基于蛋白质组学探讨电针治疗血管性痴呆的作用机制  

Mechanism of electroacupuncture for vascular dementia based on proteomics

作　　者：陈英华[1] 孙玮 王浩宇 秦瑞琦 苏晓庆 李俊峰 吴彤 赵红旭 宋元毓 杨建宇[3] CHEN Yinghua;SUN Wei;WANG Haoyu;QIN Ruiqi;SU Xiaoqing;LI Junfeng;WU Tong;ZHAO Hongxu;SONG Yuanyu;YANG Jianyu(Fifth Section of Department of Acupuncture and Moxibustion,First Affiliated Hospital of Heilongjiang University of CM,Harbin 150040,China;Heilongjiang University of CM;Guangming TCM Journal)

机构地区：[1]黑龙江中医药大学附属第一医院针灸五科,哈尔滨150040 [2]黑龙江中医药大学 [3]光明中医杂志社

出　　处：《中国针灸》2024年第7期807-820,共14页Chinese Acupuncture & Moxibustion

基　　金：国家自然科学基金资助项目:82074530;黑龙江省自然科学基金项目:ZD2021H007;黑龙江省中医药科研项目:ZHY2023-189、ZHY2023-209。

摘　　要：目的:基于串联质谱标签(TMT)定量蛋白质组学技术,探讨电针治疗血管性痴呆(VD)的可能作用机制。方法:选用SPF级雄性SD大鼠80只,采用Morris水迷宫实验筛选出符合标准的78只SD大鼠,将其随机分为假手术组(18只)和手术组(60只)。手术组采用四血管阻断(4-VO)法复制VD模型,将造模成功的36只大鼠随机分为模型组(18只)和电针组(18只)。每组进一步按干预时间分为3个亚组,每亚组6只。造模后7 d对电针组大鼠行电针干预,穴取左右“四神聪”和双侧“风池”,予连续波,频率2 Hz,电流强度1 mA,每日1次,每次30 min,3个亚组分别干预7、14、21 d。采用Morris水迷宫实验于干预前后评估大鼠学习记忆能力,并选择干预后学习记忆能力最优的电针组亚组及相应假手术组、模型组亚组进行蛋白质组学检测,鉴定电针干预VD相关差异表达蛋白并进行生物信息学分析,采用平行反应监测(PRM)技术和Western blot法验证差异表达的目标蛋白。结果:与假手术组比较,模型组大鼠逃避潜伏期延长、穿越原平台次数减少(P<0.01);与模型组比较,电针组大鼠干预7、14、21d逃避潜伏期缩短、穿越原平台次数增加(P<0.01,P<0.05)。与干预7、14 d比较,电针组大鼠干预21 d逃避潜伏期缩短、穿越原平台次数增加(P<0.01,P<0.05),选择该亚组进行后续蛋白质组学检测、PRM分析及Western blot验证。与假手术组比较,模型组大鼠差异表达蛋白有71种,其中上调的有50种,下调的有21种;与模型组比较,电针组大鼠差异表达蛋白有54种,其中上调的有30种,下调的有24种。功能富集分析、聚类分析发现,差异表达蛋白主要与细胞过程、代谢过程、吞噬识别、免疫反应、细胞外基质组织的调节等生物过程有关。在哺乳动物雷帕霉素靶蛋白(mTOR)信号通路及神经营养因子信号通路中均富集到了糖原合成酶激酶3β(GSK3β)和丝裂原活化蛋白激酶激酶2(Map2k2)蛋Objective To explore the potential mechanism of electroacupuncture(EA)for vascular dementia(VD)using tandem mass tag(TMT)quantitative proteomics technology.Methods Among 80 male SPF SD rats,78 rats which met the selection criteria through the Morris water maze test were selected and randomly divided into a sham surgery group(18 rats)and a surgery group(60 rats).VD model was established by four-vessel occlusion(4-VO)method in the surgery group,and36 rats with successful modeling were randomly assigned to a model group(18 rats)and an EA group(18 rats).Each group was further divided into three subgroups based on intervention duration,with each subgroup containing 6 rats.Seven days after model establishment,the EA group received EA intervention at left and right"Sishencong"(EX-HN 1)and bilateral"Fengchi"(GB 20),with continuous wave at a frequency of 2 Hz and current intensity of 1 mA,daily for 30 min,with subgroups receiving EA for 7,14,or 21 d respectively.Cognitive function before and after interventions was assessed using Morris water maze.Proteomic analysis was conducted on the optimal EA subgroup and corresponding sham surgery and model subgroups,identifying differentially expressed proteins and analyzing them through bioinformatics.Differentially expressed target proteins was performed using parallel reaction monitoring(PRM)and Western blot techniques.Results Compared to the sham surgery group,the model group exhibited prolonged escape latency and reduced number of platform crossings(P<0.01);compared with model group,the EA group showed reductions in escape latency and increased platform crossings after 7,14,and 21 days of intervention(P<0.01,P<0.05).Compared to the 7 and 14-day intervention,the rats in the EA group of 21-day intervention showed the most significant improvements in reductions of escape latency and increased platform crossings(P<0.01,P<0.05),and was selected for further proteomic,PRM analyses,and Western blot validation.Compared to the sham surgery group,the model group displayed 71 differential

关 键 词：血管性痴呆 电针 串联质谱标签(TMT)定量蛋白质组学 生物信息学分析 作用机制 

分 类 号：R245[医药卫生—针灸推拿学]