[논문]병풀(Centella asiatica) 및 아시아티코사이드는 미토콘드리아 호흡 및 TLR4 경로를 통해 H2O2 유도 세포염증 조절

지주리; 남영선; 강상모

doi:10.5352/jls.2021.31.4.389

병풀(Centella asiatica) 및 아시아티코사이드는 미토콘드리아 호흡 및 TLR4 경로를 통해 H2O2 유도 세포염증 조절
Centella asiatica and Asiaticoside Regulate H2O2-induced Cellular Inflammation via Mitochondrial Respiration and the TLR4 Pathway 원문보기

생명과학회지 = Journal of life science, v.31 no.4, 2021년, pp.389 - 399

지주리 (시바산) , 남영선 (신경대학교 뷰티디자인학과) , 강상모 (건국대학교 생물공학과)

초록
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이 연구는 인간진피섬유아(HDF)세포에서 병풀 및 아시아티코사이드가 H₂O₂ 유래 세포주기 정지기, 미토콘드리아 활성 및 염증성 사이토카인에 미치는 영향을 조사하였다. 병풀 80% 메탄올 추출물, 에틸아세티이트 분획물 및 병풀의 대표물질인 아시아티코사이드를 사용하였다. 병풀 추추물, 에틸아세테이트 분획 및 아시아티코사이드로 처리한 세포는 낮은 수준의 TNF-α 및 IL-6을 분비하였고, 아시아티코사이드의 항산화 효과는 병풀 추출물 및 에틸아세테이트 분획물보다 높았다. 아시아티코사이드 처리는 미토콘드리아의 막포텐셜을 증가시키고, 미토콘드리아를 정상으로 되돌렸다. 스트레스 유도 후 에틸아세테이트 분획물 및 아시아티코사이드에 의해 미토콘드리아 산소 소비율이 증가하였고, TLR4-MyD88-TRAF6-p65 경로가 재감소하였다. 이러한 결과는 병풀 추출물, 에틸 아세테이트 분획 및 아시아티코사이드가 HDF 세포의 미토콘드리아 활성을 조절할 뿐 아니라 항산화 및 항염증에 효과 있음을 시사한다.

Abstract ▼ AI-Helper

This study determined the effects of Centella asiatica leaf on H2O2 induced cell cycle arrest, mitochondrial activity, and proinflammatory cytokine production in human dermal fibroblast (HDF) cells. We used an 80% methanol extract of C. asiatica, its ethyl acetate fraction, and asiaticoside, the major constituent of C. asiatica. The C. asiatica extract, its ethyl acetate fraction, and asiaticoside attenuated G1 cell cycle-arrest and the apoptotic effect caused by H2O2-induced oxidative stress. The cells treated with C. asiatica extract, its ethyl acetate fraction, and asiaticoside secreted lower levels of TNF-α and IL-6. The antioxidant effect of asiaticoside was higher than that of C. asiatica extract and its ethyl acetate fraction. Treatment with C. asiatica extract, its ethyl acetate fraction, and asiaticoside also increased the mitochondrial membrane potential and restored normal mitochondrial morphology. Following H2O2 stress induction, cells treated with C. asiatica extract, its ethyl acetate fraction, and asiaticoside showed increased mitochondrial oxygen consumption rates and decreases in the TLR4-MyD88-TRAF6-p65 pathway activity. These findings suggest that C. asiatica extract, its ethyl acetate fraction, and asiaticoside have antioxidant and anti-inflammatory effects, as well as the ability to control the mitochondrial activities of HDF cells.

주제어

표/그림 (5)

