• Journal of Embryo Transfer
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• v.30 no.3
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• pp.121-128
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• 2015

The objective of this study was to evaluate the efficiency of sperm cryosurvival in boar sperm separated by Percoll containing antioxidant enzymes. The boar semen was collected into a pre-warmed ($37^{\circ}C$) thermos bottle by gloved-hand method and was separated by 65% Percoll with superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) before freezing. The frozen sperm was thawed at $38.5^{\circ}C$ for 45 sec in water-bath for sperm characteristic analysis. The sperm were estimated with SYBR14/PI double staining for viability, FITC-PNA/PI double staining for acrosome reaction, Rhodamine123/PI double staining for mitochondrial integrity and were analyzed using flow cytometry. In results, sperm viability, acrosome reaction and mitochondrial integrity were improved in separated sperm groups compared with unseparated sperm by Percoll (UP) group. Especially, viability was significantly higher in sperm separated by Percoll containing 400 IU CAT group compared with other groups (P<0.05). And acrosome reaction was decreased in sperm separated by Percoll with 300 IU SOD, 400 IU CAT and 0.5 mM GSH groups compared with other groups, however, there were no significantly difference mitochondrial integrity among sperm separated by Percoll with antioxidant enzymes. In conclusion, we suggest that use of Percoll containing antioxidant enzymes for sperm separation will be beneficial for sperm cryopreservation in pigs.

• Korean Journal of Animal Reproduction
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• v.15 no.1
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• pp.67-77
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• 1991

本 試驗은 受精初期段階에서 생쥐卵子 透明帶의 機能을 糾明하고 種特異的인 精子受容體의 存在 및 構造理解를 위한 基礎硏究로서 ICR 생쥐의 透明帶를 免疫原으로 하여 토끼에서 抗透明帶抗體를 生産하고 免疫分析法 및 生物學的 檢査法으로 生産된 抗體가 생쥐 透明帶에 特異性이 있음을 確認한 후 이 抗體가생쥐 未受精卵의 體外受精에 미치는 影響과 또 이 抗體를 생쥐에 受動免疫하였을 때 受精에 미치는 影響을 調査하였던 바 다음과 같은 結果를 얻었다. 1. 處理區로서 抗血淸을, 對照區로서 對照血淸을 卵子培養液에 各各 0.3~10%로 含有한 培養液에서 생쥐의 未受精卵을 2時間 培養한 後 觀察하였을 때 對照區 卵子들의 透明帶가 正常的인 形態를 나타내었던 反面에, 處理區의 卵子들은 모두 透明帶 表面에 뚜렷한 沈澱層을 나타내었다. 이어 對照區와 處理區의 卵子를 0.1% pronase가 含有된 培養液으로 옮긴 후 觀察하였을 때, 對照區 卵子들의 透明帶가 5~7分만에 完全히 融解한 反面에, 處理區에서는 2~3時間까지 殘存하였다. 2. 處理區와 對照區 卵子를 FITC-goat anti-rabbit IgG가 含有된 培養液에서 養液한 후 螢光顯微鏡下에서 觀察하였을 때 對照區에서 對照血淸을 10% 含有한 培養液에서 培養한 일부 卵子들의 透明帶에서 희미한 螢光이 나타났으나 血淸稀釋度가 增加함에 따라 전혀 螢光을 나타내지 않았던 反面에, 處理區에서는 抗血淸이 1 : 1,000으로 含有된 培養液에서 培養한 卵子의 透明帶에서도 강한 螢光이 나타났다. 3. 帶抗體를 抗原으로 하여 間接酵素免疫分析法을 實施하였을 때 抗血淸과 對照血淸이 PBS에 1 : 200까지 稀釋되었을 때의 O.D.價는 별다른 差異를 나타내지 않았으나 血淸의 稀釋度가 增加함에 따라 O.D.價가 有意한 差異를 나타냄으로써 透明帶에 特異性을 갖는 抗體임을 確認할 수 있었다. 4. 抗血淸과 對照血淸이 各各 0.3~10%까지 含有된 培養液으로 생쥐의 未受精卵을 豫備培養한 다음 體外受精을 實施하였을 때의 受精率은 對照區에서 77.7%~87.7%이었던 反面에, 處理區에서는 0~29.8%로서 抗透明帶抗體가 생쥐卵子의 體外受精을 完全히 또는 顯著히 抑制하였다. 그러나 抗血淸과 對照血淸이 各各 2.5~10%까지 含有된 培養液으로 透明帶를 除去한 未受精卵의 體外受精을 하였을 때의 受精率은 處理區와 對照區에서 各各 93.0~98.3% 및 93.3~94.3%로서 受精抑制現象을 나타내지 않았다. 5. 抗血淸과 正常血淸, 그리고 兩 血淸으로부터 protein A-affinity chromatography에 의해 分離한 immunoglubulin G를 各各 생쥐에 受動免疫하였을 때, 對照區로서 對照血淸을 0.1~0.3ml 또는 control IgG를 1~3ml 投與했을 때의 各各의 受精率이 80.4~90.2%, 82.2~94.4%이었던 反面에, 處理區로서 抗血淸을 0.3ml 또는 immune IgG를 3mg 投與한 생쥐에서는 完全한 受精抑制現象이 確認되었다.

