[논문]섬유아세포에서 세포 활성 촉진 및 광노화 억제 효능을 보이는 신규 헵타펩타이드

이응지; 강한아; 황보별; 정용지; 김은미

doi:10.15230/scsk.2022.48.2.157

[국내논문] 섬유아세포에서 세포 활성 촉진 및 광노화 억제 효능을 보이는 신규 헵타펩타이드
A Novel Heptapeptide that Promotes Cellular Activity and Inhibits Photoaging in Fibroblasts 원문보기

大韓化粧品學會誌 = Journal of the society of cosmetic scientists of Korea, v.48 no.2, 2022년, pp.157 - 167

이응지 ((주)케어젠 중앙연구소) , 강한아 ((주)케어젠 중앙연구소) , 황보별 ((주)케어젠 중앙연구소) , 정용지 ((주)케어젠 중앙연구소) , 김은미 ((주)케어젠 중앙연구소)

초록
AI-Helper

본 연구에서는 7 개의 아미노산으로 이루어진 헵타펩타이드의 섬유아세포 활성 증가 및 광노화 조건에서의 세포 손상 억제 효과를 확인하였다. 실험 결과 헵타펩타이드 처리 시 섬유아세포 증식 및 세포외기질(extracellular matrix, ECM) 구성 인자의 발현이 증가되었다. 그리고 자외선 A (ultraviolet A, UVA) 조사에 의해 유도된 광노화조건에서 감소된 세포 생존율이 헵타펩타이드에 의해 증가되었고, UVA 조사에 의해 유도된 세포 사멸, 기질금속단백질분해효소-1(matrix metalloproteinases-1, MMP-1) 발현 및 세포 내 활성산소종(reactive oxygen species, ROS) 수준이 헵타펩타이드에 의해 감소되었다. UVA 조사 시 나타나는 transforming growth factor-β (TGF-β)/smad 기전 억제와 그에 따른 ECM 구성 인자 발현 감소 또한 헵타펩타이드에 의해 회복되었다. 또 다른 광노화 유도 조건으로 heat shock을 주었고 헵타펩타이드를 전 처리 하였을 때 heat shock에 의한 mitogen-activated protein kinase (MAPK) 인산화 및 MMP-1 발현이 억제됨을 확인할 수 있었다. 이 결과를 종합해 볼 때, 본 연구의 헵타펩타이드는 섬유아세포의 활성을 촉진하며, 광노화 유도 모델로 사용된 UVA 조사 및 heat shock 조건에서도 세포 내 ROS 억제 효과를 보여 세포 손상에 대한 회복 및 보호 효과를 나타내는 것으로 보인다. 이러한 진피 보호 효과를 갖는 헵타펩타이드는 향 후 신규 화장품 소재로 응용될 수 있을 것으로 기대된다.

Abstract ▼ AI-Helper

In this study, we investigated the effects of heptapeptide on cellular activation and inhibition of cellular damage induced by photoaging condition in NIH3T3 fibroblasts. Cell proliferation and extracellular matrix (ECM) expression were induced by heptapeptide. The reduced cell viability under photoaging condition through ultraviolet A (UVA) irradiation was increased by heptapeptide. And UVA-induced apoptosis, matrix metalloproteinases-1 (MMP-1) expression, and reactive oxygen species (ROS) level were decreased by heptapeptide. In addition, the inhibition of transforming growth factor-β (TGF-β)/smad signaling under UVA irradiation which resulting in reduction of ECM expression was also recovered by heptapeptide. We also tested the effect of heptapeptide under another photoaging condition through heat shock, and pre-treatment of heptapeptide prevented the phosphorylation of mitogen-activated protein kinase (MAPK) and MMP-1 expression induced by heat shock. From these results, it has been shown that the heptapeptide has protective effects on fibroblasts through the up-regulation of cellular activity and through the decreasing of intracellular ROS level induced by UVA irradiation or heat shock. It is expected that the dermal protection effect of heptapeptide can be applied as a new cosmetic material in the future.

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표/그림 (7)

그림 Figure 1. Analysis of synthetic heptapeptide. (A) The purity of heptapeptide was confirmed by HPLC analysis and it was more than 96%. (B) The estimated molecular weight of heptapeptide was confirmed by LC-MS/MS analysis.
표 Table 1. Synthesized Heptapeptide
표 Table 2. Primer Sequences
그림 Figure 2. Effects of heptapeptide on NIH3T3 fibroblast proliferation and ECM production. (A) Cells were treated with various concentrations of heptapeptide for 72 h and cell viability was measured by MTT assay. (B) Cells were treated with various concentrations of heptapeptide for 15 min and the levels of phospho-AKT and phospho-ERK were analyzed by western blotting. (C) Cells were treated with various concentrations of heptapeptide for 24 h and the expressions of COLIA1, FN1 and ELN genes were analyzed by RT-PCR. (D) Cells were treated with various concentrations of heptapeptide for 72 h and pro-collagen type I α1 protein level was analyzed by ELISA. The results were expressed as the mean ± SD from the three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.
그림 Figure 3. Inhibitory effects of heptapeptide on UVA-induced damage in NIH3T3 fibroblasts. (A) Cells were treated with various concentrations of heptapeptide for 72 h immediately after UVA exposure. Cell viability was measured by MTT assay. (B)(C) Cells were treated with various concentrations of heptapeptide for 24 h immediately after UVA exposure. Protein levels of bax, cleaved caspase-3 and MMP-1 were analyzed by western blotting. (D) Cells were treated with various concentrations of heptapeptide for 24 h immediately after UVA exposure. DCF-DA-stained cells were analyzed by flow cytometry. The results were expressed as the mean ± SD from the three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.
그림 Figure 4. Recovery effects of heptapeptide on TGFR signaling pathway inhibited by UVA irradiation in NIH3T3 fibroblasts. (A) Cells were treated with various concentrations of heptapeptide for 5 h immediately after UVA exposure. Protein levels of phospho-smad2 and smad7 were analyzed by western blotting. (B) Cells were treated with various concentrations of heptapeptide for 6 h immediately after UVA exposure. The expressions of COLIA1, FN1 and ELN genes were analyzed by RT-PCR. The results were expressed as the mean ± SD from the three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.
그림 Figure 5. Protecting effects of heptapeptide on heat shock-induced damage in NIH3T3 fibroblasts. (A) Cells were treated with various concentrations of heptapeptide for 24 h followed by 44 ℃, 30 min heat shock. After 15 min incubation in serum-free DMEM, cells were harvested and protein levels of phospho-ERK, phospho-JNK and phospho-p38 were analyzed by western blotting. (B) Cells were treated with various concentrations of heptapeptide for 24 h followed by 44 ℃, 30 min heat shock. After 8 h incubation in serum-free DMEM, cells were harvested and MMP-1 protein level was analyzed by western blotting.

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