[논문]JAK/STAT 신호전달 경로를 통한 LPS 유도 RAW 264.7 세포의 염증에 대한 노니의 항염증 효과

조범길; 방인석

doi:10.5352/jls.2022.32.2.125

JAK/STAT 신호전달 경로를 통한 LPS 유도 RAW 264.7 세포의 염증에 대한 노니의 항염증 효과
Anti-inflammatory Effect of Morinda citrifolia on LPS-induced Inflammation in RAW 264.7 Cells Through the JAK/STAT Signaling Pathway 원문보기

생명과학회지 = Journal of life science, v.32 no.2, 2022년, pp.125 - 134

조범길 (호서대학교 기초과학연구소) , 방인석 (호서대학교 기초과학연구소)

초록
AI-Helper

본 연구는 노니(Morinda citrifolia)에 함유된 주요 생리활성물질이 RAW 264.7 세포에서 JAK/STAT 신호전달 경로를 통하여 항염증 작용에 관여하는 것을 조사하였다. 건조된 M. citrifolia 열매의 MeOH 추출에 의한 유기용매의 순차 분획에서 얻은 EtOAc 분획물(Mc-EtOAc)에서 가장 높은 항산화 활성을 확인하였고, H₂O₂로 유도된 RAW 264.7 세포의 산화적 스트레스에 대한 세포보호 효과는 처리 농도의존적으로 세포독성을 차단하였다. LPS 처리로 71.6%의 RAW 264.7 세포 생존율에서 240 ㎍/ml의 Mc-EtOAc를 전처리한 군에서 84.5%의 세포 생존율 증가를 보였다. LPS로 유도된 RAW 264.7 세포의 NO 생성 저해활성은 240 ㎍/ml의 Mc-EtOAc에서 양성 대조군의 절반 수준으로 NO의 생성양을 감소시켰다. Mc-EtOAc 처리로 iNOS의 발현량은 농도의존적으로 유의하게 감소하였고, COX-2의 발현은 LPS 유도로 3배 정도로 증가되었으나, 120, 240 ㎍/ml의 Mc-EtOAc를 전처리시 농도의존적으로 유의하게 감소시켰다. 또한 pro-inflammatory cytokine IL-1β와 TNF-α의 mRNA 발현도 억제하였다. 이러한 Mc-EtOAc의 항염증 작용이 상위 신호전달 경로인 JAK/STAT 신호전달 경로 통해 일어나지를 조사한 결과, Mc-EtOAc의 전처리로 LPS로 유도된 RAW 264.7 세포에서 pJAK1과 pSTAT3의 인산화 정도가 유의성 있게 감소하였다. 따라서 RAW 264.7 세포에서 Mc-EtOAc의 항염증 효과는 JAK1/STAT3 신호전달 경로를 통해 염증반응을 억제하는 것으로 사료된다.

Abstract ▼ AI-Helper

This study investigated whether or not the major bioactive compounds of Noni (Morinda citrifolia) are involved in anti-inflammatory activity through the JAK/STAT upper signaling pathway in RAW 264.7 cells. The experimental results show that the M. citrifolia ethyl acetate fraction (Mc-EtOAc) obtained by sequential fractionation with organic solvents from the plant's dried fruits exhibits the highest antioxidant activity. In addition, the cytoprotective effects of Mc-EtOAc against H2O2-induced oxidative stress in the RAW 264.7 cells suppressed cytotoxicity in a dose-dependent manner. The group pretreated with Mc-EtOAc at a concentration of 240 ㎍/ml showed higher cell viability of 84.5%, compared to 71.6% in the LPS-treated group, and LPS-induced NO production decreased to half the amount in the positive control group. Mc-EtOAc treatment also led to a significant dose-dependent reduction in iNOS expression. Although COX-2 expression was increased by 300% following LPS induction, it was significantly decreased in a dose-dependent manner by pretreatment with Mc-EtOAc at concentrations of 120 and 240 ㎍/ml. An inhibition of the mRNA expression of pro-inflammatory cytokines IL-1β and TNF-α was observed. The investigation also revealed that the phosphorylation levels of pJAK1 and pSTAT3 in LPS-induced RAW 264.7 cells were significantly reduced by Mc-EtOAc treatment.

