| ObjectiveTo observe the regulating effect of Yishen Huazhuo Prescription on key targets in the APP metabolic pathway in the hippocampus of 7-month-old SAMP8 mice,to preliminarily explore its mechanism of reducing A?production,and to provide some experimental basis for the application of Yishen Huazhuo Prescription for the treatment of Alzheimer's disease.Method45 healthy male 7-month-old SAMP8 mice?SPF 2?,weight?25-35?g,license number?SCXK?Jin?2015-0003?provided by the First Affiliated Hospital of Tianjin University of Traditional Chinese Medicine,were randomized into 3 groups,15 in the model?P8?group,15in the Yishen Huazhuo Prescription?YSHZ?group,and 15 in the positive LY2811376 control group.In addition,15 normal aged 7-month-old SAMR1 mice of the same homology were selected as normal control group.Mice in YSHZ group were intragastrically given Yishen Huazhuo Prescription,and the amount of crude drug was 6.24g·kg-1·d-1 according to the‘Equivalent dose ratio table between humans and animals converted by body surface area'in Pharmacological Experimental Methodology,and mice in the positive control group were intragastrically given LY2811376,with the dosage was 10mg·kg-1·d-1,and mice in R1 and P8 groups were given the same amount of distilled water once a day,for 4 weeks.?1?The Morris water maze was used to detect the changes in the time of escape latency and the number of crossing platforms in each group,t observe the effect of Yishen Huazhuo Prescription on the learning and memory abilities of SAMP8 mice.?2?Nissl staining was used to observe the effect of Yishen Huazhuo Prescription on the arrangement and number of neurons in hippocampal CA1 area of SAMP8 mice.?3?Observe the expression of A?in hippocampal neurons of SAMP8 mice by laser confocal microscope,enzyme-linked immunosorbent assay and western blotting.?4?Enzyme-linked immunosorbent assay and western blotting were used to observe the expression of key target proteins in the APP metabolic pathway in the hippocampus of each group of mice.Result?1?The results of the Morris water maze showed that:the average swimming speed of mice on the first day among the groups was found to be no statistically significant?P>0.05?by one-way analysis of variance.The 5-day escape latency period showed a gradual downward trend with the number of days,and the downward trend was roughly the same.Compared with the mice in the R1 group,the mice in the P8 group and the LY2811376 group had significantly longer escape latency?P<0.05?;compared with that in the P8 group,the the escape latency in the YSHZ group significantly reduced?P<0.05?,and there was no significant difference in the escape latency of the mice in the LY2811376 group?P>0.05?;compared with that in the LY2811376 group,the escape latency in the YSHZ group was reduced?P<0.05?.The daily escape latency between the groups analyzed by multivariate analysis of variance showed that:on day 1,there was no significant difference between the groups?P>0.05?;on day 2,compared with that in the P8 group and the LY2811376 group,the escape latency of the YSHZ group reduced?P<0.05?;on day 3,compared with that in R1 group,the escape latency of mice in P8group and LY2811376 group increased?P<0.05?,compared with that in P8 group,the escape latency of mice in YSHZ group decreased?P<0.05?;on day 4,compared with that in R1 group,the escape latency of mice in LY2811376 group increased?P<0.05?,and on day 5,compared with that in R1 group,the escape latency of mice in P8 group and LY2811376 group increased?P<0.05?.The results of the number of crossing the platform showed that:compared with that in the R1 group,the number of crossing the platform significantly reduced in the P8 group and the LY2811376 group?P<0.05?.Compared with that in the P8 group,the number of mice crossing the platform in the YSHZ group significantly increased?P<0.05?;there was no significant difference in the LY2811376 group?P>0.05?.Compared with that in the LY2811376group,the number in the YSHZ group significantly increased?P<0.05?.?2?Nissl staining results showed that compared with that in R1 group,the number of neurons in hippocampal CA1 area of mice in P8 group and LY2811376 group significantly reduced?P<0.05?.Compared with that in the P8 group,the number of neurons in the hippocampal CA1 area of the