[논문]LPS로 활성화된 RAW 264.7 cell에서 NF-𝜅B억제를 통한 육일산(六一散) 물추출물의 염증억제효과

이창욱; 박상미; 김은옥; 변성희; 김상찬

doi:10.14374/hfs.2022.30.2.45

LPS로 활성화된 RAW 264.7 cell에서 NF-𝜅B억제를 통한 육일산(六一散) 물추출물의 염증억제효과
Anti-inflammatory Effect of Yukil-san Water Extract on LPS-induced RAW 264.7 Cells 원문보기

大韓韓醫學方劑學會誌 = Herbal formula science, v.30 no.2, 2022년, pp.45 - 57

이창욱 (대구한의대학교 한의과대학 방제학교실) , 박상미 (대구한의대학교 한의과대학 방제학교실) , 김은옥 (대구한의대학교 한의과대학 방제학교실) , 변성희 (대구한의대학교 한의과대학 방제학교실) , 김상찬 (대구한의대학교 한의과대학 방제학교실)

Abstract ▼ AI-Helper

Objectives : Yukil-san (YIS, 六一散; Liu yi san) is composed of Talcum and Glycyrrhizae Radix, the name is said to be derived from the proportion of the two herbal components of the formula. The YIS originated from 'Formulas from the discussion illuminating the Yellow Emperor's Basic Question'(黃帝素問宣明論方; Huang di su wen xuan ming lun fang) written by Liu Wan-Su (劉完素). YIS could clear summerheat, resolve dampness, and augment the qi. This formula may be used to treat the common cold, influenza, acute gastroenteritis, cystitis, urethritis and bacillary dysentery. But, there is insufficient of study about the effects of YIS on the anti-inflammatory activities. The present study evaluated the anti-inflammatory effects of YIS on lipopolysaccharide (LPS)-activated RAW 264.7 cells. Methods : Cell viability was assessed by MTT assay and nitric oxide (NO) was evaluated by measuring the nitrite content in culture medium. Pro-inflammatory cytokines such as tumor necrosis factor-α, interleukin-1β and IL-6 were quantified by ELISA kit. The expression of proteins related with nuclear factor-κB (NF-κB) pathway and inducible NO synthase (iNOS) were assessed by western blot analysis. Results : YIS significantly inhibited the expression of iNOS increased by LPS, and thus significantly inhibited the production of NO. In addition, YIS significantly inhibited pro-inflammatory cytokines. In the regulation of inflammation, NF-κB pathway plays a crucial role. YIS inhibited the expression of p-IκBα and thus inhibited the translocation of NF-κB to the nucleus. Conclusions : These results suggest that YIS ameliorates inflammatory response in LPS-activated RAW 264.7 cells through the inhibition of inflammatory mediators, via suppression of the NF-κB pathway. Therefore, this study provides objective evidence for the anti-inflammatory effect of YIS including the underlying mechanisms.

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표/그림 (8)

