电针“百会”“涌泉”对6月龄APP/PS1双转基因小鼠行为学及海马Dynein、CTSD水平影响的研究

Effects of Electro-Needling DU20 and KI1 on Behavior and Levels of Dynein and CTSD in Hippocampus of 6-Month-Old APP/PS1 Double Transgenic Mice

目的:从细胞内β-淀粉样蛋白(Aβ)及自噬溶酶体角度探讨电针干预阿尔茨海默病(AD)的作用机制。方法:将20只6月龄雄性APP/PS1双转基因小鼠随机分为电针组和模型组;10只6月龄雄性C57BL/6野生小鼠作为空白对照组。电针组针刺“百会”“涌泉”穴,双“涌泉”连接电针,留针15 min/次;空白对照组和模型组小鼠用与电针组相同的方法束缚15 min/次。隔日1次,共治疗6周。利用Morris水迷宫检测小鼠空间学习和记忆能力;透射电镜观察小鼠海马自噬体、自噬溶酶体情况;免疫荧光双标法观察小鼠海马CA1区CTSD和Aβ的共表达情况;Western blot(WB)法检测小鼠海马内Dynein和CTSD的相对表达量。结果:Morris水迷宫检测结果显示,在定位航行实验中,与空白对照组比较,模型组小鼠平均逃避潜伏期增加,差异有统计学意义(P<0.05);与模型组比较,电针组小鼠平均逃避潜伏期减少,差异有统计学意义(P<0.05)。在空间探索实验中,与空白对照组比较,模型组小鼠平均穿越平台次数减少,平台象限游泳路程减少,差异均有统计学意义(P<0.05);与模型组相比,电针组小鼠平均穿越平台次数增加,平台象限游泳路程增加,差异均有统计学意义(P<0.05)。透射电镜实验结果显示,空白对照组海马可观察到少量的自噬溶酶体;模型组海马有大量的自噬体堆积,主要是在神经元轴突末端,胞体核周可见许多自噬溶酶体;电针组海马神经元轴突中也可以观察到部分自噬体堆积,胞体核周可见部分自噬溶酶体。免疫荧光双标法结果显示,CTSD和Aβ在小鼠海马CA1区神经元内存在共表达。Western blot结果显示,与空白对照组比较,模型组动力蛋白中间链DIC表达量降低,CTSD表达量增加,差异均有统计学意义(P<0.05);与模型组比较,电针组动力蛋白中间链DIC表达量增加,CTSD表达量降低,差异均有统计学意义(P<0.05)。结论:8月龄APP/PS1双转基因小鼠海马神经元自噬功能紊乱;电针可能通过调节小鼠紊乱自噬状态,促进自噬体运输及自噬溶酶体降解,改善AD小鼠的空间学习和记忆功能。

Objective:To explore the mechanism of electro-acupuncture(EA)in the treatment of Alzheimers disease(AD)from the perspective of intracellularβ-Amyloid(Aβ)and autophagy lysosome.Methods:20 APP/PS1 double transgenic mice were randomly divided into the EA group(n=10)and the model group(n=10).10 C57BL/6 wild mice were used as the blank control.The EA group was treated with electro-needling Baihui(DU20)and Yongquan(KI1)for 15min,once every other day for 6 weeks;the mice in control group and in the model group were bound for 15 minutes in the same way as in the EA group.The spatial learning and memory ability were detected by Morris water maze testing,hippocampal autophagosomes and autophagy lysosomes were observed by transmission electron microscope,the co-expression of CTSD and Aβin hippocampus CA1 was observed by immunofluorescence,and the relative expressions of Dynein and CTSD in hippocampus were detected by Western blot.Results:The results of Morris water maze test showed that the average escape latency of the model group increased compared to that in the control group(P<0.05);the average escape latency was decreased in the EA group compared to that in the model group(P<0.05).The average times of crossing the platform decreased and the swimming distance in the platform quadrant reduced in the model group compared to those in the control group(P<0.05);the average times of crossing the platform rose and the swimming distance in the platform quadrant increased in the EA group compared to those in the model group(P<0.05).The results of transmission electron microscope experiment showed that a small amount of autophagy lysosome in the hippocampus was observed in the control group;a large number of autophagosome and autophagy lysosome accumulation in the hippocampus were seen in the model group,mainly at the end of the axon of neurons;some autophagosome and autophagy lysosome accumulation also could be observed in the axon of hippocampal neurons of the EA group.The results of immunofluorescence showed that CTSD and Aβwere co-expressed in the hippocampal CA1 region of mice.Western blot results showed that the expression of DIC decreased and the expression of CTSD increased in the model group compared to those in the control group(P<0.05);the expression of DIC increased and the expression of CTSD decreased in the EA group compared to those in the model group(P<0.05).Conclusion:The function of autophagy of hippocampal neuron is disordered in 8-month-old APP/PS1 double transgenic mice,and EA may improve the ability of spatial learning-memory by regulating the disordered autophagy in mice and promoting autophagosome transport and autophagy lysosome degradation.