• Journal of Applied Biological Chemistry
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• v.57 no.2
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• pp.103-111
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• 2014

A1E is an extract from traditional Asian medicinal plants that has therapeutic activities against cancers, metabolic disease, and other intractable conditions. However, its mechanism of action on cervical cancer has not been studied. In order to ascertain if A1E would have pronounced anti-cervical cancer effect, cervical cancer cells were incubated with A1E and apoptosis was detected by nuclear morphological changes, annexin V-FITC/PI staining, cell cycle analysis, western blotting, Reverse-transcription polymerase chain reaction, and measurement of mitochondrial membrane potential. Expression of human papiloma virus E6 and E7 oncogenes was down-regulated in A1E-treated cervical cancer cells, while p53 and retinoblastoma protein levels were enhanced. A1E also perturbed cell cycle progression at sub-G1 and altered cell cycle regulatory factors in SiHa cervical cancer cells. A1E activated apoptotic intrinsic pathway markers such as caspase-9, caspase-3 and poly ADP-ribose polymerase, and down-regulated expression of Bcl-2 and Bcl-xl. A1E induced mitochondrial membrane potential collapse and cytochrome c release, and inhibited phosphatidylinositol 3-kinase (PI3K)/Akt, key factors involved in cell survival signaling. Taken all these results, A1E induced apoptosis via activation of the intrinsic pathway and inhibition of the PI3K/Akt survival-signaling pathway in SiHa cervical cancer cells. In conclusion, A1E exerts anti-proliferative action growth inhibition on cervical cancer cells through apoptosis which demonstrates its anti-cervical cancer properties.

• The Journal of Korean Obstetrics and Gynecology
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• v.25 no.2
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• pp.78-88
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• 2012

