h and after model building.High dose NGR1 group was intragastric with the dose of 50mg/kg,low dose NGR₁ group was intragastric with the dose of 25mg/kg,and continuous intragastric for 4 weeks.Morris water maze test was used to detect the learning and memory abilities of mice;HE staining was used to observe the morphological changes of brain cells;PCR method was used to detect the expression of apoptosis-related genes such as downstream of JAK2/STAT3pathway BL-2,survivin and P53 and JAK2/STAT3;Immunohistochemistry and Western blot were used to detect the phosphorylation levels of JAK2 and STAT3 in hippocampus.Results:1 In vitro experimental results show:1.1 CCK-8 result showed that:Compared with control group,A?_25-35group could significantly inhibition the activity of PC12 cells?P<0.05?;Compared with A?_25-35 group,A?_25-35+AG490 group could significantly increase the activity of PC12 cells?P<0.05?.1.2 Flow cytometer result showed that:Compared with control group,A?_25-35 group could significantly increase the early apoptosis rate of PC12cells?P<0.05?;Compared with A?_25-35 group,A?_25-35+AG490 group could significantly reduce the early apoptosis rate of PC12 cells?P<0.05?;Compared with A?_25-35+NGR₁ group,A?_25-35+AG490+NGR₁ group could significantly reduce the early apoptosis rate of PC12 cells?P<0.05?.1.3 RT-PCR result showed that:Compared with the control group,the mRNA expression of JAK2 and STAT3 in the A?_25-35 group were significantly increased?P<0.05?;Compared with the A?_25-35 group,the mRNA expression of JAK2 and STAT3 in the A?_25-35+AG490 group and A?_25-35+NGR₁ were significantly reduced?P<0.05?;Compared with the A?_25-35+NGR₁ group,the mRNA expression of JAK2 and STAT3 in the A?_25-35+AG490+NGR₁ were reduced?P<0.05?.1.4 Western blot result showed that:Compared with the control group,protein expression levels of JAK2 and STAT3 were increased in the A?_25-35group?P<0.05?;Compared with the A?_25-355-35 group,the protein expression levels of JAK2 and STAT3 were decreased in A?_25-35+AG490 group and A?_25-35+NGR1 group?P<0.05?;Compared with the A?_25-35+NGR₁ group,protein expression levels of JAK2 and STAT3 were decreased in A?_25-35+AG490+NGR₁ group?P<0.05?.2.In vivo experimental results show that:2.1.Morris water maze experiment showed that:Compared with the control group,the percentage of the original platform quadrant time of the A?_25-35group decreased significantly?P<0.05?.Compared with A?_25-35 group,the percentage of original platform quadrant residence time in A?_25-35+NGR₁high and low dose group and A?_25-35+AG490 group increased significantly?P<0.05?.Compared with the high dose group of A?_25-35+NGR₁ high dose group,the high dose group of A?_25-35+AG490+NGR1 showed that the percentage of the original platform quadrant time of the AD mice increased?P<0.05?.2.2 HE staining result showed that:the hippocampus abnormal structure is abnormal in the A?_25-35 group,morphology of neurons also changed,the number of cells decreased clearly,the cell morphology and arrangement changed obviously,the arrangement is loose and disordered,the visible nucleus shrink,the staining deepens,and the cytoplasm and nucleus boundary is not clear;Compared with the A?_25-355-35 group,the number of the hippocampus neuron was obviously increased in the A?_25-35+AG490 group,the high and low dose group of A?_25-35+NGR₁ and the high and low dose group of A?_25-35+AG490+NGR_1.And the hippocampus neuron structural abnormalities improved.The distribution of the cells is uniform and in line.And the number of the neurons and pyramidal cell increases2.3 RT-PCR result showed that:Compared with the control group,the expression of JAK2 and STAT3 mRNA increased significantly in the A?_25-35group?P<0.05?,and the expression of BCL-2 and Survivin mRNA decreased significantly?P<0.05?,while P53 mRNA expression increased significantly?P<0.05?.compared with the A?_25-35 group,the expression level of A?_25-35+NGR₁ high and low dose group and high dose group JAK2 and STAT3 mRNA decreased significantly?P<0.05?,the expression of BCL-2and Survivin mRNA were significantly increased?P>0.05?,while the high and low dose of P53 mRNA the expression was decreased significantly?P<0.05?;Correspondingly,Compared with the A?_25-35+NGR1 high and low dose group,the expression level of JAK2 mRNA in the high and low dose of A?_25-35+AG490+NGR₁ reduced significantly?P<0.05?,the expression level of STAT3 mRNA in the high and low dose of A?_25-35+AG490+NGR₁decreased but no significant difference?P<0.05?.Compared with the A?_25-35+NGR1 high and low dose group,the expression level of BCL-2mRNA significantly increased in the high and low dose of A?_25-35+AG490+NGR₁ group?P<0.05?,the expression level of survivin mRNA increased in the high dose of A?_25-35+AG490+NGR₁ group?P<0.05?,the expression level of P53 mRNA in the high and low dose of A?_25-35+AG490+NGR₁ group decreased?P<0.05?.2.4 Immunohistochemical result showed that:Compared with the control group,the percentage of positive cells number and total cells within each vision of p-JAK2 and p-STAT3 proteins in hippocampus of A?_25-355-35 group were all significantly increased?P<0.05?;Compared with the A?_25-35 group,the percentage of positive cells number and total cells within each vision of p-JAK2 and p-STAT3 proteins in hippocampus of A?_25-35+NGR₁ high and lose dose group were all significantly reduced?P<0.05?;Correspondingly,Compared with the A?_25-35+NGR₁ high and lose dose group,the percentage of positive cells number and total cells within each vision of p-JAK2 and p-STAT3 proteins in hippocampus of A?_25-35+NGR₁+AG490 high and lose dose group were all reduced?P<0.05?.2.5 Western blot result showed that:Compared with the control group,protein expression levels of p-JAK2 and p-STAT3 were increased significantly in the A?_25-35 group?P<0.05?;the levels of these proteins expression of A?_25-35+NGR₁ high and lose dose group were all significantly reduced compared to A?_25-35 group?P<0.05?;Correspondingly,Compared with the A?_25-35+NGR₁ high and lose dose group,protein expression levels of p-JAK2 and p-STAT3 were increased significantly in the A?_25-35+NGR₁+AG490 high and lose dose group?P<0.05?.Conclusion:The JAK2/STAT3 pathway can promote A?-induced neuronal apoptosis;NGR1 can inhibit the apoptosis of A?-induced neuronal by inhibiting the activity of JAK2/STAT3 pathway.The lnhibition of JAK2/STAT3 pathway could significantly increase learning and memory ability in AD model mice;NGR1 can improve the learning and memory of the AD model mice by inhibiting the JAK2/STAT3pathway.