• Journal of Microbiology and Biotechnology
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• v.9 no.1
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• pp.106-112
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• 1999

The therapeutic potential of phosphodiesterase 4(PDE4) inhibitors in inflammatory diseases including some autoimmune diseases has been explored recently with some hopeful results. These PDE4 inhibitors are thought to show their anti-inflammatory effect by down-regulating tumor necrosis factor-a (TNF-$\alpha$) production in lymphocytes and macrophages. A high concentration of TNF-$\alpha$has been found in rheumatoid arthritis (RA) synovium and reducing TNF-$\alpha$using biological agents was proven to be an effective RA treatment. To test the possibility of using PDE4 inhibitors for RA treatment, the effects of a newly synthesized PDE4 inhibitor, DWP205505, on TNF-$\alpha$ and IL-10 production was tested in cells isolated from normal peripheral blood and rheumatoid arthritis synovial fluid. Cytokine production was assayed at the protein level by sandwich enzyme-linked immunosorbent assay (ELISA) and at the mRNA expression level by semi-quantitative RT-PCR. Another PDE4 inhibitor, RP73401, was used for comparison. DWP205505 and RP73401 had no harmful effect on cell viability up to 10 $\mu$M concentration during the 24 h culture period. DWP205505 as well as RP73401 significantly reduced TNF-$\alpha$ secretion from lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells (pBMC) and synovial fluid mononuclear cells (SFMC). The effect of DWP205505 or RP73401 treatment on the mRNA expression of TNF-$\alpha$ was also studied in LPS-stimulated PBMC and SFMC. TNF-$\alpha$ mRNA expression was increased by LPS stimulation and both of the PDE4 inhibitors suppressed TNF-$\alpha$ mRNA expression. For interleukin-l0 (IL-l0), a little different results were obtained from PBMC and SFMC; IL-l0 secretion was unaffected by LPS stimulation and only minimally affected by both of the PDE4 inhibitors in PBMC. In unstimulated SFMC, DWP205505 and RP73401 slightly enhanced IL-10 secretion, while they reduced IL-l0 secretion from LPS-stimulated SFMC where IL-l0 secretion was a lot higher than unstimulated SFMC. These results suggest that the newly synthesized PDE4 inhibitor DWP205505 may have anti-rheumatoid arthritis activity.

• Korean Journal of Ecology and Environment
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• v.55 no.2
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• pp.99-110
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• 2022

We investigated physicochemical properties and isotopic compositions of organic matter (δ¹³C_TOC and δ ¹⁵N_TN) in the old fish farming (OFF) site after the cessation of aquaculture farming. Based on this approach, our objective is to determine the organic matter origin and their relative contributions preserved at sediments of fish farming. Temporal and spatial distribution of particulate and sinking organic matter(OFF sites: 2.0 to 3.3 mg L^-1 for particulate matter concentration, 18.8 to 246.6 g m^-2 day^-1 for sinking organic matter rate, control sites: 2.0 to 3.5 mg L^-1 for particulate matter concentration, 25.5 to 129.4 g m^-2 day^-1 for sinking organic matter rate) between both sites showed significant difference along seasonal precipitations. In contrast to variations of δ¹³C_TOC and δ¹⁵N_TN values at water columns, these isotopic compositions (OFF sites: -21.5‰ to -20.4‰ for δ¹³ C_TOC, 6.0‰ to 7.6‰ for δ¹⁵N_TN, control sites: -21.6‰ to -21.0‰ for δ¹³C_TOC, 6.6‰ to 8.0‰ for δ¹⁵N_TN) investigated at sediments have distinctive isotopic patterns(p<0.05) for seawater-derived nitrogen sources, indicating the increased input of aquaculture-derived sources (e.g., fish fecal). With respect to past fish farming activities, representative sources(e.g., fish fecal and algae) between both sites showed significant difference (p<0.05), confirming predominant contribution (55.9±4.6%) of fish fecal within OFF sites. Thus, our results may determine specific controlling factor for sustainable use of fish farming sites by estimating the discriminative contributions of organic matter between both sites.

