• Molecules and Cells
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• v.39 no.2
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• pp.103-110
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• 2016

As a degenerative joint disease, osteoarthritis (OA) constitutes a major cause of disability that seriously affects the quality of life of a large population of people worldwide. However, effective treatment that can successfully reverse OA progression is lacking until now. The present study aimed to determine whether two small non-coding RNAs miR-29a and miR-140, which are significantly down-regulated in OA, can be applied together as potential therapeutic targets for OA treatment. MiRNA synergy score was used to screen the miRNA pairs that potentially synergistically regulate OA. An in vitro model of OA was established by treating murine chondrocytes with IL-$1{\beta}$. Transfection of miR-29a and miR-140 via plasmids was investigated on chondrocyte proliferation and expression of nine genes such as ADAMTS4, ADAMTS5, ACAN, COL2A1, COL10A1, MMP1, MMP3, MMP13 and TIMP metallopeptidase inhibitor 1 (TIMP1). Western blotting was used to determine the protein expression level of MMP13 and TIMP1, and ELISA was used to detect the content of type II collagen. Combined use of miR-29a and miR-140 successfully reversed the destructive effect of IL-$1{\beta}$ on chondrocyte proliferation, and notably affected the MMP13 and TIMP1 gene expression that regulates extracellular matrix. Although co-transfection of miR-29a and miR-140 did not show a synergistic effect on MMP13 protein expression and type II collagen release, but both of them can significantly suppress the protein abundance of MMP13 and restore the type II collagen release in IL-$1{\beta}$ treated chondrocytes. Compared with single miRNA transfection, cotransfection of both miRNAs exceedingly abrogated the suppressed the protein production of TIMP1 caused by IL-$1{\beta}$, thereby suggesting potent synergistic action. These results provided1novel insights into the important function of miRNAs' collaboration in OA pathological development. The reduced MMP13, and enhanced TIMP1 protein production and type II collagen release also implies that miR-29a and miR-140 combination treatment may be a possible treatment for OA.

• Journal of Nutrition and Health
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• v.49 no.6
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• pp.429-436
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• 2016

Purpose: Solar ultraviolet (UV) radiation causes inflammation and matrix metalloproteinase (MMP) overexpression and extracellular matrix depletion, leading to skin photoaging such as wrinkle formation, dryness, and sagging. Activation of MMP is influenced by various molecules such as reactive oxygen species (ROS), proinflammatory cytokines, and transient receptor potential vanilloid type (TRPV)-1, which are increased in UV-irradiated skin cells. Aralia elata (AE) ethanolic extract was reported to inhibit ROS generation caused by UVB-irradiation in keratinocytes. In this study, we investigated the photoprotective effect of AE ethanolic extract on UVB-irradiated human keratinocytes (HaCaT) and human dermal fibroblasts (HDF). Methods: AE was freeze-dried, extracted in 70% ethanol, and concentrated. Skin cells were treated with AE extract for 24 h and then exposed to UVB ($55mJ/cm^2$). After 48 h of incubation, proinflammatory cytokines, MMP-1, type-1 procollagen, and TRPV-1 levels were measured by ELISA or Western blotting. Results: Treatment with AE extract ($100{\mu}g/mL$) significantly inhibited UVB-induced IL-6, IL-8, and $PGE_2$ production in HaCaT by 25.6%, 5.3%, and 70.2%, respectively, and also inhibited elevation of MMP-1 and TRPV-1 caused by UVB irradiation by 20.0% and 41.9%, respectively (p < 0.05). In HDF, AE extract treatment significantly inhibited both elevation of MMP-1 and reduction of type-1 procollagen caused by UVB irradiation (p < 0.05). In addition, type-1 procollagen was elevated by AE extract treatment in normal HDFs (p < 0.05). Conclusion: AE 70% ethanol extract has photoprotective ability via reduction of proinflammatory mediators, TRPV-1 and MMP-1 production, and elevation of collagen synthesis. Our findings suggest that AE extract might be a good natural material to protect against UVB-induced premature skin aging.

• Journal of Periodontal and Implant Science
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• v.38 no.sup2
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• pp.317-324
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• 2008

