[논문]치주염 원인균 LPS-PG로 유도된 인체 치은섬유아세포에서 연뿌리 추출물에 대한 항염증 및 항산화 효과

이영경; 김철환; 정대원; 이기원; 오영택; 김정일; 정진우

doi:10.7732/kjpr.2022.35.5.565

치주염 원인균 LPS-PG로 유도된 인체 치은섬유아세포에서 연뿌리 추출물에 대한 항염증 및 항산화 효과
Anti-inflammatory and Antioxidative Effects of Lotus Root Extract in LPS-PG-Stimulated Human Gingival Fibroblast-1 Cells 원문보기

韓國資源植物學會誌 = Korean journal of plant resources, v.35 no.5, 2022년, pp.565 - 573

이영경 (국립낙동강생물자원관) , 김철환 (국립낙동강생물자원관) , 정대원 (국립낙동강생물자원관) , 이기원 (동아대학교 의과대학 직업환경의학교실) , 오영택 (국립낙동강생물자원관) , 김정일 (동아대학교 의과대학 직업환경의학교실) , 정진우 (국립호남권생물자원관)

초록
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치주조직에 존재하는 주요한 세포의 한 형태인 인체 치은섬유아세포는 다양한 구강유해세균으로부터 염증이 유발되어지며, 그중 대표적으로 치주염 원인균인 P. gingivalis의 내독소인 LPS-PG로부터 염증성 자극에 반응하여 다양한 염증매개 물질을 분비한다. 본 연구에서는 치주염을 일으키는 주요한 원인균 중 하나인 P. gingivalis로 부터 분리한 LPS-PG를 이용하여 인체 치은섬유아세포주인 HGF-1 세포에 염증을 유도한 후 LRE에 대한 항염증 및 항산화 효과를 분석하였다. 실험 결과, LRE는 LPS-PG 유도에 따라 iNOS에 의한 NO 생성과 COX-2에 의한 PGE₂와 같은 염증 매개 인자의 발현 및 생성 억제와 함께 염증성 싸이토카인(TNF-α, IL-1β및 IL-6)의 생성 또한 억제하였다. 신호전달계에서 염증성 전사인자의 발현 경로를 확인하기 위하여 TLR4/Myd88/NF-κB의 활성을 확인한 결과, LRE 처리에 따라 농도 의존적으로 억제되는 것을 확인하였다. 또한 산화 환원 효소로 항염증효과를 나타내는 것으로 알려진2상 효소 중 하나인 NQO-1과 이의 전사인자인 Nrf2를 분석 한 결과 LRE 처리에 의해 효소의 활성이 높아지는 것을 확인할 수 있었다. 결론적으로 LRE는 TLR4/Myd88/NF-κB 신호전달 경로를 억제하고 NQO1/Nrf2 활성을 유도함으로써 HGF-1 세포에서 LPS-PG에 의해 유도된 염증을 억제하는 것으로 사료되며, 향후 LRE는 식·의약품 소재 개발에서 치주질환 개선의 가능성이 있는 후보물질이 될 수 있을 것으로 사료된다.

Abstract ▼ AI-Helper

Gingival inflammation is one of the main causes that can be related to various periodontal diseases. Human gingival fibroblast (HGF) is the major constituent in periodontal connective tissue and secretes various inflammatory mediators, such as nitric oxide (NO) and prostaglandin E2 (PGE2), upon lipopolysaccharide stimulation. This study is aimed at investigating the anti-inflammatory and antioxidative activities of Lotus Root extract (LRE) in Porphyromonas gingivalis derived lipopolysaccharide (LPS-PG)-stimulated HGF-1 cells. The concentration of NO and PGE2, as well as their responsible enzymes, inducible NO synthase (iNOS), and cyclooxygenase-2 (COX-2), was analyzed by Griess reaction, ELISA, and western blot analysis. LPS-PG sharply elevated the production and protein expression of inflammatory mediators, which were significantly attenuated by LRE treatment in a dose-dependent manner. LRE treatment also suppressed activation of Toll-like receptor 4 (TLR4)/myeloid differentiation primary response gene 88 (MyD88) and nuclear factor-κB (NF-κB) in LPS-PG-stimulated HGF-1 cells. In addition, one of phase II enzyme, NAD(P)H quinone dehydrogenase (NQO)-1, and its transcription factor, Nuclear factor erythroid 2-related factor 2 (Nrf2), were significantly induced by LRE treatment. Consequently, these results suggest that LRE ameliorates LPS-PG-induced inflammatory responses by attenuating TLR4/MyD88-mediated NF-κB, and activating NQO-1/Nrf2 antioxidant response element signaling pathways in HGF-1 cells.