• Journal of Life Science
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• v.34 no.7
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• pp.509-519
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• 2024

A previous study showed that krill oil improved recognition and memory through anti-oxidative effects in an amyloid β model, but the authors noted that further investigations are necessary of alterations to neurotransmitters' states and of serum lipid profile improvements related to serum lipid peroxidation. Accordingly, in this study, ICR mice were pre-treated intraperitoneally with scopolamine prior to induced neurotransmission impairment, and the effects of krill oil provision on their capabilities of cognition were tested by performing a passive avoidance test (PAT), water maze test (WMT), and novel object recognition test. Then, parameters including the acetylcholine (ACh) concentration, acetylcholinesterase activity (AChE), lipid peroxidation, serum lipid levels, and nerve cell proliferation were investigated. The results showed that krill oil improved the mice's abilities in recognition and memory as the times taken to complete the PAT and WMT were reduced compared to the mice in a comparison scopolamine-treated group. Krill oil produced an increased concentration of Ach, and this was accompanied by a decrease in AChE. As shown in a scopolamine-treated SH-SY5Y cell line, krill oil reduced the activity of AChE. Moreover, the suppression of lipid peroxidation-reflected in the finding that malondialdehyde was decreased with krill oil provision-is speculated to affect the recorded serum triglyceride and cholesterol decreases and LDL cholesterol increase. The intake of krill oil was also found to produce an improvement in brain-derived neurotrophic factor expression by stimulating the activation of cyclic AMP response element binding protein in the brain tissue. Overall, the current results imply that the provision of krill oil raises the cognition and memory by elevating neurotransmitters and by improving the serum lipid profile and nerve cell proliferation, which occur as lipid peroxidation is suppressed in the brain tissue.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.50 no.1
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• pp.37-48
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• 2024

Songla (Usnea diffracta Vain.) is one of the lichens belonging to the genus Usnea, and pharmacological activities such as antioxidant, antimicrobial, anti-inflammatory, anti-tumor and cardiovascular protection have been reported in previous studies, but its efficacy in skin and hair is not well known. In this study, the effect of Usnea diffracta extract (UDE) on anti-aging and dermal papilla cell proliferation was verified in vitro. As a result of the experiment, it was confirmed that the UDE significantly reduced the expression of MMP-1 and the activity of MAPKs (ERK, p38, JNK) and AP-1 (c-Fos, c-Jun), which were increased by UVA in HDFn. In addition, the UDE significantly increased the proliferation of HFDPC and significantly increased the mRNA expression of VEGF and KGF, which are hair growth factors. Accordingly, the phosphorylation of ERK/CREB involved in hair proliferation and expression of growth factors was increased in a concentration-dependent manner. The main component represented by the main peak was separated and purified using Prep LC by concentrating the UDE, which was confirmed as diffractaic acid through NMR and Mess analysis. Isolated diffractaic acid significantly reduced the expression of MMP-1 increased by UVA in HDFn and increased the proliferation of HFDPC in a concentration-dependent manner. The result suggest that UDE proved its usability as a natural cosmetic material with anti-aging and dermal papilla cell activation effects.

• Journal of Chest Surgery
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• v.37 no.3
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• pp.210-219
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• 2004

