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

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个时间点海马神经元Caspase-12蛋白表达均明显减少(P<0.01,P<0.05)。结论:针刺血清能明显减少海马神经元凋亡数,上调GRP 78蛋白表达,下调CHOP和Caspase-12蛋白表达,发挥维持内质网稳定性的重要作用。

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/F 12（1 ： 1） plus non-acupuncture serum （9 ： 1） for non-acu- puncture serum group. The cell apoptosis was assessed at 2, 12 and 48 h after application of acupuncture serum or non-acupunc- ture serum by using TUNEL method and the expression levels of GRP 78, CHOP and Caspase-12 proteins of the cultured cells at the 3 time-points detected using Western blot. Results The number of apoptotic hippocampal neurons was significantly higher in the Mg2＋ -free medium group than in the normal medium group at 2, 12 and 48 h （P〈0.01）, and considerably decreased in the acupuncture serum group （but not in the non-acupuncture serum group） relevant to the Mg2＋ -free medium group at the same time- points after application of acupuncture serum （P〈0.05, P〈0.01 ）. The expression levels of GRP 78 protein at 2 h, CHOP and Caspase-12 proteins at 2, 12 and 48 h were significantly up-regulated in the Mg2＋ -free medium group relavant to the normal me- dium group （P〈0.05, P〈0.01）. After application of acupuncture serum to the culture medium, the expression levels of GRP 78 protein at the three time-points were significantly increased in comparison with the Mg2＋-free medium group （P〈0. 01, P〈0.05）, while those of CHOP at 12 and 48 h, and Oaspase-12 at the three time-points were notably down-regulated （ P〈0.05, P〈0.01 ）. No significant differences were found between the non-acupuncture serum and Mg2 ＋ -free medium groups in the expression levels of GRP 78, CHOP and Caspase-12 proteins at the three time-points （P〉0.05）. Conclusion Acupunc- ture serum can significantly reduce apoptosis of the cultured hippocampal neurons, which may be related to its effects in increasing the expression of GRP 78 protein and down-regulating the expression of CHOP and Caspase-12 proteins, suggesting an important role of acupuncture serum in maintaining the stability of the neuronal endoplasmic reticulum.