嗅三针对帕金森病痴呆小鼠海马CA1区载脂蛋白E和病理底物表达的影响

Effects of olfactory three needle on apolipoprotein E and pathological substrates in the hippocampus of mice with Parkinson's disease dementia

目的:观察嗅三针干预对帕金森病痴呆(Parkinson’s disease dementia,PDD)小鼠海马CA1区载脂蛋白E(apolipoprotein E,Apo E)、胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)以及相关核心病理底物表达的影响,探讨嗅三针改善PDD认知功能的部分作用机制。方法:将雄性C57BL/6小鼠随机分为空白组(Control)、假手术组(Sham)、模型组(Model)和电针组(AE),每组10只;采用单侧内侧前脑束(medial forebrain tract,MFB)注射6-羟多巴胺(6-hydroxydopamine,6-OHDA)建立PD模型并筛选PDD小鼠。建模筛选成功后,电针组选取嗅三针进行电针治疗,各组小鼠干预14天后采用Morris水迷宫实验和穿梭箱实验评估各组小鼠学习记忆能力;HE染色观察海马CA1区细胞病理改变;免疫印迹法检测海马CA1区α-syn、Aβ、Apo E蛋白的表达;免疫荧光双标观察海马CA1区Apo E与GFAP的共定位率。结果:与模型组比较,电针组小鼠水迷宫逃避潜伏期缩短(P<0.01),穿越平台次数增加(P<0.05),穿梭箱主动回避次数增加(P<0.05),电击刺激平均时间减少(P<0.01)。模型组小鼠海马CA1区神经细胞排列稀疏、变性坏死,细胞核体积变小、浓染、结构不清,呈核固缩表现;而电针组小鼠海马CA1区神经元病变有明显改善,大部分细胞排列规则,细胞核圆而大,染色浅,形态清晰。电针组小鼠海马CA1区α-syn、Aβ、Apo E蛋白以及Apo E与GFAP共定位率均较模型组减少(P<0.01,P<0.01,P<0.01,P<0.05)。结论:嗅三针可提高PDD小鼠认知能力并改善神经元形态结构与功能,机制可能与其抑制星形胶质细胞Apo E表达从而减少海马CA1区α-syn、Aβ沉积有关。

Objective:To explore the mechanism of olfactory three needle therapy on Parkinson's disease dementia(PDD)by observing its effects on expression of apolipoprotein E(ApoE)、glial fibrillary acidic protein(GFAP)and related core pathology substrates in the hippocampus of PDD model mice.Method:Male C57BL/6 mice were randomly divided into four groups:control group(Control),sham operation group(Sham),model group(Model)and acupuncture electrotherapy group(AE),with 10 mice in each group.The PD model was established by injecting 6-OHDA into the medial forebrain tract(MFB)and PDD mice were selected.After successful modeling and selection,the AE group received"olfactory three needle"electroacupuncture treatment.After 14 days of intervention,Morris water maze test and shuttle box test were used to evaluate the learning and memory ability of mice in each group.Hematoxylin-eosin(HE)staining was used to observe the pathological changes of hippocampal CA1 region.Western blotting was used to detect the expression ofα-syn,Aβ and ApoE proteins in hippocampal CA1 region.The co-location rate of ApoE and GFAP in hippocampal CA1 region was observed by double immunofluorescence markers.Result:Compared with Model group,the AE group exhibited a shortened escape latency in water maze(P<0.01),increased platform crossing(P<0.05),increased active escape times of shuttle box(P<0.05),and reduced the average total time of electric stimulation(P<0.01).In the Model group,the neurons in the hippocampal CA1 area were sparsely arranged and showed signs of degeneration and necrosis;and cell nuclei were small,hyperchromatic and had unclear structures,indicating the appearance of nuclear pyrosis.In contrast,the AE group showed significant improvements in neuronal pathology,with most cells regularly arranged,round and large nuclei,lightly stained and clearly shaped.Compared with the Model group,the expression levels ofα-syn,Aβ,ApoE protein and the co-localization rate of ApoE and GFAP in hippocampal CA1 region in AE group were decreased(P<0.01,P<0.01,P<0.01,P<0.05).Conclusion:The"Olfactory three needle"acupuncture can improve the cognitive ability and restore the morphological structure and function of neurons in PDD mice.The mechanism may be related to the inhibition of ApoE expression in astrocytes and the reduction ofα-syn and Aβdeposition in hippocampal CA1 region.