Extracellular $K^{+}$ concentration ([ $K^{+}$]$_{0}$ ) can be increased within several mM by the efflux of intracellular $K^{+}$. To investigate the effect of an increase in [ $K^{+}$]$_{0}$ on vascular contractility, we attempted to examine whether extracellular $K^{+}$ might modulate vascular contractility, endothelium-dependent relaxation (EDR) and intracellular $Ca^2$$^{+}$ concentration ([C $a^2$$^{+}$]$_{i}$ ) in endothelial cells (EC). We observed isometric contractions in rabbit carotid, superior mesenteric, basilar arteries and movse aorta. [C $a^2$$^{+}$]$_{i}$ was recorded by microfluorimeter using Fura-2/AM in EC. No change in contractility was recorded by the increase in [ $K^{+}$]$_{0}$ from 6 to 12 mM in conduit artery such as rabbit carotid artery. whereas resistant vessels, such as basilar and branches of superior mesenteric arteries (SMA), were relaxed by the increase. In basilar artery, the relaxation by the increase in [ $K^{+}$]$_{0}$ to from 1 to 3 mM was bigger than that by the increase from 6 to 12 mM. In contrast, in branches of SMA, the relaxation by the increase in [ $K^{+}$]$_{0}$ to from 6 to 12 mM is bigger than that by the increase from 1 to 3 mM. $Ba^2$$^{+}$ (30 $\mu$M) did not inhibit the relaxation by the increase in [ $K^{+}$]$_{0}$ from 1 to 3 mM but did inhibit the relaxation by the increase from 6 to 12 mM. In the mouse aorta without the endothelium or treated with $N^{G}$_nitro-L-arginine (30 $\mu$M), nitric oxide synthesis blocker, the increase in [ $K^{+}$]$_{0}$ from 6 to 12 mM did not change the magnitude of contraction induced either norepinephrine or prostaglandin $F_2$$_{\alpha}$. The increase in [ $K^{+}$]$_{0}$ up to 12 mM did not induce contraction of mouse aorta but the increase more than 12 mM induced contraction. In the mouse aorta, EDR was completely inhibited on increasing [ $K^{+}$]$_{0}$ from 6 to 12 mM. In cultured mouse aorta EC, [C $a^2$$^{+}$]$_{i}$ , was increased by acetylcholine or ATP application and the increased [C $a^2$$^{+}$]$_{i}$ , was reduced by the increase in [ $K^{+}$]$_{0}$ reversibly and concentration-dependently. In human umbilical vein EC, similar effect of extracellular $K^{+}$ was observed. Ouabain, a N $a^{+}$ - $K^{+}$ pump blocker, and N $i^2$$^{+}$, a N $a^{+}$ - $Ca^2$$^{+}$ exchanger blocker, reversed the inhibitory effect of extracellular $K^{+}$. In resistant arteries, the increase in [ $K^{+}$]$_{0}$ relaxes vascular smooth muscle and the underlying mechanisms differ according to the kinds of the arteries; $Ba^2$$^{+}$-insensitive mechanism in basilar artery and $Ba^2$$^{+}$ -sensitive one in branches of SMA. It also inhibits [C $a^2$$^{+}$]$_{i}$ , increase in EC and thereby EDR. The initial mechanism of the inhibition may be due to the activation of N $a^{+}$ - $K^{+}$pump. activation of N $a^{+}$ - $K^{+}$pump.p.p.p.

• Tuberculosis and Respiratory Diseases
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• v.48 no.6
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• pp.922-931
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• 2000

Background : It has been well known that bronchia1 asthma is a chronic airway inflammatory disorder. Recently, sputum induced with hypertonic saline was introduced as a simple and useful nonivasive medium to investigate airway inflammation and symptom severity in patients with asthma. We examined the eosinophil, eosinophil cationic protein (ECP), interleukin(IL)-3, IL-5, granulocyte-macrophage colony-stimulating facta (GM-CSF), and nitric oxide (NO) derivatives in induced sputum from patients with bronchia1 asthma in order to determine the role of NO and various inflammatory cytokines as a useful markers of airway inflammation or changes in pulmonary function tests and symptoms. Methods : A total 30 patients with bronchia1 asthma received oral prednisolone 30 mg daily for 2 weeks. Forced expiratory volume in one second ($FEV_1$), total blood eosinophil count and induced sputum eosinophil count, ECP, IL-3, IL-5, GM-CSF, and NO derivatives were determined before and after the administration of prednisolone. Results : Of the 30 patients, 13 (43.3%) were male and 17 (56.7%) were female. The mean age of patients was 41.8 years (range 19-64 years). Two patients could not produce sputum at the second study and 3 could not be followed up after their first visit. Two weeks after the prednisolone administration, there was a significant increase in $FEV_1$ (% of predicted value) from 78.1$\pm$20.6 % to 90.3$\pm$ 18.3 % (P<0.001). The eosinophil percentages in induced sputum were significantly decreased after treatment with prednisolone, with values of 56.1$\pm$27.2 % versus 29.6$\pm$21.3 % (P<0.001), and ECP were $134.5\pm68.1\;{\mu}g/L$ versus $41.5\pm42.4\;{\mu}g/L$ (P<0.001) respectively. After the prednisolone treatments, the eotaxin concentration also showed a decreasing tendency from 26.7$\pm$12.8 pg/ml to 21.7$\pm$8.7 pg/ml. There was a decreasing tendency but no significant differences in total blood eosinophil count (425.7$\pm$265.9 vs 287.7$\pm$294.7) and in the concentration of NO derivatives ($70.4\pm44.6{\mu}mol/L$ vs $91.5\pm48.3\;{\mu}mol/L$) after the prednisolone treatments. IL-3, IL-5, GM-CSF were undetectable in the sputum of most subjects either before the prednisolone treatments or after the treatments. Before the prednisolone treatments, a significant inverse correlation was observed between FEV1 and sputum ECP (r=-D.364, P<0.05) and there was a significant correlation between sputum eosinophils and eotaxin (r=0.369, P<0.05) Conclusion : The eotaxin and ECP concentration in induced sputum may be used as markers of airway inflammation after treatments in bronchia1 asthma. In addition, the measurement of sputum eosinophil percent ages is believed to be a simple method displaying the degree of airway inflammation and airway obstruction before and after the prednisolone treatment in bronchia1 asthma. However, unlike exhaled NO, the examination of NO derivatives with Griess reaction in induced sputum is considered an ineffective marker of changing airway inflammation and obstructing symptoms.