Objectives: Paeonia japonica has been widely used for gynecopathy and analgesic effects in Korean Traditional Medicine. The aim of the present study is to determine the antioxidant effect of Paeonia japonica extracts(PJE) by using mouse embryonic fibroblast cells(MEF cells). Methods: We evaluated Radical Scavenging Activity of PJE by the DPPH assay. Protective effect of the PJE on the hydrogen peroxide($H_2O_2$) induced oxidative damage of MEF cells was analyzed by the MTT assay. The Morphological changes of MEF cells induced by P. japonica, $H_2O_2$ and P. japonica+$H_2O_2$ was evaluated by DAPI staining. And effect of PJE on the rate of apoptosis in MEF cells was measured using flow cytometry with Annexin V-FITC and PI double staining. Results: We observed that PJE contain significant DPPH radical scavenging activity. Cell viability of oxidative damaged cells treated with various concentrations of $H_2O_2$ was increased by treatment with PJE. Flow cytometric analysis of the cells treated with $H_2O_2$ in the absence or presence of PJE showed that the crumbled G1 peak was accumulated by the treatment with $H_2O_2$ alone, but restored by addition of PJE. Portion of cells that undergo apoptosis mediated by oxidative stress was decreased by treatment of PJE. The nuclear fragmentation occurred in the oxidative damaged MEF cells was also decreased by PJE treatment. Conclusions: Taken together, our results suggest that PJE exhibits significant antioxidant activity and functions to inhibit cell death mediated by oxidative damage induced apoptotic pathways.

• Journal of Chest Surgery
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• v.30 no.6
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• pp.553-558
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• 1997

Arterial allografts have known advantages over prosthetic vascular conduit for treatment of heart valvular disease, congenital heart disease and aortic disease. Cell viability may play a role in determining the longterm outcome of allografts. Endothelial cell is one important part in determining the allograft viability. To evaluate the viability of endothelial cells using current allograft preservation technique, porcine heart valve leaflets and arterial wall were subjected to collagenase digestion. Single endothelial cell suspension was labeled with GSA-PITC(Griffonia simplicifolia agglutininfluorescein isothiocyan te), a vascular, endothelial cell specific marker. The cell suspension was washed and incubated with Pl(Propidium iodide), which does not bind with viable cells, Endothelial cell viability was evaluated by calculating the percentage of GSA-FITC(+) and Pl(-) group using flowcytometric analysis. Allografts were treated with $4^{\circ}C$ antibiotic solo!ion for 24 hours for sterilization. After this, half of allografts were stored in $4^{\circ}C$ RPMI 1640 with HEPES buffer culture medium with 10% fetal bovine serum for 1 to 14 days(Group I). Another half of allografts were cryopreserved with a currently used technique (Group II). During the procurement and sterilization of arterial allografts, 22.8% and 24.4% of endothelial cell viability declined, respectively. In Group I, 11.9% of endothelial cell viability declined further steadily during 14 days of storage. In Group II, 13.7% of endothelial cell viability declined. These results show that largest loss of endothelial cell viability occurs during the nitial process. After 14 days of arterial allograft storage under $4^{\circ}C$ nutrient medium or cryopreservation, about 40% of endothelial cell viability is maintained. There were no differences between the endothelial cell viability from aortic valve leaflet, pulmonic valve leaflets, aortic wall and pulmonic wall.

• Biomolecules & Therapeutics
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• v.23 no.4
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• pp.367-373
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• 2015

Cordyceps species including Cordyceps bassiana are a notable anti-cancer dietary supplement. Previously, we identified several compounds with anti-cancer activity from the butanol fraction (Cb-BF) of Cordyceps bassiana. To expand the structural value of Cb-BF-derived anti-cancer drugs, we employed various chemical moieties to produce a novel Cb-BF-derived chemical derivative, KTH-13-amine-monophenyl [4-isopropyl-2-(1-phenylethyl) aniline (KTH-13-AMP)], which we tested for anti-cancer activity. KTH-13-AMP suppressed the proliferation of MDA-MB-231, HeLa, and C6 glioma cells. KTH-13-AMP also dose-dependently induced morphological changes in C6 glioma cells and time-dependently increased the level of early apoptotic cells stained with annexin V-FITC. Furthermore, the levels of the active full-length forms of caspase-3 and caspase-9 were increased. In contrast, the levels of total forms of caspases-3, caspase-8, caspase-9, and Bcl-2 were decreased in KTH-13-AMP treated-cells. We also confirmed that the phosphorylation of STAT3, Src, and PI3K/p85, which is linked to cell survival, was diminished by treatment with KTH-13-AMP. Therefore, these results strongly suggest that this compound can be used to guide the development of an anti-cancer drug or serve as a lead compound in forming another strong anti-proliferative agent.