그림 Fig.1. Effects of YIS on NO in LPS-activated RAW 264.7 cells. RAW 264.7 cells were treated with 10 ~ 300 μg/mL of YIS dissolved in DMEM for 1 h before the treatment of LPS (1 μg/mL), and then the cells were incubated for 18 h. The concentration of nitric oxide in culture medium was evaluated as described in materials and methods part. Values represent the mean ± SD with four separate experiments for each condition. *: vs. control, ** p＜0.01, #: vs. LPS alone, ## p＜0.01.
그림 Fig. 2. Effects of YIS on cell viability in LPS-activated RAW 264.7 cells. RAW 264.7 cells were treated with 10 ~ 300 μg/mL of YIS dissolved in DMEM for 1 h before the treatment of LPS (1 μg/mL), and then the cells were incubated for 18 h. After the incubation, the cells were treated with MTT solution. Values represent the mean ± SD with four separate experiments for each condition. *: vs. control, ** p＜0.01
그림 Fig. 3. Effect of YIS on iNOS expression in LPSactivated RAW 264.7 cells. The levels of iNOS protein were evaluated 18 h after treatment of LPS (1 μg/mL) with or without YIS (100, 300 μg/mL) pretreatment (i.e. 1 h before LPS). Equal amounts of protein were separated by SDS-PAGE. β -actin was used as an equal loading control (A). The relative density level of the protein was measured by scanning densitometry (B). Values represent the mean ± SD with three separate experiments for each condition. *: vs. control, ** p＜0.01, #: vs. LPS alone, # p＜0.05, ## p＜0.01
그림 Fig. 4. Effect of YIS on TNF-α in LPS-activated RAW 264.7 cells. The concentration of TNF-α was evaluated in the medium of RAW 264.7 cells cultured with LPS (1 μg/mL) in the presence or absence of YIS for 18 h. The amount of TNF-α was measured by ELISA kit as described in materials and methods part. Values represent the mean ± SD with three separate experiments for each condition. *: vs. control, ** p＜0.01, #: vs. LPS alone, # p＜0.05, ## p＜0.01
그림 Fig. 5. Effect of YIS on IL-1β in LPS-activated RAW 264.7 cells. The concentration of IL-1β was evaluated in the medium of RAW 264.7 cells cultured with LPS (1 μg/mL) in the presence or absence of YIS for 18 h. The amount of IL-1β was measured by ELISA kit as described in materials and methods part. Values represent the mean ± SD with three separate experiments for each condition. *: vs. control, ** p＜0.01, #: vs. LPS alone, ## p＜0.01
그림 Fig. 6. Effect of YIS on IL-6 in LPS-activated RAW 264.7 cells. The concentration of IL-6 was evaluated in the medium of RAW 264.7 cells cultured with LPS (1 μg/mL) in the presence or absence of YIS for 18 h. The amount of IL-6 was measured by ELISA kit as described in materials and methods part. Values represent the mean ± SD with three separate experiments for each condition. *: vs. control, ** p ＜0.01, #: vs. LPS alone, ## p＜0.01
그림 Fig. 7. Effect of YIS on p-IκBα expression in LPS-activated RAW 264.7 cells. The levels of p-IκBα protein were evaluated 1 h after treatment of LPS (1 μg/mL) with or without YIS (100, 300 μg/mL) pretreatment (i.e. 1 h before LPS). Equal amounts of protein were separated by SDS-PAGE. β-actin was used as an equal loading control (A). The relative density level of the protein was measured by scanning densitometry (B). Values represent the mean ± SD with three separate experiments for each condition. *: vs. control, ** p＜0.01, #: vs. LPS alone, ##p＜0.01
$Fig. 8. Effect of YIS on NF-κB expression in LPS-activated RAW 264.7 cells. The levels of NF-κB protein in nuclear fraction were evaluated 1 h after treatment of LPS (1 μg/mL) with or without YIS (100, 300 μg/mL) pretreatment (i.e. 1h before LPS). Equal amounts of protein were separated by SDS-PAGE. Lamin A/C was used as an equal loading control (A). The relative density level of the protein was measured by scanning densitometry (B). Values represent the mean ± SD with three separate experiments for each condition. *: vs. control, ** p＜0.01, #: vs. LPS alone, ## p＜0.01$ 그림 Fig. 8. Effect of YIS on NF-κB expression in LPS-activated RAW 264.7 cells. The levels of NF-κB protein in nuclear fraction were evaluated 1 h after treatment of LPS (1 μg/mL) with or without YIS (100, 300 μg/mL) pretreatment (i.e. 1h before LPS). Equal amounts of protein were separated by SDS-PAGE. Lamin A/C was used as an equal loading control (A). The relative density level of the protein was measured by scanning densitometry (B). Values represent the mean ± SD with three separate experiments for each condition. *: vs. control, ** p＜0.01, #: vs. LPS alone, ## p＜0.01

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