환경오염에 의한 미세먼지의 증가로 피부는 산화적 손상과 노화가 가속화된다. 본 연구에서는 선발된 한약재 추출물의 항산화, hyaluronic acid, filaggrin, MMP-1, ROS 항목을 평가함으로써 PM10으 로 부터의 각질형성세포 보호 효능을 확인하였다. 그 결과 1,1-diphenyl-2-picrylhydrazyl(DPPH), 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid(ABTS), FRAP assay에서 농도의존적으로 항산화능 이 증가하는 것을 확인하였다. 각질형성세포에 PM10 300 ㎍/㎖을 단독으로 처리한 군에서는 hyaluronic acid 및 filaggrin이 50% 이상 감소하였으며, 고량강, 유백피, 토복령 추출물을 처리한 군에서는 증가하였 다. MMP-1의 경우 PM10 단독처리군에는 55% 이상 증가하였으나, 추출물을 처리한 경우 감소하여 콜라 겐, 엘라스틴의 분해를 저해하는 것으로 평가된다. 또한 제브라피쉬 배아를 이용한 ROS 측정의 경우 추출 물을 처리하였을 때 감소되는 것을 확인하였다. 특히 토복령 추출물의 25 μg/ml에서 음성대조군과 유사 한 형광의 세기를 나타내어 ROS의 생성이 유의적으로 감소한 것을 확인하였다. 본 연구를 통하여 선별된 한약재 소재인 고량강, 유백피, 토복령은 미세먼지로부터 피부를 보호하거나 개선할 수 있는 소재로서 피 부 개선을 위한 안티에이징 제품으로 활용될 수 있을 것으로 사료된다.

This study evaluated cell viability and cytokine release in immortalized human oral fibroblasts (hTERT-hNOFs) and keratinocytes (IHOK) exposed to a dental-impregnated gingival retraction cord. To prepare the extracts, dental gingival retraction cords impregnated with aluminum chloride hexahydrate were immersed in a cell culture medium for 24 h at 37 °C. hTERT-hNOFs and IHOK were cultured for 24 h. The cell culture medium was removed and extracts of the dental gingival retraction cords were added. After incubation with the extract solution, cell viability was evaluated using an MTT assay. The levels of the cytokines IL-1α and IL-8 were measured in the supernatants of each cell type. The cell viability after exposure to the extract solution for 10 min exceeded 70 % in both cell types. The ET50 values for hTERT-hNOF and IHOK were 35.75 and 28.98 min, respectively. For IHOK, the IL-1α level was (5.35 ± 5.22) pg/mL at 10 min, (3.58 ± 5.38) pg/mL at 20 min, and (2.85 ± 4.28) pg/mL at 60 min of exposure (p > 0.05). The IL-8 level in IHOK was (67.16 ± 18.70) pg/mL at 10 min, (78.36 ± 7.50) pg/mL at 20 min, and (111.9 ± 26.10) pg/mL at 60 min of exposure (p > 0.05). Cytokine release was not observed from hTERThNOFs. Based on these results, cell viability and cytokine release were confirmed in cells exposed to the impregnated gingival retraction cord. In addition, the application of the extracts to hTERT-hNOF and IHOK during the actual contact time and determination of ET50 may be beneficial for evaluating the biocompatibility of dental-impregnated gingival retraction cords.

산화적 스트레스는 세포 및 조직 손상을 통해 피부의 탄력 및 보습 기능 저하, 피부 노화 촉진 을 비롯한 다양한 피부질환을 일으킨다. 본 연구의 목적은 인간 피부각질세포 (HaCaT keratinocyte)에서 산화적 스트레스에 대한 붉은 토끼풀 추출물의 효능을 검토하여, 피부에 효과적으로 사용할 수 있는 기능 성 소재로서의 활용 여부를 확인하고자 하였다. 본 연구에서는 붉은 토끼풀 추출물이 인간 피부각질세포에 서 산화적 스트레스에 따른 세포사를 억제시키는 것을 확인하여, 이를 조절하는 보호기전을 규명하였다. 이는 붉은 토끼풀 추출물이 Caspase-3 비활성, 세포사 촉진단백질 Bax 발현 억제, 세포생존 촉진단백질 Bcl-2 발현 증가 및 MAPK 신호전달계 단백질의 인산화 억제를 통해 H2O2에 의해 유도된 산화적 스트레 스를 보호할 수 있다는 것을 확인하였다. 따라서 붉은 토끼풀 추출물은 피부의 산화적 손상을 감소시키는 유용한 소재로 평가되며, 이는 피부보호 및 미용을 위한 다양한 제품 및 산업에 활용 가능성이 높은 것으로 판단된다.