• Journal of the Korean association of regional geographers
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• v.3 no.2
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• pp.163-193
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• 1997

Medical services are a fundamental and essential service in all urban areas. The location and accessibility of medical service facilities and institutions are critical to the diagnosis, control and prevention of illness and disease. The purpose of this paper is to present the results of a study on the location of medical facilities in Kwangju and the utilization of these facilities by the inhabitants. The following information is a summary of the findings: (1) Korea, like many countries, is now witnessing an increase in the age of its population as a result of higher living standards and better medical services. Korea is also experiencing a rapid increase in health care costs. To ensure easy access to medical consultation, diagnosis and treatment by individuals, the hierarchical efficient location of medical facilities, low medical costs, equalized medical services, preventive medical care is important. (2) In Korea, the quality of medical services has improved significantly as evident by the increased number of medical facilities and medical personnel. However, there is still a need for not only quantitative improvements but also for a more equitable distribution of and location of medical services. (3) There are 503 medical facilities in Kwangju each with a need to service 2,556 people. This is below the national average of 1,498 inhabitants per facility. The higher locational quotient and satisfactory population per medical facility showed at the civic center. On the other hand, problem regions such as the traditional residential area in Buk-Gu, Moo-deung mountain area and the outer areas of west Kwangju still maintain rural characteristics. (4) In the study area there are 86 general medicine clinics which provide basic medical services. i. e. one clinic per every 14,949 residents. As a basic service, its higher locational quotient showed in the residential area. The lower population concentration per clinic was found in the civic center and in the former town center, Songjeong-dong. In recently build residential areas and in the civic center, the lack of general medicine clinics is not a serious medical services issue because of the surplus of medical specialists in Korea. People are inclined to seek a consultation with a specialist in specific fields rather than consult a general practitioner. As a result of this phenomenon, there are 81 internal medicine facilities. Of these, 32.1% provide services to people who are not referred by a primary care physician but who self-diagnose then choose a medical facility specializing in what they believe to be their health problem. Areas in the city, called dongs, without any internal facilities make up 50% of the total 101 dongs. (5) There are 78 surgical facilities within the area, and there is little difference at the locational appearance from internal medicine facilities. There are also 71 pediatric health clinics for people under 15 years of age in this area, represents one clinic per 5,063 people. On the quantitative aspect, this is a positive situation. Accessibility is the most important facility choice factor, so it should be evenly located in proportion to demander distribution. However, 61% of 102 dongs have no pediatric clinics because of the uneven location. (6) There are 43 obstetrical and gynecological clinics in Kwangju, and the number of residents being served per clinic is 15,063. These services need to be given regularly so it should increase the numbers. There are 37 ENT clinics in the study area with the lower concentration in Dong-gu (32.4%) making no locational differences by dong. There are 23 dermatology clinics with the largest concentration in Dong-Gu. There are 17 ophthalmic clinics concentrated in the residential area because of the primary function of this type of specialization. (7) The use of general medicine clinics, internal medicine clinics, pediatric clinics, ENT clinics by the inhabitants indicate a trend toward primary or routine medical services. Obstetrics and gynecology clinics are used on a regular basis. In choosing a general medicine clinic, internal medicine clinic, pediatric clinic, and a ENT clinic, accessibility is the key factor while choice of a general hospital, surgery clinic, or an obstetrics and gynecology clinic, thes faith and trust in the medical practitioner is the priority consideration. (8) I considered the efficient use of medical facilities in the aspect of locational and management and suggest the following: First, primary care facilities should be evenly distributed in every area. In Kwangju, the number of medical facilities is the lowest among the six largest cities in Korea. Moreover, they are concentrated in Dong-gu and in newly developed areas. The desired number of medical facilities should be within 30 minutes of each person's home. For regional development there is a need to develop a plan to balance, for example, taxes and funds supporting personnel, equipment and facilities. Secondly, medical services should be co-ordinated to ensure consistent, appropriate, quality services. Primary medical facilities should take charge of out-patient activities, and every effort should be made to standardize and equalize equipment and facility resources and to ensure ongoing development and training in the primary services field. A few specialty medical facilities and general hospitals should establish a priority service for incurable and terminally ill patients. (9) The management scheme for the inhabitants' efficient use of medical service is as follows: The first task is to efficiently manage medical facilities and related services. Higher quality of medical services can be accomplished within the rapidly changing medical environment. A network of social, administrative and medical organizations within an area should be established to promote information gathering and sharing strategies to better assist the community. Statistics and trends on the rate or occurrence of diseases, births, deaths, medical and environment conditions of the poor or estranged people should be maintained and monitored. The second task is to increase resources in the area of disease prevention and health promotion. Currently the focus is on the treatment and care of individuals with illness or disease. A strong emphasis should also be placed on promoting prevention of illness and injury within the community through not only public health offices but also via medical service facilities. Home medical care should be established and medical testing centers should be located as an ordinary service level. Also, reduced medical costs for the physically handicapped, cardiac patients, and mentally ill or handicapped patients should be considered.

