眞武湯이 H_2O_2로 유도된 C6 Glial 세포사에 미치는 영향
Protective Effect of Jinmu-tang on H_2O_2-induced Cell Death in C6 Glial Cells
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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 H_2O_2 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 H_2O_2-induced C6 glial cell death. ROS formation, DNA fragmentation, IκBα phosphorylation, NF-κB activation, and secretion of TNF-α induced by H_2O_2 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 H_2O_2-induced C6 glial cell death in various pathways.
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 H_2O_2 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 H_2O_2-induced C6 glial cell death. ROS formation, DNA fragmentation, IκBα phosphorylation, NF-κB activation, and secretion of TNF-α induced by H_2O_2 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 H_2O_2-induced C6 glial cell death in various pathways.
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