• Journal of The Korean Dental Society of Anesthesiology
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• v.14 no.2
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• pp.101-106
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• 2014

Background: Remifentanil, an ultra-short-acting mu-opioid receptor agonist, is unique from other opioids because of its esterase-based metabolism, minimal accumulation, and very rapid onset and offset of clinical action. Remifentanil can prevent the inflammatory response and can suppress inducible nitric oxide synthase expression in a septic mouse model. However, the effects of remifentanil on human keratinocyte and autophagy have yet to be fully elucidated during hypoxia-reoxygenation. Here we investigated whether remifentanil confers protective effect against hypoxia-reoxygenation in human keratinocyte and, if so, whether autophagy mediates this effect. Methods: The human keratinocytes were cultured under 1% oxygen tension. The cells were gassed with 94% $N_2$, and 5% $CO_2$ and incubated for 24 h at $37^{\circ}C$. To determine whether the administration of affects human keratinocytes hypoxia-reoxygenation injury, cells were then exposed to various concentrations of remifentanil (0.01, 0.1, 0.5 and 1 ng/ml) for 2 h. After remifentanil treatment, to simulate reoxygenation and recovery, the cells were reoxygenated for 12 h at $37^{\circ}C$. Control group did not receive remifentanil treatment. Normoxia group did not receive hypoxia and remifentanil treatment for 36 h. 3-MA group was treated 3-methyladenine (3-MA) for 1h before remifentanil treatment. Cell viability was measured using a quantitative colorimetric assay with MTT, showing the mitochondrial activity of living cells. Cells were stained with fluorescence and analyzed with Western blot analysis to find out any relations with activation of autophagy. Results: Prominent accumulation of autophagic specific staining MDC was observed around the nuclei in RPT group HaCaT cells. Similarly, AO staining, red fluorescent spots appeared in RPT group HaCaT cells, while the Normoxia, control and 3-MA groups showed mainly green cytoplasmic fluorescence. We here examined activation of autophagy related protein under H/R-induced cells by Western blotting analysis. Atg5, Beclin-1, LC3-II (microtubule-associated protein 1 light chain 3 form II) and p62 was elevated in RPT group cells. But they were decreased when autophagy was suppressed by 3-MA (Fig. 5). Conclusions: Although the findings of this study are limited to an in vitro interpretation, we suggest that remifentanil may have a beneficial effect in the recovery of wound from hypoxia-reoxygenation injury.

• Korean Chemical Engineering Research
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• v.43 no.1
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• pp.103-109
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• 2005

Isolation and primary culture technique of rabbit oral keratinocytes, and the study for effect of environmental factors on the cell growth were carried out in T75-flask. $1.92{\pm}0.59{\times}10^6$ viable cells were isolated by trypsin enzymatic digestion method from $0.25cm^2$ biopsy of rabbit oral mucosa. Primary culture with 10 mL of K-SFM containing 50 mg/L BPE, $5.0{\mu}g/L$ EGF and 0.15 mM $Ca^{2+}$ showed confluence after 8 days and doubling time was 2.54 days. Effect of medium types, medium volume and supplement types on the cell growth was investigated after the cultured keratinocytes had been harvested from primary confluence. Serum addition showed adverse effect and the increase of serum concentration didn't have an effect on the cell growth. The increase of medium volume decreased the cell growth. The increase of calcium concentration increased the cell growth and 2.0 mM was optimum value. In conclusion, when rabbit oral keratinocytes was cultured in T75-flask, the most effective conditions was to use 10 mL of K-SFM containing 50 mg/L BPE, $5.0{\mu}g/L$ EGF and 2.0 mM $Ca^{2+}$, and doubling time was 1.32 days. This study can provide the useful informations to develop a process and design a bioreactor for the culture of keratinocytes in human body like skin and cornea, as well as mucosa.

• Journal of Ginseng Research
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• v.40 no.2
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• pp.169-175
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• 2016

Background: Chemotherapy-induced alopecia (CIA) is one of the most distressing side effects for patients undergoing chemotherapy. This study evaluated the protective effect of Korean Red Ginseng (KRG) on CIA in a well-established in vitro human hair follicle organ culture model as it occurs in vivo. Methods: We examined whether KRG can prevent premature hair follicle dystrophy in a human hair follicle organ culture model during treatment with a key cyclophosphamide metabolite, 4-hydroperoxycyclophosphamide (4-HC). Results: 4-HC inhibited human hair growth, induced premature catagen development, and inhibited proliferation and stimulated apoptosis of hair matrix keratinocytes. In addition, 4-HC increased p53 and Bax protein expression and decreased Bcl2 protein expression. Pretreatment with KRG protected against 4-HC-induced hair growth inhibition and premature catagen development. KRG also suppressed 4-HC-induced inhibition of matrix keratinocyte proliferation and stimulation of matrix keratinocyte apoptosis. Moreover, KRG restored 4-HC-induced p53 and Bax/Bcl2 expression. Conclusion: Overall, our results indicate that KRG may protect against 4-HC-induced premature catagen development through modulation of p53 and Bax/Bcl2 expression.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.30 no.2
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• pp.227-233
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• 2004

