| Objective:To observe the effect of H102 on the expression of related proteins and the ability of learning and memory in the AMPK-mTOR autophagy-related pathway of APP/PS1 double transgenic AD mice,and to explore whether H102 can clear Aβ through AMPK-mTOR autophagy-related pathway.method:1.30-year-old APP/PS1 AD mice were randomly divided into two groups,15 in each group.15 C57BL/6J normal male mice of the same age and the same age were used as the control group.All animals were fed and administered in the SPF animal center.The mice in the H102 administration intervention group were given 5 μl(5.8 mg/kg)of H102 solution through the nasal cavity at the same time every day.The mice in the control group and AD group were given the same dose daily.Excipient solution.Water maze and new object recognition experiments were performed 30 days after continuous administration,and the learning and memory abilities of the three groups of mice were tested.2.At the end of the behavioral experiment,25% of urethane(4 ml/Kg)was anesthetized by intraperitoneal injection,perfused with phosphate buffered heart,brain was taken on decapitated ice,and half of the brain tissue was removed and 4% paraformaldehyde was added.In the paraffin-embedded,sectioned,immunohistochemical method to detect the expression of p-AMPK,p-mTOR,p-ULK1,P62,LC3II/I,Aβ protein;the other half of the brain tissue separates the hippocampus from the cortex The package was temporarily placed in liquid nitrogen for cooling,then stored in a-80 ° C refrigerator,and the expression of p-AMPK,p-mTOR,p-ULK1,P62,LC3II/I protein was detected by Western blot.The expression of Aβ protein was examined.Result:1.Positioning navigation experiment: The escape latency of AD group was significantly increased compared with normal control group(P<0.05);the escape latency of H102 group was significantly lower than that of AD group(P<0.05).There was no significant difference in escape latency between H102 mice compared with the normal control group(P>0.05).2.Space exploration experiment: compared with the normal control group,the time of staying in the first quadrant of the AD group was significantly shorter(P<0.05),the number of crossing the hidden platform was significantly reduced(P<0.05),and the initial angle was significant.Increased(P<0.05);H102 group mice stayed in the first quadrant and the number of times across the hidden platform was significantly increased compared with AD group mice(P<0.05),the initial angle was significantly higher than that of AD group mice.The decrease was(P<0.05).Compared with the normal control group,there was no significant difference in the time of staying in the first quadrant,the number of crossing the hidden platform,and the initial angle of the H102 group(P>0.05).3.New object recognition experiment: The new object recognition index(RI)of mice in AD group was significantly lower than that in normal control group(P<0.05).The RI of H102 group was significantly higher than that of AD group(P<0.05).<0.05);Compared with the normal control group,there was no significant difference in RI between the H102 group(P>0.05).4.Immunohistochemical detection: Compared with the normal control group,the expression of p-AMPK,p-ULK1,LC3II/I protein in brain tissue of AD group was significantly decreased(P<0.05),p-mTOR,P62 protein expression Significantly increased(P<0.05);compared with AD group,the expression of p-AMPK,p-ULK1,LC3II/I protein in brain tissue of H102 group was significantly increased(P<0.05),p-mTOR,The expression of P62 protein was significantly decreased(P<0.05).Compared with the normal control group,the p-mTOR protein in the brain tissue of H102 group was significantly decreased(P<0.05),and the expression of LC3II/I protein was significantly increased(P< 0.05),the expression levels of p-AMPK,p-ULK1,and P62 protein were not statistically significant(P>0.05).5.Western blot analysis: Compared with the normal control group,the expression of p-AMPK,p-ULK1,LC3II/I protein in brain tissue of AD group was significantly decreased(P<0.05),p-mTOR,P62 protein expression The expression of p-AMPK,p-ULK1 and LC3II/I protein in brain tissue of H102 group was significantly higher than that in AD group(P<0.05),p-The expression of mTOR and P62 protein was significantly decreased(P<0.05).Compared with the normal control group,the expression of LC3II/I protein in the brain tissue of H102 group was significantly increased(P<0.05).The expression of-mTOR protein was significantly lower than that of the normal control group(P<0.05).There was no significant difference in the expression of p-mTOR protein in the cortex compared with the normal control group(P>0.05).There was no significant difference in the expression of p-AMPK,p-ULK1 and P62 proteins in the tissues compared with the normal group(P>0.05).6.The results of Aβ protein detection: immunohistochemistry and ELISA results showed that the expression of Aβ protein in brain tissue of AD group was significantly higher than that of normal control group(P<0.05);Compared with the normal control group,the expression of Aβ protein in the brain tissue of H102 group was significantly lower than that in the normal control group(P<0.05).(P>0.05).Conclusion:The β-sheet blocking peptide H102 may promote the clearance of Aβ in the brain through the AMPK-mTOR autophagy pathway and improve the learning and memory ability of mice. |
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