• Journal of Acupuncture Research
• /
• v.18 no.3
• /
• pp.69-78
• /
• 2001

목적 : 본 연구는 최근 임상에서 많이 사용하는 자하차(紫河車) 약침액(藥鍼液)이 과산화수소($H_2O_2$)로 야기된 송과선 세포의 apoptosis에 있어서 세포 보호에 미치는 영향과 그 기전을 분석하였다. 방법 및 결과 : MTT assay를 이용하여 분석한 결과 처리 시간 및 농도에 따른 세포 독성의 효과가 $H_2O_2$ 투여로부터 관찰되었다. 또한 자하거 약침액은 세포 증식 효과를 나타내었고 자하거 약침액을 전처치하고 $H_2O_2$를 처치하였을 때 세포 독성이 크게 감소되었다. DAPI staining에서 자하거 약침액 투여군은 $H_2O_2$ 투여군에 비해 fragmentation이 억제되었다. TUNEL assay를 통하여 자하거 약침액 투여군은 $H_2O_2$ 투여군에 비하여 TUNEL 양성세포의 발현이 감소되었다. Flow cytometry를 통하여 자하거 약침액 투여군은 $H_2O_2$ 투여군에 비하여 세포주기 중 sub G1 분획의 증가가 억제되었다. 결론 : 이상의 결과를 통하여 자하거 약침액이 $H_2O_2$로 유발된 apoptosis에서 세포보호 효과가 있음이 확인되었다.

• Journal of Pharmacopuncture
• /
• v.13 no.1
• /
• pp.15-35
• /
• 2010

연구목적 : 이 연구는 봉약침의 봉독이 NF-${\kappa}B$ 활성억제와 안드로겐 수용체 조절 단백질 및 세포자멸사 조절 단백질의 발현을 통하여 세포자멸사를 유도하고, 전립선 암세포를 이식한 쥐에서의 세포자멸사 유도 효과를 확인함으로써, 봉약침의 봉독이 생체 내에서도 세포자멸사를 유도하여 전립선암에 효과를 나타냄을 확인하고자 하였다. 실험방법 : 세포자멸사의 관찰에는 DAPI, TUNEL staining assay를 시행하였으며, 세포자멸사 조절 단백질의 변동 관찰에는 western blot analysis를 시행하였고, 세포자멸사와 연관된 NF-${\kappa}B$의 활성 변화를 관찰하기 위해 EMSA를 시행하였다. 결과 : 1. DAPI, TUNEL staining assay 결과 봉독 및 melittin을 처리한 LNCaP 세포 모두에서 세포자멸사 유도율이 유의한 증가를 나타내었다. 2. LNCaP 세포에 봉독이나 melittin을 처리한 결과, 안드로겐 수용체 조절 단백질 중 p-Akt, COX-2, calpain은 봉독과 melittin 모두에서 유의한 감소를 나타내었고, Akt는 melittin에서 유의한 감소를 나타냈으며, 봉독에서 증가하는 경향을 보였고, MMP-9은 증가하였다. 3. 생체 내에서의 봉독의 항암효과를 확인하기 위해 전립선암세포가 이식된 쥐에 봉독을 처리한 후 암세포의 부피와 무게, 쥐의 체중을 측정한 결과, 봉독을 처리한 군에서 암 세포 부피비율 및 무게는 감소하였고, 쥐의 체중은 증가하였다. 4. 전립선암세포가 이식된 쥐에 봉독을 처리한 결과, NF-${\kappa}B$ 활성에서 유의한 감소를 나타내었다. 5. 전립선암세포가 이식된 쥐에 봉독을 처리한 결과, 세포자멸사 조절 단백질 중 Bax/Bcl-2, p53, caspase-3, caspase-9, calpain은 유의한 증가를, COX-2는 유의한 감소를 나타냈으며, MMP-9는 증가를 나타내었다. 결론 : 이상의 결과는 봉독이 시험관 내에서 뿐만 아니라 생체 내에서도 NF-${\kappa}B$의 활성을 억제하고 안드로겐 수용체 조절 단백질 및 세포자멸사 조절 단백질의 조절을 통하여 인간 전립선암 세포주인 LNCaP의 세포자멸사를 유도함으로써 전립선암 세포 증식억제 효과 및 호르몬 비의존적인 전립선암으로의 전이를 지연시키는 경향이 있을 것으로 사료되고, 봉독이 전립선암의 예방과 치료에 효과적으로 활용될 수 있을 것으로 기대된다.

