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LPS가 처리된 RAW 264.7 대식세포에서 Nrf2/HO-1 경로 조절을 통한 매실 추출물의 NO 생성 억제 효과
강혜주,최은옥,정진우,박신형,박철,홍수현,신순식,정재훈,최영현,Kang, Hye-Joo,Choi, Eun Ok,Jeong, Jin-Woo,Park, Shin-Hyung,Park, Cheol,Hong, Su Hyun,Shin, Soon Shik,Cheong, Jae-Hun,Choi, Yung Hyun 대한한의학방제학회 2017 大韓韓醫學方劑學會誌 Vol.25 No.1
Objectives : The fruit of Prunus mume Siebold & Zucc. has been used as an alternative medicine and functional food in Korea and Japan for preventive and therapeutic purposes. However, its molecular actions and mechanism on anti-inflammatory activity have not been clearly investigated. The aim of this study was to clarify the anti-inflammatory activity of the ethanol extract of P. mume fruit (EEPM) in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells, and sought to understand the associated molecular mechanisms. Methods : Cytotoxicity was assessed by an MTT assay. The amount of nitric oxide (NO) production was determined by nitrite assay. The mRNA expression of inducible nitric oxide synthase (iNOS) was analyzed by RT-PCR. In addition, expression levels of iNOS, nuclear factor-erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) protein were detected by Western blotting. Results : Our data indicated that EEPM inhibited NO production in LPS-stimulated RAW264.7 cells in a concentration-dependent manner. At the mRNA and protein levels, EEPM suppressed LPS-induced iNOS expression. On the other hand, EEPM markedly enhanced HO-1 expression, which was associated with an induction and nuclear translocation of Nrf2. Moreover, the inhibitory effect of EEPM against LPS?induced NO production was significantly enhanced by hemin, a HO-1 inducer; however, EEPM's effect on the production of NO was abolished by zinc protoporphyrin IX, a HO-1 inhibitor. Conclusion : The results suggest that EEPM can act as a suppressor agent on NO production through an activation of Nrf2/HO-1 signaling pathway, and may be a promising candidate for the treatment of inflammatory diseases.
권다혜(Da Hye Kwon),강혜주(Hye-Joo Kang),최영현(Yung Hyun Choi),정경태(Kyung Tae Chung),이종환(Jong Hwan Lee),강경화(Kyung Hwa Kang),현숙경(Sook Kyung Hyun),김병우(Byung Woo Kim),황혜진(Hye Jin Hwang) 한국생명과학회 2016 생명과학회지 Vol.26 No.1
왕느릅나무(Ulmus macrocarpa)의 껍질을 말린 유근피는 오랫동안 부종, 감염 및 염증 제어의 목적으로 사용되어져 왔음에도 불구하고 잠재적 면역조절 효과에 관해서는 연구가 이루어진 바 없다. 본 연구에서는 전통 약용자원에서 새로운 면역기능 증가 신소재 발굴의 일환으로 유근피 열수 추출물의 면역 조절 효능을 RAW 264.7 대식세포 모델을 이용하여 조사하였다. 이를 위한 대식세포의 활성화 관련 지표로서 NO, TNF-α, IL-1β 및 IL-10의 생성량 변화를 조사하였다. 비록 유근피 추출물이 처리된 RAW 264.7 대식세포에서 IL-1β의 유의적인 유리는 관찰할 수 없었으나, NO, TNF-α 및 IL-10의 생성은 세포독성을 나타내지 않는 범위에서 유근피 추출물 처리 농도 의존적으로 증가되었으며, 이는 또한 iNOS, TNF-α 및 IL-10의 단백질 발현 증가와 연관되어 있었다. 아울러 유근피 추출물은 LPS에 의한 과도한 NO의 생성 억제능도 함유하고 있었으며, 유근피 추출물에 의한 대식세포의 활성화에는 NF-κB와 PI3K/Akt 및 MAPKs 등과 같은 면역 활성을 유도하는 신호전달계의 활성화가 연관되어 있음을 알 수 있었다. 따라서 본 연구의 결과는 유근피 추출물이 대식세포 활성화를 통한 면역 증강제로서의 개발 가능성이 매우 높음을 시사한다. The root bark of Ulmus macrocarpa has been used in traditional medicine for the treatment of various diseases such as edema, infection and inflammation. Nevertheless, the biological activities and underlying mechanisms of the immunomodulatory effects remain unclear. In this study, as part of our ongoing screening program to evaluate the immunomodulatory potential of new compounds from traditional medicinal resources, we investigated the effects of U. macrocarpa water extract (UME) on immune modulation in a murine RAW 264.7 macrophage model. As immune response parameters, the productions of as nitric oxide (NO) and cytokines such tumor necrotic factor (TNF)-α, interleukin (IL)-1β and IL-10 were evaluated. Although the release of IL-1β remained unchanged in UME-treated RAW 264.7 macrophages, the productions of NO, TNF-α and IL-10 were significantly increased, along with the increased expression of inducible NO synthase, TNF-α and IL-10 expression at concentrations with no cytotoxicity. UME treatment also induced the nuclear translocation of nuclear factor κB (NF-κB), and phosphorylation of Akt and mitogen-activated protein kinases (MAPKs) indicating that UME activated macrophages through the activation of NF-κB, phosphoinositide-3-kinase (PI3K)/Akt and MAPKs signaling pathways in RAW 264.7 macrophages. Furthermore, pre-treatment with UME significantly attenuated the production of NO, but not TNF-α, IL-1β and IL-10, in lipopolysaccharidestimulated RAW 264.7 cells suggesting that UME may be useful in preventing inflammatory diseases mediated by excessive production of NO. These findings suggest that the beneficial therapeutic effects of UME may be attributed partly to its ability to modulate immune functions in macrophages.