• Journal of Ginseng Research
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• v.46 no.3
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• pp.444-453
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• 2022

Background: Compound K (CK) is among the protopanaxadiol (PPD)-type ginsenoside group, which produces multiple pharmacological effects. Herein, we examined the effects of CK on muscle atrophy under hyperlipidemic conditions along with its pro-myogenic effects. Further, the molecular pathways underlying the effects of CK on skeletal muscle have been justified. Methods: C2C12 myotubes were treated with palmitate and CK. C2C12 myoblasts were differentiated using CK for 4-5 days. For the in vivo experiments, CK was administered to mice fed on a high-fat diet for 8 weeks. The protein expression levels were analyzed using western blotting analysis. Target protein suppression was performed using small interfering (si) RNA transfection. Histological examination was performed using Jenner-Giemsa and H&E staining techniques. Results: CK treatment attenuated ER stress markers, such as eIF2a phosphorylation and CHOP expression and impaired myotube formation in palmitate-treated C2C12 myotubes and skeletal muscle of mice fed on HFD. CK treatment augmented AMPK along with autophagy markers in skeletal muscle cells in vitro and in vivo experiments. AMPK siRNA or 3-MA, an autophagy inhibitor, abrogated the impacts of CK in C2C12 myotubes. CK treatment augmented p38 and Akt phosphorylation, leading to an enhancement of C2C12 myogenesis. However, AMPK siRNA abolished the effects of CK in C2C12 myoblasts. Conclusion: These findings denote that CK prevents lipid-induced skeletal muscle apoptosis via AMPK/autophagy-mediated attenuation of ER stress and induction of myoblast differentiation. Therefore, we may suggest the use of CK as a potential therapeutic approach for treating muscle-wasting conditions associated with obesity.

• Anatomy and Cell Biology
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• v.57 no.1
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• pp.85-96
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• 2024

Glioblastoma is the most common primary malignant brain tumor in adults. Temozolomide (TMZ) is an FDA-approved drug used to treat this type of cancer. Cinnamaldehyde (CIN) is a derivative of cinnamon extract and makes up 99% of it. The aim of this study was to investigate the in vitro combined effect of CIN and TMZ on human glioblastoma multiforme T98G cell line viability. In this study, we used 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl-tertazolium bromide (MTT) method to evaluate the extent of IC₅₀, acridine orange, Giemsa and Hoechst staining to evaluate the manner of apoptosis and the Western blotting method to examine the expression change of apoptotic proteins. Our results show that TMZ has an inhibitory effect on CIN when both used in combination at concentrations of 300 and 100 µM (P<0.05) and has a cytotoxic effect when used alone at the same concentrations (P<0.05). The western blotting result showed that TMZ at concentrations of 2,000 and 1,000 µM significantly increased Bax expression and decreased Bcl2 expression (P<0.05), indicating that TMZ induced apoptosis through the mitochondrial pathway. However, CIN had no effect on Bax and Bcl2 expressions, thus causing apoptosis from another pathway. Also, the Bax:Bcl2 expression ratio at concentrations combined was lower than that for TMZ 1,000 µM and higher than that for CIN 150 and 100 µM (P<0.05), which confirms the inhibitory effect of TMZ on CIN. From the present study, we conclude that TMZ in combination with CIN has an inhibitory effect on increasing the cytotoxicity rate.

• Tuberculosis and Respiratory Diseases
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• v.45 no.1
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• pp.176-183
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• 1998

Background: The total and differential cell count of bronchoalveolar lavage(BAL) fluid are useful assessing activity, prognosis and response to therapy in diffuse interstitial lung disease. But controversy exist as to the appropriate method in processing BAL fluid. Therefore we investigated the effect of gauze filtration, centrifugation and different storage time of BAL fluid on the total and differential cell count. Methods: We obtained BAL fluid from 6 persons with no active lung lesion and divided pooled BAL fluid into several siliconized glass tubes and filtered through 0, 1, 2, 4 folds of cotton guaze(pore size: 1mm), and compared total cell count using hemocytometer after trypan blue staining and differential cell count after Wright-Giemsa staining of cytocentrifuged preparations. And we also counted total and differential cell count after centrifugation(400g for 30 min) and various storage time(2hr, 24hr, and 48hr). Results: There was no difference in total and differential cell count according to folds of gauze filtraion. But without gauze filtration, mucus threads that hampered total and differential cell count were found in 2 cases (33%). Centrifugation resulted in loss of total cell count($24{\pm}18%$) without change in differential cell count. There was no change in total cell count after 2hr storage but significant cell loss was found after 24hr storage time(24hr : $28{\pm}21%$, 48hr : $41{\pm}24%$). However there was no change in differential cell count with 48hr storage time. Conclusion: Total and differential cell count of BAL fluid may be best performed after cotton gauze filtration without centrifugation and within 2 hours.

