• 한국식품영양과학회지
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• 제43권11호
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• pp.1695-1700
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• 2014

본 연구에서는 한방 발효주 주박의 피부 생리 기능 활성을 알아보기 위해 주박 추출물 및 분획물을 준비하고 항산화, 미백 및 주름 개선 효과를 조사하였다. 그 중 한방 발효주 주박의 에틸아세테이트 분획물(KSD E4-3)이 가장 우수한 항산화 효과를 나타내었으며 이는 다양한 약재의 발효에 의한 효과로 사료된다. KSD E4-3은 tyrosinase 활성 억제 및 tyrosine을 기질로 melanin이 형성되는 pathway에 관여하는 주요한 인자인 TRP-1과 TRP-2를 저해하는 작용 기전을 통해 피부 색소침착의 주요 원인 물질인 melanin 생합성을 농도 의존적으로 저해하는 것을 확인하였다. 또한 KSD E4-3은 피부의 keratinocyte가 생성, 분비하는 MMP-1, -2, -9의 발현을 억제하여 UV 조사에 의한 피부 광노화에 따른 피부 주름 생성을 억제할 수 있음을 확인하였다. 이상의 결과를 종합하면 한방 발효주 주박에서 추출한 KSD E4-3은 피부 미백 및 주름 개선에 우수한 효과를 나타내고 있으므로 기능성 화장품의 주요한 소재로 이용 가치가 높을 것으로 사료된다.

• 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.

• 대한본초학회지
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• 제36권5호
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• pp.93-99
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• 2021

Objective : Caryophylli Flos has been used in Korean medicine to relieve vomiting and pains caused by chills that make fluid circulation difficult. This study was designed to investigate the protective effect of ethanol extract of Caryophylli Flos (CF) in hydrogen peroxide (H₂O₂)-induced apoptotic cell death in human keratinocyte HaCaT cells. Methods : CF was prepared by extracting 200 g of Caryophylli Flos in 2 L of ethanol for 48 h. Cell viability was measured by MTT assay, and the protein expression was monitored by Western blot analysis. Apoptosis was determined by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Reactive oxygen species (ROS) was measured using fluorescent dye, and reduced glutathione (GSH) was determined with a colorimetric commercial kit. Results : CF protected HaCaT cells from cell death caused by oxidative stress after H₂O₂ treatment. H₂O₂ amplified generation of ROS and induced depletion of GSH, whereas these changes in ROS and GSH were inhibited by GF treatment. In addition, H₂O₂ resulted in apoptosis as assessed by TUNEL assay and the expression of apoptosis regulator proteins. However, cells treated with CF showed a decrease in TUNEL-positive cells and restored the reduced expression of procaspase-9, -3 and PARP. Conclusion : This study showed cytoprotective effects of CF by anti-apoptotic activity while exerting antioxidative activity in H₂O₂-treated HaCaT cells. These results suggest that CF could be beneficial in skin damage caused by oxidative stress.

• Journal of Ginseng Research
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• 제45권3호
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• pp.456-463
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• 2021

Background: Keratinocytes form a physical barrier and act as an innate immune cell in skin. Keratinocytes secrete pro-inflammatory cytokines, such as interleukin (IL)-1β, resulting from inflammasome activation when exposed to ultraviolet (UV) irradiation. Korean Red Ginseng extracts (RGE) have been well-studied as modulators of inflammasome activation in immune cells, such as macrophages. In the study, we elucidated the role of RGE on the UV-mediated inflammasome activation in keratinocytes compared with that in macrophages. Methods: Human skin keratinocyte cells (HaCaT), human epidermal keratinocytes (HEK), human monocyte-like cells (THP-1), and mouse macrophages were treated with RGE or a saponin fraction (SF) or non-saponin fraction (NS) of RGE before and after UV irradiation. The secretion levels of IL-1β, as an indicator of inflammasome activation, were analyzed. Results: The treatment of RGE or SF in macrophages after UV irradiation inhibited IL-1β secretion, but similar treatment in HaCaT cells did not. However, the treatment of RGE or SF in HaCaT cells in the presence of poly I:C, a toll-like receptor (TLR) 3 ligand, before UV exposure elicited the inhibition of the IL-1β secretion. The inhibition was caused by the disruption by RGE or SF of the TLR mediating up-regulation of the pro-IL-1β and NLRP3 genes during the priming step. Conclusion: RGE and its saponins inhibit IL-1β secretion in response to UV exposure in both keratinocytes and macrophages. In particular, RGE treatment interrupted only the priming step in keratinocytes, although it did attenuate both the priming and activation steps in macrophages.

