针刺研究2017，Vol.42Issue(2)：95-101,7.DOI:10.13702/j.1000-0607.2017.02.001
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
摘要
Abstract
Objective To observe the effect of acupuncture serum on the number of apoptosis of the cultured hippocampal 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(Caspase-12),so as 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 displayed 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 pentylenetetrazol (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 hippocampal tissue isolated from 24 h-old SD rats.The isolated neurons were incubated normally in Dulbecco's Modified Eagle Medium (DMEM) / Nutrient Mixture F 12(1 ∶ 1) for 10 days,followed by exposure to the extracellular fluid {composed of NaCl,KCl,CaCl2,MgCl2,HEPES [4-(2-hydroxyethyl)-1-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 returned to a regular culture medium [DMEM/F 12(1 ∶ 1)] plus acupuncture serum (9 ∶ 1) for acupuncture serum group,and by exposure to Mg2+-free extracellular fluid for 3 h and returned to DMEM/F 12(1 ∶ 1) plus non-acupuncture serum (9 ∶ 1) for non-acupuncture serum group.The cell apoptosis was assessed at 2,12 and 48 h after application of acupuncture serum or non-acupuncture 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 medium 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 Caspase-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 Acupuncture 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.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Key words
Acupuncture serum/Convulsions/Cell apoptosis/GRP 78 protein/CHOP protein/Caspase-12 protein/Endoplasmic reticulum homeostasis分类
医药卫生引用本文复制引用
张慧,杨帆,吴旭,沈德凯,陈浩,吴生兵,昂文平,宋小鸽,解明月..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[J].针刺研究,2017,42(2):95-101,7.基金项目
国家自然科学基金(No.81072872) （No.81072872）
安徽高校科研平台创新团队项目(No.2015 TD 033) （No.2015 TD 033）
安徽省高校自然科学研究重点项目(No.KJ 2016 A 858) （No.KJ 2016 A 858）
