[논문]탕화지통산(湯火止痛散)의 NF-κB 및 MAPK 억제를 통한 항염증 효과

고민정; 지선영; 황보민

doi:10.6114/jkood.2023.36.1.001

[국내논문] 탕화지통산(湯火止痛散)의 NF-κB 및 MAPK 억제를 통한 항염증 효과
Anti-inflammatory Effects of Tanghwajitong-san through Inhibition of NF-κB and MAPK 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.1 - 20

고민정 (우보한의원 창원점) , 지선영 (대구한의대학교 한의과대학 안이비인후피부과학교실) , 황보민 (대구한의대학교 한의과대학 안이비인후피부과학교실)

Abstract ▼ AI-Helper

Objectives : The purpose of this study was to evaluate the anti-inflammatory effect of Tanghwajitongsan(THJTS) through inhibition of NF-κB and MAPK. Methods : We evaluated cell survival rate by MTT assay, NO production by nitrite content in the culture medium. We quantified TNF-α, IL-1β, IL-6 and PGE2 of the cultured supernatant by ELISA. And we evaluated the effect of THJTS on protein expression of NF-κB, MAPK, iNOS and COX-2 by Western blot analysis. THJTS ameliorates LPS-activated alterations in protein expression of NF-κB, p-38, iNOS and COX-2 and production of NO, pro-inflammatory cytokines and PGE2. Also, THJTS ameliorates LOX, PGN and FLA-activated alterations in protein expression of NO, iNOS. THJTS ameliorates only PGN-activated alterations in protein expression of COX-2. Results : THJTS ameliorates LPS-activated alterations in protein expression of NF-κB, p-38, iNOS and COX-2 and production of NO, pro-inflammatory cytokines and PGE2. Also, THJTS ameliorates LOX, PGN and FLA-activated alterations in protein expression of NO, iNOS. THJTS ameliorates only PGN-activated alterations in protein expression of COX-2. Conclusions : This study can provide scientific evidence for the anti-inflammatory effects and underlying mechanisms of THJTS.

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표/그림 (14)

