• 대한화장품학회지
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• 제27권1호
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• pp.17-27
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• 2001

A cell-based assay system for monitoring NF-$textsc{k}$B activity was developed to determine the influence of activated NF-$textsc{k}$B in human HaCaT cells. The pNF-$textsc{k}$B-SEAP-NPT plasmid that permits expression of the secreted alkaline phosphatase (SEAP) reported gene in response to the NF-$textsc{k}$B activity and contains neomycin phosphotransferase (NPT) gene for the geneticin resistance in host cells was constructed and transfected into human keratinocyte cell line HaCaT. Human HaCaT transfectant cells secreted the SEAP enzyme into the culture medium in a time-dependent manner until 72h. NF-$textsc{k}$B activities were measured in the SEAP reporter gene assay using a fluorescent detection method. The treatment of HaCaT cell transfectants with known antioxidants [e.g., N-acetyl-L-cysteine and vitamin C] showed inhibition of NF-$textsc{k}$B activity in a time-and concentration-dependent manner. The phorbol 12-myristate 13-acetate (PMA) known as a stimulator of NF-$textsc{k}$B expression demonstrated that it increased NF-$textsc{k}$B activity in a time- and concentration-dependent manner. This assay system could be used to determine the quantitative measurement of NF-$textsc{k}$B activity in the human skin and allow the screening of anti-inflammatory agents from various synthetic chemicals and natural products for dermatological purpose. Abbrevitions used: NF-$textsc{k}$B, nuclear factor kappa B; I-$textsc{k}$B, Inhibitory kappa B; SEAP, secreted alkaline phosphatase; NPT, neomycin phosphotransferease; PCR, polymerase chain reaction: dNTP, deoxynucleoside triphosphates; DMEM, dulbecco’s modified eagle medium; FBS, fetal bovine serum; PBs, phosphate-buffered saline; MUP, 4-methylumbellifery phosphate; NAC, N-acetyl-L-cysteine; DMSO, dimethyl sulfoxide; PMA, phorbol 12-myristate 13-acetate.

• 대한약침학회지
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• 제15권2호
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• pp.11-14
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• 2012

Objectives: The purpose of this study was to compare the antioxidant and anti-inflammatory effects of Alum (AL) and Burnt Alum (BAL), which are commonly used as external ointments. Methods: Extracts of AL and BAL were classified into three groups: 20, 50, and $100mg/{\mu}{\ell}$. The cytotoxicity was measured by using MTT assays in human keratinocyte cell line (HaCaT). The anti-oxidant effect was measured by using the DPPH (1, 1-diphenyl-2-picryl-hydrazyl-hydrate) radical scavenger. The anti-inflammatory effect was measured by using the inhibitory efficacy for the amount of nitric-oxide (NO) produced in mouse macrophage cell line (RAW 264.7). Results: BAL showed a higher level of cytotoxicity than AL. The AL groups showed a concentration-dependent scavenging effect on DPPH radicals, but no significant relevance was found. The BAL groups showed a concentration-dependent scavenging effect on DPPH radicals. The scavenging effects of the BAL groups were almost insignificant, but the values for the 20, 50, and $100{\mu}g/m{\ell}$ trials were different. The BAL groups showed significant concentration-dependent inhibitory effects on NO production, but the AL groups did not. Conclusions: AL showed an anti-oxidant effect more efficiently than BAL did, which demonstrated a superior anti-inflammatory effect. Therefore, for external usage, AL must be distinguished from BAL.

• 대한본초학회지
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• 제26권2호
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• pp.45-49
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• 2011

Objectives : The purpose of this study was to investigate the anti-aging effects on cultured human keratinocytes with Nelumbo nucifera extracts. Methods : Each parts of leaves, flowers and stamen were extracted with water or 70% ethanol. These extracts were tested for cell viability on HaCaT cells (human keratinocyte line) by MTT assay. We investigated the effects of Ultraviolet-B (UVB) irradiation on cytotoxicity and lipid peroxidation in cultured skin keratinocytes. Results : The ethanol extract of Nelumbo nucifera flowers showed maximun cell viability as 111.39% in 30 ug/ml concentration. The water extracts of stamen, flowers, leaves showed cell viability as 107.12, 101.65, 101.46%, respectively. HaCaT keratinocytes were survived 63.06% at $20mJ/cm^2$ UVB irradiation. The cell membrane lipid peroxidation was measured by accumulation malondialdehyde (MDA). The levels of MDA were decreased by the ethanol extract of Nelumbo nucifera flowers and the water extracts of stamen. Conclusions : These finding suggest that the ethanol extract of Nelumbo nucifera flowers prevent anti-aging effects on cultured human keratinocytes during UVB irradiation.

