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 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 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 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 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). CONCLUSIONS: 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.
