| Objective:To observe the intervention effect of concise prescriptions of Chinese medicine (Huannao Yicongfang, HNYCF) extract on APH-1, PEN-2 regulatory pathway of y secretase in brain tissue of APP/PS1 double transgenic dementia mice model.To explore the mechanism of HNYCF extract and provide a scientific basis for development an effective drug for prevention and treatment of Alzheimer’s disease (AD).Methods:60 APP/PS1 double transgenic model mice (3 months old) were randomly divided into 5 groups, especially the normal group, the model group (n=15), Donepezil group (n=15), HNYCD low-dose group (n=15) and HNYCD high-dose group (n=15). Another 15 C57BL/6J mice of 3-month-old were selected into the normal control group. The normal group and the model group were given equal-volume distilled water, other groups were respectively given Donepezil (0.65mg/kg), Huannao Yicongfang low dosage extract (5.46g/kg) and Huannao Yicongfang high dosage extract (10.92g/kg) through gastric infusion. The administration was once a day, lasting for 3 months. The ability of memory and learning were tested by Morris water gaze, the content of amyloid precursor protein (APP) and β amyloid protein (Aβ) was detected by ELISA, the activity of gamma was detected by Dual Luciferase Reporter Gene Assay Kit. And the levels of APH-1 a, HIF-1α, PEN-2, CREB mRNA and protein in hippocampus were detected by RT-PCR and western-blot.Results:The first part:In the place navigation test of Morris water maze showed, compared with the normal control group, the time of sailing were prolonged obviously (P<0.01), and the swimming distance in the fourth quadrant was increased significantly in the model group and the low dosage of HNYCF group (P<0.01). After medication of 3 months, compared with the model group, the time of sailing were shortened obviously the western medicine control group and the control group (P<0.01), the swimming distance in the fourth quadrant was shortend significantly in the model group and the low dosage of HNYCF group (P<0.01). In the space probe trial of Morris water maze, compared with the normal control group, the average speed and the first cross-platform were significant difference between the control group and the low dosage of HNYCF group (P< 0.01), the first cross-platform were significant difference between the western medicine control group. After medication of 3 months, compared with the model group, the average speed and the first cross-platform were significant difference between the western medicine control group (P< 0.01), the first cross-platform were significant difference between the high dosage of HNYCF group (P< 0.05), there was significant difference in average speed between the western medicine control group and the low dosage of HNYCF group.The second part:ELISA showed that compared with the normal control group, the expression of APP, Aβ1-40 and A β1-42 all were increased significantly in hippocampal CA1 region of mice in the model group (P<0.01), the expression of Aβ1-42 was increased significantly in the low dosage of HNYCF group (P<0.01). After medication of 3 months, compared with the model group, the expression of APP, Aβ1-40 and A β1-42 were all reduced significantly in the western medicine control group (P<0.01), the expression of APP and Aβ1-42 in the low dosage of HNYCF group were reduced significantly (P<0.05). Compared with the western medicine control group, the expression of Aβ1-42 was increased significantly in the low dosage of HNYCF group (P<0.05).The third part:Dual Luciferase Reporter Gene Assay Kit showed that with the normal control group, the activity of gamma was increased significantly the model group (P<0.01). After medication of 3 months, compared with the model group, the activity of gamma was no significantly different in the western medicine control group (P>0.05), the activity of gamma was reduced in high dosage HNYCF group (P<0.05). There were no significantly different between the other groups (P>0.05).The fourth part:RT-PCR showed that compared with the normal control group, the expression of APH-la mRNA, HIF-1α mRNA all increased significantly in the model group and each medicine group (P<0.05 or P<0.01), the expression of PEN-2 mRNA, CREB mRNA increased significantly in the western medicine control group (P< 0.01), After medication of 3 months, compared with the model group, the expression of PEN-2 mRNA, CREB mRNA increased significantly in the low and high dosage HNYCF groups (P<0.01).The fifth part:RT-PCR showed that compared with the normal control group, the expression of APH-la, HIF-1α all increased significantly in the model group and each medicine group (P< 0.05 or P< 0.01), the expression of PEN-2, CREB increased significantly in the western medicine control group (P<0.05). After medication of 3 months, compared with the model group, the expression of PEN-2, CREB increased significantly in the low and high dosage HNYCF groups (P<0.01).Conclusion:HNYCF extract can improve learning and memory ability and spatial cognitive ability of APP/PS1 double transgenic mice obviously, its mechanism may be related to inhibit the activity of y secretase by down-regulating the activity of CREB protein and the expression of PEN-2 protein, and reduce the decomposition of APP and the production of A (3.This shows that HNYCF extract has promissing research and application as an effective drug for prevention and treatment of AD. |
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