• The Korean Journal of Physiology and Pharmacology
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• v.21 no.2
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• pp.197-204
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• 2017

In the present study, we tried to examine whether oleanolic acid regulates the activity, secretion and gene expression of matrix metalloproteinase-3 (MMP-3) in primary cultured rabbit articular chondrocytes, as well as the production of MMP-3 in the knee joint of rat to evaluate the potential chondroprotective effect of oleanolic acid. Rabbit articular chondrocytes were cultured in a monolayer, and reverse transcription-polymerase chain reaction (RT-PCR) was used to measure interleukin-$1{\beta}$ (IL-$1{\beta}$)-induced gene expression of MMP-3, MMP-1, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4), ADAMTS-5 and type II collagen. In rabbit articular chondrocytes, the effects of oleanolic acid on IL-$1{\beta}$-induced secretion and proteolytic activity of MMP-3 were investigated using western blot analysis and casein zymography, respectively. The effect of oleanolic acid on in vivo MMP-3 protein production was also examined, after intra-articular injection to the knee joint of rat. The results were as follows: (1) oleanolic acid inhibited the gene expression of MMP-3, MMP-1, MMP-13, ADAMTS-4, and ADAMTS-5, but increased the gene expression of type II collagen; (2) oleanolic acid reduced the secretion and proteolytic activity of MMP-3; (3) oleanolic acid suppressed the production of MMP-3 protein in vivo. These results suggest that oleanolic acid can regulate the activity, secretion and gene expression of MMP-3, by directly acting on articular chondrocytes.

• Journal of the Korean Society of Food Science and Nutrition
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• v.33 no.9
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• pp.1486-1491
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• 2004

The estrogenicities of six heavy metal compounds, which contaminate frequently in foods, were assayed using a combination of in vitro and in vivo assays. The assays were 1) estrogen receptor dependent transcriptional expression assay, 2) E-screen assay and, 3) the uterotropic assay in mice. The chemicals studied were 17$\beta$ -estradiol, diethylstilbestrol (DES), arsenic oxide, bis(tri-n-butyltin), cadmium chloride, chromium chloride, lead acetate, and mercuric chloride. Using the estrogen receptor dependent transcriptional expression assay, the following estrogenicity ranking was measured: bis(tri-n-butyltin) > cadmium chloride > chromium chloride >> mercuric chloride >lead acetate = arsenic oxide. Using E-screen test, the following estrogenicity ranking was measured: bis(tri-n-butyltin) > cadmium chloride > chromium chloride >> mercuric chloride > lead acetate = arsenic oxide. Results from the uterotropic assay showed that bis(tri-n-butyltin), cadmium chloride, chromium chloride caused an increase in uterine wet weight, while lead acetate, mercuric chloride, and arsenic oxide failed to do so. Bis(tri-n-butyltin), cadmium chloride and chromium chloride showed the highest estrogenicity in three assay systems. Recent studies suggesting that bis(tri-n-butyltin), cadmium chloride have estrogenicities are compatible with the present finding. Furthermore, our study is suggesting that chromium chloride may be estrogenic. The results demonstrate that this three level-assay combination (transcriptional activation, cell proliferation, and an in vivo effect in an estrogen-responsive tissue) could serve as a useful method to assess the estrogenicity of heavy metals.

• The Journal of Pediatrics of Korean Medicine
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• v.21 no.1
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• pp.117-137
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• 2007

