[논문]LPS로 자극한 RAW 264.7 세포에서 중국 연변에 자생하는 약용 식물 에탄올 추출물의 항염증 효과 연구

박예진; 서종환; 길태영; 천세윤; 박인철; 이상우; 차윤엽; 안효진

doi:10.6116/kjh.2018.33.6.71.

LPS로 자극한 RAW 264.7 세포에서 중국 연변에 자생하는 약용 식물 에탄올 추출물의 항염증 효과 연구
Anti-inflammatory Effects of Ethanol Extract of Chinese Medicinal Plants in Yanjin on LPS-stimulated RAW 264.7 Macrophages 원문보기

大韓本草學會誌 = The Korea journal of herbology, v.33 no.6, 2018년, pp.71 - 78

박예진 (상지대학교 한의과대학 약리학교실) , 서종환 (상지대학교 한의과대학 약리학교실) , 길태영 (상지대학교 한의과대학 약리학교실) , 천세윤 (상지대학교 한의과대학 약리학교실) , 박인철 (옌벤대학교 농과대학) , 이상우 (한국생명공학연구원 해외생물소재센터) , 차윤엽 (상지대학교 한의과대학 재활의학교실) , 안효진 (상지대학교 한의과대학 약리학교실)

Abstract ▼ AI-Helper

Objectives : This study was fulfilled to investigate nominee materials as anti-inflammatory agent from ethanol extract of Chinese medicinal plants in Yanjin. Among the 20 candidates, we selected most effective one, the ethanol extract of Cicuta virosa L. (CVL). The mechanism underlying the anti-inflammatory effects of CVL is not clearly identified as yet. Accordingly, we clarified the anti-inflammatory effects of CVL and its underlying molecular mechanisms in LPS-stimulated RAW 264.7 macrophages. Methods : RAW264.7 macrophages were incubated with CVL (12.5, 25, or $50{\mu}M$) and/or lipopolysaccharide (LPS) ($1{\mu}g/m{\ell}$). Cytotoxicity was determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay and the level of nitric oxide (NO) production was measured with Griess reagent. The prostaglandin $E_2$ ($PGE_2$) production was measured with enzyme immunoassay kits and the protein expression of inducible nitric oxide synthase (iNOS) was determined using Western blot analysis. Results : Among the 20 ethanol extract of Chinese medicinal plants of Yanjin tested, CVL significantly reduced the production of NO in a dose-dependent manner via inhibition the protein expressions of iNOS without cytotoxicity on the LPS-stimulated RAW 264.7 macrophages. In addition, CVL also effectively declined the production of $PGE_2$ in LPS-simulated RAW 264.7 macrophages. Conclusions : Taken together, these data presented in this study demonstrate that CVL possesses anti-inflammatory activity by suppressing the production of pro-inflammatory mediators NO and $PGE_2$, and pro-inflammatory protein iNOS expression in LPS-stimulated RAW 264.7 macrophages.

주제어

표/그림 (6)

표 Table 1. Effect of Ethanol extract of Chinese medicinal plants in Yanjin on the cell viability in RAW 264.7 macrophages.
표 Table 2. Effect of Ethanol extract of Chinese medicinal plants in Yanjin on LPS-stimulated NO production level in RAW 264.7 macrophages.
그림 Figure 1. Effect of CVL on the viability of RAW 264.7 cells.
그림 Figure 2. Effect of CVL on production of NO in LPS-stimulated RAW 264.7 cells.
그림 Figure 3. Effect of CVL on production of PGE2 in LPS-stimulated RAW 264.7 cells.
그림 Figure 4. Effect of CVL on iNOS protein expression in LPS-stimulated RAW 264.7 cells.

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제안 방법

(CVL) is a member of the Apiaceae family and the study on anti-inflammatory effect of CVL is not clearly identified as yet. Therefore, in this current study, we explored the anti-inflammatory effects of CVL in RAW 264.7 macrophages, which can be activated with LPS to mimic the situation of inflammation.

대상 데이터

The RAW 264.7 macrophage cell line was obtained from Korea Cell Line Bank (KCLB, Seoul, Republic of Korea). The cells were cultured in DMEM supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (100 ㎍/㎖) in a 37℃ and humidified atmosphere of 5% CO₂.

이론/모형

Cell viability was assessed using the MTT assay. Briefly, RAW 264.
7 cells. The cells were treated with different concentrations of CVL for 24 h, and their viability were determined using MTT assay. Values represent mean ± SD of three independent experiments.

성능/효과

In conclusion, the data presented in this study demonstrate that CVL possesses anti-inflammatory activity via suppression on production of pro-inflammatory mediators NO and PGE₂ and expression of pro-inflammatory protein iNOS in LPS-stimulated RAW 264.7 macrophages.
Therefore, screening of anti-inflammatory materials from natural products through estimation of their suppression effect on NO production has been extensively performed¹⁷⁾. Our results indicated that four plants (Cirsium viassovianum Fisch.ex DC.; CVF, Carex neurocarpa Maxim.; CNM, Cicuta virosa L.; CVL, and Circaea caulescens Kom.; CCK) of them showed strong inhibitory ratio (more than 30%) on NO production (Table 2). Moreover, we selected the ethanol extract of CVL, a most effective one, as anti-inflammatory candidate according to these results of Table 1 and 2.
Statistically significant differences were determined using analysis of variance and Dunnett’s post hoc test, and P-values of less than 0.05 were considered statistically significant.
In table 2, ‘IC50’ is the concentration of the individual plants required to inhibit 50% of NO production and the ‘inhibitory ratio’ is measured to assess the inhibition effects in maximum concentration of individual plants. The data showed that only 4 plants (Cirsium viassovianum Fisch.ex DC.; CVF, Carex neurocarpa Maxim.; CNM, Cicuta virosa L.; CVL, and Circaea caulescens Kom.; CCK) had the inhibitory ratio of more than 30% on NO production. In addition, Cicuta virosa L.
5, 25, or 50 ㎍/㎖). The six concentrations of CVL simultaneously inhibited LPS-stimulated NO production and these results indicated that high concentration (50 ㎍/㎖) of CVL is capable of decreasing NO production in RAW 264.7 macrophages better than NIL. Because NO is synthesized by the iNOS¹⁸⁾ , the iNOS has been also considered as promising targets for testing the capability of molecules against inflammatory disorders¹⁹⁾ .

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