[논문]LPS로 자극한 RAW264.7 대식세포에서 흰점박이꽃무지 유충 유래 Protaetiamycine 9의 항염증 효과

최라영; 서민철; 이준하; 김인우; 김미애; 황재삼

doi:10.5352/jls.2021.31.11.987

LPS로 자극한 RAW264.7 대식세포에서 흰점박이꽃무지 유충 유래 Protaetiamycine 9의 항염증 효과
Inhibitory Effect of Protaetiamycine 9 Derived from Protaetia brevitarsis seulensis Larvae on LPS-mediated Inflammation in RAW264.7 Cells 원문보기

생명과학회지 = Journal of life science, v.31 no.11, 2021년, pp.987 - 994

최라영 (농촌진흥청 국립농업과학원 농업생물부 곤충양잠산업과) , 서민철 (농촌진흥청 국립농업과학원 농업생물부 곤충양잠산업과) , 이준하 (농촌진흥청 국립농업과학원 농업생물부 곤충양잠산업과) , 김인우 (농촌진흥청 국립농업과학원 농업생물부 곤충양잠산업과) , 김미애 (농촌진흥청 국립농업과학원 농업생물부 곤충양잠산업과) , 황재삼 (농촌진흥청 국립농업과학원 농업생물부 곤충양잠산업과)

초록
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이전 연구에서 흰점박이꽃무지 유충 유래 항균 펩타이드 Protaetiamycine 2와 6의 항염증 효과를 입증하였다. 본 연구에서는 lipopolysaccharide (LPS)로 염증을 유도한 RAW264.7 대식세포에서 흰점박이꽃무지 유충의 새로운 항균 펩타이드인 Protaetiamycine 9의 염증 조절 기전을 검토하였다. 항염증 활성을 확인하기 위하여 RAW 264.7 세포에 독성이 나타나지 않는 범위(25-100 ㎍/ml)로 Protaetiamycine 9를 1시간 동안 전처리한 후, 24시간 동안 LPS (100 ng/ml)로 염증을 유도하였다. Protaetiamycine 9 (25-100 ㎍/ml)는 LPS 자극으로 증가된 nitric oxide (NO) 분비를 농도의존적으로 감소시켰고, 염증 매개 인자의 생성에 관여하는 inducible NO synthase (iNOS) 및 cyclooxygenase-2 (COX-2)의 발현을 유의적으로 억제하였다. Protaetiamycine 9는 LPS로 유도된 inhibitory kappa B alpha (IκB-α)의 분해를 저해하고, extracellular signal regulated kinase (ERK), c-Jun N-terminal kinase (JNK) 및 p38을 포함하는 mitogen-activated protein kinases (MAPKs)의 인산화를 억제함으로써 염증성 사이토카인(interleukin (IL)-6와 IL-1β)의 생성과 유전자 발현을 효과적으로 억제하였다. 따라서, Protaetiamycine 9는 염증반응의 신호전달경로인 NF-κB와 MAPKs의 활성화를 억제함으로써 항염증 효과를 나타내는 것으로 사료된다.

