电针对阿尔茨海默病不同病理阶段自噬状态影响差异性研究

Study of the effect differences of electroacupuncture on autophagy state in different pathological stages of Alzheimer's disease

目的 观察电针对APP/PS1双转基因小鼠老年斑出现前后不同病理阶段自噬状态影响的差异性,从自噬角度探讨电针干预阿尔茨海默病(Alzheimer’s disease,AD)的作用机制。方法 4月龄和8月龄APP/PS1双转基因雄性小鼠各20只,随机分为模型组(4月龄和8月龄模型组)和电针组(4月龄和8月龄电针组),每组10只;以同月龄同性别C57BL/6野生型小鼠为正常组(4月龄和8月龄正常组)各10只。电针组取双侧涌泉穴直刺后接电针仪,百会穴平刺后单向滞针再留针,共20 min。干预隔日1次,共6周。采用Morris水迷宫实验评价小鼠空间学习记忆能力;通过免疫组化技术观察小鼠海马淀粉样斑块沉积情况;用免疫荧光技术观察小鼠海马LC3和β-淀粉样蛋白(β-amyloid,Aβ)表达情况;用酶联免疫吸附法检测小鼠海马Aβ表达水平;用Western blot法检测小鼠海马自噬相关蛋白LC3和p62相对表达量。结果 Morris水迷宫结果显示,6月龄和10月龄模型组小鼠平均逃避潜伏期均较同月龄正常组增加(P<0.01),平台象限停留时间均减短(P<0.01);与6月龄和10月龄模型组相比,同月龄电针组小鼠平均逃避潜伏期均减少(P<0.05),平台象限停留时间均延长(P<0.01)。免疫组化结果显示,6月龄模型组海马区域可见少量老年斑,10月龄模型组海马区域可见大量老年斑。免疫荧光技术结果显示,LC3和Aβ阳性表达主要位于海马神经元核周,LC3阳性还表达在细胞突起中。酶联免疫吸附法检测结果显示,同月龄模型组海马Aβ水平均高于正常组(P<0.01);同月龄电针组海马Aβ水平均低于同月龄模型组(P<0.01);6月龄模型组海马Aβ水平明显低于10月龄模型组(P<0.01)。Western blot法检测结果显示,与同月龄正常组比较,6月龄模型组LC3Ⅱ/Ⅰ比值和p62表达均降低(P<0.01);与同月龄模型组比较,电针组LC3Ⅱ/Ⅰ比值增加(P<0.01),p62差异无统计学意义(P>0.05)。与同月龄正常组比较,10月龄模型组LC3Ⅱ/Ⅰ比值和p62表达均增加(P<0.01,P<0.05);与同月龄模型组比较,电针组LC3Ⅱ/Ⅰ比值和p62表达均降低(P<0.01,P<0.05)。结论6月龄和10月龄APP/PS1转基因鼠老年斑出现前后不同病理阶段的自噬状态不同。电针可提高6月龄APP/PS1双转基因鼠海马自噬功能,调节10月龄双转基因鼠自噬功能紊乱状态,电针改善不同病理阶段APP/PS1双转基因小鼠的学习记忆能力,降低海马Aβ水平的机制存在差异性。

Objective To observe the effect differences of electroacupuncture(EA)on the autophagy state in APP/PS1 double transgenic mice in different pathological stages,before and after the occurrence of senile plaques,and to explore the mechanism of EA treatment of Alzheimer’s disease(AD)from the perspective of autophagy.Method Four-month-old and 8-month-old APP/PS1 double transgenic male mice,20 in each age group,were randomized into model groups(4-month-old model group and 8-month-old model group)and EA groups(4-month-old EA group and 8-month-old EA group),with 10 mice in each group.Wild-type C57BL/6 mice of the same ages and gender were taken as control groups(4-month-old and 8-month-old normal groups),with 10 mice in each group.The EA groups received EA at bilateral Yongquan(KI1)and Baihui(GV20)was horizontally punctured with the needle twirled till stuck and then retained for 20 min.The intervention was performed once every other day for 6 weeks.Morris water maze test was used to evaluate the spatial learning and memory abilities of the mice;immunohistochemistry was taken to observe the deposition of amyloid plaques in the hippocampus of mice;the immunofluorescence technique was employed to detect the expression of LC3 andβ-amyloid(Aβ)in the hippocampus of mice;the enzyme-linked immunosorbent assay(ELISA)was used to determine the expression level of Aβin the hippocampus of mice;Western blot(WB)was used to detect the relative expression levels of autophagy-related proteins LC3 and p62 in the hippocampus.Result Morris water maze results showed that compared with the normal groups,the 6-month-old and 10-month-old model groups had a longer average escape latency(P<0.01)and shorter platform dwelling time(P<0.01);compared with the 6-month-old and 10-month-old model groups,mice in the same age EA groups had a reduced average escape latency(P<0.05)and extended platform dwelling time(P<0.01).The immunohistochemistry results showed a small amount of senile plaques in the hippocampus of 6-month-old model mice and a large amount of senile plaques in the hippocampus of 10-month-old model mice.The immunofluorescence results revealed that LC3 and Aβwere mainly expressed around the nucleus of hippocampal neurons,and LC3 was also found in axons.ELISA showed that the hippocampal Aβlevel in the model mice was higher than that in the same age normal groups(P<0.01);the hippocampal Aβlevel in the EA groups was lower than that in the same age model groups(P<0.01);the hippocampal Aβlevel was significantly lower in the 6-month-old model mice than in the 10-month-old model group(P<0.01).The WB results demonstrated that LC3 II/I and the expression of p62 decreased in the 6-month-old model group compared with the same age normal group(P<0.01);compared with the same age model groups,the EA groups had higher LC3 II/I(P<0.01),while the expression of p62 showed no significant difference(P>0.05).Compared with the same age normal group,the 10-month-old model group had increased LC3 II/I and p62 expression(P<0.01,P<0.05);compared with the same age model groups,the EA groups showed decreased LC3II/I and p62 expression(P<0.01,P<0.05).Conclusion Before and after the appearance of senile plaques,the autophagy status differs depending on the different pathological stages in APP/PS1 transgenic mice.EA can enhance the hippocampal autophagy function in 6-month-old APP/PS1 double transgenic mice and regulate autophagy dysfunction in 10-month-old double transgenic mice.Further,the mechanism of EA varies in improving the learning and memory abilities and reducing the hippocampal Aβlevel in APP/PS1 double transgenic mice in different pathological stages.