당귀용회환(當歸龍?丸)의 glutamate에 의한 청신경세포(聽神經細胞) 손상(損傷) 보호효과(保護效果)

유동희,박래길,소홍섭,이기남,정명수,Yu, Dong-Hee,Park, Rae-Gil,So, Hong-Seob,Lee, Ki-Nam,Chong, Myong-Soo 대한예방한의학회 2012 대한예방한의학회지 Vol.16 No.2

Objective : The water extract of Danguiyonghoihwan (DGYHW) has been traditionally used in treatment of tinnitus in Oriental Medicine. However, little is known about the mechanism by which DGYHW rescues auditory neuronal cells from injury damages. Therefore, in this study I effort to elucidate the mechanism of the cytoprotective effect of the DGYHW extract on glutamate-induced auditory sensorineuronal cell death. Methods : I determined the elevated cell viability by DGYHW extract on glutamate-induced auditory sensorineuronal cell death. Glutamate induced neuronal damage in oranotypic explant culture also, glutamate decreased cell viability on VOT-33 cells but pretreatment with DGYHW inhibited cell death. Results : One of the main mediator of glutamate-induced cytotoxicity was known to generation of reactive oxygen species (ROS). Pretreatment with DGYHW inhibited this ROS generation from glutamated-stimulated VOT-33 cells. Also, I identified that the ROS-induced DCF-DA green fluorescence is reduced by DGYHW pretreatment. The critical markers of apoptotic cell death were cleavages of procaspase-3 protease protein. So I checked the expression level and cleavage of procaspase-3 protease protein. Glutamate-treated VOT-33 cells were shown to have cleavage of procaspase-3 protease proteins and following reduction of expression of these proteins. But I found that pre-treatment with DGYHW protects glutamate-induced changes of biochemical marker protein, caspase-3. Conclusion : These findings indicated that DGYHW may prevent cell death from glutamate induced VOT-33 cell death by inhibiting the ROS generation and modulation of protein expressions in procaspase-3, catalase and Bcl-2.

Cisplatin에 의한 耳毒性에서 苓桂朮甘湯의 보호 효과

전호성(Ho Seong Jeon),박래길(Rae Gil Park),소홍섭(Hong Seob So),정명수(Myong Soo Chong),이수경(Su Kyung Lee) 한의병리학회 2012 동의생리병리학회지 Vol.26 No.5

팔진탕합화적환(八珍湯合化積丸)과 Adriamycin의 병용처리시 나타나는 synergistic 항종양(抗腫瘍) 효과(效果)에 관(關)한 작용기전 연구(硏究)

문구,문석재,원진희,조정연,박상구,송봉길,박래길,이병구,Moon, Gu,Moon, Seok-Jae,Won, Jin-Hee,Cho, Jung-Yun,Park, Sang-Gu,Song, Bong-Gil,Park, Rae-Gil,Lee, Byung-Gu 대한한방내과학회 2000 大韓韓方內科學會誌 Vol.21 No.3