Repetitive or excessive exposure to ultraviolet (UV) radiation causes oxidative stress-mediated skin photoaging through the overproduction of reactive oxygen species. Actinidia polygama is known as a medical plant used in oriental medicine for treating several diseases such as abdominal pain, stroke and rheumatoid arthritis. Recently, it was reported that A. polygama extract had anti-wrinkle and skin hydrating properties in ultraviolet B (UVB)-exposed hairless mice. However, the molecular biological mechanism of this extract on alleviating skin photoaging is still unknown. Therefore, we investigated the anti-photoaging effects of PB203, which is the powder of A. polygama extract, in the in vivo and in vitro photoaging models. First, PB203 showed 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) and 2,2-diphenyl-1-picrylhydrazyl radical scavenging activities due to the presence of anti-oxidant components including flavonoids and polyphenols. In UVB-irradiated hairless mice, oral administration of PB203 (100 mg/ kg) significantly improved wrinkle formation, skin dehydration, elasticity and skin barrier function by decreasing the levels of matrix metalloproteinases (MMPs) and increasing those of collagen I, filaggrin, involucrin and loricrin. Especially, the reduced production of p-p38, p-c-Jun and p-c-Fos by PB203 reversed the elevated levels of MMPs mediated by UVB exposure, resulting in the upregulation of collagen I expression. Consistent with these animal data, PB203 remarkably enhanced the mRNA expression of collagen I, filaggrin, involucrin and loricrin, while suppressed that of MMPs in UVB-irradiated HaCaT cells. And PB203 increased the wound recovery rate of cells by promoting their proliferation and migration. Moreover, PB203 significantly recovered the activity of superoxide dismutase inhibited by UVB in both mice and cells. In conclusion, PB203, which protects skin from UVB-induced photodamage by exerting antioxidant properties, can be considered to have sufficient potential as a functional ingredient or therapeutic agent improving skin photoaging and related skin symptoms.

This study aimed to investigate whether neurotransmitter receptors in the nervous system were also expressed in oral keratinocytes. Expressions of various neurotransmitter receptor genes in immortalized mouse oral keratinocyte (IMOK) cells were examined by reverse transcriptase polymerase chain reaction. IMOK cells expressed calcitonin gene-related peptide (CGRP) receptor subunit genes Ramp1 and Ramp3 and glutamate receptor subunit genes Grina , Gria3 , Grin1 , Grin2a , and Grin2d . Moreover, IMOK cells expressed Adrb2 and Chrna5 that encode beta 2 adrenergic receptor and cholinergic receptor nicotinic alpha 5 for sympathetic and parasympathetic neurotransmitters, respectively. The expression of Bdkrb1 and Ptger4 , which encode receptors for bradykinin and prostaglandin E2 involved in inflammatory responses, was also observed at low levels. Expressions of Ramp1 and Grina in the mouse gingival epithelium were also confirmed by immunohistochemistry. When the function of neurotransmitter receptors expressed on IMOK cells was tested by intracellular calcium response, CGRP, glutamate, and cholinergic receptors did not respond to their agonists, but the bradykinin receptor responded to bradykinin. Collectively, oral keratinocytes express several neurotransmitter receptors, suggesting the potential regulation of oral epithelial homeostasis by the nervous system.

본 연구에서는 기능성 화장품의 소재로서의 금전초(Lysimachia christinae Hance)의 항산화와 항주름 효과를 조사하였다. 최근 천연물의 주름 개선 개발의 연구가 지속적인 관심을 받고 있어 본 연구를 통해 활성산소종(reactive oxygen species, ROS) 생성과 pro-collagen 합성 및 MMPs의 연관성에 대해 알아보았다. 금전초는 70% 에탄올(LcHE)과 열수(LcHW)로 각각 추출하여 실험을 진행하였다. HaCaT cells에서 LcHE가 LcHW보다 ROS 저해효능이 더 우수하고 세포독성 결과 250 μg/mL 농도 까지 독성을 보이지 않아 LcHE를 선택하여 주름 개선 소재연구를 진행하였다. pro-collagen 합성실험을 통하여 UVB에 의해 감소된 type-1 pro-collagen의 합성 활성을 유의미하게 확인하였다. Western blot 실험을 통하여 피부세포에서 UVB에 의해 유도된 MMPs 중 MMP-1 -3 -9의 증가를 억제함을 확인하였으며, Real time PCR을 통하여 상위단계인 mRNA levels에서도 MMP-1, MMP-2, MMP-3, MMP-9의 mRNA levels가 농도 의존적으로 유의미한 감소를 보여 추출물의 효능을 확인하였다. 위의 실험결과에 따라 UVB에 의한 주름생성과 피부 광노화를 효과적으로 예방할 수 있는 화장품의 천연소재로서의 이용이 기대된다.