주제어

표/그림 (13)

$Fig. 1. Schematic diagram of extraction and fractionation of M. citrifolia.$ 그림 Fig. 1. Schematic diagram of extraction and fractionation of M. citrifolia.
표 Table 1. Realtime PCR primer sequences
$Fig. 2. DPPH free radical scavenging activity in various solvents fraction from M. citrifolia. ED<sub>50</sub> values were defined as the sample concentrations required for 50% the radical to be scavenged.$ 그림 Fig. 2. DPPH free radical scavenging activity in various solvents fraction from M. citrifolia. ED₅₀ values were defined as the sample concentrations required for 50% the radical to be scavenged.
$Table 2. Yields of each organic solvent fraction obtained from the total MeOH extract<sup>1</sup> of M. citrifolia$ 표 Table 2. Yields of each organic solvent fraction obtained from the total MeOH extract¹ of M. citrifolia
$Table 3. Absorbance change by DPPH radical scavenging activity of each fraction of M. citrifolia$ 표 Table 3. Absorbance change by DPPH radical scavenging activity of each fraction of M. citrifolia
$Fig. 3. Quantification of EtOA fraction from M.-citrifolia extract by UPLC Q-TOF.$ 그림 Fig. 3. Quantification of EtOA fraction from M.-citrifolia extract by UPLC Q-TOF.
그림 Fig. 4. Effects of Mc-EtOAc on cell viability in RAW 264.7 cells. Cell viability was measured by the MTT assay as described in materials and methods. Data are presented as the mean ± SD (n=3).
그림 Fig. 5. Effects of Mc-EtOAc against oxidative stress induced by H2O2 in RAW 264.7 cells. Cells were treated with various concentration of Mc-EtOAc and 50 μM H₂O₂ as described in materials and methods. Cell viability was analyzed using MTT assay. Data are presented as the mean ± SD (n=3). ***p<0.001 compared to un-treated control; ^##p<0.01, ^###p<0.001 compared with H₂O₂ alone.
그림 Fig. 6. Effects of Mc-EtOAc on cell viability in LPS-induced RAW 264.7 cells. Cells were treated with Mc-EtOAc and LPS (1 μg/ml) as described in materials and methods. Cell viability was analyzed using MTT assay. Data are presented as the mean ± SD (n=3). *p<0.05 compared to control; #p<0.05 compared with LPS alone.
그림 Fig. 7. Effects of Mc-EtOAc on NO production in LPS-induced RAW 264.7 cells. Cells were treated with Mc-EtOAc and LPS as described in materials and methods. NO production was measure using the Griess reagent in culture media. Data are presented as the mean ± SD (n=3). *p< 0.05 compared to control; ^#p<0.05, ^##p<0.01 compared to LPS alone.
그림 Fig. 8. Effects of Mc-EtOAc on LPS-induced iNOS and COX-2 production in RAW 264.7 cells. Cells were treated with Mc-EtOAc plus LPS or LPS alone for indicated time. Total cellular proteins were prepared and subjected to Western blotting to determined protein expression levels of iNOS and COX-2 (A). Levels of iNOS and COX-2 were normalized against actin expression. The relative density levels of protein bands were measured by scanning densitometry (B, C). Data are presented as the mean ± SD (n=3). *p<0.05, **p<0.01 compared to control; ^#p<0.05 compared to LPS alone.
그림 Fig. 9. Effects of Mc-EtOAc on LPS-induced pro-inflammatory cytokine in RAW 264.7 cells. Cells were treated with Mc- EtOAc plus LPS or LPS alone for indicated time. Total cellular RNA was prepared and subjected to qRT-PCR to determined mRNA expression levels of IL-1β (A), IL-6 (B), and TNF-α (C). Levels of IL-1β, IL-6, and TNF-α were normalized against GAPDH expression. Data are presented as the mean ± SD (n=3). **p<0.01, ***p<0.001 compared to control; ^#p<0.05, ^###p<0.001 compared to LPS alone.
그림 Fig. 10. Effects of Mc-EtOAc on LPS-induced JAK/STAT signaling pathways. The levels of protein expression were determined by Western blotting by treatment with LPS and Mc-EtOAc combined with LPS in time-and dosedependent manner. The data presented are representative of three independent experiments.