$Fig. 1. Centella asiatica extract, ethyl acetate fraction and asiaticoside leads to cell cycle recovery from oxidative in HDF cells. (A) Flow cytometric analysis of cell cycle distribution in Centella asiatica extract (20 μg/ml), ethyl acetate fraction (13 μg/ml) and asiaticoside (100 μM). HDF cells were collected and fixed with 70% ethanol for 30 min at 4℃, then subjected to FACS after propidium iodide (PI, 5 μg/ml) staining. The area parameter histogram was used to determine the percentage of cells in apoptosis (M1), G1 (M2), S (M3), G2/M (M4) phases. (B) Results of cell viability assay different concentrations of Centella asiatica extract, ethyl acetate fraction and asiaticoside on HDF cells. (C) The percentage of G1 (M2) phase by FACS analysis of cell cycle distribution. Error bars represent averages ± SD from at least three independent groups (D) The percentage of apoptosis (M1) phase by FACS analysis of cell cycle distribution. (E) ethyl acetate fraction, (AS) asiaticoside, (ME) 80% Methanol extract. Error bars represent averages ± SD from at least three independent groups. #p<0.05 versus control; ##p<0.01 versus control; ###p<0.001 versus control; **p<0.01 versus H2O2-treated cells, ***p<0.001 versus H2O2-treated cells.$ 그림 Fig. 1. Centella asiatica extract, ethyl acetate fraction and asiaticoside leads to cell cycle recovery from oxidative in HDF cells. (A) Flow cytometric analysis of cell cycle distribution in Centella asiatica extract (20 μg/ml), ethyl acetate fraction (13 μg/ml) and asiaticoside (100 μM). HDF cells were collected and fixed with 70% ethanol for 30 min at 4℃, then subjected to FACS after propidium iodide (PI, 5 μg/ml) staining. The area parameter histogram was used to determine the percentage of cells in apoptosis (M1), G1 (M2), S (M3), G2/M (M4) phases. (B) Results of cell viability assay different concentrations of Centella asiatica extract, ethyl acetate fraction and asiaticoside on HDF cells. (C) The percentage of G1 (M2) phase by FACS analysis of cell cycle distribution. Error bars represent averages ± SD from at least three independent groups (D) The percentage of apoptosis (M1) phase by FACS analysis of cell cycle distribution. (E) ethyl acetate fraction, (AS) asiaticoside, (ME) 80% Methanol extract. Error bars represent averages ± SD from at least three independent groups. #p<0.05 versus control; ##p<0.01 versus control; ###p<0.001 versus control; **p<0.01 versus H2O2-treated cells, ***p<0.001 versus H2O2-treated cells.
$Fig. 2. Effects of Centella asiatica extract, ethyl acetate fraction and asiaticoside on TNF-α and IL-6 cytokine secretion in H2O2-induced HDF cells. (A) TNF-α and (B) IL-6 protein levels were detected by ELISA. HDF cells were treated with Centella asiatica extract (20 μg/ml), ethyl acetate fraction (13 μg/ml) and asiaticoside (100 μM). The experiment was carried out according to the manual. Error bars represent averages ± SD from at least five independent groups. (E) ethyl acetate fraction, (AS) asiaticoside, (ME) 80% Methanol extract. Error bars represent averages ± SD from at least three independent groups. ###p<0.001 versus control; ***p<0.001 versus H2O2-treated cells.$ 그림 Fig. 2. Effects of Centella asiatica extract, ethyl acetate fraction and asiaticoside on TNF-α and IL-6 cytokine secretion in H₂O₂-induced HDF cells. (A) TNF-α and (B) IL-6 protein levels were detected by ELISA. HDF cells were treated with Centella asiatica extract (20 μg/ml), ethyl acetate fraction (13 μg/ml) and asiaticoside (100 μM). The experiment was carried out according to the manual. Error bars represent averages ± SD from at least five independent groups. (E) ethyl acetate fraction, (AS) asiaticoside, (ME) 80% Methanol extract. Error bars represent averages ± SD from at least three independent groups. ###p<0.001 versus control; ***p<0.001 versus H₂O₂-treated cells.
$Fig. 3. Effect of Centella asiatica extract, ethyl acetate fraction and asiaticoside on H2O2-induced loss of mitochondrial membrane potential in HDF cells. (A) Scatter diagram of JC-1 staining, as measured by flow cytometry HDF cells were treated Centella asiatica extract (20 μg/ml), ethyl acetate fraction (13 μg/ml) and asiaticoside (100 μM). (B) Quantitative analysis of the Q2 ratio of fluorescence intensity (Red : FL2, >103 intensity / Green : FL1, >104 intensity). (E) ethyl acetate fraction, (AS) asiaticoside, (ME) 80% Methanol extract. Error bars represent averages ± SD from at least three independent groups. ###p<0.001 versus control; ***p<0.001 versus H2O2-treated cells.$ 그림 Fig. 3. Effect of Centella asiatica extract, ethyl acetate fraction and asiaticoside on H₂O₂-induced loss of mitochondrial membrane potential in HDF cells. (A) Scatter diagram of JC-1 staining, as measured by flow cytometry HDF cells were treated Centella asiatica extract (20 μg/ml), ethyl acetate fraction (13 μg/ml) and asiaticoside (100 μM). (B) Quantitative analysis of the Q2 ratio of fluorescence intensity (Red : FL2, >10³ intensity / Green : FL1, >10⁴ intensity). (E) ethyl acetate fraction, (AS) asiaticoside, (ME) 80% Methanol extract. Error bars represent averages ± SD from at least three independent groups. ###p<0.001 versus control; ***p<0.001 versus H₂O₂-treated cells.