Chronic exposure to solar radiation, particularly ultraviolet (UV) light, causes a variety of adverse reactions on human skin, such as sunburn, photoaging and photocarcinogenesis. Free radicals and reactive oxygen species (ROS) caused by UV exposure or other environmental facts play critical roles in cellular damage. And, repeated-UV irradiation activated the expression of the matrix metalloproteinase (MMP) and induced skin irritation. Therefore, the development of effective and safe photoprotectants that can reduce and improve the skin damage has been required. The purpose of this study was to investigate the photo-protective effect of several chinese medical plants (Juniperus chinensis) on the UV -induced skin cell damages. We tested free radical and superoxide scavenging effect in vitro. Fluorometric assays of the proteolytic activities of MMP-1 (collagenase) were performed using fluorescent collagen substrates. UVA induced MMP-1 synthesis and activity were analyzed by enzyme-linked immunosorbent assay (ELISA) and gelatin-based zymography in skin fibroblasts. We also examined anti-inflammatory effects by the determination test of proinflammatory cytokine, interleukin 6 in HaCaT keratinocytes. Expression of prostaglandin E$_2$ (PGE$_2$) after UVB irradiation was measured by competitive enzyme immunoassay(EIA) using PGE$_2$ monoclonal antibody. In the human skin we tested anti-irritation effect on the SLS-induced damage skin after appling the extract containing emulsion. We found that Juniperus chinensis extract had potent radical scavenging effect by 98% at 100$\mu\textrm{g}$/mL. The extract of Juniperus chinensis showed strong inhibitory effect on MMP-1 activities by 97% at 100 $\mu\textrm{g}$/mL and suppressed the UVA induced expression of MMP-1 by 79% at 25$\mu\textrm{g}$/mL. This extract also showed strong inhibition on MMP-2 activity in UVA irradiated fibroblast by zymography. In the test of proinflammatory cytokines of human keratinocytes Juniperus chinensis extract decreased expression of interleukin 6 about 30%. The amount of PGE$_2$ by HaCaT keratinocytes was significantly increased at the doses of above 10 mJ/$\textrm{cm}^2$ of UVB (p < 0.05). At the concentrations of 3.2-25$\mu\textrm{g}$/mL of this extract, the production of PGE$_2$ by HaCaT keratinocytes (24 h after 10mJ/$\textrm{cm}^2$ UVB irradiation) was significantly inhibited in culture supernatants (p < 0.05). In SLS-induced skin irritation model in vivo, we found to reduce skin erythema and improve barrier recovery after appling Juniperus chinensis extract containing emulsion when compared to irritated non-treated and placebo-treated skin. Our results suggest that Juniperus chinensis extract can be effectively used for the prevention of UV and SLS-induced adverse skin reactions and applied as anti-aging and anti-irritation cosmetics.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.42 no.4
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• pp.393-401
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• 2016

Ultraviolet (UV) irradiation from the sun is the primary environmental factor that causes skin damages including skin cancer and premature skin aging. Because, even the most powerful sunscreen can't always afford enough protection, it is necessary to enhance the defensive power of skin against UV. Recently, constitutive photomorphogenic protein-1 (COP1) has shown to contribute to the regulation of UVB response of keratinocytes. In this study, we represent that COP1 and its associated protein, de-etiolated 1 (DET1), might participate in photoaging process in human skin as Arabidopsis COP1 does sun-protective function in plants. After UVB irradiation, the decrease of COP1 and DET1 mRNA expression was followed by the increase of c-Jun total protein. Moreover, transfection with DNA vectors expressing COP1 and DET1 down-regulated the c-Jun total protein. We found that Zanthoxylum piperitum extract (ZE) up-regulated the expression of COP1 and DET1 on human keratinocytes, and inhibited the expression of MMP1 which is one of the genes regulated by c-Jun signal. In addition, ZE has been reported to stimulate PPAR-${\alpha}$ and strengthen the skin barrier. We found that ZE decreased the UVB-induced IL-6 and IL-8 in NHEK cells. In human study, ZE protected skin against UV-B induced erythema and erythema-induced pigmentation. These results indicate that ZE could be useful for the protection against the adverse effects of UV irradiation through various mechanisms.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.33 no.1 s.60
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• pp.17-22
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• 2007

In this study, we investigated the effects of Sedum sarmentosum extract on the expression of hyaluronan synthase (HAS) genes and hyaluronan (HA) production in HaCaT keratinocytes. We also assessed water content (electrical capacitance) and transepidermal water loss (TEWL) in human skin after topical treatment with Sedum sarmentosum extract. Sedum sarmentosum extract increased expression of HAS-2 and HAS-3 genes in HaCaT cells, when assayed by real-time reverse-transcriptase polymerase chain reactions (RT-PCR). Sedum sarmentosum extract induced HA production in HaCaT cells, when determined by enzyme-linked immunosorbent assay (ELISA). Finally, treatment of Sedum sarmentosum extract on human skin increased the skin hydration and decreased TEWL when measured using Corneometer and Tewameter. Our study suggests that Sedum sarmentosum extract should be a very useful cosmetic ingredient, as a skin moisturizer.