Immortalization is an essential process of the transformation of cells to a neoplastic growth. High risk human papillomavirus (hrHPV) infection has been the major cause of head and neck squamous cell carcinoma (HNSCC). The aim of this study was to search for a novel pathway causing immortalization in HPV16 E6/E7 transfected immortalized oral keratinocytes (IHOK). hrHPV integration sites were identified through DNA sequencing. HPV16 E6/E7 genes were integrated into 1q32.2, 12q21.2, 15q15.2, and 19q13.43 in IHOKs. Array-CGH was conducted to examine the deranged sites of the genes of IHOK. Of the 587 amplification genes, 70 genes were resided on chromosome 20. We selected PLAGL2 and MAPRE1 as the most amplified genes. PLAGL2 and MAPRE1 mRNA showed higher expression in IHOK than in normal keratinocytes. Knockdown of MAPRE1 significantly reduced telomerase activity. The analysis using a public database substantiated our data, showing the amplification of chromosome 20 and MAPRE1. In conclusion, our results suggest that MAPRE1 could play a crucial role in activating telomerase activity in hrHPV-infected cells. This finding may provide basic data to develop a novel target therapy for hrHPV-related HNSCC.

Vinca alkaloids from plant Vinca minor have been investigated for their effects of tyrosinase inhibition, stimulation of ROS generation and increasement of cell migration activity. The methanolic crude extract and the water-soluble fraction exhibited IC50 value of 3.1 mg/mL and 2.1 mg/mL. Vinca minor extract treatment significantly increased ROS levels in HaCaT cells, in a concentration-dependent manner. Treatments of Vinca minor extract led to increase wound closure when compared with non-treatment. Low dose (0.1% or 0.3%) of extracts have not significantly affected, compared with that in controls. By contrast, 0.5% extract have dramatic effect on wound healing activity of keratinocytes. Effects of Vinca minor extract in a filter-based cell mobility assay appear similar to that of wound closure assay, which suggests that the Vinca minor extract have wound healing effects on skin.

Mushroom is known for anti-inflammatory and anti-oxidative potential. This study provides evidence that theinhibitory effect of mushroom on the expression of pro-inflammatory cytokines in human keratinocytes, HaCaT cells. To definethe underlying mechanisms of action, tumor necrosis factorα/IFNγ-activated human keratinocytes model was used. Mushroomsignificantly inhibited the expression of cytokines in HaCaT cells. Taken together, the results demonstrate that mushroom inhibitedinflammtion, suggesting that mushroom (DW extract: Grifola frondosa Cordyceps militaris), (Ethanol extract: Ganoderma lucidum,Lentinus edodes, Cordyceps militaris, Flammulina velutipes) might be a candidate for the treatment of skin inflammation.

The aim of this study was to determine the beneficial effect of propofol on human keratinocytes that have undergone hypoxia reoxygenation (H/R) injury and to investigate whether autophagy is associated with the protective mechanism. Thus, we evaluated how propofol influences the intracellular autophagy and apoptosis during the H/R process in the HaCaT cells. The cultured human keratinocyte cells were exposed to 24 h of hypoxia (5% CO2, 1% O2, 94% N2) followed by 12 h of reoxygenation (5% CO2, 21% O2, 74% N2). The experiment was divided into 4 groups: (1) Control=Normoxia ; (2) H/R=Hypoxia Reoxygenation ; (3) PPC+H/R=Propofol Preconditioning+Hypoxia Reoxygenation; (4) 3-MA+PPC+ H/R=3-MA-Methyladenine+Propofol Preconditioning+ Hypoxia Reoxygenation. In addition, Western blot analysis was performed to identify the expression of apoptotic pathway parameters, including Bcl-2, Bax, and caspase 3 involved in mitochondrial-dependent pathway. Autophagy was determined by fluorescence microscopy, MDC staining, AO staining, and western blot. The H/R produced dramatic injuries in keratinocyte cells. In our study, the viability of Propofol in H/R induced HaCaT cells was first studied by MTT assay. The treatment with 25, 50, and 100 μM Propofol in H/R induced HaCaT cells enhanced cell viability in a dose-dependent manner and 100 μM was the most effective dose. The Atg5, Becline-1, LC3-II, and p62 were elevated in PPC group cells, but H/R-induced group showed significant reduction in HaCaT cells. The Atg5 were increased when autophagy was induced by Propofol, and they were decreased when autophagy was suppressed by 3-MA. These data provided evidence that propofol preconditioning induced autophagy and reduced apoptotic cell death in an H/R model of HaCaT cells, which was in agreement with autophagy playing a very important role in cell protection.