• 대한화장품학회지
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• 제44권3호
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• pp.343-347
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• 2018

화장품에 방부제(살균/보존제)로 사용되는 1, 2-hexanediol (HD)로 인한 부작용을 극복하기 위하여, Escherichia coli (E. coli)의 ${\beta}-galactosidase$ (${\beta}-gal$)를 이용하여 transgalactosylation 반응으로 1, 2-hexanediol galactoside (HD-Gal)를 합성하였다. 본 연구에서는 합성된 HD-Gal의 인간 피부세포에 대한 독성이 어느 정도인지를 HD와 비교하여 관찰하였다. HD-Gal과 HD의 세포독성은 인간 피부각질형성세포(HaCaT cell line)에 HD와 HD-Gal을 처리한 후, cell proliferation assay를 이용하여 비교 분석하였다. 또한 이때, 위상차 현미경으로 HD-Gal과 HD로 처리한 세포의 상태를 비교 관찰하였다. 그 결과, HD-Gal은 42.2 mM에서 211 mM의 농도 범위에서 세포독성이 관찰되지 않았으며, 현미경 관찰에서도 큰 변화를 관찰할 수 없었다. 그러나, HD의 경우에는 저농도에서(42.2 mM and 84.4 mM)는 세포독성이 관찰되지 않았으나, 고농도(168.8 and 211 mM)에서 매우 높은 세포독성을 나타내었고, 현미경 관찰에서는 고농도에서는 물론이고, 세포독성이 관찰되지 않은 HD의 저농도에서도 세포모양과 세포 수에서의 변화가 관찰되었다. 앞으로 세포독성이 감소된 HD-Gal이 HD의 대체제로서 안전, 건강 및 웰빙 개념의 새로운 용도로 개발될 수 있을 것으로 생각된다.

• 문화기술의 융합
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• 제8권6호
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• pp.849-854
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• 2022

본 연구에서는 참당귀(Angelica gigas Nakai)의 핵심 유효 성분인 decursin과 decursinol angelate를 높은 수율로 추출한 초임계 이산화탄소 선택 추출물의 항염증 효능을 에탄올 추출물과 비교하여 측정하였다. 항염증 효능을 측정하기 위해 염증 매개 인자인 nitric oxide(NO), 염증성 사이토카인인 interleukin(IL)-6, IL-8 생성량을 측정하였다. NO 생성량은 lipopolysaccharide(LPS)로 염증 반응을 유도한 Raw 264.7 세포를 대상으로 Griess assay를 통해 측정하였고, IL-6 및 IL-8 생성량은 tumor necrosis factor(TNF)-α로 염증 반응을 유도한 human keratinocyte cell line(HaCaT) 세포를 대상으로 enzyme-linked immunoadsorbent assay(ELISA)를 통해 측정하였다. NO 생성량은 초임계 이산화탄소 추출물이 에탄올 추출물에 비해 뛰어나게 억제하였다. IL-6 및 IL-8 생성량 또한 에탄올 추출물은 오히려 증가시키는 반면, 초임계 이산화탄소 추출물은 6.25 ㎍/mL의 농도에서 통계적으로 유의하게 억제하였다(P<0.01). 우리는 이러한 결과를 통해 참당귀 초임계 이산화탄소 선택 추출물이 아토피 피부염을 완화시키는 항염증 코스메슈티컬 소재로써 활용 가능함을 확인하였다.

• 대한한방소아과학회지
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• 제30권2호
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• pp.96-106
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• 2016

Objectives The purpose of this study is to investigate the effects of Gleditsiae Fructus 70% EtOH extract (JS_E) on anti-inflammatory action in HaCaT cells (A spontaneously immortalized human keratinocyte cell line) and inhibiting triglyceride genesis in SEB-1 cells (Immortalized human sebocyte). Methods The anti-inflammatory effect of JS_E was analyzed by enzyme-linked immunosorbent assays (ELISA) which measured levels of IP-10, RANTES and MDC in HaCaT cells. Also the effect on secretion of sebum of JS_E was analyzed by TG-S kit which measured the quantity of triglyceride in SEB-1 cells. Results JS_E inhibited IP-10, RANTES and MDC expression in a dose dependent manner. IP-10 expression was inhibited significantly in comparison to TNF-${\alpha}$ and IFN-${\gamma}$ recombination (TI) control group at concentration of JS_E $200{\mu}g/ml$ and RANTES and MDC expressions were inhibited significantly at concentration of JS_E 100, $200{\mu}g/ml$. JS_E also inhibited triglyceride secretion of SEB-1 cells significantly in comparison to the control group in a dose dependent manner. Conclusions This study shows that JS_E has the effects of anti-inflammatory action and inhibiting sebum secretion. According to these results, JS_E can be used for treating skin diseases such as acne and dermatitis caused by inflammation and excessive secretion of sebum by controlling the activity of the HaCaT and SEB-1 cells.