주제어

표/그림 (6)

그림 Fig. 1. High-performance liquid chromatography (HPLC) analysis of Lotus Root extract (LRE).
그림 Fig. 2. Effects of LRE and LPS-PG on the cell viability of HGF-1 cells. The cells were treated with the indicated concentrations of LRE alone or pretreated with LRE for 2 h before LPS-PG (1 μg/mL) treatment. After 24 h, cell viability was assessed using an MTT assay. Data are expressed as the means ± standard deviation of the three independent experiments (N.S., no significance).
그림 Fig. 3. Inhibition of NO and PGE2 production by LRE in LPS-PG-stimulated HGF-1 cells. The cells were pretreated with the indicated concentrations of LRE for 2 h prior to incubation with LPS-PG (1 μg/mL) for 24 h. The levels of NO (A) and PGE2 (B) in culture media were measured by Griess assay and a commercial ELISA kit, respectively. Each value indicates the mean ± SD and is representative of the results obtained from three independent experiments. (C, D, E and F) Cell lysates were prepared for Western blot analysis, with antibodies specific for murine iNOS and COX-2, and for an ECL detection system. The experiment was repeated three times and similar results were obtained. Actin was used as the internal controls for the Western blot analysis, respectively. The significance was determined by the Student's t-test (*p < 0.05, compared with control; #p < 0.05, compared with LPS-PG).
그림 Fig. 4. Effects of LRE on LPS-PG--stimulated cytokines production in HGF-1 cells. Cells were pre-treated with the indicated concentrations of LRE 2 h prior to incubation with LPS-PG (1 μg/mL). After incubation for 24 h, the levels of TNF-α (A), IL-1β (B), IL-6 (C) and IL-8 (D) present in the supernatants were measured using ELISA kits. Each value is presented as the mean ± standard deviation and is representative of results obtained from four independent experiments. The significance was determined by the Student's t-test (*p < 0.05, compared with control; #p < 0.05, compared with LPS-PG).
$Fig. 5. Effects of LRE on the LPS-PG-induced expression of TLR4 and NF-κB in HGF-1 cells. After 1 μg/mL LPS-PG treatment with or without 80 μg/mL LRE for the indicated times, the nuclear and cytosolic proteins were isolated, and the expression of TRL4/MyD88 (A and C) and NF-κB/IκBα (B and D) was determined by Western blot analysis using an enhanced chemiluminescence (ECL) detection system. Nucleolin and β-actin were used as internal controls for the nuclear and cytosolic fractions, respectively.$ 그림 Fig. 5. Effects of LRE on the LPS-PG-induced expression of TLR4 and NF-κB in HGF-1 cells. After 1 μg/mL LPS-PG treatment with or without 80 μg/mL LRE for the indicated times, the nuclear and cytosolic proteins were isolated, and the expression of TRL4/MyD88 (A and C) and NF-κB/IκBα (B and D) was determined by Western blot analysis using an enhanced chemiluminescence (ECL) detection system. Nucleolin and β-actin were used as internal controls for the nuclear and cytosolic fractions, respectively.
그림 Fig. 6. Effects of LRE on protein expression of NQO1 and Nrf2 in LPS-PG-stimulated HGF-1 cells. Protein expression levels of NQO1 and Nrf2 by LRE were analyzed by western blot analysis. All signals were normalized to protein levels of Actin, an internal control, and expressed as a ratio. Data represent the mean ± SD of triplicate experiments. Actin was used as the internal controls for the Western blot analysis, respectively.

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