Objectives : In the present study, the author intended to investigate Gagam-jeonggitang(GJT), Gami-hwajeongjeon(GHJ) and Gami-tonggyutang(GTT) significantly affect in vivo and in vitro mucin secretion from airway epithelial cells. Methods : In vivo experiment, the author induced hypersecretion of airway mucin, hyperplasia of tracheal goblet cells and the increase in intraepithelial mucosubstances by exposing rats to SO2 during 3 weeks. Effects of orally-administered GJT, GHJ and GTT during 1 week on in vivo mucin secretion and hyperplasia of tracheal goblet cells were assesed using ELISA and staining goblet cells with alcian blue. For in vitro experiment, confluent HTSE cells were metabolically radiolabeled with 3H-glucosamine for 24 hrs and chased for 30 min in the presence of each agent to assess the effects of each agent on 3H-mucin secretion. Possible cytotoxicities of each agent were assessed by measuring lactate dehydrogenase release. Also, the effects of each agent on contractility of isolated tracheal smooth muscle and effects of each agent on MUC5AC gene expression in cultured HTSE cells were investigated. Results : GJT, GHJ and GTI inhibited hypersecretion of in vivo mucin: GJT and GHJ inhibited the increase of number of goblet cells. However, GTT did not affect the increase of number of goblet cells; GJT and GTT significantly increased mucin secretion from cultured HTSE cells, without significant cytotoxicity. GHJ increased mucin secretion and showed mild cytotoxicity at the highest concentration: GJT, GHJ and GTT chiefly affected the 'mucin' secretion; GJT, GHJ and GTT did not affect Ach-induced contraction of isolated tracheal smooth muscle; GTT did not significantly affect the expression levels of MUC5AC gene. However, GJT significantly. inhibit the expression levels of MUC5AC gene and GHJ significantly increased the expression levels of MUC5AC gene. These results suggest that GJT, GHJ and GTI can increase mucin secretion during short-term treatment(in vitro), whereas it can inihibit hypersecretion of mucin during long-term treatment(in vivo) and GJT and GHJ can not only affect the secretion of mucin but also affect the expression of mucin gene. Conclusions : The author suggests that the effects GJT, GHJ and GTT with their components should be further investigated and it is valuable to find, from oriental medical prescriptions, novel agents which might regulate hypersecretion of mucin from airway epithelial cells.

• Journal of fish pathology
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• v.16 no.3
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• pp.147-152
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• 2003

The CMV promoter driven luciferase reporter gene coding plasmid (pcDNA-luc) was constructed and used as a model for DNA immunization study. Expression of the recombinant luciferase protein was confirmed in vitro in RTG-2 cell line before using in vivo study in olive flounder. In dose response study, the maximum expression of the luciferase gene was found in the group injected with 10-15μg of plasmid DNA. The kinetic study showed that the luciferase gene expression was reached at the maximum level at one day after injection and slightly decreased after then but significantly high level of expression was sustained until the conducted experiment of 7 days. In the study of tissue distribution of gene expression, it was found that luciferase gene was expressed at the significant level in immune organs such as gill and spleen, located far from the injected site, suggesting the systemic distribution of the intramuscularly injected DNA in olive flounder.

• Molecules and Cells
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• v.39 no.11
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• pp.790-796
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• 2016

Dental pulp is a highly vascularized tissue requiring adequate blood supply for successful regeneration. In this study, we investigated the functional role of stem cells from human exfoliated deciduous teeth (SHEDs) as a perivascular source for in vivo formation of vessel-like structures. Primarily isolated SHEDs showed mesenchymal stem cell (MSC)-like characteristics including the expression of surface antigens and in vitro osteogenic and adipogenic differentiation potentials. Moreover, SHEDs were positive for NG2, ${\alpha}$-smooth muscle actin (SMA), platelet-derived growth factor receptor beta ($PDGFR{\beta}$), and CD146 as pericyte markers. To prove feasibility of SHEDs as perivascular source, SHEDs were transplanted into immunodeficient mouse using Matrigel with or without human umbilical vein endothelial cells (HUVECs). Transplantation of SHEDs alone or HUVECs alone resulted in no formation of vessel-like structures with enough red blood cells. However, when SHEDs and HUVECs were transplanted together, extensive vessel-like structures were formed. The presence of murine erythrocytes within lumens suggested the formation of anastomoses between newly formed vessel-like structures in Matrigel plug and the host circulatory system. To understand underlying mechanisms of in vivo angiogenesis, the expression of angiogenic cytokine and chemokine, their receptors, and MMPs was compared between SHEDs and HUVECs. SHEDs showed higher expression of1VEGF, SDF-$1{\alpha}$, and $PDGFR{\beta}$ than HUVECs. On the contrary, HUVECs showed higher expression of VEGF receptors, CXCR4, and PDGF-BB than SHEDs. This differential expression pattern suggested reciprocal interactions between SHEDs and HUVECs and their involvement during in vivo angiogenesis. In conclusion, SHEDs could be a feasible source of perivascular cells for in vivo angiogenesis.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.3
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• pp.1163-1169
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• 2014