• Microbiology and Biotechnology Letters
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• v.24 no.3
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• pp.290-298
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• 1996

In an effort to understand the role of the conserved domain and of the heterologous one-third part of the carboxy terminal domain of transglutaminase C (TGase II), attempts were made to express TGase II cDNA of human origin in yeast Saccharomyces cerevisiae as in a full-length form as well as in a form of C-terminal truncation. The 2$\mu$-based expression plasmids which contained the TGase II cDNA under the gal inducible promoter were introduced into yeast and the maintenance of the full-length and truncated form of the TGase II gene plasmids were confirmed by Southern blot. The expression of the TGase II gene was analysed by reverse transcription polymerase chain reaction (RT-PCR), and western blot analyses. As assayed by [1,4$^{14}$C]-putrescine incorporation into succinylated casein, the full-lenth as well as the truncated forms of recombinant TGase II showed some catalytic activity. These results indicate that the N-terminal homologous domain of human TGase II retains a catalytically active domain. The level of TGase II expressed in yeast, however, was far lower than satisfactory and other expression system should be sought further chracterization of the enzyme. The negative effect of TGase II on the growth of yeast is interesting with respect to the physiological effect of TGase II in cornification of epidermal keratinocytes.

• Korean Journal of Pharmacognosy
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• v.39 no.4
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• pp.300-304
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• 2008

The root of Astragalus membranaceus Bunge (Leguminosae) has been used in the Korean oriental medicine for strengthening the vital energy. UV irradiation has been suggested as a major cause of photo aging in skin. In order to investigate protective effects against UV induced cellular damage, Astragali Radix was extracted with 70% ethanol and dissolved in DMSO. The protective effect was detected by MTT assay, LDH assay, and Comet assay in immortalized human keratinocyte cell line, HaCaT cell system after UV irradiation. Astragli Radix 70% EtOH extract reduced UV induced cellular damage in cell survival, membrane integrity and DNA damage.

• Journal of Microbiology and Biotechnology
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• v.25 no.1
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• pp.119-126
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• 2015

Among the various human growth factors, epidermal growth factor (hEGF, consisting of 53 amino acids) has various effects on cell regeneration, stimulation of proliferation, migration of keratinocytes, formation of granulation tissues, and stimulation of fibroblast motility, which are important for wound healing. Owing to their multiple activities, EGFs are used as pharmaceutical and cosmetic agents. However, their low productivity, limited target specificity, and short half-life inhibit their application as therapeutic agents. To overcome these obstacles, we fused the collagen-binding domain (CBD) of Vibrio mimicus metalloprotease to EGF protein. About 18 or 12 amino acids (aa) (of the 33 total amino acids), which were essential for collagen-binding activity, were combined with the N- and C-termini of EGF. We constructed, expressed, and purified EGF (53 aa)-CBD (18 aa), EGF (53 aa)-CBD (12 aa), CBD (18 aa)-EGF (53 aa), and CBD (12 aa)-EGF (53 aa). These purified recombinant proteins increased the numbers of cells in treated specimens compared with non-treated specimens and control hEGF samples. The collagen-binding activities were also evaluated. Furthermore, CBD-hybridized hEGF induced phosphorylation of the EGF receptor. These results suggested that these fusion proteins could be applicable as small therapeutic agents in wound tissue healing.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.26 no.1
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• pp.17-40
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• 2000

Cytotoxicity assays using artificial skins have been proposed as in vitro alternatives to minimize animal ocular and dermal irritation testing. Accordingly, the responses of artificial skins to the well-characterized chemical irritants toluene, glutaraldehyde, and sodium lauryl sulfate (SLS), and the nonirritant polyethylene glycol were studied. The evaluation of the irritating and non-irritating test chemicals was also compared with the responses observed in human dermal fibroblasts and human epidermal keratinocytes grown in a monolayer culture. The responses monitored included an MTT mitochondrial functionality assay. In order to better understand the local mechanisms involved in skin damage and repair, the production of several mitogenic proinflammatory mediators, interleukin-l$\alpha$, 12-HETE, and 15-HETE, was also investigated. Dose-dependent increases in the levels of かIn and the HETEs were observed in the underlying medium of the skin systems exposed to the two skin irritants, glutaraldehyde and SLS. The results of the present study show that both human artificial skins can be used as efficient in vitro testing models for the evaluation of skin toxicity and for screening contact skin irritancy.