표 Table 1. The Composition & Amount of Tanghwajitong-san
표 Table 2. Solvent Grade Conditions for UPLC-PDA Analysis
표 Table 3. Experimental Groups in This Study
그림 Fig. 1. Analysis of 5 Standard Compounds in THJTS by UPLC-PDA (A) UPLC chromatograms of 5 standard compounds, Decursin, Decursinol, Ferulic acid, Sennoside A & Rhein (B) UPLC chromatograms of Decursin, Decursinol, Ferulic acid, Sennoside A & Rhein in THJTS(Decursin: 330nm, Decur sinol: 330nm, Ferulic acid: 280nm, Sennoside A: 340nm, Rhein: 254nm)
그림 Fig. 2. Effect of THJTS on NO Production and Cell Survival Rate in LPS-stimulated Raw 264.7 Cell Raw 264.7 cells were treated with 10, 30, 100 and 300㎍/㎖ of THJTS for 1 hour and then treated with 1㎍/㎖ of LPS for 18 hours. (A) Relative NO production(fold) was determined by using Griess reagent and (B) relative cell survival rat e(%) was measured by MTT assay. Data represent the mean±SD of 3 separate experiments. *: significant as compared to control, **p<0.01, ^#: significant as compared to LPS-stimulated cells, ^##p<0.01
그림 Fig. 3. Inhibitory Effect of THJTS on LPS, LOX, PGN and FLA-induced NO Production and Cell Survival Rate in Raw 264.7 Cell Raw 264.7 cell were pretreated with 300㎍/㎖ of THJTS for 1 hour, before cultured with 30㎍/㎖ of LPS, LOX, PGN and FLA for 18 hours. (A) The relative levels of NO production were analyzed by measuring the absorbance and (B) the relative cell viability were examined by MMT assay. Data represent as mean±SD of 3 independent experiments. *: sig nificant as compared to control, *p<0.05, **p<0.01
그림 Fig. 4. Effect of THJTS on the iNOS and COX-2 in LPS-stimulated Raw 264.7 Cell The expressions of iNOS and COX-2 proteins were monitored 18 hours after treatment of LPS 1㎍/㎖ with or without THJTS 100, 300㎍/㎖ pretreatment(i. e. 1 hour before LPS). Same amounts of protein were separated by SDS-PAGE. Th e expressions of iNOS and COX-2 proteins were analyzed by immunoblot analysis using specific iNOS and COX-2 ant ibody. (A) The expression of iNOS and COX-2 confirmed same loading of the total lysates. (B) The relative iNOS densi ty was measured by scanning densitometer. (C) The relative COX-2 density was measured by scanning densitometer. Data represent the mean±SD of 3 separate experiments for each condition. *: significant as compared to control, **: p<0.01, ^#: compared to LPS alone, ^##: p<0.01
그림 Fig. 5. Inhibitory Effect of THJTS on LPS, LOX, PGN and FLA-induced iNOS and COX-2 Production in Raw 264.7 Cell (A) Raw 264.7 cell were pretreated with 300㎍/㎖ of THJTS for 1 hour, and then, further cultured with 30㎍/㎖ of LPS, LOX, PGN and FLA for 18 hours. (B) Relative levels of iNOS production were assayed by measuring the absorbance and (C) relative levels of COX-2 production were assayed by measuring the absorbance. Data were expressed as mean ±SD of 3 independent experiments. *:　significant as compared to control, *p<0.05, **p<0.01
그림 Fig. 6. Effect of THJTS on the Cytokine Production in LPS-induced Raw 264.7 Cell Pro-inflammatory cytokines were assayed in the medium of Raw 264.7 cells treated with LPS 1㎍/㎖ for 18 hours in the presence or absence of THJTS. The amounts of (A)TNF-α, (B)IL-1β and (C)IL-6 were measured by ELISA kit as des cribed in materials and methods section. Data represent the mean±SD of 3 separate experiments for each condition. *: vs control group, **p<0.01, ^#: LPS alone, ^##p＜0.01
그림 Fig. 7. Effect of THJTS on PGE2 Production in LPS-stimulated Raw 264.7 Cell Raw 264.7 were assayed in the medium of Raw 264.7 cell s treated with LPS 1㎍/㎖ for 18 hours in the presence or absence of THJTS. Production of PGE2 was measured by the PGE2 assay kit. Data represent as mean±SD of 3 sep arate experiments. *: significant as compared to control, **p<0.01, ^#: significant as compared to LPS-stimulated cell s, ^##P<0.01
$Fig. 8. Effect of THJTS on the NF-κB Signaling Pathway in Raw 264.7 Cell Stimulated with LPS The levels of IκBα proteins were monitored 30 minutes, p-IκBα and NF-κB proteins were 60 minutes after treatment of LPS 1㎍/㎖ with or without THJTS pretreatment(i. e. 1 hour before LPS). (A) The β-actin confirmed same loading of the whole cell lysate, and (D) Lamin A/C confirmed same loading of the nuclear fraction. (B, C, E) The relative den sity levels of the bands were measured with scanning densitometer. Data represent the mean±SD with 3 separate exp eriments. *: vs control group, **p<0.01, <sup>#</sup>: vs LPS alone, <sup>##</sup>p＜0.01$ 그림 Fig. 8. Effect of THJTS on the NF-κB Signaling Pathway in Raw 264.7 Cell Stimulated with LPS The levels of IκBα proteins were monitored 30 minutes, p-IκBα and NF-κB proteins were 60 minutes after treatment of LPS 1㎍/㎖ with or without THJTS pretreatment(i. e. 1 hour before LPS). (A) The β-actin confirmed same loading of the whole cell lysate, and (D) Lamin A/C confirmed same loading of the nuclear fraction. (B, C, E) The relative den sity levels of the bands were measured with scanning densitometer. Data represent the mean±SD with 3 separate exp eriments. *: vs control group, **p<0.01, ^#: vs LPS alone, ^##p＜0.01
그림 Fig. 9. Effect of THJTS on the Phosphorylation of MAPKs (A) The phosphorylated forms and total levels of MAPK proteins(p38, ERK and JNK). The proteins were measured 60 minutes after LPS(1μg/㎖) treatment with or without THJTS pretreatment(i.e. 1 hour before LPS). (B, C, D) The relative density levels of the bands were measured with a scanning densitometer. Values represent the mean±SD of 3 separat e experiments for each condition. *: vs control group, **p<0.01, ^#: vs LPS alone, ^##p＜0.01
그림 Fig. 11. Effect of THJTS on the iNOS in CA-induced Rat THJTS was administered orally to rats at dose of 0.3 and 1.0g/㎏/day for 5 days before the induction of paw edema. (A) The level of iNOS protein was determined by immunoblot analysis using an iNOS specific primary antibody. Same amounts of total protein were separated by SDS-PAGE. The expression of β-actin confirmed same loading of the lysat e. (B) The relative density levels of the bands were measured with a scanning densitometer. Data represent mean±SD of 3 independent experiments. *: vs normal group, **p<0.01, ^#: vs CA alone, ^##p＜0.01
그림 Fig. 10. Effect of THJTS on the Paw Edema in CA-stimulated Rat THJTS was administered orally to rats at dose of 0.3 and 1.0g/㎏/day for 5 days prior to occurrence of paw edem a. Paw edema was occurred by subcutaneous shot of CA (s.c. 1% solution of CA dissolved in saline, 0.1㎖/rat) into the right hind paw. The paw edema volume was measure d at 0, 1, 2, 3, 4 hours after CA subcutaneous shot. DEX A(1㎎/㎏ p.o.) was used as a positive control. Data repres ent the mean±SD of 5 animals. *: vs normal group, **p<0. 01, ^#: vs CA alone, ^#p<0.05, ^##p<0.01

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