• Journal of Korean Neurosurgical Society
• /
• v.50 no.6
• /
• pp.481-485
• /
• 2011

Objective : The purpose of this study is to investigate serial changes of hypoxia-inducible factor $1{\alpha}$ (HIF-$1{\alpha}$), as a key regulator of hypoxic ischemia, and apoptosis of hippocampus induced by bilateral carotid arteries occlusion (BCAO) in rats. Methods : Adult male Wistar rats were subjected to the permanent BCAO. The time points studied were 1, 2, 4, 8, and 12 weeks after occlusions, with n=6 animals subjected to BCAO, and n=2 to sham operation at each time point, and brains were fixed by intracardiac perfusion fixation with 4% neutral-buffered praraformaldehyde for brain section preparation. Immunohistochemistry (IHC), western blot and terminal uridine deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay were performed to evaluate HIF-$1{\alpha}$ expression and apoptosis. Results : In IHC and western blot, HIF-$1{\alpha}$ levels were found to reach the peak at the 2nd week in the hippocampus, while apoptotic neurons, in TUNEL assay, were maximal at the 4th week in the hippocampus, especially in the cornu ammonis 1 (CA1) region. HIF-$1{\alpha}$ levels and apoptosis were found to fluctuate during the time course. Conclusion : This study showed that BCAO induces acute ischemic responses for about 4 weeks then chronic ischemia in the hippocampus. These in vivo data are the first to show the temporal sequence of apoptosis and HIF-$1{\alpha}$ expression.

• YAKHAK HOEJI
• /
• v.48 no.2
• /
• pp.147-152
• /
• 2004

A wide variety of cancer chemotherapeutic agents have been shown to induce programmed cell death (PCD, APOPTOSIS) in various tumor cell lines in vitro. cis-Malonato [(4R,5R)-4,5-bis(aminomethyl)-2-isoprpopyl-1,3-dioxolane] platinum(II) (heptaplatin), which is a new drug approved by KFDA in 1999, in a novel platinum-based antitumor agent with clinical potential against stomach cancer and the 3rd generation of the cisplatin. This study was performed to know how heptaplatin and cisplatin and sunpla (mixture of heptaplatin and mannitol) affect on SK-MEL-28 cell line, and how they induce the apoptosis. At EM analysis, the morphology of the cell was changed by treatment of the cisplatin, heptaplatin and sunpla. Apoptotic body formed around plasma membrane, and chromatin condensation represented in nucleus. This phenomenon is one of the characteristic of the apoptosis. The DNA of SK-MEL-28 cell line truncated by cisplatin and sunpla treatment was identified on 2% agarose gel electrophoresis. TUNEL assay was performed to know whether SK-MEL-28 cell die as apoptosis or necrosis by cisplatin, heptaplatin and sunpla. At this result, fluorescence intensity increased according to increase of time and concentration. Therefore, it was identified that cislatin, heptaplatin and sunpla induced apoptosis. Fas expressed on SK-MEL-28 cell membrane by cisplatin, heptaplatin and sunpla was identified by using flow cytometer and the expression of bcl-2(anti-apoptotic gene) decreased according to increase of concentration of the cisplatin, heptaplatin and sunpla. Cisplatin, heptaplatin and sunpla induced apoptosis against SK-MEL-28 cell line, and the apoptotic mechanism was identified as Fas-mediated apoptosis and decreased bcl-2 expression.

• The Journal of Internal Korean Medicine
• /
• v.29 no.3
• /
• pp.582-594
• /
• 2008