• Journal of the Korean Society of Marine Environment & Safety
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• v.19 no.4
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• pp.334-344
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• 2013

In order to understand the characteristics of oceanic environment in the coastal aquaculture waters of the East Sea, the observation of the CTD (temperature and salinity), dissolved oxygen, chlorophyll a and N/P (DIN ($NO_2$-N, $NO_3$-N, $NH_4$-N) : DIP($PO_4$-P)) ratio was carried out at Sokcho, Jukbyon and Gampo in February, April, June, August, October, December 2013. Based on T(temperature)-S(salinity) diagram analysis, the water masses in the study area were divided into 3 groups; Tsushima Surface Water (TSW: $20-28.3^{\circ}C$ temperatures and 31.04-33.75 salinities), Tsushima Middle Water (TMW: $8.1-16.3^{\circ}C$ and 33.00-34.49), and North Korean Cold Water (NKCW: $1.8-9.4^{\circ}C$ and 33.78-34.42). In winter, DO concentrations in the northern part were higher than those in southern part. In spring and fall, they were low in the surface layer, and increased in summer. Chl-a concentrations < $0.4{\mu}g/L$ dominated in February, April, October and December. Chl-a concentrations were higher in June and August. In particular, the highest Chl-a concentration > $2{\mu}g/L$ was observed in the middle layer of Gampo in August. In February, April, June and December, the N/P ratio in the most of the water masses was less than the Redfield ratio (16), indicating that nitrate did act as a limiting factor in phytoplankton growth. On the contrary, in August and October, the N/P ratio in surface and sub-surface layer was greater than the Redfield ratio, suggesting that phosphate was a limiting factor.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.28 no.3
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• pp.202-212
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• 2006