• Journal of Life Science
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• v.33 no.3
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• pp.277-286
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• 2023

Sarcopenia is the age-related loss of muscle mass and function. It is a natural part of aging and can lead to decreased mobility and increased frailty. The ubiquitin-proteasome pathway, which is involved in muscle protein degradation, is closely linked to sarcopenia. Germinated Rhynchosia nulubilis hydrolysate (GRH) has been reported to have anti-inflammatory and antioxidant properties, but there have been no reports on its inhibitory effect on muscle reduction. However, no study has yet explored the relationship between GRH and muscle loss inhibition. In this study, we evaluated the effects of GRH on muscle atrophy inhibitory activity in dexamethasone (Dexa)-induced muscle atrophy C2C12 myotubes and mouse models. Moreover, we identified a molecular pathway underlying the effects of GRH on skeletal muscle. May Grunwald-Giemsa staining showed that the length and area of myotubes increased in the groups treated with GRH. In addition, the GRH-treated group significantly reduced the expression of muscle ring finger protein 1 and muscular atrophy F-box (MAFbx) in the Dexa-induced muscular atrophy C2C12 model. GRH also improved muscle strength in C57BL/6 mice with Dexa-induced muscle atrophy, resulting in prolonged running exhaustive time and increased grip strength. We found that muscle strengthening by GRH was correlated with a decreased expression of the MAFbx gene in mouse muscle tissue. In conclusion, GRH can attenuate Dexa-induced muscle atrophy by inhibiting the ubiquitin-proteasome pathway via downregulation of the MAFbx gene expression.

• Journal of Pharmacopuncture
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• v.20 no.2
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• pp.127-131
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• 2017

Objectives: Polymorphonuclear neutrophils (PMNs) constitute the first line of defense against invading microbial pathogens. Early events in inflammation involve the recruitment of neutrophils to the site of injury or damage where changes in intracellular calcium can cause the activation of pro-inflammatory mediators from neutrophils including superoxide generation, degranulation and release of myeloperoxidase (MPO), productions of interleukin (IL)-8 and tumor necrosis factor ${\alpha}$ ($TNF-{\alpha}$), and adhesion to the vascular endothelium. To address the anti-inflammatory role of flavonoids, in the present study, we investigated the effects of the flavonoids quercetin and vitexin on the stimulus-induced nitric oxide (NO), $TNF-{\alpha}$, and MPO productions in human neutrophils. Methods: Human peripheral blood neutrophils were isolated, and their viabilities were determined by using the Trypan Blue exclusion test. The polymorphonuclear leukocyte (PMNL) preparations contained more than 98% neutrophils as determined by morphological examination with Giemsa staining. The viabilities of cultured neutrophils with various concentrations of quercetin and vitexin ($1-100{\mu}M$) were studied using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays. Neutrophils were cultured in complete Roswell Park Memorial Institute (RPMI) medium, pre-incubated with or without quercetin and vitexin ($25{\mu}M$) for 45 min, and stimulated with phorbol 12-myristate 13-acetate (PMA) ($10^{-7}M$). NO production was carried out through nitrite determination by using the Griess method. Also, the $TNF-{\alpha}$ and the MPO productions were measured using enzyme-linked immunosorbent assay (ELISA) kits and MPO assay kits. Results: Neutrophil viability was not affected up to a concentration of $100{\mu}M$ of quercetin or vitexin. Both quercetin and vitexin significantly inhibited $TNF-{\alpha}$, NO, and MPO productions in human neutrophils (P < 0.001). Conclusion:The present study showed that both quercetin and vitexin had significant anti-inflammatory effects. Thus, treatment with either quercetin or vitexin may be considered as a therapeutic strategy for treating patients with neutrophil-mediated inflammatory diseases.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.1
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• pp.125-131
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• 2015

Currently, taxol is mainly extracted from the bark of yews; however, this method can not meet its increasing demand on the market because yews grow very slowly and are a rare and endangered species belonging to first-level conservation plants. Recently, increasing efforts have been made to develop alternative means of taxol production; microbe fermentation would be a very promising method to increase the production scale of taxol. To determine the activities of the taxol extracted from endophytic fungus N. sylviforme HDFS4-26 in inhibiting the growth and causing the apoptosis of cancer cells, on comparison with the taxol extracted from the bark of yew, we used cellular morphology, cell counting kit (CCK-8) assay, staining (HO33258/PI and Giemsa), DNA agarose gel electrophoresis and flow cytometry (FCM) analyses to determine the apoptosis status of breast cancer MCF-7 cells, cervical cancer HeLa cells and ovarian cancer HO8910 cells. Our results showed that the fungal taxol inhibited the growth of MCF-7, HeLa and HO8910 cells in a dose-and time-dependent manner. IC50 values of fungal taxol for HeLa, MCF-7 and HO8910 cells were $0.1-1.0{\mu}g/ml$, $0.001-0.01{\mu}g/ml$ and $0.01-0.1{\mu}g/ml$, respectively. The fungal taxol induced these tumor cells to undergo apoptosis with typical apoptotic characteristics, including morphological changes for chromatin condensation, chromatin crescent formation, nucleus fragmentation, apoptotic body formation and G2/M cell cycle arrest. The fungal taxol at the $0.01-1.0{\mu}g/ml$ had significant effects of inducing apoptosis between 24-48 h, which was the same as that of taxol extracted from yews. This study offers important information and a new resource for the production of an important anticancer drug by endofungus fermentation.

