• The Korean Journal of Physiology and Pharmacology
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• 제21권1호
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• pp.19-26
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• 2017

We investigated whether betulin affects the gene expression, secretion and proteolytic activity of matrix metalloproteinase-3 (MMP-3) in primary cultured rabbit articular chondrocytes, as well as in vivo production of MMP-3 in the rat knee joint to evaluate the potential chondroprotective effect of betulin. Rabbit articular chondrocytes were cultured 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. Effect of betulin on IL-$1{\beta}$-induced secretion and proteolytic activity of MMP-3 was investigated using western blot analysis and casein zymography, respectively. Effect of betulin on MMP-3 protein production was also examined in vivo. The results were as follows: (1) betulin 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) betulin inhibited the secretion and proteolytic activity of MMP-3; (3) betulin suppressed the production of MMP-3 protein in vivo. These results suggest that betulin can regulate the gene expression, secretion, and proteolytic activity of MMP-3, by directly acting on articular chondrocytes.

• The Korean Journal of Physiology and Pharmacology
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• 제22권2호
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• pp.173-182
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• 2018

Recent studies have provided several lines of evidence that peripheral administration of oxytocin induces analgesia in human and rodents. However, the exact underlying mechanism of analgesia still remains elusive. In the present study, we aimed to identify which receptor could mediate the analgesic effect of intraperitoneal injection of oxytocin and its cellular mechanisms in thermal pain behavior. We found that oxytocin-induced analgesia could be reversed by $d(CH_2)_5[Tyr(Me)^2,Dab^5]$ AVP, a vasopressin-1a (V1a) receptor antagonist, but not by $desGly-NH_2-d(CH_2)_5[D-Tyr^2,Thr^4]OVT$, an oxytocin receptor antagonist. Single cell RT-PCR analysis revealed that V1a receptor, compared to oxytocin, vasopressin-1b and vasopressin-2 receptors, was more profoundly expressed in dorsal root ganglion (DRG) neurons and the expression of V1a receptor was predominant in transient receptor potential vanilloid 1 (TRPV1)-expressing DRG neurons. Fura-2 based calcium imaging experiments showed that capsaicin-induced calcium transient was significantly inhibited by oxytocin and that such inhibition was reversed by V1a receptor antagonist. Additionally, whole cell patch clamp recording demonstrated that oxytocin significantly increased potassium conductance via V1a receptor in DRG neurons. Taken together, our findings suggest that analgesic effects produced by peripheral administration of oxytocin were attributable to the activation of V1a receptor, resulting in reduction of TRPV1 activity and enhancement of potassium conductance in DRG neurons.

• 대한약침학회지
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• 제18권4호
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• pp.20-25
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• 2015

Objectives: Recently, thread-embedding therapy (TET) has been widely applied in Korean medicine for cosmetic purposes such as reducing skin wrinkles. An inserted thread was reported to have induced continuous stimulation, followed by support for connective tissue regeneration. However, the potential role of TET in hair-growth has not yet been reported. Methods: We designed this study to evaluate whether TET has a hair-growth-promoting effect. C57 black 6 (C57BL/6) mice were divided into three groups: normal saline-treated, minoxidil-treated, and thread-embedded groups. Normal saline or 5% minoxidil was topically sprayed on the dorsal skin of the mice once a day for 16 days. Medical threads were embedded into the dorsal skin of the mice in a single application. Hair growth activity was evaluated by using dermoscopic and microscopic observations. Sections of the dorsal skin were stained with hematoxylin and eosin. Expressions of bromodeoxyuridine (BrdU), proliferating cell nuclear antigen (PCNA), fibroblast growth factor-7 (FGF-7), and fibroblast growth factor-5 (FGF-5) were detected by using immunohistochemical staining. A reverse transcription-polymerase chain reaction (RT-PCR) analysis was adopted to measure the messenger RNA (mRNA) expressions of FGF-7 and FGF-5. Results: TET enhanced anagen development in the hair follicles of C57BL/6 mice. The expressions of BrdU and PCNA, both of which imply active cellular proliferation, were increased by using TET. Moreover, TET increased the expression of FGF-7, an anagen-inducing growth factor, while decreasing the expression of FGF-5, an anagen-cessation growth factor, both at the protein and the mRNA levels. Conclusion: TET enhanced hair re-growth in C57BL/6 mice. TET regulated the expressions of anagen-associated growth factors and activated the proliferation of hair follicular cells in depilated skin lesions. Considering its long-lasting effect, TET may be a good alternative therapeutic for the treatment of alopecia.