• Journal of Life Science
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• v.17 no.3 s.83
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• pp.340-344
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• 2007

Avian influenza viruses cause a considerable threat to humans and animals. In this study, we investigated whether alkaline disinfectant solution can inactivate H5N1, H3N2, H6N1, and H9N2 subtypes of avian influenza virus. When H5N1, H3N2, H6N1, and H9N2 avian influenza viruses were treated with alkaline solution diluted with PBS (pH 7.2) prior to infection into MDCK cells, alkaline disinfectant solution (at dilutions up to $10^{-2}$) completely inactivated all avian influenza subtypes tested. To confirm the inactivation of avian influenza viruses by alkaline disinfectant solution, we used an immunofluorescence assay with influenza A anti-nucleoprotein antibody and FITC-labeled secondary antibody to stain MDCK cells infected with avian H9N2 influenza viruses. No staining was observed in MDCK rells infected with H9N2 viruses that were pre-treated with a $10^{-2}$ dilution of alkaline disinfectant solution, while strong staining was observed in MDCK cells infected with H9N2 viruses without pre-treatment. Our results indicate that alkaline solution could help to control avian influenza viruses including the highly pathogenic H5N1 subtype.

• Nutrition Research and Practice
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• v.11 no.6
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• pp.470-478
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• 2017

BACKGROUND/OBJECTIVE: Orostachys japonicus A. Berger (Crassulaceae) has been used in traditional herbal medicines in Korea and other Asian countries to treat various diseases, including liver disorders. In the present study, the anti-fibrotic effects of O. japonicus extract (OJE) in cellular and experimental hepatofibrotic rat models were investigated. MATERIALS/METHODS: An in vitro hepatic stellate cells (HSCs) system was used to estimate cell viability, cell cycle and apoptosis by MTT assay, flow cytometry, and Annexin V-FITC/PI staining techniques, respectively. In addition, thioacetamide (TAA)-induced liver fibrosis was established in Sprague Dawley rats. Briefly, animals were divided into five groups (n = 8): Control, TAA, OJE 10 (TAA with OJE 10 mg/kg), OJE 100 (TAA with OJE 100 mg/kg) and silymarin (TAA with Silymarin 50 mg/kg). Fibrosis was induced by treatment with TAA (200 mg/kg, i.p.) twice per week for 13 weeks, while OJE and silymarin were administered orally two times per week from week 7 to 13. The fibrotic related gene expression serum biomarkers glutathione and hydroxyproline were estimated by RT-PCR and spectrophotometry, respectively, using commercial kits. RESULTS: OJE (0.5 and 0.1 mg/ mL) and silymarin (0.05 mg/mL) treatment significantly (P < 0.01 and P < 0.001) induced apoptosis (16.95% and 27.48% for OJE and 25.87% for silymarin, respectively) in HSC-T6 cells when compared with the control group (9.09%). Further, rat primary HSCs showed changes in morphology in response to OJE 0.1 mg/mL treatment. In in vivo studies, OJE (10 and 100 mg/kg) treatment significantly ameliorated TAA-induced alterations in levels of serum biomarkers, fibrotic related gene expression, glutathione, and hydroxyproline (P < 0.05-P < 0.001) and rescued the histopathological changes. CONCLUSIONS: OJE can be developed as a potential agent for the treatment of hepatofibrosis.