• Journal of Pharmacopuncture
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• v.20 no.1
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• pp.52-56
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• 2017

Objectives: Neutrophils represent the front line of human defense against infections. Immediately after stimulation, neutrophilic enzymes are activated and produce toxic mediators such as pro-inflammatory cytokines, nitric oxide (NO) and myeloperoxidase (MPO). These mediators can be toxic not only to infectious agents but also to host tissues. Because flavonoids exhibit antioxidant and anti-inflammatory effects, they are subjects of interest for pharmacological modulation of inflammation. In the present study, the effects of rutin on stimulus-induced NO and tumor necrosis factor $(TNF)-{\alpha}$ productions and MPO activity in human neutrophils were investigated. Methods: Human peripheral blood neutrophils were isolated using Ficoll-Hypaque density gradient centrifugation coupled with dextran T500 sedimentation. The cell preparations containing > 98% granulocytes were determined by morphological examination through Giemsa staining. Neutrophils were cultured in complete Roswell Park Memorial Institute (RPMI) medium, pre-incubated with or without rutin ($25{\mu}M$) for 45 minutes, and stimulated with phorbol 12-myristate 13-acetate (PMA). Then, the $TNF-{\alpha}$, NO and MPO productions were analyzed using enzyme-linked immunosorbent assay (ELISA), Griess Reagent, and MPO assay kits, respectively. Also, the viability of human neutrophils was assessed using tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), and neutrophils were treated with various concentrations of rutin ($1-100{\mu}M$), after which MTT was appended and incubated at $37^{\circ}C$ for 4 hour. Results: Rutin at concentrations up to $100{\mu}M$ did not affect neutrophil viability during the 4-hour incubation period. Rutin significantly decreased the NO and $TNF-{\alpha}$ productions in human peripheral blood neutrophils compared to PMA-control cells (P < 0.001). Also, MPO activity was significantly reduced by rutin (P < 0.001). Conclusion: In this in vitro study, rutin had an anti-inflammatory effect due to its inhibiting NO and $TNF-{\alpha}$ productions, as well as MPO activity, in activated human neutrophils. Treatment with rutin may be considered as a therapeutic strategy for neutrophil-mediated inflammatory/autoimmune diseases.

• Animal Bioscience
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• v.37 no.6
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• pp.1001-1006
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• 2024

Objective: This study aimed to investigate the effect of Codonopsis pilosula polysaccharide (CPP) on the motility, mitochondrial integrity, acrosome integrity rate, and antioxidant ability of sheep sperm after preservation at 4℃. Methods: Semen from healthy adult rams were collected and divided into four groups with separate addition of 0, 200, 400, and 1,000 mg/L CPP. Sperm motility was analyzed using the Computer-Assisted Semen Analysis software after preservation at 4℃ for 24, 72, 120, and 168 h. Sperm acrosome integrity rate was analyzed by Giemsa staining at 24, 72, and 120 h, and mitochondrial membrane integrity was analyzed by Mito-Tracker Red CMXRos. The total antioxidant capacity (T-AOC) and malondialdehyde (MDA) content of spermatozoa were measured after 120 h of preservation. Results: The sperm viability and forward-moving sperm under 200 mg/L CPP were significantly higher than that in the control group at 72 h (61.28%±3.89% vs 52.83%±0.70%, 51.53%±4.06% vs 42.84%±1.14%), and 168 h (47.21%±0.85% vs 41.43%±0.37%, 38.68%±0.87% vs 31.68%±0.89%). The percentage of fast-moving sperm (15.03%±1.10% vs 11.39%±1.03%) and slow-moving sperm (23.63%±0.76% vs 20.29%±1.11%) in the 200 mg/L group was significantly higher than control group at 168 h. The mitochondrial membrane integrity of the sperm in the group with 200 mg/L CPP was significantly higher than those in the control group after storage at 4℃ for 120 h (74.76%±2.54% vs 65.67%±4.51%, p<0.05). The acrosome integrity rate in the group with 200 mg/L (87.66%±1.26%) and 400 mg/L (84.00%±2.95%) was significantly higher than those in the control group (80.65%±0.16%) after storage for 24 h (p<0.05). CPP also increased T-AOC and decreased the MDA concentration after preservation at 4℃ (p<0.05). Conclusion: Adding CPP could improve the T-AOC of sperm, inhibit lipid peroxidation, and facilitate semen preservation.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.12
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• pp.4773-4780
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• 2014