YSHZ group increased significantly?P<0.05?;compared with that in the LY2811376 group,the number of neurons in the hippocampal CA1 area of the YSHZ group increased significantly?P<0.05?.?3?A?expression level in hippocampus:?1?confocal laser detection results showed that:Compared with R1 group,P8 group and LY2811376 group have more immunofluorescence yellow co-expression areas,and the average optical density value significantly increased?P<0.05?;compared with that in P8 group,immunofluorescence yellow co-expression area was less in the YSHZ group,and the average optical density value significantly reduced?P<0.05?,and it did not change significantly in LY2811376 group?P>0.05?;compared with that in the LY2811376 group,the fluorescence co-expression area decreased in the YSHZ group,and the average optical density value decreased significantly?P<0.05?.?2?ELISA results showed that the relative content of A?o protein in the P8,YSHZ and LY2811376 groups significantly increased compared with that in the R1 group?P<0.05?;compared with that in the P8 group,the relative content of A?o protein in the YSHZ and LY2811376 groups significantly decreased?P<0.05?;compared with that in the LY2811376 group,the relative content of A?o protein in the YSHZ group significantly increased?P<0.05?.?3?Western blot results showed that:compared with that in R1 group,the relative expression of A?o protein in P8 group,YSHZ group and LY2811376 group increased?P<0.05?;compared with that in P8 group,the relative expression of A?o protein in YSHZ group and LY2811376 group significantly reduced?P<0.05?;compared with thtat in the LY2811376 group,the relative expression of A?o protein in the YSHZ group significantly reduced?P<0.05?.?4?The expression level of key target protein in APP metabolic pathway:?1?Western blot results showed:Compared with that in R1 group,the relative expression of APP protein in P8,YSHZ and LY2811376 groups increased significantly?P<0.05?,and the relative expression of SORL1 protein Significantly decreased?P<0.05?;Compared with that in the P8 group,the relative expression of APP protein in the YSHZ group and the LY2811376 group decreased significantly?P<0.05?,and the relative expression of SORL1 protein increased significantly?P<0.05?;compared with that in the LY2811376 group,the relative expression of APP protein decreased significantly in YSHZ group?P<0.05?,and the relative expression of SORL1 protein increased significantly?P<0.05?.Compared with that in the R1 group,the relative expression of BACE1 and QPCT protein in the P8,YSHZ and LY2811376 groups increased?P<0.05?;compared with that in the P8 group,the relative expression of BACE1 and QPCT protein in the YSHZ and LY2811376 groups significantly decreased?P<0.05?;Compared with that in the LY2811376 group,the relative expression of BACE1 protein in the YSHZ group significantly increased?P<0.05?,and the relative expression of QPCT protein significantly reduced?P<0.05?.Compared with that in the R1 group,the ADAM10 protein expression in the P8,YSHZ and LY2811376 groups significantly reduced?P<0.05?;compared with that in the P8 group,the ADAM10 protein expression in the YSHZ group significantly reduced;compared with that in the LY2811376 group,the ADAM10 protein in the YSHZ group increased significantly?P<0.05?.?3?The ELISA results showed that the relative content of s APP?protein in the P8,YSHZ and LY2811376 groups significantly reduced compared with the R1 group?P<0.05?;compared with that in the P8 group,the relative content of s APP?in the YSHZ and LY2811376groups significantly reduced?P<0.05?;Compared with that LY2811376 group,the relative content of s APP?protein in YSHZ group significantly increased?P<0.05?Conclusion?1?Yishen Huazhuo Prescription can effectively improve the learning and memory decline of7-month-old SAMP8 mice,and the effect may be related to the reduction of hippocampal A?production,thereby reducing its self-aggregation to form toxic A?o and reducing hippocampal neuron loss.?2?The mechanism of Yishen Huazhuo Prescription to reduce A?production might be by regulating the expression of key target proteins in APP metabolism,promoting APP reverse transport and non-amyloid pathway metabolism,and inhibiting APP amyloid pathway metabolism. |
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