Astragalus, a commonly used traditional Chinese medicine, has exhibited antitumor actions in patients. In this study, in vitro and in vivo antitumor effects of astragalus and synergistic antitumor efficacy in combination with pterostilbene were investigated. Melanoma cells were treated with pterostilbene (Pt), graduated doses of astragalus injection (AI), or these in combination. Cell viability was measured using a MTT assay. Released nucleosomes and caspase activity were measured using enzyme-linked immunosorbent assay. Growth inhibition in vitro and in vivo was also assessed. Analysis of variance and t tests were used for statistical analysis. Significant reduction (p<0.05) in cellular proliferation were observed with AI and AI-Pt in a time- and concentration-dependent manner. Apoptosis and caspase-3/7 activity were significantly increased by AI and AI-Pt treatment (p<0.05). In vivo, AI inhibited melanoma tumor growth, with inhibition rates ranging from 36.5 to 62.3%, by inducing apoptosis via up-regulation Bax expression and the Bax/Bcl-2 ratio and down-regulating Bcl-2 expression. AI significantly inhibits the growth of melanoma in vitro and in vivo by inducing apoptosis. These data suggest that combined treatment of astragalus with pterostilbene enhances antitumor efficacy.

• Nutritional Sciences
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• v.8 no.1
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• pp.23-27
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• 2005

The major garlic component, Allicin [diallylthiosulfinate, or (R, S)-diallyldissulfid-S-oxide] is known for its medicinal effects, such as antihypertensive activity, microbicidal activity, and antitumor activity. Allicin and diallyldisulfide, which is a converted form of allicin, inhibited the cholesterol level in hepatocytes, in vivo and in vitro. The metabolism of allicin reportedly occurs in the microsomes of hepatocytes, predominantly with the contribution of cytochrome P-450. However, little is known about how allicin affects the genes involved in the activity of hepatocytes in vivo. In the present study, we used the short-term intravenous injection of allicin to examine the in vivo genetic profile of hepatocytes. Allicin up-regulate ten genes in the hepatocytes. For example, the interferon regulator 1 (IRF-I), the wingless-related MMTV (mouse mammary tumor virus) integration site 4 (wnt-4), and the fatty acid binding protein 1. However, allicin down-regulated three genes: namely, glutathione S-transferase mu6, a-2-HS glycoprotein, and the corticosteroid binding globulin of hepatocytes. The up-regulated wnt-4, IRF-1, and mannose binding lectin genes can enhance the growth factors, cytokines, transcription activators and repressors that are involved in the immune defense mechanism. These primary data, which were generated with the aid of the Atlas Plastic Mouse 5 K Microarray, help to explain the mechanism which enables allicin to act as a therapeutic agent, to enhance immunity, and to prevent cancer. The data suggest that these benefits of allicin are partly caused by the up-regulated or down-regulated gene profiles of hepatocytes. To evaluate the genetic profile in more detail, we need to use a more extensive mouse genome array.

• The Journal of Pediatrics of Korean Medicine
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• v.31 no.2
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• pp.1-13
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• 2017

Objectives In this study, the author intended to investigate whether Gami-cheongpetang (GCP), Gagam-jeongkitang (GJG), Gami-samooltang (GSM) and Gami-ijoongtang (GIJ) significantly affect in vivo (animal model) and in vitro (cultured cells) mucin secretion and MUC5AC gene expression in airway epithelial cells. Methods For in vivo experiment, the author induced hypersecretion of airway mucin in rats by introducing SO2 for 3 weeks. Enzyme-linked immunosorbent assay (ELISA) was used to assess the effects of orally-administered GCP, GJG, GSM and GIJ in vivo mucin secretion from tracheal goblet cells of rats after 1 week. Also, the effects of the agents on TNF- or EGF-induced MUC5AC gene expression in human airway epithelial cells (NCI-H292) were investigated. Possible cytotoxicities of the agents were assessed by examining the rate of survival and proliferation of NCI-H292 cells. Results (1) GCP and GJG significantly inhibited hypersecretion of in vivo mucin, although GSM and GIJ did not affect hypersecretion of in vivo mucin; (2) GCP and GJG significantly increased in vitro mucin secretion from cultured HTSE cells. However, GSM and GIJ did not affect in vitro mucin secretion from HTSE cells; (3) GCP and GJG significantly inhibited the expression levels of EGF-induced MUC5AC gene in NCI-H292 cells. However, GSM and GIJ increased the expression levels of EGF-induced MUC 5AC gene in NCI-H292 cells; (4) GCP, GJG, GSM and GIJ did not significantly inhibit the survival and proliferation of NCI-H292 cells. Conclusions These results suggest that GCP, GJG, GSM and GIJ can not only affect the secretion of mucin but also affect the expression of mucin gene. The author suggests that the effects of GCP, GJG, GSM and GIJ with their components should be further investigated by using animal experimental models that simulate the diverse pathophysiology of pulmonary diseases.

