“补肾活血”针刺法对SAMP8小鼠海马蛋白质表达的影响

Effect of protein expression in hippocampus tissue of SAMP8 by Kidney-reinforcing and Blood-activating Acupuncture Method

目的运用蛋白质组学技术观察"补肾活血"针刺法对SAMP8小鼠海马蛋白质表达的影响及探讨其治疗阿尔茨海默病(AD)的作用机制。方法将5月龄SAMP8 20只随机分为针刺治疗组(n=10)和模型空白对照组(n=10),针刺治疗组予以"补肾活血"针刺法干预,取穴百会、肾腧、血海、膈腧;模型空白对照组未予特殊处理。干预4 w后,取各组小鼠海马组织,应用双向凝胶电泳(2-DE)技术分别分离治疗组和对照组SAMP8海马区组织总蛋白,经胶体考马斯亮蓝染色,利用ImageScanner图像扫描仪及ImageMaster双向凝胶电泳软件对图像进行扫描分析,得到差异蛋白质点,通过质谱分析得到相应肽质量指纹图谱,经蛋白质数据库查询,鉴定差异蛋白质点。结果针刺治疗组与模型对照组存在13个差异点,初步鉴定了浮舰蛋白1(Flotillin-1)、胶质纤维酸性蛋白(GFAP)、脑红蛋白(NGb)、α-烯醇酶(α-enolase)等9个差异蛋白质,其中6个表达上调,3个下调;这些蛋白的功能涉及Aβ代谢的调节、氧化应激的调节、神经炎症的调节以及细胞骨架的修复等方面。结论 "补肾活血"针刺法可调节SAMP8海马组织的多种蛋白质表达,提示其具有多靶点治疗的作用,可能通过调节Aβ的代谢、抗氧化应激、抑制神经反应、修复细胞骨架等途径来实现其治疗AD的作用。

Objective To investigate the effect of Kidney-reinforcing and Blood-activating Acupuncture Method on hippocampal protein expressions in senescence accelerated mouse-prone/8（SAMP8） and explore the probable remedial mechanism and potential remedial protein targets of AD by this acupuncture method.Methods 20 five-month-old male SAMP8 were randomly divided into acupuncture treatment and model groups（n=10）.Mice in acupuncture treatment group were treated by acupuncture with reinforcing kidney and activating blood method for 4 weeks and Baihui（GV20）,Shenshu（BL23）,Xuehai（SP10） and Geshu（BL17） were selected,mice in model control group were raised without special treatment.After 4 weeks,the total proteins of hippocampus tissue of SAMP8 in each group were separated by two-dimensional gel electrophoresis（2-DE） respectively.The gels of each group were stained by Coomassie brilliant blue,scanned by ImageScanner and analyzed in PDQuest software.Differential expression protein spots between the two groups were identified by peptide mass fingerprint（PMF） based on the technology of matrix-assisted laser desorption /ionization time of flight mass spectrometry（MALDI-TOF-MS） and MSDB or NCBInr database searching.Results There were 13 differentially expressed protein spots in acupuncture treatment group compared with model control group,and 9 differential expression proteins were preliminarily identified,including Flotillin-1,glial fibrillary acidic protein,neuroglobin,α-enolase,etc.,of which 6 protein spots were up-regulated and 3 protein spots were down-regulated.The functions of the identified proteins involved in regulating Aβ metabolism,inhibiting neuroinflammation,anti-oxidative stress,repairing cytoskeleton,and so on.Conclusions Kidney-reinforcing and blood-activating zcupuncture method could regulate multiple protein expressions in hippocampus tissues of SAMP8,which suggests this method has multi-target therapeutic action and its remedial mechanism is possibly realized by way of reducing production of Aβ,inhibiting neuroinflammation,antagonizing oxidation stress,repairing cytoskeleton,and so on.