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The Journal of Internal Korean Medicine > Volume 33(3); 2012 > Article
The Journal of Internal Korean Medicine 2012;33(3): 272-283.
眞武湯이 H2O2로 유도된 C6 Glial 세포사에 미치는 영향
최정훈1, 신용진2, 하예진2, 조문영2, 유주연2, 이숭인3, 신선호2
1함소아한의원
2원광대학교 한의과대학 내과학교실
3생생한의원
Protective Effect of Jinmu-tang on H2O2-induced Cell Death in C6 Glial Cells
Jung-hoon Choi1, Yong-jeen Shin2, Ye-jin Ha2, Mun-young Cho2, Ju-yeon You2, Soong-in Lee3, Sun-ho Shin2
1Hamsoa Oriental Medical Clinic
2Dept. of Internal Medicine, College of Oriental Medicine, Won-Kwang University
3Sangsang Oriental Medical Clinic
Correspondence  Sun-ho Shin ,Tel: 063-270-1013, Fax: 063-270-1594, Email: shshin@wonkwang.ac.kr
  Published online: September 30, 2012.
ABSTRACT
Objectives:
The purpose of this study was to investigate the mechanism of protective effect of Jinmu-tang (JMT, Zhenwu-tang) extract on H2O2-induced cell death in C6 glial cells.

Methods :
Cultured C6 glial cells of white mice were pretreated with JMT extract and exposed to H2O2 for inducing cell death. We measure the cell viability by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay and investigate the cell morphology using a light microscope after crystal violet (CV) staining. Reactive oxygen species (ROS) formation was analyzed using a flow cytometer and a fluorescent microscope after staining with 2'7'-dichlorofluorescein diacetate (DCF-DA). DNA fragmentation was analyzed using a flow cytometer after propidium iodide (PI) staining and nuclei morphology was investigated using a fluorescent microscope after 2-[4-amidinophenyl]-6-indo-lecarbamidine dihydrochloride (DAPI) staining. We analyzed expression of Bax, processing of procaspase-3 and poly (ADP-ribose) polymerase (PARP), and activation of nuclear factor-kB (NF-κB) by western blot method. Tumor necrosis factor-α (TNF-α) secretion was analyzed using Quantikine kit.

Results:
We determined the elevated cell viability by JMT extract on H2O2-induced C6 glial cell death. ROS formation, DNA fragmentation, IκBα phosphorylation, NF-κB activation, and secretion of TNF-α induced by H2O2 are inhibited by JMT extract pre-treatment. JMT extract inhibits Bax expression, processing of caspase-3 and PARP that are critical biochemical markers of apoptotic cell death.

Conclusions:
These results suggest that JMT extract has a protective effect on H2O2-induced C6 glial cell death in various pathways.
Key words: H2O2, C6 glial cell, Protective effect, Jinmu-tang (Zhenwu-tang)
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