RAW 264.7 세포에서 말오줌나무 추출물의 iNOS, COX-2 단백질 및 mRNA 발현 억제 효과
Inhibitory Effect of Sambucus sieboldiana var. pendula (Nakai) Extract on the mRNA and Protein Expression of iNOS and COX-2 in Raw 264.7 Cells
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초록
본 연구는 새로운 기능성 화장품 소재를 개발하기 위해 천연물 재료인 말오줌나무 추출물 활용 가능성 연구하였다. 이목적을 이루기 위하여, 말오줌나무의 세포독성효과를 MTT assay를 통해 확인한 결과, 500 μg/ml 농도에서 100% 이상의 세포 생존율을 나타내었다. 항염증 활성을 효과적으로 확인하기 위하여, LPS로 유도된 대식세포 내 NO 생산을 억제하는 효과를 griess의 방법으로 조사하였다. 그 결과 NO의생성이 말오줌나무 추출물의 농도 의존적으로 저해되었음을확인하였다. 말오줌나무 추출물을 LPS로 유도된 RAW 264.7 대식세포에서 전염증성 인자(iNOS, COX-2)들을 생성하여측정하였다. 그 후, iNOS와 COX-2의 단백질 발현 억제 효과를 측정하기 위해 50, 100, 500 μg/ml 농도에서 western blot을 수행하였고, β-actin를 양성대조군으로 사용하였다. iNOS와 COX-2의 mRNA 발현 억제 효과를 측정하기 위해50, 100, 500 μg/ml 농도에서 RT-PCR을 수행하였고, 양성대조군으로 GAPDH를 사용하였다. 결과적으로, western blot으로 iNOS와 COX-2의 단백질 발현 억제 효과를 측정한결과 500 μg/ml 농도에서 각각 31.2%, 54.7%의 감소 효과를보였으며, iNOS, COX-2의 mRNA 발현 억제 효과를 RTPCR로측정한 결과 500 μg/ml 농도에서 각각 72.2%, 89% 정도로 감소하였다. 이러한 결과들을 통해 말오줌나무 추출물은 항염증 효과를 가진 천연물 소재로 활용 가능할 것으로 생각된다.
Abstract
This study examined a new functional cosmetic material possessing application possibility of Sambucus sieboldiana var. pendula (Nakai) (SS) extract. For this, we analyzed the toxic effect of the SS extract on macrophages (RAW 264.7 cells) by performing MTT assay. Results of the MTT assay showed ≥100% cell viability after treatment with 500 μg/ml SS extract. To determine the anti-inflammatory activity of the SS extract, we examined its inhibitory effect on lipopolysaccharide (LPS)-induced NO production in RAW 264.7 cells by performing Griess assay. Result of the Griess assay showed that the SS extract inhibited LPSinduced NO production in a concentration-dependent manner. Next, we examined the effect of the SS extract on the production of proinflammatory factors inducible NOS (iNOS) and cyclooxygenase-2 (COX-2) in LPS-stimulated RAW 264.7 cells. First, we determined the inhibitory effect of 50, 100, and 500 μg/ml SS extract on iNOS and COX-2 protein expression by performing western blot analysis, with β-actin as a positive control. Results of western blotting showed that treatment with 500 μg/ml SS extract decreased iNOS and COX-2 protein expression by 31.2% and 54.7%, respectively. Next, we determined the inhibitory effect of 50, 100, and 500 μg/ml SS extract on iNOS and COX-2 mRNA expression by performing reverse transcription- polymerase chain reaction (PCR), with GAPDH as a positive control. Results of reverse transcription- PCR showed that treatment with 500 μg/ml SS extract decreased the mRNA expression of iNOS and COX-2 by 72.2% and 89%, respectively. These results suggest that the SS extract is a highly valuable natural compound because of its functional components and anti-inflammatory activity.
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