[논문]괴화산(槐花散)의 NF-κB 기전을 통한 항염증 효과 연구

조현희; 최지영; 황보민; 지선영

doi:10.6114/jkood.2023.36.1.021

[국내논문] 괴화산(槐花散)의 NF-κB 기전을 통한 항염증 효과 연구
Anti-inflammatory Effects of Goihwa-san Water Extract via NF-κB Inhibition 원문보기

韓方眼耳鼻咽喉皮膚科學會誌 = The journal of Korean Medicine Ophthalmology & Otolaryngology & Dermatology, v.36 no.1, 2023년, pp.21 - 39

조현희 (가원한의원) , 최지영 (대구한의대 부속 대구한방병원 안이비인후피부과) , 황보민 (대구한의대 부속 대구한방병원 안이비인후피부과) , 지선영 (대구한의대 부속 대구한방병원 안이비인후피부과)

Abstract ▼ AI-Helper

Objectives : The purpose of this study was to investigate the anti-inflammatory effect of Goihwa-san water extract(GHS) in vitro & in vivo. Methods : In vitro, we evaluated the anti-inflammatory effect of GHS by comparing the Raw 264.7 cells with 10, 30, 100, 300㎍/㎖ of GHS for 1 hour before Lipopolysaccharide(LPS) to the single LPS treated group. We examined the relative cell viability by MTT assay and the relative level of LPS, Loxoribine(LOX), Peptidoglycan(PGN), Flagellin(FLA)-induced NO production by using Griess reagent and measured relative iNOS protein level and COX-2 protein level by using western blot and Image analyzing system. We measured the production of TNF-α, IL-1β, and IL-6 by each ELISA kits and then measured the relative levels of IκBα, p-IκBα in whole-cell lysate fraction and NF-κB in nuclear fraction by using western blot and Image analyzing system. In vivo, we induced the paw edema by subcutaneous injection of 100㎕/rat CA and measured the swelling volume of paw by using a plethysmometer and then measured the relative iNOS protein level by using western blot. Results : As a result, in vitro, LPS, PGN-induced NO production was significantly inhibited by pretreatment with GHS. GHS reduced LPS, PGN-induced iNOS expression, PGN-induced COX-2 expression and LPS-induced production of cytokine(TNF-α, IL-1β, IL-6). Expression of IκBα was increased by pretreatment with GHS 100㎍/㎖. And the expression of p-IκBα and NF-κB were decreased by pretreatment with GHS 100㎍/㎖. In vivo, CA-induced inflammation rat model was used for the evaluation of the anti-inflammatory effect of GHS. 0.3 or 1.0g/kg of GHS significantly reduced the increases of paw swelling and iNOS expression in paw tissues. Conclusions : These results show that GHS can decrease inflammatory response via inhibition of the NF-κB pathway in vitro. And in vivo, the anti-inflammatory effect suggest the clinical basis of GHS for the treatment of inflammatory diseases.

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표/그림 (14)