Objective : This study was designed to evaluate the synergistic effect on cytotoxicity of combination with adriamycin and Palginhonhapwhajucwhan, a traditional prescription for cancer treatment in oriental medicine, in Chang, HL-60, Hep-3B and Alexander cells. Methods : We observed cell viability in Chang, HL-60, Hep-3B, and Alexander cells by crystal violet staining. Those cells were treated with various concentrations of adriamycin alone, Palginhonhapwhajucwhan alone and combination of two medications for 10 hr. On condition of $0.5{\mu}l/ml$ adriamycin alone, $15.6{\mu}l/ml$ Paljintanghapwhajucwhan alone and combination of two medications, at first, we observed colony forming of Chang and HL-60 cells. Second, we observed DNA fragmentation by agarose electrophoresis in Chang, HL-60, Hep-38 and Alexander cells. Third, we measured the catalytic activation of caspase-1, 2, 3, 6, 8, and 9 protease in Chang cells and caspase-3 protease in Chang, HL-60, Hep-3B and Alexander cells by using fluorogenic substrate. Finally, we isolated mRNA of Fas in Chang, HL-60, Hep-38 and Alexander cells and observed that Fas gene was amplified by RT-PCR Results : 1. The combination of adriamycin and Palginhonhapwhajucwhan synergistically augmented the cytotoxicity of Chang and HL-60 cells whereas did not in Hep-38 and Alexander cells. 2. Cotreatment of two drugs also markedly inhibited the colony forming ability both in Chang and HL-60 cells. 3. The cytotoxicity of these medicatons was revealed as apoptosis characterized by high molecular wight DNA fragmentaton. 4. The apoptotic cytotoxicity was mediated by activation of caspase-3 protease in Chang cells. 5. Synergistic increase in apoptotic cytotoxicity by combination of two medications was dependent on the expression of Fas in cancer cells. Conclusions : Combination of adriamycin and Palginhonhapwhajucwhan significantly augmented apoptotic cytotoxicity of Fas-positive cells such as Chang and HL-60 cells via acticaton of apoptosis signaling pathway.

백화사설초(白花蛇舌草) 메탄올 추출물(抽出物)의 항종양(抗腫瘍) 효과(效果) 및 항암(抗癌) 기전(機轉)에 관(關)한 연구(硏究)

노훈정,문구,문석재,원진희,문영호,박래길,No, Hoon-Jeong,Moon, Gu,Moon, Seok-Jae,Won, Jin-Hee,Moon, Young-Ho,Park, Rae-Gil 대한암한의학회 2000 大韓癌韓醫學會誌 Vol.6 No.1

Objectives: This experimental study was carried out to evaluate the effects of aqueous and methanol extracts of Hedyotis diffusa which has long been used for cancer treatment in oriental medicines on the induction of apoptotic cell death in human lymphoid leukemia cell line, HL-60. Methods: Cells were treated with various concentrations (200 to $0.4{\mu}g$) and periods (6 to 30 hr) of $H_2O$ and methanol extracts of Hedyotis diffusa. Then, cells were tested for viability by MTT assay. Cells wrere treated with $200{\mu}g/ml$ of methanol extract fork various periods. Genomic DNA was isolated, separated, on 1.5% agarose gels, stained with ethidium bromide and visualized under UV light. Cells were treated with $200{\mu}g/ml$ of each extract for 16 hr. Then, cells were treated with Hoechst dye 33342 and observed by fluorescence microscopy. Cells were treated with various doses of each for 12 hr and $100{\mu}g/ml$ of methanol extract for various periods. Lysate from the cells used to measure the activity of Caspase-1 and-3 proteases by using fluorogenic peptide substrates including acetyl-YVAD-AMC and acetyl-DEVD-AMC, respectively. Cells were treated with $200{\mu}g/ml$ of each extract for various periods. Cell lysates were immunoprecipated with anti-JNKl antibodies. The immune complex was reacted with $32^p-ATP$ and c-Jun as a substrate. The phosphotransferase activity of JNKI was measured by using PhosphoImage analyzer (Fuji Co., Japan). Nuclear extracts were isolated and incubated with oligonucleotide probe of $NF-{\kappa}B$. Transcriptional activation of ${\kappa}B$ was measured by using EMSA and visualized by PhosphoImage analyzer (Fuji Co, Japan). Cell lysates were prepared and analyzed by Western blotting with anti-Bc12 antibodies and anti-Bax antibodies. Cells were pretreated with various doses of methanol extract for 2 hr. Then, the extract was removed by centrifugation. Cells were resuspended with RPMI-1640 media containing 0.3% agarose, 10% FBS, overlayred onto bottom layer agarose and incubated at $CO_2$ incubator for 6 days. The number of colony was counted under light microscopy ($\time100$). Results: The death of HL-60 cells was markedly induced by the addition of methanol extract of Hedyotis diffusa in a dose and time-dependent manners. The apoptotic characteristic ladder pattern of DNA strand break was observed in death of HL-60 cells. In addition, it was shown nucleus chromatin condensation and fragmentation under Hoechst staining. Therefore, Hedyotis diffusa extract-induced death of HL-60 cells is mediated by apoptotic signaling processes. The activity of Caspase 3-like proteases remained in a basal level in HL-60 cells treated with aqueous extract of Hedyotis diffusa. However, it was markedly increased in HL-60 cells treated with methanol extract of Hedyotis diffusa. In addition, the phosphotransferase activity of JNKl was increased in HL-60 cells treated with methanol extract of Hedyotis diffusa. Furthermore, the activation of transcriptional activator, $NF-{\kappa}B$ was markedly induced by methanol extract of Hedyotis diffusa. Anti-apoptotic Bc12 was cleaved into 23Kda fragment by treatment of methanol extract of Hedyotis diffusa. However, expression of proapoptotic Bax protein was increased by treatment of methanol extract of Hedyotis diffusa in a time-dependent manner. Furthermore, methanol extract markedly inhibited the colony forming efficiency of HL-60 cells in semisolid agar culture. Conclusions: Above results suggest that methanol extract of Hedyotis diffusa induces the apoptotic death of human leukemic HL-60 cells via activations of Caspase-3 proteases, JNKI, transcriptional activator $NF-{\kappa}B$, In addition, our results also suggest that methanol extract of Hedyotis diffusa reduces the malignant potential of HL-60 cells via down regulation of colony forming

