Protective Effect of Jinmu-tang on H2O2-induced Cell Death in C6 Glial Cells

Choi, Jung-hoon; Shin, Yong-jeen; Ha, Ye-jin; Cho, Mun-young; You, Ju-yeon; Lee, Soong-in; Shin, Sun-ho

The Journal of Internal Korean Medicine > Volume 33(3); 2012 > Article

Original 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)