Purpose: Aggregatibacter actinomycetemcomitans was associated with localized aggressive periodontitis, endocarditis, meningitis, and osteomyelitis. The cytolethal distending toxin (CDT) of A. actinomycetemcomitans was considered as a key factor of these diseases is composed of five open reading frames (ORFs). Among of them, An enzymatic subunit of the CDT, CdtB has been known to be internalized into the host cell in order to induce its genotoxic effect. However, CdtB can not be localized in host cytoplasm without the help of a heterodimeric complex consisting of CdtA and CdtC. So, some studies suggested that CdtC functions as a ligand to interact with GM3 ganglioside of host cell surface. The precise role of the CdtC protein in the mechanism of action of the holotoxin is unknown at the present time. The aim of this study was to generate recombinant CdtC proteins expression from A. actinomycetemcomitans, through gene cloning and protein used to investigate the function of Cdt C protein in the bacterial pathogenesis. Materials and Methods: The genomic DNA of A. actinomycetemcomitans Y4 (ATCC29522) was isolated using the genomic DNA extraction kit and used as template to yield cdtC genes by PCR. The amplifed cdtC genes were cloned into T-vector and cloned cdt C gene was then subcloned to pET28a expression vector. The pET28a-cdtC plasmid expressed in BL21 (DE3) Escherichia coli system. Diverse conditons were tested to opitimize the expression and purification of functional CdtC protein in E. coli. Results: In this study we reconstructed CdtC subunit of A. actinomycetemcomitans Y4 and comfirmed the recombinant CdtC expression by SDS-PAGE and Western Blotting. The expression level of the recombinant CdtC was about 2% of total bacterial proteins. Conclusion: The lab condition of procedure for the purification of functionally active recombinant CdtC protein is established. The active recombinant CdtC protein will serve to examine the role of CdtC proteins in the host recognition and enzyme activity of CDT and investigate the pathological process of A. actinomycetemcomitans in periodontal disease.

• Journal of dental hygiene science
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• v.18 no.2
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• pp.76-84
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• 2018

Wet wipes are being increasingly used because of their convenience. Particularly, oral wet wipes are useful for regular cleaning of a baby's mouth after birth. Therefore, the consumption of oral wet wipes has increased over the past few years and a variety of products are commercially available. However, product information on safety is not sufficiently provided and still raises doubts regarding adverse effects. To confirm the safety of wet wipes as an oral hygiene item and provide information for their use, we investigated the cytotoxicity of oral wet wipes and verified the underlying mechanism. The anti-bacterial effect of oral wet wipes was analyzed using the disk diffusion method. The cytotoxic effects of oral wet wipes were observed based on morphological changes using microscopy and determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in gingival epithelial cells and gingival fibroblasts. Evaluation of apoptosis by oral wet wipes was explored using propidium iodide flow cytometric analysis and a terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) assay. Apoptosis-related molecules were also analyzed using western blotting. Five types of oral wet wipes were tested, and two products from Fisher-Price and Dr. Kennedy revealed strong cytotoxic effects on gingiva epithelial cells and gingiva fibroblasts, although they also showed intense anti-bacterial effects on oral bacteria. Cell cycle arrest in the G2/M phase and apoptosis were observed based on treatment of extracts from Fisher-Price and Dr. KENNEDY. Relatively high TUNEL levels, reduction of proliferating cell nuclear antigen and cyclin-dependent kinase 4 expression, and fragmentation of poly (ADP-ribose) polymerase were also elucidated. These results suggest that commercial oral wet wipes could exert cytotoxic influences on oral tissue, although there are anti-bacterial effects, and careful attention is required, especially for infants and toddlers.

• Journal of Ginseng Research
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• v.38 no.4
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• pp.256-263
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• 2014

Background: Korean Red Ginseng (KRG) is known to have antianxiety properties. This study was conducted to investigate the anxiolytic effects of KRG extract (KRGE) during ethanol withdrawal (EW) and the involvement of the mesoamygdaloid dopamine (DA) system in it. Methods: Rats were treated with 3 g/kg/d of ethanol for 28 d, and subjected to 3 d of withdrawal. During EW, KRGE (20 mg/kg/d or 60 mg/kg/d, p.o.) was given to rats once/d for 3 d. Thirty min after the final dose of KRGE, anxiety-like behavior was evaluated in an elevated plus maze (EPM), and plasma corticosterone (CORT) levels were determined by a radioimmunoassay (RIA). In addition, concentrations of DA and 3,4-dihydroxyphenylacetic acid (DOPAC) in the central nucleus of the amygdala (CeA) were also measured by high performance liquid chromatography (HPLC). Results: The EPM test and RIA revealed KRGE inhibited anxiety-like behavior and the over secretion of plasma CORT during EW. Furthermore, the behavioral effect was blocked by a selective DA D2 receptor (D2R) antagonist (eticlopride) but not by a selective DA D1 receptor (D1R) antagonist (SCH23390). HPLC analyses showed KRGE reversed EW-induced decreases of DA and DOPAC in a dose-dependent way. Additionally, Western blotting and real-time polymerase chain reaction (PCR) assays showed that KRGE prevented the EW-induced reductions in tyrosine hydroxylase (TH) protein expression in the CeA and TH mRNA expression in the ventral tegmental area (VTA). Conclusion: These results suggest that KRGE has anxiolytic effects during EW by improving the mesoamygdaloid DA system.