멜라닌 合成의 信號傳達機轉에 미치는 西施玉容散의 效果

우원홍,남우열,정우열,박래길,김지수,김원신,전병훈 대한동의병리학회 2001 동의생리병리학회지 Vol.15 No.1

Melanocytes are specialized cells located at the basal layer of the epidermis that synthesize and transfer melanin pigments to surrounding keratinocytes leading thereby to a uniform skin pigmentation. There are three melanocyte-specific enzymes, tyrosinase, tyrosinase-related protein 1 (TRP1), tyrosinase-related protein 2 (TRP2). Syntheses of melanin start from the conversion of the amino acid then the oxidations of L-dopa yield dopaquinone by the enzyme tyrosinase. Syntheses of melanin start from the conversion of the amino acid then the oxidations of L-dopa yield dopaquinone by the enzyme tyrosinase. Dopaquinone is converted to a relatively unstable intermediate, dopachrome, which successively undergoes to yield 5,6-dihydroxyindole. It leads to the formation of melanin polymer. Tyrosinase is a key enzyme in melanogenesis. The melanogenic effects of α-melanocyte stimulating hormone(MSH) can be mimicked by cAMP-elevating agents such as forskolin, cholera toxin, and isobuthylmethyxanthine. Recently many efforts were focused to understand the mechanical insights of melanogenesis to develop the therapeutic agents for hyper- and hypo-pigmentation. This study is designed to evaluate the efficiencies of Seosiokyoungsan(SOS), which had been used against face skin disease as pimple & freckles for many decades. Seosiokyoungsan markedly suppressed the increase of α -MSH(10nM)-induced melanogenesis as well as tyrosinase activity. α -MSH significantly increased the phosphotransferase activity of c-Jun N-terminal kinase1 (JNK1) which was returned to control level by pretreatment of Seosiokyoungsan. In addition, pretreated Seosiokyoungsan profoundly inhibited the ability of the nuclear proteins from B16 melanoma cells to bind with activated protein-1 (AP-1) nucleotide binding consensus sequences. These results indicate that Seosiokyoungsan inhibits α -MSH induced melanogenesis of B16 melanoma cells via suppression of phosphotransferase activity of JNK1 and transcriptional activation of AP-1. Key word : Seosiokyoungsan, α -MSH, melanin, tyrosinase activity, JNK1 , AP-1