$Fig. 4. Effects of Centella asiatica extract, ethyl acetate fraction and asiaticoside on intracellular ROS and mitochondrial oxygen consumption rate in HDF cells. (A) Representative images of mito-YFP signal intensity are shown in HDF cells. HDF cells were treated Centella asiatica extract (20 μg/ml), ethyl acetate fraction (13 μg/ml) and asiaticoside (100 μM). Scale bar, 50 μm. (B) Determination of cellular ROS by DCF-DA assay. Histograms were generated to show cells stained with DCF-DA in comparison to the H2O2 treated control (Expression of FL1 >103 intensity). (C) Oxygen consumption rate (OCR) measurements of HDF cells mitochondria provided with oligomycin (0.5 uM), FCCP (1 μM) and antimycin A (1 μM) were subsequently added as indicated (arrows). (E) ethyl acetate fraction, (AS) asiaticoside, (ME) 80% Methanol extract. Error bars represent averages ± SD from at least three independent groups. ###p<0.001 versus control; *p<0.05 versus H2O2-treated cells, ***p<0.001 versus H2O2-treated cells.$ 그림 Fig. 4. Effects of Centella asiatica extract, ethyl acetate fraction and asiaticoside on intracellular ROS and mitochondrial oxygen consumption rate in HDF cells. (A) Representative images of mito-YFP signal intensity are shown in HDF cells. HDF cells were treated Centella asiatica extract (20 μg/ml), ethyl acetate fraction (13 μg/ml) and asiaticoside (100 μM). Scale bar, 50 μm. (B) Determination of cellular ROS by DCF-DA assay. Histograms were generated to show cells stained with DCF-DA in comparison to the H₂O₂ treated control (Expression of FL1 >10³ intensity). (C) Oxygen consumption rate (OCR) measurements of HDF cells mitochondria provided with oligomycin (0.5 uM), FCCP (1 μM) and antimycin A (1 μM) were subsequently added as indicated (arrows). (E) ethyl acetate fraction, (AS) asiaticoside, (ME) 80% Methanol extract. Error bars represent averages ± SD from at least three independent groups. ###p<0.001 versus control; *p<0.05 versus H₂O₂-treated cells, ***p<0.001 versus H₂O₂-treated cells.
$Fig. 5. Effects of Centella asiatica extract, ethyl acetate fraction and asiaticoside mediated expression levels of TLR4-MyD88-TRAF6-p65 proteins in H2O2-induced HDF cells. (A) Protein expression level was analyzed by western blotting with indicated antibodies. HDF cells were treated Centella asiatica extract (20 μg/ml), ethyl acetate fraction (13 μg/ml) and asiaticoside (100 μM). (B) Quantitative analysis of expression was scanned and analyzed using Image J software. Error bars represent averages ± SD from at least three independent groups. (E) ethyl acetate fraction, (AS) asiaticoside, (ME) 80% Methanol extract. Error bars represent averages ± SD from at least three independent groups. **p<0.01 versus H2O2-treated cells, ***p<0.001 versus H2O2-treated cells.$ 그림 Fig. 5. Effects of Centella asiatica extract, ethyl acetate fraction and asiaticoside mediated expression levels of TLR4-MyD88-TRAF6-p65 proteins in H₂O₂-induced HDF cells. (A) Protein expression level was analyzed by western blotting with indicated antibodies. HDF cells were treated Centella asiatica extract (20 μg/ml), ethyl acetate fraction (13 μg/ml) and asiaticoside (100 μM). (B) Quantitative analysis of expression was scanned and analyzed using Image J software. Error bars represent averages ± SD from at least three independent groups. (E) ethyl acetate fraction, (AS) asiaticoside, (ME) 80% Methanol extract. Error bars represent averages ± SD from at least three independent groups. **p<0.01 versus H₂O₂-treated cells, ***p<0.001 versus H₂O₂-treated cells.

AI 본문요약
AI-Helper

문제 정의

본 연구에서는 병풀 추출물, 분획물(아시아티코사이드를 포함한 분획물) 및 아시아티코사이드로부터 ROS 자극 시 세포의 데미지를 조절하는 영향을 TLR4신호전달 체계를 통해 분석하여 염증완화 매커니즘을 확인하고자 하며, 병풀이 갖는 항산화 매커니즘에 아시아티코사이드가 대표적 물질임을 밝히고자 하였다.

대상 데이터

병풀은 신선약초에서 판매하는 제주 서귀포에서 채취한 건조 시료를 사용하였다. 병풀 잎 건조물을 분쇄하여 813.
87 g)을 얻어 농축 및 동결건조 후 실험에 이용하였다. 아시아티코사이드는 Sigma-Aldrich에서 구매하여 실험에 이용하였다.

데이터처리

모든 생리활성 실험은 3회 반복 실험을 통해 수치화 하였으며, 통계분석은 one-way ANOVA로 신뢰구간 p<0.05으로 검정을 실시하였다
05으로 검정을 실시하였다. 통계프로그램은 Graph Pad Prism 5 software (Graph Pad Software, Inc., La Jolla, CA, USA)를 이용하였다.

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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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