[논문]지모 주정 추출물이 염증으로 손상된 피부장벽 기능에 미치는 영향

정미림; 이규영; 홍철희

doi:10.6114/jkood.2018.31.2.011

[국내논문] 지모 주정 추출물이 염증으로 손상된 피부장벽 기능에 미치는 영향
Effects of Ethanol Extracts of Anemarrhena asphodeloides on Skin Barrier Function by Inflammation 원문보기

韓方眼耳鼻咽喉皮膚科學會誌 = The journal of Korean Medicine Ophthalmology & Otolaryngology & Dermatology, v.31 no.2, 2018년, pp.11 - 23

정미림 (상지대학교 한의과대학 안이비인후피부과학교실) , 이규영 (상지대학교 한의과대학 안이비인후피부과학교실) , 홍철희 (상지대학교 한의과대학 안이비인후피부과학교실)

초록
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목적 : 본 연구에서는 $TNF-{\alpha}$와 $IFN-{\gamma}$로 자극한 인간피부각질형성세포 (HaCaT keratinocytes) 모델을 사용하여 지모가 피부장벽 기능에 미치는 영향을 알아보고자 하였다. 방법 : MTT assay를 통하여 지모 주정(70% 에탄올) 추출물 (EAA)이 HaCaT keratinocytes의 세포생존율에 미치는 영향을 확인하였으며 wound healing assay를 통해 EAA가 HaCaT 세포의 이주 능력에 영향을 주는지 관찰하였다. 또한 western blot analysis와 qRT-PCR을 통하여 EAA가 $TNF-{\alpha}/IFN-{\gamma}$로 자극한 HaCaT 세포에서 iNOS의 단백질 발현 및 IL-4, IL-13, IL-6의 mRNA 발현, filaggrin의 단백질과 mRNA 발현에 미치는 영향을 조사하였다. 결과 : EAA는 처리 농도 $500{\mu}g/ml$까지 HaCaT keratinocytes의 세포생존율에 영향을 미치지 않았다. EAA는 wound healing assay에서 HaCaT 세포의 이주 능력을 증가시켰으며, $TNF-{\alpha}/IFN-{\gamma}$로 자극한 HaCaT 세포에서 iNOS의 단백질 수준을 감소시켰다. 또한 EAA가 IL-4, IL-13, IL-6의 mRNA 발현을 억제하는 것 역시 확인할 수 있었다. 뿐만 아니라 EAA는 $TNF-{\alpha}/IFN-{\gamma}$ 자극에 의해 감소했던 filaggrin을 단백질과 mRNA 수준에서 회복시켰다. 결론 : EAA가 HaCaT 세포에서 Th2 type cytokines, pro-inflammatory cytokine의 억제와 filaggrin 회복을 통해 피부장벽 기능 손상에 대한 억제활성을 갖는 것을 확인하였으며, 이를 통해 EAA가 염증으로 인해 손상된 피부장벽 기능 개선에 효과적일 것으로 사료된다.

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문제 정의

The defects of epidermal barrier function is considered as a key event in allergic sensitization²⁾. This study was carried out to investigate the effects of extracts of Anemarrhena asphodeloides (EAA) on skin barrier which has these important functions.
asphodeloides (EAA) on skin barrier function remains unclear. In this study, we examined the effects of EAA on the skin barrier function in HaCaT keratinocytes.

가설 설정

In this study, we determined the mechanisms of the protective effects of EAA on skin barrier function in TNF-α/IFN-γ stimulated HaCaT keratinocytes.

제안 방법

The wound closure distance was evaluated using an Eclipse TE2000U inverted microscope with twin CCD cameras (magnification, ×200; Nikon, Tokyo, Japan; n = 3).
During wound re-epithelialisation stage, skin lesions close the wound and remodel the cytoskeleton by enhancing the proliferation and migration of keratinocytes¹²⁾. To evaluate whether EAA can influence keratinocytes migration, we assessed the effects of EAA on HaCaT keratinocytes motility using wound healing assay. The migration of HaCaT keratinocytes was evaluated by wound closure distance.

대상 데이터

The roots of A. asphodeloides were purchased in Nanum herb (Yeongchen, Gyeongbuk, Korea). Dried roots of A.
The HaCaT keratinocytes were provided by Prof. Seung-Heon Hong (Wonkwang University, Republic of Korea). The cells were cultured in DMEM supplemented with 10% FBS, penicillin (100U/㎖), and streptomycin (100㎍/㎖) in a 37°C and 5% CO₂ incubator.

데이터처리

Data were analyzed using one-way analysis of variance (ANOVA) with Dunnett’s test, and p-values <0.05 were considered statistically significant.
05 were considered statistically significant. All statistical analyses were performed using GraphPad Prism (version 5.00 for Windows, San Diego, CA, USA).

이론/모형

All primer sequences are shown in (Table 1). Gene expression was calculated according to the comparative threshold cycle (Ct) method. The results are expressed as the ratio of the optical density of the target locus to GAPDH.
To measure the cytotoxic effects of EAA on HaCaT keratinocytes, cell viability was investigated by using MTT assay. Treatment with different concentration of EAA ranging from 7.
HaCaT keratinocytes were treated with different concentrations of EAA for 24 hrs. and their viability were determined using MTT assay. (The data shown represent mean ± SD of three independent experiments.

성능/효과

Likewise, excessive generation of Prostaglandin E2 (PGE2) by cyclo-oxygenase-2 (COX-2) is a crucial physiological factor contributing inflammation development³³⁾. In this study, it is found that treatment with EAA increased the migration of HaCaT keratinocytes and inhibited expression levels of iNOS proteins. It is possible to assume that EAA facilitates the wound healing in skin barrier through inhibition of excessive expression of NO.
1. The inhibitory effects of EAA is observed in the expression of Th2 type cytokine, such as IL-4 and IL-13 and pro-inflammatory cytokine such as IL-6 at mRNA expression levels.
2. EAA increased the migration of HaCaT keratinocytes.

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