• Journal of Ginseng Research
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• v.40 no.4
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• pp.423-430
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• 2016

Background: The induction of cellular defensive genes such as phase II detoxifying and antioxidant enzymes is a highly effective strategy for protection against carcinogenesis as well as slowing cancer development. Transcription factor Nrf2 (nuclear factor E2-related factor 2) is responsible for activation of phase II enzymes induced by natural chemopreventive compounds. Methods: Red ginseng oil (RGO) was extracted using a supercritical $CO_2$ extraction system and chemical profile of RGO was investigated by GC/MS. Effects of RGO on regulation of the Nrf2/antioxidant response element (ARE) pathway were determined by ARE-luciferase assay, western blotting, and confocal microscopy. Results: The predominant components of RGO were 9,12-octadecadienoic acid (31.48%), bicyclo[10.1.0] tridec-1-ene (22.54%), and 22,23-dihydrostigmasterol (16.90%). RGO treatment significantly increased nuclear translocation of Nrf2 as well as ARE reporter gene activity, leading to upregulation of heme oxygenase-1 and NAD(P)H:quinone oxidoreductase 1. Phosphorylation of the upstream kinases such as apoptosis signal-regulating kinase (ASK)1, mitogen-activated protein kinase (MAPK) kinase (MKK)4/7, c-Jun N-terminal kinase (JNK), and p38 MAPK were enhanced by treatment with RGO. In addition, RGO-mediated Nrf2 expression and nuclear translocation was attenuated by JNK inhibitor SP600125 and p38 MAPK inhibitor SB202190. Conclusion: RGO could be used as a potential chemopreventive agent, possibly by induction of Nrf2/ARE-mediated phase II enzymes via ASK1-MKK4/7-JNK and p38 MAPK signaling pathways.

• Korean Journal of Acupuncture
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• v.32 no.3
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• pp.108-115
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• 2015

Objectives : Acupuncture is frequently used as an alternative therapy for Parkinson's disease(PD) in Korea. Using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)-induced Parkinson's disease mouse model, the present study investigated a possible role of acupuncture stimulation at GB34 in suppressing dopaminergic neuronal death and regulating the phosphorylation of protein kinase B(Akt) in substantia nigra(SN) and striatum(ST). Methods : Eight-week-old male C57BL/6 mice were administered intraperitoneally with 30 mg/kg of MPTP at 24-h intervals for 5 days. Acupuncture stimulation at GB34 or SI3 was performed once a day for 12 days consecutively from the first MPTP injection. After the last acupuncture stimulation, pole test was performed to assess the effect of the acupuncture stimulations. Dopaminergic neuronal survival in the SN and the ST, dopamine transporter( DAT) and caspase-3 expression in the ST were evaluated by immunohistochemistry. The phosphorylations of Akt in the SN and the ST were measured by Western blotting. Results : MPTP administration caused behavioral impairment and dopaminergic neuronal death in the SN and the ST. It also decreased DAT expression and increased caspase-3 expression in the ST. Acupuncture stimulation at GB34 alleviated these MPTP-induced impairments. Moreover, MPTP suppressed Akt phosphorylation in the SN and the ST, whereas acupuncture stimulation at GB34 alleviated the phosphorylation in the SN. Conclusions : These results indicate that acupuncture stimulation at GB34 can inhibit MPTP-induced dopaminergic neuronal death and alleviate the Akt phosphorylation in the SN, suggesting a possible role for acupuncture in the treatment of PD.

• Radiation Oncology Journal
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• v.18 no.4
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• pp.329-336
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• 2000

Backgrounds : The purpose of this study was to identify drugs that can enhance radioresponse of murine fepatocarcinorna. Methods : CSH/HeJ mice bearing 8 mm tumors of murine fepatocarcinorna, HCa-1, were treated with 25 Gy radiation and one of the following drugs: 5-Fu, 150 mghg; adriamycin, 8 mg/kg; cisplatin, 6 mg/kg; paclitaxel, 40 mg/kg; and gemcitabine, 50 mg/kg. Tumor response to the treatment was determined by tumor growth delay assay and by enhancement factor. Apoptotic level was assessed in tissue sections. Expression of regulating molecules was analyzed by western blotting for p53, Bcl-2, Bax, Bcl-XL, Bcl-XS, and p21$^{WAF1/CIP1}$. Results :Among the drugs tested, only gemcitabine enhanced the antitumor effect of radiation, with enhancement factor of 1.6. Induction of apoptosis by a combination of gemcitabine and radiation was shown as only additive level. In analysis of radiation-induced expression of regulating molecules, the most significant change by combining gemcitabine was activation of p21$^{WAF1/CIP1}$ Conclusion :Gemcitabine is the first drug showing an enhancement of radioresponse in murine hepatocarcinoma, when combined with radiation. The key element of enhancement is thought to be p21$^{WAF1/CIP1}$.