표 Table 1. The Composition and Amount of Goihwa-san
표 Table 2. Solvent Gradient Conditions for UPLC-PDA Analysis
표 Table 3. Experimental Groups
그림 Fig. 1. Analysis of Four Standard Compounds by UPLC-PDA (A) UPLC chromatogram of four marker compounds (B) The chromatograms were obtained 5.915㎚(Rutin), 6.439㎚ (Sophoricoside), 3.59㎚(Sophocarpine) and 1.846㎚(Galic acid).
표 Table 4. Contents of the Four Marker Components
그림 Fig. 2. Effects of GHS on (A)Cell Viability and (B)NO Production in LPS-induced Raw 264.7 Cell (A) Raw 264.7 cells were treated with 10, 30, 100 and 300㎍/㎖ of GHS dissolved in DMEM for 1 hour before to the treatment of LPS(1㎍/㎖), and then the cells were incubated for 18 hours. After the incubation, the cells were treated with MTT solution. Cell viability was measured by MTT assay. (B) Raw 264.7 cells were treated with 10, 30, 100 and 300㎍/㎖ of GHS dissolved in DMEM for 1 hour before to the treatment of LPS(1㎍/㎖), and then the cells were incubated for 18 hours. After the incubation, the concentration of nitric oxide in culture medium was evaluated as described in materials and methods part. Values represent the mean±SD of four separate experiments for each condition(*: significant as compared with control, **p<0.01, ^#: significant as compared with LPS alone, ^#p<0.05, ^##p<0.01). GHS: Goihwa-san Water Extract, NO: nitric oxide
그림 Fig. 3. Effect of GHS on NO-production and Cell Survival in LOX- in PGN-, and in FLA- induced in Raw 264.7 Cell Raw 264.7 cells were pre-treated with 100㎍/㎖ of GHS for 1 hour and then further treated with 30㎍/㎖ of LOX, PGN and FLA for 18 hours. (A) The relative NO production was evaluated by absorbance. (B) And relative cell viability were examined by MMT assay. Data were expressed as mean±SD of three separate experiments(*: Significant as compared with control group, **p<0.01). GHS: Goihwa-san Water Extract, NO: nitric oxide, LOX: loxoribine, PGN: peptidoglycan, FLA: flagellin
그림 Fig. 4. Effect of GHS on iNOS in LPS-induced Raw 264.7 Cells The levels of iNOS proteins were measured by treated GHS(0, 30, 100㎍/㎖) pretreatment for 1 hour with LPS(1㎍/㎖) during 18 hours. (A) iNOS expression was measured by western blot. (B) Relative iNOS protein was measured by scanning densitometry. Values are represented the mean±SD of three independent experiments(Significant as compared with control, *: vs control group, **p<0.01, ^#: vs LPS alone, ^##p<0.01).
그림 Fig. 5. Effect of GHS on iNOS and COX-2 Protein Level in Four TLR Ligand-induced Raw 264.7 Cells (A) iNOS and COX-2 proteins were measured through western blot analysis using specific primary antibodies. The same amount of total protein was isolated by SDS-PAGE. The β-actin level was used as an internal control for the loading protein. (B, C) Relative (B)iNOS and (C)COX-2 protein was measured by scanning densitometry. Values are represented the mean±SD of three independent experiments(*: significant compared to control, *p<0.05, **p<0.01). GHS: Goihwa-san Water Extract, iNOS: Inducible nitric oxide synthase, COX-2: Cyclooxygenase-2, NO: nitric oxide, LOX: loxoribine, PGN: peptidoglycan, FLA: flagellin
그림 Fig. 6. Effects of GHS on the Cytokines Production in LPS-induced Raw 264.7 Cells Pro-inflammatory cytokines were evaluated in the medium of Raw 264.7 cells treated with LPS(1㎍/㎖) in the presence or absence of GHS for 18 hours. The amount of (A)TNF-α, (B)IL-1β and (C)IL-6 were evaluated by ELISA kit as described in materials and methods part. Values represent the mean±SD of three separate experiments for each condition(*: vs control group, **p<0.01, ^#: LPS alone, ^#p＜0.05, ^##p＜0.01).
$Fig. 7. Effects of GHS in LPS-induced Raw 264.7 Cells on the NF-κB Pathway The IκBα protein was monitored for 30 minutes, the p-IκBα protein and the NF-κB protein was monitored for 60 minutes, after treatment of LPS(1㎍/㎖) with or without GHS pretreatment(i.e. 1 hour prior to LPS). (A, D) β-actin confirmed the constant load of whole cell lysate, and Lamin A/C confirmed the constant load of nuclear fraction. (B, C, E) The relative density levels of the bands were measured by scanning densitometry. The data represent the mean±SD through three separate experiments(*: vs control group, **p<0.01, <sup>#</sup>: vs LPS alone, <sup>##</sup>p<0.01).$ 그림 Fig. 7. Effects of GHS in LPS-induced Raw 264.7 Cells on the NF-κB Pathway The IκBα protein was monitored for 30 minutes, the p-IκBα protein and the NF-κB protein was monitored for 60 minutes, after treatment of LPS(1㎍/㎖) with or without GHS pretreatment(i.e. 1 hour prior to LPS). (A, D) β-actin confirmed the constant load of whole cell lysate, and Lamin A/C confirmed the constant load of nuclear fraction. (B, C, E) The relative density levels of the bands were measured by scanning densitometry. The data represent the mean±SD through three separate experiments(*: vs control group, **p<0.01, ^#: vs LPS alone, ^##p<0.01).
그림 Fig. 8. Effects of GHS on Phosphorylation of MAPKs (A) The MAPKs protein was monitored after 1 hour depending on the presence or absence of GHS pretreatment after LPS(1㎍/㎖). (B, C, D) The relative density level of the band was measured by a scan density measurement method. The three individual experiments for each condition represent the mean±SD(*: vs control group, *p<0.05, **p<0.01).
그림 Fig. 9. Inhibitory Effects of GHS in Acute Inflammatory Edema Induced by CA GHS was administered to mice in P.O. doses at 0.3g/㎏ /day and 1.0g/㎏/day for 4 days prior to the onset of paw edema. Paw edema was induced by injection of CA(0.1㎖ /rat) dissolved in saline the right hind paw. Paw edema was measured at 0-4 hours after CA injection. DEXA(1㎎/ ㎏ P.O.) was used as a positive control. The data represent the mean±SD of five animals. *: vs normal group, **p<0.01, ^#: vs CA, ^##p<0.01
그림 Fig. 10. Effects of GHS on iNOS Expression in CA-induced Rat GHS was administered to mice in P.O. doses of 0.3 and 1.0g/㎏/day for 4 days before paw edema was induced. (A) The expression of iNOS was measured through western blot analysis using iNOS-specific primary antibody. β-actin identified the constant load of the entire cell solution, the same amount of total protein was isolated by SDS-PAGE. (B) The relative density level of the band was measured by the scanning density measurement method. The value represents the mean±SD. These are the results of three separate experiments(*: vs Control Group, **p<0.01, ^#: vs CA alone, ^##p<0.01).

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