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 Mg

(2+)

-free medium were divided into normal extracellular fluid(medium)group(normal)

Mg(2+)-free medium were divided into normal extracellular fluid(medium)group(normal)

Mg

(2+)

-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 7days)to prepare acupuncture serum.Hippocampal neuronal cultures were prepared from hippocampal tissue isolated from 24h-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

CaCl_2

MgCl_2

HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid]

D-glucose and aminoethanoic acid(pH 7.3)}for3hand returned to normal culture for normal group

by exposure to Mg(2+)-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 7days)to prepare acupuncture serum.Hippocampal neuronal cultures were prepared from hippocampal tissue isolated from 24h-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

CaCl_2

MgCl_2

HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid]

D-glucose and aminoethanoic acid(pH 7.3)}for3hand returned to normal culture for normal group

by exposure to Mg

(2+)

-free extracellular fluid(to induce seizure-like discharges)for 3hand returned to normal culture for Mg(2+)-free extracellular fluid(to induce seizure-like discharges)for 3hand returned to normal culture for Mg

(2+)

-free medium group

by exposure to Mg(2+)-free medium group

by exposure to Mg

(2+)

-free extracellular fluid for 3hand returned to a regular culture medium [DMEM/F 12(1∶1)]plus acupuncture serum(9∶1)for acupuncture serum group

and by exposure to Mg(2+)-free extracellular fluid for 3hand returned to a regular culture medium [DMEM/F 12(1∶1)]

plus acupuncture serum(9∶1)for acupuncture serum group

and by exposure to Mg

(2+)

-free extracellular fluid for 3hand 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 48hafter 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 3time-points detected using Western blot.Results The number of apoptotic hippocampal neurons was significantly higher in the Mg(2+)-free extracellular fluid for 3hand 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 48hafter 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 3time-points detected using Western blot.Results The number of apoptotic hippocampal neurons was significantly higher in the Mg

(2+)

-free medium group than in the normal medium group at 2

12 and 48h(P

<

0.01)

and considerably decreased in the acupuncture serum group(but not in the non-acupuncture serum group)relevant to the Mg(2+)-free medium group than in the normal medium group at 2

12 and 48h(P

<

0.01)

and considerably decreased in the acupuncture serum group(but not in the non-acupuncture serum group)relevant to the Mg

(2+)

-free medium group at the same timepoints after application of acupuncture serum(P

<

0.05

P

<

0.01).The expression levels of GRP 78 protein at 2h

CHOP and Caspase-12 proteins at 2

12 and 48hwere significantly up-regulated in the Mg(2+)-free medium group at the same timepoints after application of acupuncture serum(P

<

0.05

P

<

0.01).The expression levels of GRP 78 protein at 2h

CHOP and Caspase-12 proteins at 2

12 and 48hwere significantly up-regulated in the Mg

(2+)

-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 Mg(2+)-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 Mg

(2+)

-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 Mg(2+)-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 Mg

(2+)

-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.