“醒脑开窍”针刺后局灶性脑缺血模型大鼠脑基底核组织在蛋白质水平上的变化

Changes in protein level of basal ganglia in rats with focal cerebral ischemia after“Xingnao Kaiqiao”acupuncture

目的:观察“醒脑开窍”针刺治疗后局灶性脑缺血模型大鼠基底核组织在蛋白质水平上发生的改变,以分析针刺对脑缺血后蛋白质表达的影响。方法:实验于2003-10/2004-07在天津中医学院第一附属医院分子生物学实验室和全国重点实验室国家生物医学分析中心完成。①选用40只成年雄性Wistar大鼠,按随机抽签法分为4组,每组10只:正常组、假手术组、模型组和针刺组。模型组和针刺组采用颈内动脉插入线栓法建立大脑中动脉阻塞模型。假手术组除不向颈内动脉内插线外,其余步骤同模型组。正常组不作任何处理。针刺组:选用韩氏穴位神经刺激仪以醒脑开窍针法主穴内关、人中穴进行针刺;频率2Hz,电流3mA,持续10min/次。于大鼠神经功能缺陷评分合格后,即刻针刺一次,处死前10min再针刺一次。各组在术后6h快速处死,冰上剥离缺血侧基底核。②对4组蛋白质提取物行双向凝胶电泳,采用计算机辅助图像分析技术,分析凝胶图像和找出差异表达蛋白点。选取差异表达蛋白质点用基质辅助激光解吸飞行时间质谱测定肽质量指纹谱,检索Swiss-Prot蛋白质数据库鉴定差异蛋白。结果:Wistar大鼠40只均进入结果分析。4组基底核组织采用双向凝胶电泳根据蛋白质的分子量和等电点将其分离。并用考染方法显示了凝胶上蛋白质点的位置和丰度。结果显示这种方法对Wistar大鼠基底核组织蛋白质达到了很好的分离效果,经过计算机图像软件分析凝胶图像有以下结果:经过对比分析及质谱鉴定,找到质和/或量有明显改变的15个点。其中,在模型组表达上调,针刺组治疗后下调的2个点:泛醌-细胞色素C还原酶铁硫亚单位(分子量/等电点:29427/9.04),3-磷酸甘油醛脱氢酶(35656/8.45);治疗后进一步上调有3个点:谷胱甘肽S-转移酶P(23293/7.30),组织型纤溶酶原激活物前体(62862/8.55),抗氧化蛋白2(24439/5.8)。在模型组下调、针刺组治疗后上调有4个:热休克家族Mr71000蛋白(70827/5.37),肌酸激酶(42685/5.33),组氨酸三核苷凝结蛋白1(13637/6.39),Cu-Zn超氧化剂物歧化酶(15771/5.89)。在模型组有变化、针刺组治疗后未发生明显变化有4个:电压依赖性阴离子选择性通道蛋白1(30606/8.63),泛素C-末端水解酶同工酶L1(24822/5.14),G蛋白β1亚单位(37353/5.60),蛋白酶体β4亚单位(29197/6.8)。在模型组无变化、针刺组治疗后出现变化的有2个:磷酸甘油酸变位酶1(28497/6.21),硫氧还原蛋白(11876/4.6)。结论:对大鼠基底节组织蛋白质提取物进行双向凝胶电泳并进行基质辅助激光解吸飞行时间质谱,可以有效地将局灶性脑缺血损伤大鼠异常改变及针刺治疗起效的基底节蛋白质分离开,这些差异表达的蛋白质可能为脑缺血损伤及针刺治疗作用的靶蛋白。

AIM： To observe the change of protein levei of basal ganglia of cerebral ischemia after ＂Xingnao Kaiqiao＂ （XNKQ） acupuncture method with twodimensional electrophoresis （2-DE） technique in order to analyze the effect of acupuncture on protein level in local cerebral ischemia. METHODS： From October 2003 to July 2004 the experiment was conducted at the Laboratory of Molecular Biology of the First Affiliated Hospital of Tianjin University of Traditional Chinese Medicine and the National Center of Biomedical Analysis. ①Totally 40 adult male Wistar rats were selected and divided into four groups： normal group, sham operation group, model group and acupuncture group with 10 rats in each group. The middle cerebral artery occlusion models of model group and acupuncture group were induced by internal carotid artery insert the filament method Sham operated group besides not to insert the filament to internal carotid artery, other steps were the same to the model group. The normal group did not make any processing. The acupuncture group elected main points of ＂XNKQ＂ acupuncture method： Neiguan （P 6） and Renzhong （Du 26） to carry on the acupuncture with Hanshi point neurostimulation meter. Acupuncture method： The electricity needle, frequency was 2 Hz and electric current was 3 mA for 10 minutes once. Acupuncturing was performed once when neural functional defect score was qualified. It was conducted again at 10 minutes before execnting. The rats were killed immediately 6 hours after operation. Ischemic asipodite was detached on ice. ②2-DE was performed in protein extract of the 4 groups. Gel imaging was analyzed and differential expression protein was found with computer combined with imaging technique. Differential exression protein was selected with matrix assisted laser desorption/ionization time of flight mass spectrometry （MALDI/TOF MS） so as to measure peptide mass fingerprinting. Swiss-Prot protein database was retrieved to identify differential expression protein. RESULTS： Totally 40 Wistar rats were involved in the result analysis. The protein of basal ganglia was separated by its two characteristics： isoelectric point and molecular weight based on 2-DE in the 4 groups. The position and abundance of the protein showed with coomassie brillint blue. The result showed this method had achieved to good separation effect, has the result after the computer image software analyzed the images： It was found that 15 spots had the distinct change on the quality and/or quantity after contrasting 4 groups. Among them, the levels of 2 proteins were significantly up-regulated in model group and expressed down-regulated after ＂XNKQ＂ acupuncture treatment： Ubiquinol-cytochrome C reductase ironsulfur subunit （29 427/9.04）, glyceraldehyde-3-phosphate-dehydrogenase （35 656/8.45）; After the treatment further up-regulated had 3 spots： Glutathione S-transferase P . （23 293/7.30）, tissue-type plasminogen activator precursor （62862/8.55）, anti-oxidant protein 2 （24 439/5.8）. The levels of 4 proteins were down-regulated in model group and expressed up-regulated after ＂XNKQ＂ acupuncture treatment： Heat shock cognate Mr 71 000 protein （70 827/5.37）, creatine kinase （42 685/5.33）, histidine triad nu cleotide-binding protein 1 （13 637/6.39）, Cu-Zn superoxide dismutase （15 771/5.89）. Four proteins had the change in model group, but did not have the obvious change after treatment： Voltage-dependent anion-selective channel protein 1 （30 606/8.63）, ubiquitin carboxyl-terminal hydrolase isozyme L1 （24 822/5.14）, guanine nucleotide-binding protein G（I）/G（S）/G （T） beta subunit 1 （37 353/5.60）, proteasome phosphoglycerate mutase alpha subunit alpha type 4 （29 197/6.8）. Two proteins appeared no change in the model group, but had changes after the treatment： phosphoglycerate mutase 1 （28 497/6.21）, thioredoxin （11 876/4.6）. CONCLUSION： Protein extractive of basal ganglia receives 2-DE and MALDI/TOF MS, which may separate effectively protein of basal ganglia of abnormal change and effective acupuncture treatment; These different expression proteins are possibly concerned as target protein after cerebral ischemic injury and the acupuncture treatment.