• IMMUNE NETWORK
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• 제23권3호
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• pp.29.1-29.23
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• 2023

Cholesterol (CL) is required for various biomolecular production processes, including those of cell membrane components. Therefore, to meet these needs, CL is converted into various derivatives. Among these derivatives is cholesterol sulfate (CS), a naturally produced CL derivative by the sulfotransferase family 2B1 (SULT2B1), which is widely present in human plasma. CS is involved in cell membrane stabilization, blood clotting, keratinocyte differentiation, and TCR nanocluster deformation. This study shows that treatment of T cells with CS resulted in the decreased surface expression of some surface T-cell proteins and reduced IL-2 release. Furthermore, T cells treated with CS significantly reduced lipid raft contents and membrane CLs. Surprisingly, using the electron microscope, we also observed that CS led to the disruption of T-cell microvilli, releasing small microvilli particles containing TCRs and other microvillar proteins. However, in vivo, T cells with CS showed aberrant migration to high endothelial venules and limited infiltrating splenic T-cell zones compared with the untreated T cells. Additionally, we observed significant alleviation of atopic dermatitis in mice injected with CS in the animal model. Based on these results, we conclude that CS is an immunosuppressive natural lipid that impairs TCR signaling by disrupting microvillar function in T cells, suggesting its usefulness as a therapeutic agent for alleviating T-cell-mediated hypersensitivity and a potential target for treating autoimmune diseases.

• IMMUNE NETWORK
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• 제10권2호
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• pp.76-81
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• 2010

Ultraviolet B (UVB) induces multiple inflammatory and carcinogenic reactions. In skin, UVB induces to secrete several kinds of inflammatory cytokines from keratinocytes and also increases angiogenic process via the modulation of vascular endothelial growth factor (VEGF) production. Interleukin-21 (IL-21) is an inflammatory cytokine and produced by activated T cells. The biologic functions of IL-21 have not yet extensively studied. In the present study, we investigate the production of IL-21 from human keratinocyte cell line, HaCaT and its biological effect after exposure to UVB. First, we confirmed the IL-21 production and its receptor expression in HaCaT. And then, the change of IL-21 and VEGF production in HaCaT by UVB irradiation was examined. Not only IL-21 but also VEGF production was enhanced by UVB irradiation. Next, to determine relationship of enhanced production of IL-21 and VEGF, we detected VEGF production after neutralization of IL-21. VEGF production was reduced by IL-21 neutralization, which indicates that the IL-21 is involved in the VEGF production. Taken together, our results suggest that IL-21 and VEGF production is enhanced by UVB irradiation in HaCaT. In addition, it seems that IL-21 plays a role in the angiogenic process in skin via the modulation of VEGF production.

• 한방안이비인후피부과학회지
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• 제20권3호
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• pp.82-97
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• 2007

Objective : Cortex of Ulmi pumilae(CUP) has been used to treat several diseases including boil, swelling, and scabies etc. Recently, CUP was known to have wrinkle care and whitening actions. But, It's exact mechanisms are unclear. Methods : The present study was designed to investigate effects of CUP on Human HaCaT keratinocyte and malignant melanoma cells such as SK-MEL-2 and B16F10 in terms of cell viabilities, proliferations, DPPH free radical scavenging activities, oxygen free radical productions and inhibitory action on elastase activities. Results : CUP accelerated proliferation of HaCaT keratinocytes in the lower concentration. CUP also prevented cell death of HaCaT induced by Hydrogen peroxide, which products oxygen free radicals. On the contrary, CUP did not affect proliferations of SK-MEL-2 or B16F10. Futhermore, CUP showed inhibitory action against SK-MEL-2 proliferation at the concentration of $500{\mu}g/m{\ell}$ In addition, CUP was shown to have DPPH free radical scavenging activities and also have inhibitory effects on elastase activities too. On the fluorescent examinations, the present author knows that CUP elevated production levels of oxygen free radicals in malignant melanoma cell, SK-MEL-2. Conclusions : These results suggest that CUP has possibilities of usage for functional cosmetics which have wrinkle care and whitening activities and related mechanisms are involved in inhibition of elastase action and acceleration of oxidative stress in melanoma cell.

