针刺血清调控内质网应激反应对无镁诱导培养大鼠海马神经元惊厥性损伤的保护作用  

Protective Effect of Acupuncture Serum Derived from Acute Convulsion Rats on Cultured Hippocampal Neurons with Seizure-like Discharges by Regulating Expression of Endoplasmic Reticulum Stress-inducible Molecular Chaperones

作　　者：张慧[1] 杨帆[1] 吴旭[2] 沈德凯[1] 陈浩[1] 吴生兵[3] 昂文平[1] 宋小鸽[1] 解明月 ZHANG Hui YANG Fan WU Xu SHEN De-kai CHEN Hao WU Sheng-bing ANG Wen-ping SONG Xiao-ge XIE Ming-yue(Clinical College of Acu-moxibustion and Orthopedics-traumatology , 3 Center for Scientific Research, Anhui University of Traditional Chinese Medicine, Hef ei 230038, China College of Life Science, University of Science and Technology of China, Hefei 230012)

机构地区：[1]安徽中医药大学针灸骨伤临床学院,合肥230038 [2]中国科技大学生命科学学院,合肥230012 [3]安徽中医药大学科研实验中心,合肥230038

出　　处：《针刺研究》2017年第2期95-101,共7页Acupuncture Research

基　　金：国家自然科学基金(No.81072872);安徽高校科研平台创新团队项目(No.2015TD 033);安徽省高校自然科学研究重点项目(No.KJ 2016A858)

摘　　要：目的:观察针刺血清对无镁诱导惊厥样放电海马神经元凋亡数及内质网应激伴侣葡萄糖调节蛋白78(GRP 78)、C/EBP同源蛋白(CHOP)和半胱氨酸蛋白酶蛋白-12(Caspase-12)表达的影响,探讨针刺血清对海马神经元惊厥性损伤的保护机制。方法:SD大鼠腹腔注射戊四唑诱发急性惊厥后分为针刺组及对照组。针刺组取"百会""大椎"针刺,每日1次,共7d。末次针刺后,针刺组大鼠取血清作为针刺血清,对照组取血清作为非针刺血清。以无镁诱导惊厥样放电的原代培养胎鼠海马神经元为模型,根据对培养10d海马神经元的不同处理分为正常细胞外液组(简称正常组)、无镁细胞外液组(简称无镁组)、针刺血清组和非针刺血清组。正常组将无血清培养液换成细胞外液培养3h后换液培养,其他各组用无镁细胞外液培养3h后换液培养。4组分别于换液培养2、12、48h3个时间点采用TUNEL法和Western blot法分别检测海马神经元凋亡情况及GRP 78、CHOP和Caspase-12蛋白表达情况。结果:正常组仅见少量凋亡海马神经元;无镁组3个时间点凋亡海马神经元数均较正常组明显增加(P<0.01);针刺血清组3个时间点凋亡海马神经元数与无镁组比较明显减少(P<0.05,P<0.01),与非针刺血清组12、48h比较凋亡海马神经元数明显减少(P<0.01)。与正常组比较,无镁组3个时间点海马神经元CHOP和Caspase-12蛋白表达均明显增强(P<0.05,P<0.01),2h时间点海马神经元GRP 78蛋白表达明显增强(P<0.05);与无镁组比较,针刺血清组3个时间点神经元GRP 78蛋白表达均明显增强(P<0.01,P<0.05),12、48h时间点海马神经元CHOP蛋白表达明显减少(P<0.05,P<0.01),3个时间点神经元Caspase-12蛋白表达均显著减少(P<0.05,P<0.01);与非针刺血清组相比,针刺血清组2、12h时间点海马神经元GRP 78蛋白表达均明显增强(P<0.01,P<0.05),12、48h时间点海马神经元CHOP蛋白表达明显减少(P<0.05),3个时间点海马神Objective To observe the effect of acupuncture serum on the number of apoptosis of the cultured hippocam- pal neurons with seizure-like discharges and the expression levels of endoplasmic reticulum stress-inducible molecular chaperones glucose-regulated protein 78 （GRP 78） , C/EBP homologous protein （CHOP） and cysteine protease protein-12（Oaspase-12）, soas to reveal its protective mechanism on seizure-induced injury of hippocampal neurons. Methods A regular primary culture of neurons derived from the hippocampus of the newly-born SD rats was conducted for 10 days, then these cultured neurons that dis- played seizure-like discharges in Mg2＋ -free medium were divided into normal extracellular fluid （medium） group （normal）, Mg2＋ - free medium group, acupuncture serum group and non-acupuncture serum group （n = 30）. Blood examples were taken from penty- lenetetrazol （i. p. i. ） -induced acute convulsion adult SD rats underwent manual acupuncture stimulation of ＂Baihui＂ （GV 20） and ＂Dazhui＂ （GV 14, once daily for 7 days） to prepare acupuncture serum. Hippocampal neuronal cultures were prepared from hip- pocampal tissue isolated from 24 h-old SD rats. The isolated neurons were incubated normally in Dulbecco＇s Modified Eagle Me- dium （DMEM） / Nutrient Mixture F 12（ 1 ： 1 ） for 10 days, followed by exposure to the extracellular fluid { composed of NaCI, KCI, CaCl2, MgCl2, HEPES [4-（2-hydroxyethyl）-l-piperazineethanesulfonic acid], D-glucose and aminoethanoic acid （pH 7.3）） for 3 h and returned to normal culture for normal group, by exposure to Mg2＋ -free extracellular fluid （to induce seizure-like discharges） for 3 h and returned to normal culture for Mg2＋ -free medium group, by exposure to Mg2＋ -free extracellular fluid for 3 h and re- turned to a regular culture medium I-DMEM/F 12（1 ： 1）1 plus acupuncture serum （9 ： 1） for acupuncture serum group, and by ex- posure to Mg2＋ -free extracellular fluid for 3 h and returned to DMEM/

关 键 词：针刺血清 惊厥 细胞凋亡 GRP 78蛋白 CHOP蛋白 Caspase-12蛋白 内质网应激 

分 类 号：R245.311[医药卫生—针灸推拿学]