• The Journal of Korean Medicine
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• v.30 no.1
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• pp.51-63
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• 2009

Objectives: Peroxynitrite ($ONOO^-$), superoxide anion radical (${\cdot}{O_2}^-$ and nitric oxide (NO) are cytotoxic because they can oxidize several cellular components such as proteins, lipids and DNA. They have been implicated in the aging processes, and age-related diseases such as Alzheimer's disease, rheumatoid arthritis, cancer, diabetes, obesity and atherosclerosis. The aim of this study was to investigate the $ONOO^-$, NO, ${\cdot}{O_2}^-$ scavenging and NF-${\kappa}B$ related anti-inflammatory activities of Sotosaja-hwan in ob/ob mice. Methods: Mice were grouped and treated for 5 weeks as follows. Both the normal lean (C57/BL6J black mice) and control obese (ob/ob mice) groups have received standard chow. The experimental groups were fed with a diet of chow supplemented with 30 and 90 mg Sotosaja-hwan per 1 kg of body weight for 14 days. For this study, the fluorescent probes, namely 2',7'-dichlorodihydrofluorescein diacetate (DCFDA), 4,5-diaminofluorescein (DAF-2) and dihydrorhodamine 123 (DHR 123) were used. Western blotting was performed using anti-phospho-$I{\kappa}B$-${\alpha}$, anti-IKK-${\alpha}$, anti-NF-${\kappa}B$ (p50, p65), anti-COX-2, anti-iNOS, anti-YCAM-1 and anti-MMP-9 antibodies, respectively. Results: Sotosaja-hwan inhibited the generation of $ONOO^-$, NO and ${\cdot}{O_2}^-$ in the lipopolysaccharide (LPS)-treated mouse kidney postmitochondrial fraction in vitro. The generation of $ONOO^-$, NO, ${\cdot}{O_2}^-$ and PGE2 were inhibited in the Sotosaja-hwan-administered ob/ob mice groups. The GSH/GSSG ratio was decreased in the ob/ob mice, whereas the ratio was improved in the Sotosaja-hwan-administered groups. Sotosaja-hwan inhibited the protein expression levels of phospho-$I{\kappa}B$-${\alpha}$, IKK-${\alpha}$, NF-${\kappa}B$ (p50, p65), COX-2, iNOS, YCAM-1 and MMP-9 genes. Conclusions: These results suggest that Sotosaja-hwan is an effective $ONOO^-$, ${\cdot}{O_2}^-$ and NO scavenger and has NF-kB related anti-inflammatory activity in ob/ob mice. Therefore, Sotosaja-hwan might be a potential therapeutic drug against the inflammation process and inflammation-related diseases.

• Journal of Microbiology and Biotechnology
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• v.26 no.11
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• pp.1881-1890
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• 2016

Manganese superoxide dismutase (MnSOD) is a vital enzyme that protects cells from free radicals through eliminating superoxide radicals ($O^{2-}$). Hirudin, a kind of small active peptide molecule, is one of the strongest anticoagulants that can effectively cure thrombus diseases. In this study, we fused Hirudin to the C terminus of human MnSOD with the GGGGS linker to generate a novel dual-feature fusion protein, denoted as hMnSOD-Hirudin. The hMnSOD-Hirudin gene fragment was cloned into the pET15b (SmaI, CIAP) vector, forming a recombinant pET15b-hMnSOD-Hirudin plasmid, and then was transferred into Escherichia coli strain Rosetta-gami for expression. SDS-PAGE was used to detect the fusion protein, which was expected to be about 30 kDa upon IPTG induction. Furthermore, the hMnSOD-Hirudin protein was heavily detected as a soluble form in the supernatant. The purification rate observed after Ni NTA affinity chromatography was above 95%. The hMnSOD-Hirudin protein yield reached 67.25 mg per liter of bacterial culture. The identity of the purified protein was confirmed by western blotting. The hMnSOD-Hirudin protein activity assay evinced that the antioxidation activity of the hMnSOD-Hirudin protein obtained was $2,444.0{\pm}96.0U/mg$, and the anticoagulant activity of the hMnSOD-Hirudin protein was $599.0{\pm}35.0ATU/mg$. In addition, in vitro bioactivity assay showed that the hMnSOD-Hirudin protein had no or little cytotoxicity in H9c2, HK-2, and H9 (human $CD_4{^+}$, T cell) cell lines. Transwell migration assay and invasion assay showed that the hMnSOD-Hirudin protein could suppress human lung cancer 95-D cell metastasis and invasion in vitro.