Objective : The purpose of our study was to investigate the effect of JinlnWhaChul-tang-ga-wasong (JIN) on NDEA-induced liver tumorigenesis. Materials and Methods : We investigated the possible protective effects of Jininwhachul-tang-ga-wasong (JIN) as an anticancer against NDEA-induced liver injury in mice. Experimental mice were classified into 3 groups; normal, saline administered group (control group), and JIN extract (0.15g/kg/every other day) administered group (JIN group) after being injected with NDEA over 12 weeks. We examined the state of differentiation of these tumors and the effects of JIN after 6 weeks. To confirm the induction of apoptosis, the cells were analyzed by terminal deorynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, acridine orange staining and flow cytometric analysis. To investigate inhibitory effect on the expression of COX-2 by JIN, we performed COX-2 immunohistochemistry and reverse transcription polymerase chain reaction analysis. Results : Body weights significantly decreased in the control and JIN groups compared with the normal group. The levels of cholesterol, hemoglobin and testosterone decreased in the control compared with the normal group. The level of estradiol significantly increased in the control compared with the normal group. The control group reacted with TUNEL assay more than the normal and JIN groups. Upon naked eye, light and electron microscopic examination, JIN improved the morphological and histopathological changes of the liver caused by NDEA-induced hepatic neoplasm. COX-2 immunoreactivity decreased in the JIN group compared with the control group, mRNA expression of the control group was greater than the normal and JIN groups. Conclusion : these results suggest the possibility that JIN may exert an anti-tumor effect on NDEA-induced liver tumorigenesis.

• Asian Pacific Journal of Cancer Prevention
• /
• v.15 no.20
• /
• pp.8679-8684
• /
• 2014

CD133 was recently reported to be a cancer stem cell and prognostic marker. Quercetin is considered as a potential chemopreventive agent due to its involvement in suppression of oxidative stress, proliferation and metastasis. In this study, the expression of CD133/CD44 in esophageal carcinomas and Eca109/9706 cells was explored. In immunoflurorescence the locations of $CD133^+$ and multidrug resistance 1 $(MDR1)^+$ in the same E-cancer cells were coincident, mainly in cytomembranes. In esophageal squamous cell carcinomas detected by double/single immunocytochemistry, small $CD133^+$ cells were located in the basal layer of stratified squamous epithelium, determined as CSLC (cancer stem like cells); $CD44^+$ surrounding the cells appeared in diffuse pattern, and the larger $CD44^+$ (hi) cells were mainly located in the prickle cell layer of the epithelium, as progenitor cells. In E-cancer cells exposed to nanoliposomal quercetin (nLQ with cytomembrane permeability), down-regulation of NF-${\kappa}Bp65$, histone deacetylase 1 (HDAC1) and cyclin D1 and up-regulation of caspase-3 were shown by immunoblotting, and attenuated HDAC1 with nuclear translocation and promoted E-cadherin expression were demonstrated by immunocytochemistry. In particular, enhanced E-cadherin expression reflected the reversed epithelial mesenchymal transition (EMT) capacity of nLQ, acting as cancer attenuator/preventive agent. nLQ acting as an HDAC inhibitor induced apoptotic cells detected by TUNEL assay mediated via HDAC-NF-${\kappa}B$ signaling. Apoptotic effects of liposomal quercetin (LQ, with cytomembrane-philia) combined with CD133 antiserum were also detected by CD133 immunocytochemistry combined with TUNEL assay. The combination could induce greater apoptotic effects than nLQ induced alone, suggesting a novel anti-CSC treatment strategy.

• Journal of Physiology & Pathology in Korean Medicine
• /
• v.16 no.3
• /
• pp.594-601
• /
• 2002

The current study was carried out to evaluate neuroprotective effects of Citri Pericarpium after transient global ischemia in gerbils. Male Mongolian gerbils weighing 60-80g were anesthetized with 2% isoflurane mixed with 30% oxygen and 70 % nitrogen. Bilateral common carotid arteries were occluded for 5 minute with microaneurysm dips. On 3 or 7 days after ischemic surgery, the gerbils were sacrificed. The brain were removed, embedded in paraffin and sectioned at 8㎛-thickness. Gerbils that received ischemic insult for 5 min showed extensive neuronal damage in the hippocampal CA1 region, and the number of viable neuronal cell was 51.0±2.5/mm, 32.2% of normal group at 7 days after ischemic surgery. In animals that underwent the extract of Citri Pericarpium treatment, the number of viable neuronal cell were significantly better preserved at 110.58±3.58/mm, 72.0% of normal group than those of ischemic group (P<0.01). In the immunohistochemistry of Bax and Bcl-2, the Citri Pericarpium treated group down-regulated the expression of Bax protein at 72hr after transient global ischemia. In contrast, Bcl-2 protein level was not changed. The appearance in TUNEL assay is similar to the pattern of Bax protein. The water extract of Citri Pericarpium significantly reduced the number of TUNEL-positive CA1 pyramidal neurons at 72hr. The results suggest that Citri Pericarpium has potential neuroprotective effects in the transient global ischemia and the increase in the ratio of Bcl-2 to Bax may contribute to the anti-apoptotic effect of Citri Pericarpium.