• Biomolecules & Therapeutics
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• 제25권3호
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• pp.296-307
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• 2017

In spite of frequent usage of primary human foreskin keratinocytes (HFKs) in the study of skin biology, senescence-induced block-age of in vitro proliferation has been a big hurdle for their effective utilization. In order to overcome this passage limitation, we first isolated ten HFK lines from circumcision patients and successfully immortalized four of them via a retroviral transduction of high-risk human papillomavirus (HPV) E6 and E7 oncogenes. We confirmed expression of a keratinocyte marker protein, keratin 14 and two viral oncoproteins in these immortalized HFKs. We also observed their robust responsiveness to various exogenous stimuli, which was evidenced by increased mRNA expression of epithelial differentiation markers and pro-inflammatory genes in response to three reactive chemicals. In addition, their applicability to cytotoxicity assessment turned out to be comparable to that of HaCaT cells. Finally, we confirmed their differentiation capacity by construction of well-stratified three dimensional skin cultures. These newly established immortalized HFKs will be valuable tools not only for generation of in vitro skin disease models but also for prediction of potential toxicities of various cosmetic chemicals.

• Biomolecules & Therapeutics
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• 제27권6호
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• pp.553-561
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• 2019

Rab25, a member of the Rab11 small GTPase family, is central to achieving cellular polarity in epithelial tissues. Rab25 is highly expressed in epithelial cells of various tissues including breast, vagina, cervix, the gastrointestinal tract, and skin. Rab25 plays key roles in tumorigenesis, mainly by regulating epithelial differentiation and proliferation. However, its role in skin physiology is relatively unknown. In this study, we demonstrated that Rab25 knock-out (KO) mice show a skin barrier dysfunction with high trans-epidermal water loss and low cutaneous hydration. To examine this observation, we investigated the histology and epidermal differentiation markers of the skin in Rab25 KO mice. Rab25 KO increased cell proliferation at the basal layer of epidermis, whereas the supra-basal layer remained unaffected. Ceramide, which is a critical lipid component for skin barrier function, was not altered by Rab25 KO in its distribution or amount, as determined by immunohistochemistry. Notably, levels of epidermal differentiation markers, including loricrin, involucrin, and keratins (5, 14, 1, and 10) increased prominently in Rab25 KO mice. In line with this, depletion of Rab25 with single hairpin RNA increased the expression of differentiation markers in a human keratinocyte cell line, HaCaT. Transcriptomic analysis of the skin revealed increased expression of genes associated with skin development, epidermal development, and keratinocyte differentiation in Rab25 KO mice. Collectively, these results suggested that Rab25 is involved in the regulation of epidermal differentiation and proliferation.

• 동의생리병리학회지
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• 제28권5호
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• pp.520-529
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• 2014

The purpose of this study is to investigate the protective effects of Yukmijiwhang-tang(YM) water extracts against the UVB irradiation on the human keratinocyte HaCaT cells. We observed the effects of YM on the oxidative stress, gene expression of pro-inflammatory cytokine such as TNF-${\alpha}$ and IL-$1{\beta}$, and matrix metalloproteinase-9 in UVB-irradiated HaCaT cells. On the effects of oxidative stress and antioxidant function on the treatment with YM, The activity of xanthine oxidase(XO) was significantly decreased by treatment of YM in all the concentrations(p<0.01). The activity of superoxide dismutase(SOD) and catalase(CAT) was significantly increased by treatment of YM in a dose dependent manner(p<0.05 and p<0.01). DPPH radical was erased by treatment of YM under dose of $500{\mu}g/m{\ell}$ concentration. Treatment of HaCaT cells with YM had also significantly reduced intracellular ROS produced by UVB irradiation in a dose dependent manner(p<0.05, p<0.01, p<0.001). Gelatin zymography assay showed that YM downregulated the MMP-9 activity in UVB-irradiated HaCaT cells. RT-PCR analysis revealed that YM suppressed the expression of IL-$1{\beta}$ and MMP-9 however, it has no effects on the expression of TNF-${\alpha}$ and MMP-3. Our study suggests that Yukmijiwhang-tang exert protective actions on the UVB-irradiated HaCaT cells largely by anti-oxidative and anti-inflammatory processes.