• Journal of Marine Life Science
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• v.2 no.1
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• pp.7-11
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• 2017

Sulfated polysaccharides are known to be immune-stimulators in mammals and can be used as food additives to enhance immunity. In this study, the immune-stimulating activity of water-soluble anionic macromolecules F₂ fractionation isolated from Codium fragile using ion-exchange chromatography was tested in olive flounder, Paralichythys olivaceus, in vitro and in vivo. The gene expression of interleukin (IL)-1β was adopted to check the immune-affection. As a result, in vitro study revealed that the expression of IL-1β was significantly upregulated in head kidney cells by 1 and 5 ㎍/ml of polysaccharides 4 h and by 5 ㎍/ml of polysaccharides at 24 h. In vivo, IL-1β gene expression in head kidney was significantly upregulated by 20 and 100 ㎍ of the polysaccharides at day 1 post-i.p. injection, while downregulated at day 3 but not significant. Meanwhile, in peritoneal cells, it was upregulated by 20 ㎍ of the polysaccharides at day 1 but the upregulation was sustained until day 3 though it was not significant. These results indicate that the sulfated polysaccharides from C. fragile are an immune-stimulator and might be potential feed additives for olive flounder.

• Nutrition Research and Practice
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• v.2 no.2
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• pp.74-79
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• 2008

Zinc plays a protective role in anti-atherosclerosis but the clear mechanism has not been proposed yet. In the present study, we evaluated whether zinc modulates atherosclerotic markers, VACM-1 and ICAM-1 and cell viability both in endothelial cells in vitro and mouse aortic cell viability ex vivo. In study 1, as in vitro model, endothelial EA.hy926 cells were treated with $TNF{\alpha}$ for 5 hours for inducing oxidative stress, and then treated with Zn-adequacy ($15\;{\mu}M$ Zn) or Zn-deficiency ($0\;{\mu}M$ Zn) for 6 hours. Pro-atherosclerosis factors, VCAM-1 and ICAM-1 mRNA expression and cell viability was measured. In study 2, as ex vivo model, mouse aorta ring was used. Mourse aorta was removed and cut in ring then, cultured in a 96-well plate. Aortic ring was treated with various $TNF{\alpha}$ (0-30 mg/ml) and intracellular zinc chelator, N, N, N', N', -tetrakis (2-pyridylmethyl) ethylenediamine (TPEN, $0-30\;{\mu}M$) for cellular zinc depletion for 2 days and then cell viability was measured. The results showed that in in vitro study, Zn-adequate group induced more VCAM-1 & ICAM-1 mRNA expression than Zn-deficient group during 6-hour zinc treatment post-5 hour TNF-$\alpha$ treatment, unexpectedly. These results might be cautiously interpreted that zinc would biologically induce the early expression of anti-oxidative stress through the increased adhesion molecule expression for reducing atherosclerotic action, particularly under the present 6-hour zinc treatment. In ex vivo, mouse aortic ring cell viability was decreased as TNF-$\alpha$ and TPEN levels increased, which suggests that mouse aortic blood vessel cell viability was decreased, when oxidative stress increases and cellular zinc level decreases. Taken together, it can be suggested that zinc may have a protective role in anti-atherosclerosis by cell viability in endothelial cells and aorta tissue. Further study is needed to clarify how pro-atherosclerosis molecule expression is modulated by zinc.