• Restorative Dentistry and Endodontics
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• v.25 no.2
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• pp.225-234
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• 2000

The purpose of this study was to investigate the distribution and fluorescene intensity of vasoactive intestinal polypeptide(VIP) immunoreactive cells in rat trigeminal ganglion after inferior alveolar nerve axotomy. The animals were divided into normal and two experimental groups. The experimental animals were sacrificed at 14th and 28th day after inferior alveolar nerve axotomy. The trigeminal ganglion was removed and immersed in the 4% paraformaldehyde-0.2% picric acid in 0.1M phosphate buffer. Serial frozon sections about $16{\mu}m$ in thickness were cut with a cryostat. The immunofluorescence staining was performed. The rabbit anti-VIP(1 : 8,000) was used as primary antibody and fluorescene isothiocynate(FITC)-conjugated anti-rabbit IgG(1 : 80) as secondary antibody. The slides were observed under confocal laser scanning microscope. Three-dimensional images were constructed from 9 serial images(each $1{\mu}m$ in thickness) made by automatic optical sectioning. Unprocessed optical sections were obtained and stored on a optical disk. Color picture were printed by a video copy processor. The results were as follows; 1. The appearance of VIP immunoreactive cells in the mandibular part of trigeminal ganglion was 8.79${\pm}$1.99% in normal group and 39.16${\pm}$5.62% in 14 days, 16.25${\pm}$2.39% in 28 days after inferior alveolar nerve axotomy groups. 2. The relative fluorescence intensity of VIP immunoreactive cell bodies in the mandibular part of trigeminal ganglion was 134.40${\pm}$10.39 in normal group and 192.88${\pm}$14.06 in 14 days, 143.10${\pm}$5.02 in 28 days after nerve axotomy groups. Therefore, the relative fluorescence intensity of 14 days after nerve axotomy group was 43.3% higher than intensity of normal group. 3. In optical single section analysis of VIP immunoreactive cell bodies, white cell bodies(moderate fluorescence intensity) were the most abundant in normal and 28 days after nerve axotomy groups. Whereas, in 14 days after nerve axotomy group, red cell bodies(high fluorescence intensity) were the most abundant. 4. In optical serial section analysis of VIP immunoreactive cell bodies, red cell bodies(high fluorescence intensity) were observed in a part of the 9 sections of normal and 24 days after nerve axotomy groups. Whereas, red cell bodies were observed in all of the 9 sections of 14 days after nerve axotomy group. 5. The results indicates that number and fluorescence intensity of VIP immunoreactive cells were increased in the mandibular part of trigeminal ganglion following inferior alveolar nerve axotomy.

• Journal of Ginseng Research
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• v.39 no.1
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• pp.22-28
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• 2015

Background: Sun ginseng (SG), a specific formulation of quality-controlled red ginseng, contains approximately equal amounts of three major ginsenosides (RK1, Rg3, and Rg5), which reportedly has antitumor-promoting activities in animal models. Methods: MTT assay was used to assess whether SG can potentiate the anticancer activity of epirubicin or paclitaxel in human cervical adenocarcinoma HeLa cells, human colon cancer SW111C cells, and SW480 cells; apoptosis status was analyzed by annexin V-FITC and PI and analyzed by flow cytometry; and apoptosis pathway was studied by analysis of caspase-3, -8, and -9 activation, mitochondrial accumulation of Bax and Bak, and cytochrome c release. Results: SG remarkably enhances cancer cell death induced by epirubicin or paclitaxel in human cervical adenocarcinoma HeLa cells, human colon cancer SW111C cells, and SW480 cells. Results of the mechanism study highlighted the cooperation between SG and epirubicin or paclitaxel in activating caspase-3 and -9 but not caspase-8. Moreover, SG significantly increased the mitochondrial accumulation of both Bax and Bak triggered by epirubicin or paclitaxel as well as the subsequent release of cytochrome c in the targeted cells. Conclusion: SG significantly potentiated the anticancer activities of epirubicin and paclitaxel in a synergistic manner. These effects were associated with the increased mitochondrial accumulation of both Bax and Bak that led to an enhanced cytochrome c release, caspase-9/-3 activation, and apoptosis. Treating cancer cells by combining epirubicin and paclitaxel with SG may prove to be a novel strategy for enhancing the efficacy of the two drug types.