• Reproductive and Developmental Biology
• /
• v.36 no.1
• /
• pp.71-77
• /
• 2012

The objective of this study was to examine the effect of thymidine treatment during $in$ $vitro$ maturation (IVM) of porcine follicular oocytes on blastocyst development. Porcine oocytes were treated with thymidine (10 mM, 20 mM and 30 mM) for 2 or 6 hr in the preiods of IVM I and/or II. The survival rates of the blastocysts in the 6 hr treatment groups of 10 mM and 20 mM during IVM I period were significantly higher than those of control group ($p$<0.05). However, the survival rate of the blastocysts in the 2 hr treatment group of 20 mM during IVM II period was significantly higher than control group ($p$<0.05). Furthermore, the survival rate of the blastocysts in the 6 hr treatment group of 30 mM during IVM II period was significantly lower than control group ($p$<0.05). Consistent with the previous result, blastocyst development of both IVM I and II treatment group was also showed as similar pattern. Total and apoptotic cell numbers of blastocysts derived from thymidine treated porcine oocytes were examined by using Tunel assay. The results showed that there was no significant differences in total cell number of blastocysts between thymidine treated and untreated groups. However, apoptosis-positive cells in the thymidine treated group (6 hr IVM I) were significantly lower than those of other groups ($p$<0.05). Taken together, these results indicate that high quality oocytes were selected by DNA synthesis mechanism according to high concentration thymidine treatment during porcine oocyte maturation. Therefore, we concluded that presumptive selected oocytes by thymidine treatment during maturation periods improved the further embryo development and embryonic quality of IVF embryos by decreasing the incidence of apoptosis in preimplantation porcine embryos.

• Applied Microscopy
• /
• v.42 no.2
• /
• pp.53-59
• /
• 2012

We evaluated the level of toxicity by LC50 and investigated the mechanism of brain impairment and GFAP expression by light and fluorescence microscopes in the pale chub, Zacco platypus, treated with fenvalerate. Survival rate was decreased according to the rise of fenvalerate concentration, and LC50 concentration was $27.79{\mu}g/L$. Apoptosis was increased according to the rise of fenvalerate concentration by TUNEL assay which determine apoptotic cell death population. Also, GFAP expression was increased in the periventricular zone. These results suggest that apoptosis might be a major mechanism to brain impairment of the pale chub by fenvalrerate. Increased GFAP expression in the periventricular zone would be an index of brain impairment. Taken together, this study might contribute to reveal the pathological mechanism of fish brain impairment by insecticide of pyrethroid, and to be an useful basic data for preservation of aquatic ecosystem.

• Clinical and Experimental Reproductive Medicine
• /
• v.45 no.1
• /
• pp.17-24
• /
• 2018

Objective: To investigate sperm chromatin/DNA integrity, global DNA methylation, and DNMT mRNA transcription in men with oligoasthenoteratozoospermia (OAT) compared with normozoospermic men. Methods: Semen samples from 32 OAT patients who comprised the case group and 32 normozoospermic men who comprised the control group were isolated and purified using a standard gradient isolation procedure according to World Health Organization criteria. DNMT1, DNMT3A, and DNMT3B transcripts were then compared between groups using real-time quantitative reverse-transcription polymerase chain reaction. Global DNA methylation in sperm was determined by an enzyme-linked immunosorbent assay. Protamine deficiency and the proportion of apoptotic spermatozoa were evaluated using chromomycin A3 (CMA3), aniline blue (AB), and toluidine blue (TB) staining, as well as the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The p-values < 0.05 were considered to indicate statistical significance. Results: Significantly higher proportions of AB+, TB+, CMA3+, and TUNEL+ spermatozoa, as well as DNMT3A and DNMT3B transcription, were found in the OAT group. Positive correlations were detected between sperm parameters, DNA/chromatin damage, and DNMT3A and DNMT3B transcripts. Global DNA methylation was significantly higher in the OAT patients and had a significant correlation with abnormal results of all sperm chromatin integrity tests, but was not associated with DNMT1, DNMT3A, or DNMT3B expression. Conclusion: Oligoasthenoteratozoospermic men showed abnormal sperm parameters, abnormal chromatin/DNA integrity, and a higher global DNA methylation rate, as well as overexpression of DNMT mRNA.