[논문]RAW 264.7 대식세포에서 마늘 유래 황 함유 화합물에 의한 요산 유도 inflammasome 활성화의 억제는 ROS 생성 차단과 연관성이 있음

김민영; 최영현

doi:10.5352/jls.2023.33.4.349

RAW 264.7 대식세포에서 마늘 유래 황 함유 화합물에 의한 요산 유도 inflammasome 활성화의 억제는 ROS 생성 차단과 연관성이 있음
Suppression of Monosodium Urate-induced NLRP3 Inflammasome Activation by Garlic-derived Sulfur-containing Phytochemicals is Associated with Blocking ROS Generation in RAW 264.7 Macrophages 원문보기

생명과학회지 = Journal of life science, v.33 no.4, 2023년, pp.349 - 356

초록
AI-Helper

만성 염증성 관절염 질환인 통풍은 고요산혈증을 특징으로 하며 NLRP3 inflammasome의 활성화에 따른 IL-1β와 같은 염증성 cytokine 방출과 연관된 MSU에 대한 염증 반응에 의해 유발될 수 있다. 마늘에 함유된 황 함유 화합물은 다양한 질병에 대한 잠재적인 유익한 약리학적 효능을 가지지만, NLRP3 inflammasome 활성화와 연관된 통풍 억제에 대한 효능은 현재까지 입증되지 않았다. 본 연구에서는 대표적인 마늘 유래 황화합물인 DADS와 DATS가 MSU에 의한 NLRP3 inflammasome 활성을 억제할 수 있는지를 조사하였다. 본 연구의 결과에 의하면, 비세포 독성 조건에서 DADS와 DATS는 LPS가 전처리된 RAW 264.7 대식세포에서 MSU에 대한 반응으로 증가된 NO의 생성과 IL-1β 유리를 유의적으로 차단하였다. DADS와 DATS는 또한 증가된 NLRP3, ASC, caspase-1 p20 및 IL-1β의 발현을 감소시켰으며, 이는 MSU로 유도된 LRP3 inflammasome 활성화가 DADS와 DATS에 의해 억제되었음을 의미한다. 아울러, DADS와 DATS는 NLRP3 inflammasome 활성화에 상위 신호로 작용하는 산화적 스트레스를 차단했으며 ROS 생성을 제거한다는 사실에서 입증되었다. 결론적으로, 본 연구의 결과는 DADS와 DATS가 ROS/NLRP3 경로를 억제하여 inflammasome 활성화를 차단함으로서 NLRP3 의존성 통풍성 관절염 치료를 위한 잠재력이 우수함을 의미한다.

Abstract ▼ AI-Helper

Gout, a chronic inflammatory arthritic disease, is characterized by hyperuricemia. Gout can be induced by an inflammatory response to monosodium urate (MSU) crystals mediated by pro-inflammatory cytokine release following activation of the NOD-like receptor protein 3 (NLRP3) inflammasome. Many sulfur-containing phytochemical compounds in garlic (Allium sativum L.) are considered active ingredients because of their potential pharmacological benefits for various diseases, but their efficacy in NLRP3 inflammasome activation-mediated gout has not been demonstrated. In this study, we investigated whether diallyl disulfide (DADS) and diallyl trisulfide (DATS), representative garlic-derived sulfur compounds, have an inhibitory effect on MSU-induced NLRP3 inflammasome activation. Our results showed that under non-cytotoxic conditions, DADS and DATS significantly blocked nitric oxide production and interleukin (IL)-1β release in response to MSU in lipopolysaccharide (LPS)-primed RAW 264.7 macrophages. DADS and DATS also attenuated enhanced expression of NLRP3 and its adapter protein, apoptosis-associated speck-like protein, which was associated with downregulation of and caspase-1 p20 and IL-1β expression, suggesting that MSU-induced LRP3 inflammasome activation was counteracted by DADS and DATS. Furthermore, DADS and DATS blocked oxidative stress, an upstream event for NLRP3 inflammasome activation, as evidenced by the fact that they scavenged reactive oxygen species (ROS) production. Taken together, our findings demonstrate that DADS and DATS suppressed NLRP3 inflammasome activation by inhibiting the ROS/NLRP3 pathway and that they have potential as treatments for NLRP3-dependent gouty arthritis.

주제어

표/그림 (5)

