미세조류 유래 astaxanthin의 항염증 및 항산화 효과

Abstract

Astaxanthin (ATX)은 다양한 생명체에서 생성되는 카로티노이드 색소이다. 본 연구에서는 ATX가 RAW264.7 cell에서 LPS에 의한 inducible nitric oxide synthase (iNOS), nitric oxide (NO), 염증성 사이토카인, nuclear factor-kappa B (NF-<TEX>${\kappa}B$</TEX>)와 reactive oxygen species (ROS)의 생성을 억제 시키는 지 또한, superoxide radical 소거능이 있는 지를 조사하였다. iNOS와 NF-<TEX>${\kappa}B$</TEX>는 immunoblot analysis로, interleukin (IL)-6와 tumour necrosis factor-<TEX>${\alpha}$</TEX> (TNF-<TEX>${\alpha}$</TEX>)는 ELISA 법으로 분석하였다. NO 양은 nitrite의 양을 측정하였고, ROS는 2',7'-dichlorodihydrofluorescin diacetate (DCFH-DA) 법으로 superoxide radical 소거능은 superoxide radical scavenging activity assay로 검증하였다. 100 <TEX>${\mu}M$</TEX>의 ATX 농도에서 LPS로 유도된 NO, IL-6 및 TNF-<TEX>${\alpha}$</TEX> 같은 염증성 사이토카인의 생성 뿐만 아니라 iNOS 및 NF-<TEX>${\kappa}B$</TEX>의 발현도 억제되었다. 특히, IL-6 및 TNF-<TEX>${\alpha}$</TEX> 생성에 있어 ATX의 최대 억제율은 각각 65.2% 및 21.2% 이었으며 LPS로 유도된 NF-<TEX>${\kappa}B$</TEX>의 전사활성을 억제하였다. 이러한 현상은 세포질에서 핵으로 NF-<TEX>${\kappa}B$</TEX>의 전위를 억제하는 것과 관련이 있다. 또한, 25-100 <TEX>${\mu}M$</TEX>의 ATX 농도에서 세포 내 ROS 생성을 억제하였으며, 5 mg/ml 농도의 ATX는 동일농도의 <TEX>${\alpha}$</TEX>-tocopherol에 비해 superoxide radical 소거능이 1.33배 높았다. 이러한 결과들은 ATX가 대식세포에서 ROS 생성 및 NF-<TEX>${\kappa}B$</TEX> 활성을 저해하므로 iNOS의 발현, NO 및 염증성 사이토카인의 생성을 억제하며, 또한 우수한 superoxide radical 소거능을 보유한다는 것을 나타내었다. 결론적으로, ATX가 항염증제 및 항산화제로서 유용하게 사용될 수 있을 것으로 사료된다. Astaxanthin (ATX) is a red-orange carotenoid pigment that occurs naturally in a wide variety of living organisms. In this study we investigated the inhibitory effects of ATX on the induction of inducible nitric oxide synthase (iNOS), nitric oxide (NO), proinflammatory cytokines, nuclear factor-kappa B(NF-<TEX>${\kappa}B$</TEX>) and reactive oxygen species (ROS) in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. In addition, we tested the superoxide radical scavenging activity of ATX by scavenging assay. iNOS and NF-<TEX>${\kappa}B$</TEX> expressions were determined by immunoblot analysis. Interleukin (IL)-6 and tumour necrosis factor-<TEX>${\alpha}$</TEX> (TNF-<TEX>${\alpha}$</TEX>) were assayed by ELISA. NO production was monitored by measuring the amount of nitrite. ROS was examined by using the 2', 7'-Dichlorodihydrofluorescin diacetate (DCFH-DA) method. At a concentration of 100 <TEX>${\mu}M$</TEX>, ATX inhibited the expression level of LPS-induced NF-<TEX>${\kappa}B$</TEX>, as well as the production of LPS-induced NO and proinflammatory cytokines (IL-6 and TNF-<TEX>${\alpha}$</TEX>), by suppressing iNOS expression. In particular, the maximal inhibition rate of IL-6 and TNF-<TEX>${\alpha}$</TEX> production by ATX (100 <TEX>${\mu}M$</TEX>) was 65.2----- and 21.2-----, respectively. In addition, ATX inhibited the LPS-induced transcriptional activity of NF-<TEX>${\kappa}B$</TEX>, and this was associated with suppressing the translocations of NF-<TEX>${\kappa}B$</TEX> from the cytosol to the nucleus. Moreover, at various concentrations (25-100 <TEX>${\mu}M$</TEX>), ATX inhibited the intracellular level of ROS. At a concentration of 5 mg/ml, the superoxide radical scavenging activity of ATX was 1.33 times higher than <TEX>${\alpha}$</TEX>-tocopherol of the same concentration. These results showed that ATX inhibited the expression of iNOS and the production of NO and proinflammatory cytokines resulting from ROS production and NF-<TEX>${\kappa}B$</TEX> activation in macrophages. Furthermore, ATX was found to be more effective in superoxide radical scavenging activities compared to <TEX>${\alpha}$</TEX>-tocopherol. These findings are expected to strengthen the position of ATX as anti-inflammatory medicine and antioxidant.