• 대한약침학회지
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• 제15권3호
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• pp.13-18
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• 2012

Objectives: The purpose of this study is to examine whether Hominis Placental pharmacopuncture solution (HPPS) combined with zinc-oxide nanoparticles (ZnO NP) activates RAW 264.7 cells. Methods: We soaked ZnO nanoparticles in the Hominis Placenta pharmacopuncture solution, thereby making a combined form (ZnO NP HPPS). The effect of ZnO NP HPPS on the intracellular reactive oxygen species (ROS) production was measured by 2', 7'-dichlorofluorescin diacetate (DCFH-DA) assay. The effect of ZnO NP HPPS on NF-${\kappa}B$ was measured by using a luciferase assay. The effect of ZnO NP HPPS on the cytokine expression was assessed by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR). The cellular uptake of ZnO NP HPPS was measured by using a flow cytometric analysis, and cellular structural alterations were analyzed by using transmission electron microscopy (TEM). Results: Neither the HPPS nor the ZnO NPs induced intracellular ROS production in RAW 264.7 cells. Neither of the materials activated NF-${\kappa}B$ or it's dependent genes, such as TNF-${\alpha}$, IL-1, and MCP-1. However, ZnO NP HPPS, the combined form of ZnO NPs and HPPS, did induce the intracellular ROS production, as well as prominently activating NF-${\kappa}B$ and it's dependent genes. Also, compared to ZnO NPs, it effectively increa-sed the uptake by RAW 264.7 cells. In addition, cellular structural alterations were observed in groups treated with ZnO NP HPPS. Conclusions: Neither ZnO NP nor HPPS activated RAW 264.7 cells, which is likely due to a low cellular uptake. The ZnO NP HPPS, however, significantly activated NF-${\kappa}B$ and up-regulated its dependent genes such as TNF-${\alpha}$, IL-1, and MCP-1. ZnO NP HPPS was also more easily taken into the RAW 264.7 cells than either ZnO NP or HPPS.

• Restorative Dentistry and Endodontics
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• 제32권5호
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• pp.459-468
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• 2007

이 연구에서는 법랑모세포 분화과정에서 APin의 기능을 알아보고자 APin-protein microarray를 시행한 후 치아발생과 관련이 있는 MEF2, Aurora kinase A, BMPR-IB와 EF-hand calcium binding protein을 분석하여 다음과 같은 결과를 얻었다. 1 CMV-APin construct를 transfection하여 APin의 과발현을 유도한 경우에는 MEF2와 Aurora kinase A 둘 모두에서 발현이 현저히 감소한 반면에, APin의 발현억제를 유도한 경우에는 둘 모두 변화가 없었다. 2. APin의 과발현을 유도한 경우에는 BMPR-IB와 EF-hand calcium binding protein 모두에서 발현이 크게 증가한 반면, APin을 발현억제 시킨 경우에는 BMPR-IB는 변화가 없었고, EF-hand calcium binding protein은 현저히 감소하였다. 위의 결과들로 보아 APin 단백질은 MEF2, Aurora kinase A, BMPR-IB, EF-hand calcium binding protein과 상호작용하여 법랑모세포의 분화와 석회화 과정 중에 중요한 역할을 하는 것으로 사료된다.

• Environmental Analysis Health and Toxicology
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• 제19권4호
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• pp.359-366
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• 2004