• 생약학회지
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• 제44권2호
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• pp.154-160
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• 2013

Ceramium boydenii belongs to euphorbia humitusa of red algae, and is distributed along the coast of Jeju island. This study was conducted to examine the anti-inflammatory effect and action mechanism of C. boydenii in the human keratinocyte cell line HaCaT. The 80% EtOH extract of C. boydenii inhibits the production of MDC (macrophage derived chemokine), an inflammatory chemokine, induced by IFN-${\gamma}$ and TNF-${\alpha}$ in a concentration dependent manner. It also suppressed the phosphorylation and nuclear translocation of STAT1, a key transcription factor in IFN-${\gamma}$ and TNF-${\alpha}$ signaling pathway, at the effective concentrations. These results suggest that C. boydenii demonstrates the anti-inflammatory activity via the suppression of STAT1 activation and has the significant value as an anti-inflammatory source.

• BMB Reports
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• 제46권8호
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• pp.410-415
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• 2013

Adhesion molecules such as ICAM-1 are important in the infiltration of leukocytes into the site of inflammation. In this study, we investigated the inhibitory effects of curcumin on ICAM-1 expression and monocyte adhesiveness as well as its underlying action mechanism in the TNF-${\alpha}$-stimulated keratinocytes. Curcumin induced expression of heme oxygenase-1 (HO-1) in the human keratinocyte cell line HaCaT. In addition, curcumin induced Nrf2 activation in dose- and time-dependent manners in the HaCaT cells. Curcumin suppressed TNF-${\alpha}$-induced ICAM-1 expression and subsequent monocyte adhesion, which were reversed by the addition of tin protoporphyrin IX (SnPP), a specific inhibitor of HO-1, or HO-1 knockdown using siRNA. Furthermore, Nrf2 knockdown using siRNA reversed the inhibitory effect of curcumin on the TNF-${\alpha}$-induced ICAM-1 expression and adhesion of monocytes to keratinocytes. These results suggest that curcumin may exert its anti-inflammatory activity by suppressing the TNF-${\alpha}$-induced ICAM-1 expression and subsequent monocyte adhesion via expression of HO-1 in the keratinocytes.

• BMB Reports
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• 제49권1호
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• pp.57-62
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• 2016

Up-regulation of adhesion molecules plays an important role in the infiltration of leukocytes into the skin during the development of various inflammatory skin diseases, such as atopic dermatitis. In this study, we investigated the modulatory effects of 2,3-dimethoxy-2′-hydroxychalcone (DMHC) on tumor necrosis factor (TNF)-α-induced intercellular adhesion molecule-1 (ICAM-1) expression and monocyte adhesiveness, as well as the molecular mechanisms underlying its action in the HaCaT human keratinocyte cell line. Pre-treating HaCaT cells with DMHC significantly suppressed TNF-α-induced ICAM-1 expression and subsequent monocyte adhesiveness. DMHC inhibited TNF-α-induced activation of NF-ᴋB. In addition, DMHC induced HO-1 expression as well as NRF2 activation. Furthermore, HO-1 knockdown using siRNA reversed the inhibitory effect of DMHC on TNF-α-induced ICAM-1 expression and adhesion of monocytes to keratinocytes. These results suggest that DMHC may inhibit TNF-α-induced ICAM-1 expression and adhesion of monocytes to keratinocytes by suppressing the signaling cascades leading to NF-ᴋB activation and inducing HO-1 expression in keratinocytes. [BMB Reports 2016; 49(1): 57-62]

• The Korean Journal of Physiology and Pharmacology
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• 제12권4호
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• pp.177-183
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• 2008

The layers of keratinocytes form an acid mantle on the surface of the skin. Herein, we investigated the effects of acidic pH on the membrane current and $[Ca^{2+}]_c$ of human primary keratinocytes from foreskins and human keratinocyte cell line (HaCaT). Acidic extracellular pH ($pH_e{\leq}5.5$) activated outwardly rectifying $Cl^-$ current ($I_{Cl,pH}$) with slow kinetics of voltage-dependent activation. $I_{Cl,pH}$ was potently inhibited by an anion channel blocker 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS, 73.5% inhibition at 1${\mu}$M). $I_{Cl,pH}$ became more sensitive to $pH_e$ by raising temperature from $24^{circ}C$ to $37^{circ}C$. HaCaT cells also expressed $Ca^{2+}$-activated $Cl^-$ current ($I_{Cl,Ca}$), and the amplitude of $I_{Cl,Ca}$ was increased by relatively weak acidic $pH_e$ (7.0 and 6.8). Interestingly, the acidic $pH_e$ (5.0) also induced a sharp increase in the intracellular [$Ca^{2+}$] (${\triangle}[Ca^{2+}]_{acid}$) of HaCaT cells. The ${\triangle}[Ca^{2+}]_{acid}$ was independent of extracellular $Ca^{2+}$, and was abolished by the pretreatment with PLC inhibitor, U73122. In primary human keratinocytes, 5 out of 28 tested cells showed ${\triangle}[Ca^{2+}]_{acid}$. In summary, we found $I_{Cl,pH}$ and ${\triangle}[Ca^{2+}]_{acid}$ in human keratinocytes, and these ionic signals might have implication in pathophysiological responses and differentiation of epidermal keratinocytes.