A 67 years old female showed diffuse erosive ulceration at left buccal mucosa. She had received tegretol to treat the patient’s pain and anxiety of trigeminal neuralgia for 18 months. Otherwise her medical history was nonspecific. Under the clinical diagnosis of lichen planus she received anti-inflammatory therapies using antibiotics and steroid ointment, which were not effective. Consequently her oral ulceration was gradually expanded and aggravated. In the biopsy examination mucosa epithelium was irregularly keratinized and focally detached from underlying connective tissue by thin cleft spaces, accompanied with inflammatory cell infiltration into the subepithelial area. The epithelium was generally acanthomatous with short rete ridges. Many spots of acantholysis were found in the basal and suprabasal layers of epithelium, into which melanocytes were migrated. Particularly, many keratinocytes not only in the spinous layer but also in the suprabasal layer contained atypical keratohyalin granules in their cytoplasms. In the immunohistochemistry the epithelium was rarely positive for PCNA and IgK, but strongly positive for HSP-70, and many keratinocytes showed strong positive reaction of lysozyme in their cytoplasms. Taken together, with the characteristic cytotoxic changes of keratinocytes, which are usually found in the oral epithelium damaged by certain drug abuse, the present case of pemphigus-like oral lesion was diagnosed as drug-induced pemphigus caused by long time intake of tegretol, carbamazepine derivative. The acute oral drug-induced pemphigus should be differentially diagnosed from oral lichen planus, recurrent aphthous ulceration, oral leukoplakia, candidiasis, autoimmune pemphigus, etc., in order to treat properly in the absence of biohazards of systemic therapeutic drugs

Propionibacterium acnes (P. acnes) cause an inflammatory acne that plays an important role in the pathogenesis of acne by inducing inflammatory mediators. Bee venom therapy has been used in oriental medicine for the relief of pain and the treatment of inflammatory diseases. However, a direct effect of bee venom in skin inflammation has not been established. The purpose of this study was to investigate anti-inflammatory properties of bee venom in skin inflammation stimulated by heat-killed P. acnes using human keratinocytes and monocytes cell line. P. acnes stimulates the production of proinflammatory cytokines such as interleukins-1β, -8, interferon-γ and tumor necrosis factor-α in HaCaT and THP-1 cells. Bee venom effectively inhibits the secretion of IL-1β, IL-8, IFN-γ, and TNF-α. P. acnes treatment activates the expression of TLR2, which results in IL-8 expression. However, bee venom treatment reduces the expression of TLR2 and IL-8. Based on these results, bee venom has effects on anti-inflammatory activity against P. acnes in HaCaT and THP-1 cells.

Rushton bodies are known to be the aberrant keratinization and calcification in the epithelium of odontogenic cyst, which are similar to the features of calcifying odontogenic cyst and pilomatricoma. However, the pathogenetic mechanism of keratinization and calcification of Rushton bodies has not been clearly elucidated. Here, a case of Rushton bodies found in dentigerous cyst was examined by immunohistochemical method using antisera of PCNA, pAKT, HIF, PIM1, and PARP. The globular keratinization in lamellate fashion showed weak birefringency under polarizing microscope, and the Rushton bodies frequently underwent the dystrophic calcification. The polygonal keratinocytes of Rushton bodies were strongly positive for HIF and PARP, and the cyst epithelium was diffusely positive for pAKT and PIM1. Particularly, the cyst epithelium was hyperplastic and focally invaginated into cyst wall with positive reaction of PCNA. These findings may indicate the active response of odontogenic epithelium against the apoptotic stress of the cyst, producing the globular keratinization and irregular calcification in the polygonal keratinocytes. Therefore, it is presumed that the lamellate keratinization and dystrophic calcification of Rushton bodies are aberrant products of retrograding keratinocytes slowly undergoing apoptotic progresses similar to the phenomena of the ghost cells in calcifying odontogenic cyst and pilomatricoma, and also may have a potential for oncogenic proliferation.

