基于蛋白质组学探讨电针心经穴对阿尔茨海默病大鼠学习记忆能力的影响及作用机制

Discussion on the Effects and Mechanism of Electroacupuncture at Heart Meridian Acupoints in Alzheimer Disease Rats Based on Proteomics

目的观察电针对阿尔茨海默病(AD)大鼠学习记忆能力的影响,基于蛋白质组学探讨其潜在作用机制。方法36只健康雄性SD大鼠随机分为假手术组、模型组、心经穴组,每组12只。于双侧海马CA1区注射β淀粉样蛋白1-42建立AD大鼠模型,假手术组注射等体积生理盐水。心经穴组予相应穴位电针刺激,20 min/次,针刺6 d休息1 d,连续7周。Morris水迷宫实验评估大鼠学习记忆能力,串联质谱标签(TMT)标记定量蛋白质组学技术和生物信息学分析筛选差异表达蛋白及潜在功能,通过平行反应监测(PRM)验证差异蛋白表达。结果各组大鼠游泳速度比较差异无统计学意义(P>0.05)。与假手术组比较,模型组Morris水迷宫实验逃避潜伏期显著延长(P<0.01);与模型组比较,心经穴组第2~4日逃避潜伏期均显著缩短(P<0.01)。TMT标记定量共鉴定出209个差异表达蛋白,其中有12个蛋白在3组间均有显著变化。GO注释涉及金属离子输运、钠离子传输、钠离子跨膜转运等生物过程,突触、突触前、突触囊泡等细胞组分,溶质:钠协同转运蛋白活性,有机酸:钠协同转运蛋白活性,氨基酸:阳离子转运体活性,氨基酸:钠转运蛋白活性等分子功能;KEGG分析显著富集于突触囊泡通路。PRM验证结果表明,电针心经穴可降低钠和氯依赖的GABA转运蛋白3(GAT3)表达,与TMT标记定量检测结果一致。结论电针心经穴可改善AD大鼠学习记忆能力,可能是通过调控突触囊泡通路上的突触转运体GAT3表达发挥神经保护作用。

Objective To observe the effects of electroacupuncture on the learning and memory abilities of Alzheimer disease(AD)rats;To explore its potential mechanism based on proteomics.Methods Totally 36 SD male rats were randomly divided into sham-operation group,model group and heart meridian acupoints group,with 12 rats in each group.Aβ1-42 were injected into the bilateral hippocampus to establish AD rat model,the sham-operation group was injected with an equal volume of normal saline.The heart meridian acupoints group received electroacupuncture treatment,accompanied by a stimulation time of 20 minutes,rest for 1 day after 6 days of electroacupuncture for 7 consecutive weeks.The Morris water maze experiment was used to evaluate the learning and memory abilities of AD rats,tandem mass tag(TMT)labeling technology and bioinformatics analysis were used to screen core differentially expressed proteins in important typical pathways,and key differentially expressed protein was verified by parallel reaction monitoring(PRM).Results There was no statistically significant difference in swimming speed between each group of rats(P>0.05).Compared with the sham-operation group,the escape latency of Morris water maze experiment in the model group increased significantly(P<0.01);compared with the model group,the escape latency of heart meridian acupoints group was significantly shortened on the 2-4th day(P<0.01).A total of 209 differentially expressed proteins were identified in different groups using TMT labeling quantification,among which 12 proteins showed significant changes among the 3 groups.GO annotation involved biological processes such as metal ion transport,sodium ion transport,and sodium ion transmembrane transport,as well as cellular components such as synapses,presynapse,and synaptic vesicle,involving solute:sodium symporter activity,organic acid:sodium symporter activity,amino acid:cation symporter activity,amino acid:sodium symporter activity,and other molecular functions;KEGG analysis significantly enriched the synaptic vesicle pathway.The PRM validation results indicated that electroacupuncture at the heart meridian acupoints could reduce the expressions of sodium and chloride dependent GABA transporter protein 3(GAT3),which was consistent with the quantitative detection results of TMT labeling quantification.Conclusion Electroacupuncture at the heart meridian acupoints can improve the learning and memory abilities of AD rats,possibly by regulating the expression of synaptic transporter GAT3 on the synaptic vesicle pathway to exert neuroprotective effects.