Abstract ▼ AI-Helper

Our previous studies have reported that antimicrobial peptides (AMPs) derived from the larvae of white-spotted flower chafer (Protaetia brevitarsis seulensis) exert anti-inflammatory and neuroprotective activities. This study explored the anti-inflammatory effects of protaetiamycine 9 (CVLKKAYFLTNLKLRG-NH2), a novel AMP, derived from P. b. seulensis against lipopolysaccharide (LPS)-mediated inflammatory response in RAW264.7 macrophage cells. Protaetiamycine 9 (25, 50, 75, and 100 ㎍/ml) did not cause cytotoxic effects against RAW264.7 cells. The RAW264.7 cells were pre-treated with various concentrations of protaetiamycine 9 (25-100 ㎍/ml) for 1 hr and then exposed to LPS (100 ng/ml) for 24 hr. Protaetiamycine 9 treatments decreased the LPS-induced secretion of inflammatory mediators, such as nitric oxide (NO), in a dose-dependent manner. Protaetiamycine 9 (25-100 ㎍/ml) effectively downregulated the LPS-induced increase in mRNA and the protein expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2), which are involved in the production of inflammatory mediators. Protaetiamycine 9 also suppressed the production and gene expression of pro-inflammatory cytokines, including interleukin (IL)-6 and IL-1β, compared to the presence of LPS alone. Furthermore, protaetiamycine 9 inhibited the degradation of inhibitory kappa B alpha (IκB-α) and the phosphorylation of mitogen-activated protein kinases (MAPKs), such as extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38. In conclusion, these results suggest that protaetiamycine 9 exhibits LPS-mediated inflammatory responses by blocking IκB-α degradation and MAPK phosphorylation.

주제어

표/그림 (5)

표 Table 1. Sequences of primers used for RT-PCR
그림 Fig. 1. Effects of Protaetiamycine 9 on cell viability (A) and nitric oxide production (B) in LPS-stimulated RAW264.7 cells. Cell viability was determined from the 24 hr culture of RAW264.7 cells in the presence of Protaetiamycine 9. To examine nitric oxide levels, RAW264.7 cells were pre-incubated for 1 hr with Protaetiamycine 9 (25, 50, 75, 100 μg/ml) and then stimulated for 24 hr with lipopolysaccharide (LPS, 100 ng/ml) alone or LPS plus Protaetiamycine 9. Data are expressed as the mean ± standard error (n=3). ###p<0.001, compared with the untreated control (CTR) group. *p<0.05 and ***p<0.001, compared with the cells treated with LPS alone.
그림 Fig. 2. Effects of Protaetiamycine 9 on mRNA expression (A) and protein expression (B) of iNOS and COX-2 in LPS-stimulated RAW264.7 cells. RAW264.7 cells were pre-incubated for 1 hr with Protaetiamycine 9 (25, 50, 75, 100 μg/ml) and then stimulated for 5 hr or 24 hr with lipopolysaccharide (LPS, 100 ng/ml) alone or LPS plus Protaetiamycine 9. Data are expressed as the mean ± standard error (n=3). ##p<0.01 and ###p<0.001, compared with the untreated control (CTR) group. **p<0.01 and ***p<0.001, compared with the cells treated with LPS alone.
그림 Fig. 3. Effects of Protaetiamycine 9 on mRNA expression (A) and production (B) of IL-6 and IL-1β in LPS-stimulated RAW264.7 cells. RAW264.7 cells were pre-incubated for 1 hr with Protaetiamycine 9 (25, 50, 75, 100 μg/ml) and then stimulated for 5 hr or 24 hr with lipopolysaccharide (LPS, 100 ng/ml) alone or LPS plus Protaetiamycine 9. Data are expressed as the mean ± standard error (n=3). #p<0.05 and ###p<0.001, compared with the untreated control (CTR) group. *p<0.05 and ***p<0.001, compared with the cells treated with LPS alone.
그림 Fig. 4. Effects of Protaetiamycine 9 on the protein expression of MAPKs (A) and IκB-α (B) in LPS-stimulated RAW264.7 cells. RAW264.7 cells were pre-incubated for 1 hr with Protaetiamycine 9 (25, 50, 75, 100 μg/ml) and then stimulated for 30 min with lipopolysaccharide (LPS, 100 ng/ml) alone or LPS plus Protaetiamycine 9.

AI 본문요약
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데이터처리

모든 실험 결과는 3회 반복하여 평균과 표준오차로 나타내었으며 실험군 간의 유의성은 Statistical Package for Social Science 프로그램(SPSS, SPSS Inc., Chicago, IL, USA)을 이용하여 Student’s t-test를 통해 검정하였고, p<0.05 수준에서 유의성을 비교하였다.

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