Iron (Fe) is an essential metal in biological processes, which maintains a homeostasis in the human body. Divalent metal transporter 1 (DMT1) has been known as an iron transporting membrane protein, which is involved in the uptake Fe at the apical portion of intestinal epithelium, and may transport Fe across the membrane of acidified endosome in peripheral tissues. In this study, we studied the tissue distribution of DMT1 in the Fe supplemented (FeS) diet fed rats, and the regulation of DMT1 expression by depleting body Fe. Sprague-Dawley rats were divided into two groups, and fed FeS (120 mg Fe/kg) diet or Fe deficient (FeD, 2∼6 mg Fe/kg) diet for 4 weeks. The evaluation of body Fe status was monitored by measuring sFe, UIBC and tissue Fe concentration. Additionally, DMT1 mRNA levels were analyzed in the peripheral tissues by using the quantitative real time RT-PCR method. In the FeS diet fed rats, the tissue Fe was maintained at a relatively high level, and DMT1 was eventually expressed in all tissues studied. DMT1 was highly expressed in the testis, kidney and spleen, while a moderate levels of DMT1 expression was detected in the brain, liver and heart. In the digestive system, the highest level of DMT1 was found in the duodenum. Feeding the FeD diet caused a reduced body weight gain and depletion of body Fe with finding of decreased sFe, increased UIBC and decreased tissue Fe concentration. The depletion of body Fe upregulated DMT1 expression in the peripheral tissue. The expression of DMT1 was very sensitive to the body Fe depletion in the small intestine, especially in the duodenum, showing dramatically higher levels in the FeD rats than those of the FeS group. In the FeD diet fed animals, the expression of DMT1 was low significantly in other tissues compared with the duodenum. The expression of DMT1, however, was 60∼120% higher in the testis, kidney and spleen, and 30∼50% higher in the lung, liver and heart, compared to the FeS diet fed rats. In summary, DMT1 expression was ubiquitous in mammalian tissue, and the level of expression was the organ-dependent. The expression of DMT1 in peripheral tissues was upregulated by depletion of body Fe. Duodenum was the most sensitive tissue among organs studied during Fe depletion, and expressed the greatest level of DMT1, while other tissues were less higher than in duodenum. This study supports that DMT1 plays a role in maintaining the body Fe level through intestinal uptake as well as homeostasis of Fe in the peripheral tissue.

• Asian Pacific Journal of Cancer Prevention
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• 제15권10호
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• pp.4331-4338
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• 2014

$N^1$-(2, 5-dimethoxyphenyl)-$N^8$-hydroxyoctanediamide (N25) is a novel SAHA cap derivative of HDACi, with a patent (No. CN 103159646). This invention is a hydroxamic acid compound with a structural formula of $RNHCO(CH_2)6CONHOH$ (wherein R=2, 5dimethoxyaniline), a pharmaceutically acceptable salt which is soluble. In the present study, we investigated the effects of N25 with regard to drug distribution and molecular docking, and anti-proliferation, apoptosis, cell cycling, and $LD_{50}$. First, we designed a molecular approach for modeling selected SAHA derivatives based on available structural information regarding human HDAC8 in complex with SAHA (PDB code 1T69). N25 was found to be stabilized by direct interaction with the HDAC8. Anti-proliferative activity was observed in human glioma U251, U87, T98G cells and human lung cancer H460, A549, H1299 cells at moderate concentrations ($0.5-30{\mu}M$). Compared with SAHA, N25 displayed an increased antitumor activity in U251 and H460 cells. We further analyzed cell death mechanisms activated by N25 in U251 and H460 cells. N25 significantly increased acetylation of Histone 3 and inhibited HDAC4. On RT-PCR analysis, N25 increased the mRNA levels of p21, however, decreased the levels of p53. These resulted in promotion of apoptosis, inducing G0/G1 arrest in U251 cells and G2/M arrest in H460 cells in a time-dependent and dose-dependent manner. In addition, N25 was able to distribute to brain tissue through the blood-brain barrier of mice ($LD_{50}$: 240.840mg/kg). In conclusion, our findings demonstrate that N25 will provide an invaluable tool to investigate the molecular mechanism with potential chemotherapeutic value in several malignancies, especially human glioma.

• Asian Pacific Journal of Cancer Prevention
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• 제14권3호
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• pp.1715-1720
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• 2013

Background: The discovery that microRNAs (miRNAs) regulate proliferation, invasion and metastasis provides a principal molecular basis of tumor heterogeneity. Microvessel distribution is an important characteristic of solid tumors, with significant hypoxia occurring in the center of tumors with low blood flow. The distribution of miR-374a in breast tumors was examined as a factor likely to be important in breast cancer progression. Methods: Breast tissue samples from 40 patients with breast cancer were classified into two groups: a highly invasive and metastatic group (HIMG) and a low-invasive and metastatic Group (LIMG). Samples were collected from the center and edge of each tumor. In each group, six specimens were examined by microRNA array, and the remaining 14 specimens were used for real-time RT-qPCR, Western blot and immunohistochemical analyses. Correlation analysis was performed for the miRNAs and target proteins. Follow-up was carried out during 28 months to 68 months after surgery, and survival data were analyzed. Results: In the LIMG, the relative content of miR-374a was lower in the center of the tumor than at its edge; in the HIMG, it was lower at the edge of the tumor, and miR-374a levels were lower in breast cancer tissues than in normal tissues. There was no difference between VEGF-A and VCAM-1 mRNA levels at the edge and center of the tumor; however, we observed a significant difference between VEGF-A and VCAM-1 protein expression levels in these two regions. There was a negative correlation between miR-374a and target protein levels. The microvessel density (MVD) was lower in the center of the tumor than at its edge in HIMG, but the LIMG vessels were uniformly distributed. There was a significant positive correlation between MVD and the number of lymph node metastases (Pearson correlation, r=0.912, P<0.01). The median follow-up time was 48.5 months. LIMG had higher rate of disease-free survival (100%, P=0.013) and longer median survival time (66 months) than HIMG, which had a lower rate of 75% and shorter median survival time (54 months). Conclusions: Our data demonstrated miR-374a to be differentially distributed in breast cancer; VEGF-A and VCAM-1 mRNA had coincident distribution, and the distribution of teh respective proteins was uneven and opposite to that for the miR-374a. These data might explain the differences in the distribution of MVD in breast cancer and variation in breast cancer prognosis.