Background: To investigate the effects of small interference RNA (siRNA) targeting BCR/ABL mRNA on proliferation and apoptosis in the K562 human chronic myeloid leukemia (CML) cell line and to provide a theoretical rationale and experimental evidence for its potential clinical application for anti-CML treatment. Materials and Methods: The gene sequence for BCR/ABL mRNA was found from the GeneBank. The target gene site on the BCR/ABL mRNA were selected according to Max-Planck-Institute (MPI) and rational siRNA design rules, the secondary structure of the candidate targeted mRNA was predicted, the relevant thermodynamic parameters were analyzed, and the targeted gene sequences were compared with BLAST to eliminate any sequences with significant homology. Inhibition of proliferation was evaluated by MTT assay and colony-formation inhibiting test. Apoptosis was determined by flow cytometry (FCM) and the morphology of apoptotic cells was identified by Giemsa-Wright staining. Western blotting was used to analyze the expression of BCR/ABL fusion protein in K562 cells after siRNA treatment. Results: The mRNA local secondary structure calculated by RNA structure software, and the optimal design of specific siRNA were contributed by bioinformatics rules. Five sequences of BCR/ABL siRNAs were designed and synthesized in vitro. Three sequences, siRNA1384, siRNA1276 and siRNA1786, which showed the most effective inhibition of K562 cell growth, were identified among the five candidate siRNAs, with a cell proliferative inhibitory rate nearly 50% after exposure to 12.5nmol/L~50nmol/L siRNA1384 for 24,48 and 72 hours. The 50% inhibitory concentrations ($IC_{50}$) of siRNA1384, siRNA1276 and siRNA1786 for 24hours were 46.6 nmol/L, 59.3 nmol/L and 62.6 nmol/L, respectively, and 65.668 nmol/L, 76.6 nmol/L, 74.4 nmol/L for 72 hours. The colony-formation inhibiting test also indicated that, compared with control, cell growth of siRNA treated group was inhibited. FCM results showed that the rate of cell apoptosis increased 24 hours after transfecting siRNA. The results of annexinV/PI staining indicated that the rate of apoptosis imcreased (1.53%, 15.3%, 64.5%, 57.5% and 21.5%) following treamtne with siRNAs (siRNA34, siRNA372, siRNA1384, siRNA1276 and siRNA1786). Morphological analysis showed td typical morphologic changes of apoptosis such as shrunken, fragmentation nucleus as well as "apoptotic bodies" after K562 cell exposure to siRNA. Western blot analysis showed that BCR/ABL protein was reduced sharply after a single dose of 50nmol/L siRNA transfection. Conclusions: Proliferation of K562 cells was remarkbly inhibited by siRNAs (siRNA1384, siRNA1276 and siRNA1786) in a concentration-dependent manner in vitro, with effective induction of apoptosis at a concentration of 50 nmol/L. One anti-leukemia mechanism in K562 cells appeared that BCR/ABL targeted protein was highly down-regulated. The siRNAs (siRNA1384, siRNA1276 and siRNA1786) may prove valuable in the treatment of CML.

• Water for future
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• v.5 no.1
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• pp.27-36
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• 1972

This thesis is the study of the rainfall probability depth in the major areas of Korea, such as Seoul, Pusan and Taegu. The purpose of the paper is to analyze the rainfall in connection with the safe planning of the hydraulic structures and with the project life. The methodology used in this paper is the statistical treatment of the rainfall data in the above three areas. The scheme of the paper is the following. 1. The complementation of the rainfall data We tried to select the maximm values among the values gained by the three methods: Fourier Series Method, Trend Diagram Method and Mean Value Method. By the selection of the maximum values we tried to complement the rainfall data lacking in order to prevent calamities. 2. The statistical treatment of the data The data are ordered by the small numbers, transformed into log, $\sqrt{}, \sqrt[3]{}, \sqrt[4], and$\sqrt[5], and calculated their statistical values through the electronic computer. 3. The examination of the distribution types and the determination of the optimum distibution types By the $x^2-Test$ the distribution types of rainfall data are examined, and rejected some part of the data in order to seek the normal rainfall distribution types. In this way, the optimum distribution types are determined. 4. The computation of rainfall probability depth in the safety project life We tried to study the interrelation between the return period and the safety project life, and to present the rainfall probability depth of the safety project life. In conclusion we set up the optimum distribution types of the rainfall depths, formulated the optimum distributions, and presented the chart of the rainfall probability depth about the factor of safety and the project life.ct life.