Cellular microenvironment is an essential issue for regulating epithelial characteristics through the alteration of intricate signaling pathways and intercellular communications in different cell types. Thus, microenvironment influences tumor initiation, progression, and metastasis. This study aimed to investigate the relationship between microenvironment and epithelial property in HPV16 E6/E7-immortalized human oral keratinocytes (IHOKs). To investigate characteristics of IHOK cultured in different media, two media were used, which included keratinocyte growth media (KGM), F-medium composed of 3:1 ratio of DMEM and F-12 (P media) supplemented with 10% FBS and 1% penicillin/streptomycin. Proliferative property and invasive and migratory activity were observed. As results, proliferating activities of IHOK in different culture condition were changed. Likewise, migratory and invasive activities were also different depending on media types. These results suggest that cellular microenvironment can affect modification of biological properties of epithelial cells.

MicroRNAs (miRNAs) are small non-coding RNAs that mediate gene expression at the post-transcriptional level by degrading or repressing targeted mRNAs. These molecules are about 21-25 nucleotides in length and exert their effects by binding to partially complementary sites in mRNAs, predominantly in the 3'-untranslated region (3'-UTR). Recent evidence has demonstrated that miRNAs can function as oncogenes or tumor suppressors through the modulation of multiple oncogenic cellular processes in cancer development, including initiation, cell proliferation, apoptosis, invasion and metastasis. In our present study, we examined the expression profile of miRNAs related to oral cancer cell growth inhibition using normal human oral keratinocytes (NHOK) and YD-38 human oral cancer cells. By miRNA microassay analysis, 40 and 31 miRNAs among the 1,769 examined were found to be up- and down-regulated in YD-38 cells compared with NHOK cells, respectively. Using qRT-PCR analysis, the expression levels of miR-30a and miR-1246 were found to be increased in YD-38 cells compared with NHOK cells, whereas miR-203 and miR-125a were observed to be decreased. Importantly, the overexpression of miR-203 and miR-125a significantly inhibited the growth of YD-38 cells. This finding and the microarray data indicate the involvement of specific miRNAs in the development and progression of oral cancer.

Glutamate-induced oxidative injury contributes to neuronal degeneration in many central nervous system (CNS) diseases, such as Parkinson’s disease, Alzheimer’s disease, epilepsy and ischemia. Inducible heme oxygenase (HO)-1 acts against oxidants that are thought to play a role in the pathogenesis of these diseases. The EtOH extracts of Viola mandshurica (NNMBS274), Viola patrinii (NNMBS275) and Viola papilionacea Pursh (NNMBS276), origin plants of Violae Herba, showed the potent neuroprotective effects on glutamate-induced neurotoxicity. Among them, NNMBS275, the extract of V. patrinii possessed the protective effects against glutamate toxicity by inducing the expression of heme oxygenase (HO)-1 in the mouse hippocampal HT22 cells. These results suggest that extracts of V. patrinii could be the effective candidates for the treatment of ROS-related neurological diseases. Furthermore, it is suggested that the protective effects of V. patrinii extract due to inducing the expression of HO-1 asAs the exfoliated keratinocytes (EKs) in oral mucosa are aging and degenerating cells, of which cytoplasms are almost replaced by cross-linked keratin materials. Consequently, the EKs become apoptotic with nuclear lysis. A question is arisen what is the biological role of these EKs in oral cavity? Are they simply degrading as aging keratinocytes or do they have some essential function still remained in the exfoliated status? The buccal smear samples from ten healthy adult subjects were observed under scanning electron microscope. On the outer surface of the EKs the features of bacterial adhesion were explored. The microorganisms attached on the surface of EKs were much deformed, shrunken and teared. Only a few microorganisms were found on the EK surface, aggregated focally. The attached microorganisms were gradually fused on the cell membrane of EKs, and subsequently endocytosed. Resultantly, many round endocytotic concave cavities similar size to the cocci were remained on the surface of EKs similar to the sequels of caveolae endocytosis. These data indicate that the degenerating EKs can actively engulf microorganisms attached on their cell surface via the processes of caveolae endocytosis. Therefore, it is presumed that the oral EKs still play a role for endocytotic scavenging of oral microorganisms using the denatured cell bodies themselves, which become highly adherent to oral microorganisms and still function for caveolae endocytosis in mixed saliva environment. an antioxidant/cytoprotective target