그림 Fig. 1. Effects of DADS, DATS, LPS and MSU treatment alone or their combination on cell viability of RAW 264.7 macrophages. (A-D) RAW 264.7 cells were treated with different concentrations of DADS, DATS and MSU alone for 24 hr (A-C), or pretreated with or without LPS (0.25 or 0.5 μg/ml) for 2 hr before 500 μg/ml MSU stimulation for 24 hr (D). (E and F) RAW 264.7 cells pretreated or not with DASA and DATS (20 μM) for 1 hr were treated with LPS (0.5 μg/ml) for 2 hr and further treated with MSU (500 μg/ml) for 24 hr. Cell viability was analyzed using the MTT assay. Each value indicates the mean ± SD of three independent experiments (*p<0.05 compared to control).
그림 Fig. 2. Inhibitory effects of DADS and DATS on MSU-induced production of NO and IL-1β in LPS-primed RAW 264.7 macrophages. RAW 264.7 cells were treated with LPS (0.5 μg/ml) for 2 hr and further stimulated with MSU (500 μg/ml) for 24 hr. DASA and DATS (20 μM) was used to treat cells for 1 hr before LPS treatment in RAW 264.7 cells. (A and D) The level of NO production was investigated using Griess reagent. (C and D) IL-1β secretion was calculated using an ELISA kit. Data are the mean ± SD from three independent experiments (***p<0.001 compared to control; ^#p<0.05 and ^###p<0.001 compared to LPS + MSU).
그림 Fig. 3. Inhibitory effects of DADS and DATS on MSU-induced activation of XO in LPS-primed RAW 264.7 macrophages. RAW 264.7 cells were treated with LPS (0.5 μg/ml) for 2 hr and further stimulated with MSU (500 μg/ml) for 24 hr. DASA and DATS (20 μM) was used to treat cells for 1 hr before LPS treatment in RAW 264.7 cells. (A and B) XO activity was measured using an XO activity assay kit. Data are the mean ± SD from three independent experiments (***p<0.001 compared to control; ^##p<0.01 and ^###p<0.001 compared to LPS + MSU).
그림 Fig. 4. Inhibitory effects of DADS and DATS on MSU-induced NLRP3 inflammasome activation in LPS-primed RAW 264.7 macrophages. RAW 264.7 cells were treated with LPS (0.5 μg/ml) for 2 hr and further stimulated with MSU (500 μg/ml) for 24 hr. DASA and DATS (20 μM) was used to treat cells for 1 hr before LPS treatment in RAW 264.7 cells. (A) After treatment, representative images of NLRP3 (green) and ASC immunofluorescence (red) observed under a fluorescence microscope are shown. The location of the nucleus was indicated by counterstaining with DAPI (blue). (B) After extracting cell lysate of each treatment group, expression levels of IL-1β and cleaved caspase-1 (p20) proteins were investigated through immunoblotting. Actin was used as a loading control.
그림 Fig. 5. Inhibitory effects of DADS and DATS on MSU-induced ROS generation in LPS-primed RAW 264.7 macrophages. RAW 264.7 cells were treated with LPS (0.5 μg/ml) for 2 hr and further stimulated with MSU (500 μg/ml) for 2 hr. DASA and DATS (20 μM) was used to treat cells for 1 hr before LPS treatment in RAW 264.7 cells. ROS generation was detected using a DCFDA probe and quantified by flow cytometry. (A) Numbers indicate the percentage of DCF-positive cells in the gate. (B) Densitometric analysis was used to quantify the levels of ROS. The results represent the mean ± SD for three experiments (***p<0.001 compared to control; ^###p<0.001 compared to LPS + MSU).

AI 본문요약
AI-Helper

문제 정의

본 연구에서는 마늘의 주요 황 함유 화합물인 DADS와 DATS가 염증성 질환의 일종인 통풍의 예방과 치료 적용성에 관한 가능성을 조사하였다. 이를 위하여 잘 확립된 LPS 자극 후 MSU를 처리하는 in vitro RAW 264.

대상 데이터

본 연구에서는 마늘의 주요 황 함유 화합물인 DADS와 DATS가 염증성 질환의 일종인 통풍의 예방과 치료 적용성에 관한 가능성을 조사하였다. 이를 위하여 잘 확립된 LPS 자극 후 MSU를 처리하는 in vitro RAW 264.7 세포 모델을 이용하였다. 본 연구의 결과에 의하면, 세포 독성이 없는 범위에서 DADS와 DATS는 MSU에 의한 NO의 생성과 특히 NLRP3 inflammasome의 활성과 깊은 연관성이 있는 IL-1β의 유리를 유의적으로 억제하였다.

이론/모형

처리가 끝난 후, 생성된 NO와 IL-1β의 양은 Griess 시약(Sigma-Aldrich Chemical Co.) 및 enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems Inc., Minneapolis, MN, USA)를 사용하여 제조사의 방법에 준하여 측정하였다

성능/효과

본 연구의 결과에 의하면, 세포 독성이 없는 범위에서 DADS와 DATS는 MSU에 의한 NO의 생성과 특히 NLRP3 inflammasome의 활성과 깊은 연관성이 있는 IL-1β의 유리를 유의적으로 억제하였다

후속연구

또한, DADS와 DATS는 XO의 활성과 ROS의 생성을 차단시켰으며, 이는 NLRP3 inflammasome 활성의 저해와 연관성이 있었다. 비록 in vivo 모델에서의 통풍 억제 효능의 확인과 ROS 생성의 중재 과정에 관여하는 세포 내 신호 전달계 역할에 관한 추가 연구가 요구되지만, 본 연구에서는 마늘에 함유된 황화합물이 통풍 처치를 위한 잠재력이 매우 우수함을 제시하고자 한다.

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저자의 다른 논문 :

표제어: PCR

동의어: Packet Collision Rate

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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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