[논문]RAW264.7 대식세포에서 미선나무 잎 추출물의 항산화, 항염증 효능 및 기전연구

유주희; 김경아

doi:10.4163/jnh.2023.56.5.455

RAW264.7 대식세포에서 미선나무 잎 추출물의 항산화, 항염증 효능 및 기전연구
Antioxidant and anti-inflammatory effects and mechanism of Abeliophyllum distichum leaf extract in RAW264.7 macrophages 원문보기

Journal of nutrition and health, v.56 no.5, 2023년, pp.455 - 468

초록
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본 연구에서는 잠재적인 기능성 소재로의 개발 가능성을 검증하기 위하여 미선나무 잎 추출물의 항산화능과 항염증 효능을 확인하였다. 시료의 총 폴리페놀함량, DPPH 및 ABTS 라디칼 소거능, FRAP value를 측정하여 미선나무 잎 추출물의 항산화 활성을 확인하였으며 또한 RAW264.7 세포에 미선나무 잎 추출물 처리 후 HO-1의 발현이 증가하는 것을 통해 미선나무 잎 추출물의 항산화 기전을 확인하였다. 한편, LPS로 유도된 염증 상태의 RAW264.7 세포에서 미선나무 잎 추출물은 NF-κB 활성 억제를 통한 NO 생성 억제, iNOS, COX-2 발현 억제 및 염증성 사이토카인의 발현과 생성을 억제하는 것을 확인하였다. 이러한 본 연구 결과는 향후 미선나무 잎 추출물의 기능성 소재로의 개발을 위한 기초자료 마련에 그 의의가 있다.

Abstract ▼ AI-Helper

Purpose: Abeliophyllum distichum (A.distichum) is a plant native to Korea. In this study, we investigated the mechanism of antioxidant and anti-inflammatory effects of the leaf extract of A.distichum. Methods: The antioxidant capacity of the A.distichum leaf extract was determined based on the total polyphenol content, 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay, and the ferric reducing antioxidant power (FRAP) assay. The anti-inflammatory effects of the A.distichum leaf extract were evaluated by measuring the production of nitric oxide (NO) and the expression levels of proinflammatory cytokines including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 using the enzyme-linked immunosorbent assay (ELISA) and reverse transcription quantitative real-time PCR (RT-qPCR). In addition, the expression of heme oxygenase-1 (HO-1), nuclear transcription factor-erythroid 2 related factor (Nrf2), inducible nitric oxide synthase (iNOS), and cyclooxygenase 2 (COX-2), as well as the activation of nuclear factorkappa B (NF-ĸB) were examined using the western blot analysis. Results: The total polyphenol content of the A.distichum leaf extract was 329.89 ± 30.17 gallic acid equivalents mg/g and the DPPH and ABTS scavenging activities were 55% and 70%, respectively. Additionally, the FRAP value of the extract was 743.68 ± 116.59 mg/mL. After 12-hour treatment with the A.distichum leaf extract, there was a tendency for the Nrf2 expression to increase, and the expression of HO-1 was significantly elevated in the RAW264.7 cells. The A.distichum leaf extract treatment resulted in decreased levels of NO, TNF-α, IL-6, and IL-1β, as well as reduced expression of iNOS and COX-2, along with inhibition of NF-κB activation in lipopolysaccharide-stimulated RAW264.7 cells. Conclusion: These results suggest that the A.distichum leaf extract exerts antioxidative and anti-inflammatory effects by upregulating the expression of HO-1 and downregulating NF-κB activation.

주제어

표/그림 (7)

표 Table 1. Antioxidant capacities of ADE
그림 Fig. 1. Effect of Abeliophyllum distichum leaf extracts on RAW264.7 cells viability. RAW264.7 cells were pretreated for 1 hour with various concentration of A. distichum leaf extract before lipopolysaccharide (100 ng/mL) treated. Values are expressed as mean ± standard deviation. CON, control. *p < 0.05 compared to CON.
그림 Fig. 2. Effect of ADEs on the expression levels of Nrf2 and HO-1 in RAW264.7 cells. Protein expression was analyzed by Western blot hybridization. Actin was used as an internal control. Results are mean ± standard deviation (n = 3-5). ADE, Abeliophyllum distichum leaf extract; Nrf2, nuclear transcription factor-erythroid 2 related factor; HO-1, heme oxygenase-1. *p < 0.05, **p < 0.01 compared to control.
그림 Fig. 3. Effect of ADEs on NO production in LPS-stimulated RAW264.7 cells. The production of NO was assayed in the culture media of cells pretreated with ADE (25, 50, 100, 200 μg/mL) or DMSO for 1 hour and then stimulated with LPS (100 ng/mL) for 24 hours. Results are mean ± standard deviation of 4 replicate determinations (n = 4). ADE, Abeliophyllum distichum leaf extract; NO, nitric oxide; LPS, lipopolysaccharide. *p < 0.05, **p < 0.01 compared to LPS. ^##p < 0.01 compared to control.
그림 Fig. 4. Effect of ADEs on TNF-α (A), IL-1β (B) and IL-6 (C) production in RAW264.7 cells. Cells were pretreated with ADE (25, 50, 100, 200 μg/mL) or DMSO'for 1 hour and then stimulation with LPS (100 ng/mL) for 24 hours. The levels of TNF-α, IL-1β and IL-6 production were measured using enzyme-linked immunosorbent assay kits. The data are presented as means ± standard deviation (n = 4). ADE, Abeliophyllum distichum leaf extract; LPS, lipopolysaccharide; IL, interleukin; TNF, tumor necrosis factor. *p < 0.05, **p < 0.01 compared to LPS. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 compared to control.
그림 Fig. 5. Effect of ADEs on LPS-induced mRNA expression levels of TNF-α (A), IL-1β (B), IL-6 (C) in RAW264.7 cells. Cells were pretreated with ADE (25, 50, 100, 200 μg/mL) or DMSO for 1 hour and then stimulation with LPS (100 ng/mL) for 24 hours. The mRNA levels of TNF-α, IL-1β and IL-6 were measured by quantitative real-time polymerase chain reaction. The data are presented as means ± standard deviation (n = 8). ADE, Abeliophyllum distichum leaf extract; LPS, lipopolysaccharide; IL, interleukin; TNF, tumor necrosis factor. *p < 0.05, **p < 0.01, ***p < 0.001 compared to LPS. ^###p < 0.001 compared to control.
그림 Fig. 6. Inhibitory effect of ADEs on protein levels of p-p65, iNOS, COX-2 in RAW264.7 cells. RAW264.7 cells were pretreated with ADE (25, 50, 100, 200 μg/mL) or DMSO or 1 hour and then stimulated with LPS (100 ng/mL) for 24 hours. The results presented as mean ± standard deviation of experiments (n = 3-5). ADE, Abeliophyllum distichum leaf extract; iNOS, inducible nitric oxide synthase; COX-2, cyclooxygenase 2; LPS, lipopolysaccharide. *p < 0.05, **p < 0.01 compared to LPS. ^###p < 0.001 compared to control.

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