It is well-known that paclitaxel($Taxol^{(R)}$), which is extracted from the pacific and English yew, has been used as a chemotherapeutic agent for ovarian carcinoma and advanced breast carcinoma and Cyclosporin A, which is highly lipophilic cyclic peptide and isolated from a fungus, has been also used as an useful immunosuppressive drug after transplantation and is associated with cellular apoptosis. Since 1953, in which James Watson, Rosalind Franklin and Francis Crick discovered the double helical structure of DNA, a few kinds of techniques for identifying gene expression have been developed. In postgenomic period, many of researchers have used the DNA microarray which is high throughput screening technique to screen large numbers of gene expression simultaneously. In this study, we searched and screened the gene expression in the oral squamous cell carcinoma cell lines treated with $Taxol^{(R)}$, cyclosporin or cyclosporin combined with $Taxol^{(R)}$ using cDNA microarray. The results were as following; 1. It was useful that the appropriate concentration of Cyclosporin A and $Taxol^{(R)}$ used in oral squamous cell carcinoma cell line was under 1${\mu}g/ml$ and 3${\mu}g/ml$. 2. In the experimental group in which $Taxol^{(R)}$ and $Taxol^{(R)}$ + Cyclosporin A were used, the cell growth was extremely decreased. 3. In the group in which Cyclosporin A was used, the MTT assay was rarely decreased which means the activity of succinyl dehydrogenase is remained in mitochondria but in the group in which the mixture of Cyclosporin A and $Taxol^{(R)}$ were used, the MTT assay was extremely decreased. 4. In the each group in which Cyclosporin A(3 ${\mu}g/ml$) and $Taxol^{(R)}$(1 ${\mu}g/ml$) were used, the cell arrest was appeared in $G_2/M$ phase and in the group in which $Taxol^{(R)}$(3 ${\mu}g/ml$) was used, the cell arrest was appeared in both S phase and $G_2/M$ phase. 5. In the oral squamous cell carcinoma cell line treated with $Taxol^{(R)}$, several genes including ANGPTL4, RALBP1 and TXNRD1, associated with apoptosis, SUI1, MAC30, RRAGA and CTGF, related with cell growth, HUS1 and DUSP5, related with cell cycle and proliferation, ATF4 and CEBPG, associated with transcription factor, BTG1 and VEGF, associated with angiogenesis, FDPS, FCER1G, GPA33 and EPHA4 associated with signal transduction and receptor activity and AKR1C2 and UGTA10 related with carcinogenesis were detected in increased levels. The genes that showed increaced expression in the oral squamous cell carcinoma cell line treated with Cyclosporin A were CYR61, SERPINB2, SSR3 and UPA3A which are known as genes associated with cell growth, carcinogenesis, receptor activity and transcription factor. The genes expressed in the HN22 cell line treated with cyclosporin combined with $taxol^{(R)}$ were ALCAM and GTSE1 associated with cancer invasiveness and cell cycle regulation.

• The Journal of Korean Obstetrics and Gynecology
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• v.30 no.2
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• pp.49-70
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• 2017

Objectives: The present study is to observe the skin-regeneration, anti-wrinkle, whitening and skin moisturizing effects of Cheongsangbangpung-tang (CSBPT) with cytotoxicity. Methods: In the present study, cytotoxicity of CSBPT lyophilized aqueous extracts (yield=18.71%) was experimented against human normal fibroblast cells and B16F10 murine melanoma cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT) assay, and skin regeneration and anti-wrinkle effects were also showed through the assay of collagen type I synthesis by an enzyme immunoassay (EIA) kit as comparing with transforming growth factor (TGF)-${\beta}1$, hyaluronidase, collagenase and matrix metalloproteinase (MMP)-1 inhibitory assays as comparing with oleanolic acid (OA), and elastase inhibitory effects as comparing with phosphoramidon disodium salt (PP). In addition, whitening effects of CSBPT were observed by tyrosinase inhibitory assay and melanin formation test in B16/F10 melanoma cells as comparing with arbutin, and skin moisturizing effects were measured through mouse skin water contents test, respectively. Results: No CSBPT treatment related cytotoxic effects were demonstrated against human normal fibroblast cells and B16/F10 murine melanoma cells. CSBPT concentration-dependent increased collagen type I synthesis at human normal fibroblast cells. It also effectively suspreessed hyaluronidase, collagenase, elastase and MMP-1 activities, which were enzymes that related to declining of ECM and formation of wrinkle. CSBPT supressed B16/F10 melanoma cells's melanin productions with tyrosinase activity, which was an enzyme connected with melanin formation, and dose-dependent and significant increases of skin water contents were detected in CSBPT treated mouse skin as compared with vehicle control skins. Conclusions: CSBPT showed favorable and enough skin regeneration, anti-wrinkle, whitening and skin moisturizing effects at least in a condition of this experiment. However, more detail mechanism and in vivo skin protective efficacy studies should be conducted in future with the screening of the biological active compounds in individual herbs of Cheongsangbangpung-tang.