• Clinical and Experimental Reproductive Medicine
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• 제38권4호
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• pp.193-202
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• 2011

Objective: We found previously that $interferon$ $regulatory$ factor ($Irf$)-1 is a germinal vesicle (GV)-selective gene that highly expressed in GV as compared to metaphase II oocytes. To our knowledge, the function of $Irf-1$ in oocytes has yet to be examined. The present study was conducted to determine the relationship between retinoic acid (RA) and RA-mediated expression of $Irf-1$ and the mouse oocyte maturation. Methods: Immature cumulus-oocyte-complexes (COCs) were collected from 17-day-old female mice and cultured $in$ $vitro$ for 16 hours in the presence of varying concentrations of RA (0-10 ${\mu}M$). Rate of oocyte maturation and activation was measured. Gene expression was measured by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) and cytokine secretion in the medium was measured by Bio-Plex analysis. Apoptosis was analyzed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Results: The rates of oocyte maturation to metaphase II and oocyte activation increased significantly with RA treatment (10 nM-1 ${\mu}M$). With 100 nM RA treatment, lowest level of $Irf-1$ mRNA and cumulus cell's apoptosis was found. Among 23 cytokines measured by Bio-Plex system, the substantial changes in secretion of tumor necrosis factor-${\alpha}$, macrophage inflammatory protein-$1{\beta}$, eotaxin and interleukin-12 (p40) from COCs in response to RA were detected. Conclusion: We concluded that the maturation of oocytes and $Irf-1$ expression are negatively correlated, and RA enhances the developmental competence of mouse immature oocytes $in$ $vitro$ by suppressing apoptosis of cumulus cells. Using a mouse model, results of the present study provide insights into improved culture conditions for $in$ $vitro$ oocyte maturation and relevant cytokine production and secretion in assisted reproductive technology.

• The Journal of Advanced Prosthodontics
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• 제5권4호
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• pp.402-408
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• 2013

PURPOSE. The aim of this study was to evaluate the surface properties and in vitro bioactivity to osteoblasts of magnesium and magnesium-hydroxyapatite coated titanium. MATERIALS AND METHODS. Themagnesium (Mg) and magnesium-hydroxyapatite (Mg-HA) coatings on titanium (Ti) substrates were prepared by radio frequency (RF) and direct current (DC) magnetron sputtering.The samples were divided into non-coated smooth Ti (Ti-S group), Mg coatinggroup (Ti-Mg group), and Mg-HA coating group (Ti-MgHA group).The surface properties were evaluated using scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). The surface roughness was evaluated by atomic force microscopy (AFM). Cell adhesion, cell proliferation and alkaline phosphatase (ALP) activity were evaluated using MC3T3-E1 cells. Reverse transcription polymerase chain reaction (RT-PCR) analysis was performed. RESULTS. Cross-sectional SEM images showed that Mg and Mg-HA depositionson titanium substrates were performed successfully. The surface roughness appeared to be similaramong the three groups. Ti-MgHA and Ti-Mg group had improved cellular responses with regard to the proliferation, alkaline phosphatase (ALP) activity, and bone-associated markers, such as bone sialoprotein (BSP) and osteocalcin (OCN) mRNA compared to those of Ti-S group. However, the differences between Ti-Mg group and Ti-MgHA group were not significant, in spite of the tendency of higher proliferation, ALP activity and BSP expression in Ti-MgHA group. CONCLUSION. Mg and Mg-HAcoatings could stimulate the differentiation into osteoblastic MC3T3-E1 cells, potentially contributing to rapid osseointegration.