AI 본문요약
AI-Helper

문제 정의

7 세포에서 ROS 수준에 대한 M. citrifolia 열매 추출 분획물의 세포보호 효과 및 LPS로 자극된 RAW 264.7 세포 염증 모델에서 NO 생성 억제와 전염증 매개체와 cytokine 유전자 및 단백질 발현에 미치는 영향, 그리고 염증반응의 주요 신호전달 경로인 JAK/STAT 신호전달 과정과의 관련성에 대해 연구하였다.

제안 방법

RAW 264.7 세포를 6 well plate에 1 ml씩 분주하여 농도별 시료를 처리하고 2시간 후 염증반응을 유도하기 위하여 LPS를 처리하여 16시간(COX-2/iNOS), 45분(JAK), 그리고 3 시간(STAT) 동안 각각 배양하였다. 세포를 PBS로 3회 세척한 후 원심분리하여 pellet에 RIPA buffer (Thermo Fisher, USA) 를 첨가한 다음, 4℃, 14, 000× g에서 15분간 원심분리하고 상등액을 Bradford 방법[3]으로 단백질을 정량하였다.
citrifolia의 항염증 효과에 주목하여 건조된 M. citrifolia 열매를 methanol (MeOH) 추출에 의한 유기용매별 분획물의 항산화 효과를 확인하고, RAW 264.7 세포에서 ROS 수준에 대한 M. citrifolia 열매 추출 분획물의 세포보호 효과 및 LPS로 자극된 RAW 264.

데이터처리

대조군과 실험군사이의 차이를 SPSS 프로그램의 one-way ANOVA로 분석한 후 통계적 유의성이 있는 경우, 군 사이의 차이를 Post-Hoc (Tukey) multiple range tests로 검증하여 p<0.05을 유의한 것으로 간주하였다.
모든 실험은 3회 반복하여 평균 ± 표준편차(mean±SD)로 표시하였다

이론/모형

Pro-inflammatory cytokine, IL-1β, IL-6, 그리고 TNF-α 유전자 발현 분석은 AriaMX system (Agillent Technologies, USA)을 이용하여 유전자 특이적 primer (Table 1) 1 pmole, cDNA 5 ng, AccuPower® 2X Green star qPCR Master Mix (Bioneer) 10 ul, Rnase Free water (Sigma-aldrich) 4 ul로 각 well 당 20 ul로 혼합하는 조건으로 quantitative real-time polymerase chain reaction (qRT- PCR) 을 수행하였다
M. citrifolia의 MeOH 추출에 의한 유기용매 별 분획물의 항산화 활성은 DPPH의 자유라디칼 소거효과(free radical scavenging effect)를 측정하는 Blois [2]의 방법에 따라 측정하였다. MeOH에 녹인 0.
유전자 상대정량 분석은 glyceraldehyde 3-phosphate dehydrogenase (GAPDH)의 발현양을 기준으로 normalize를 수행하였으며 상대정량은 2−∆∆Ct 방법을 사용하여 사용하여 분석하였다[21].

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표제어: PCR

동의어: Packet Collision Rate

용어 설명 출처 목록 (6)

용어 설명: PCR은 세균 특이성이 있는 primer를 이용하여 적은 수의 세균이 있을지라도 쉽게 검출할 수 있는 유용한 방법이며, 이를 이용하여 구강 내 치면세균막이나 타액에서 직접 세균을 검출할 수 있게 되었다[8].

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