[논문]작약이 HaCaT 세포에서 아토피 피부염 관련 염증 억제에 미치는 영향

이혜인; 김엄지; 손동빈; 주병덕; 손영주; 김은영; 정혁상

doi:10.14406/acu.2022.007

작약이 HaCaT 세포에서 아토피 피부염 관련 염증 억제에 미치는 영향
Effect of Paeonia Lactiflora Pallas on Atopic Dermatitis-Related Inflammation in HaCaT Cell 원문보기

Korean journal of acupuncture, v.39 no.2, 2022년, pp.43 - 53

이혜인 (경희대학교 한의과대학 해부학교실) , 김엄지 (경희대학교 한의과대학 해부학교실) , 손동빈 (경희대학교 한의과대학 해부학교실) , 주병덕 (경희대학교 한의과대학 해부학교실) , 손영주 (경희대학교 한의과대학 해부학교실) , 김은영 (경희대학교 한의과대학 해부학교실) , 정혁상 (경희대학교 한의과대학 해부학교실)

Abstract ▼ AI-Helper

Objectives : Paeonia lactiflora Pallas (PLP) have been reported to have pharmacological effects such as anti-inflammatory and analgesic. However, it is not yet known whether PLP extract has anti-inflammatory effect on HaCaT cells, human keratinocyte. Methods : To confirm the anti-inflammatory effect of PLP on keratinocyte, TNF-𝛼/IFN-𝛾-stimulated HaCaT cells were used. HaCaT cells were pre-treated with PLP for 1h before stimulation with TNF-𝛼/IFN-𝛾. Then HaCaT cells were stimulated with TNF-𝛼/IFN-𝛾 for 24 h, the cells and media were harvested to measure the inflammatory cytokines levels. Granulocyte-macrophage colony stimulating factor (GM-CSF), monocyte chemoattractant protein-1 (MCP-1), interleukin 1 beta (IL-1𝛽), and TNF-𝛼 were analyzed by enzyme-linked immunosorbent assay (ELISA), and the mRNA expression of thymus and activation-regulated chemokines (TARC), IL-6, and IL-8 were measured by reverse transcription-polymerase chain reaction (RT-PCR). We also investigated the inhibitory mechanism of the mitogen-activated protein kinase (MAPKs) including ERK, JNK, and p38 and nuclear factor-kappaB (NF-𝜅B) by PLP using western blot. Results : PLP did not show cytotoxicity in HaCaT cells. In TNF-𝛼/IFN-𝛾-stimulated HaCaT cells, PLP significantly inhibited the expression of GM-CSF, MCP-1 IL-1𝛽, TNF-𝛼, TARC and IL-6. PLP inhibited the phosphorylation of ERK and translocation of NF-𝜅B into the nucleus. Conclusions : These results indicate that PLP could ameliorate the TNF-𝛼/IFN-𝛾-stimulated inflammatory response through inhibition of MAPK and NF-kB signal pathway. This suggests that PLP could be used beneficial agent to improve skin inflammation.

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표/그림 (6)

그림 Fig. 1. The cytotoxicity of PLP in HaCaT cells. HaCaT cells were treated with various concentrations (12.5, 25, 50 and 100 μg/ml) of PLP for 24 h. The cytotoxicity of PLP in HaCaT cells was measured by the MTS assay. All experiments were conducted at least 3 times.
표 Table 1. Primer Sequence for RT-PCR Analysis
그림 Fig. 2. The effect of PLP on the expression of pro-inflammatory cytokines in HaCaT cells. To analyze the effect of PLP on cytokine expression, HaCaT cells were treated with PLP (12.5, 25, 50 and 100 μg/ml) for 1 h and then treated with TNF-α/IFN-γ (each 10 ng/ml) for 24 h. The concentration of (A) GM-CSF, (B) MCP-1, (C) IL-1β and (D) TNF-α in the cell culture medium was determined by ELISA. All experiments were conducted at least 3 times. All data represent the means ± SEM. ^#p＜ 0.05 and ^##p＜0.01 versus nontreated group; *p＜0.05 and **p＜ 0.01 versus only TNF-α/IFN-γ- treated group.
그림 Fig. 3. The effect of PLP on the mRNA expression of pro-inflammatory cytokines in HaCaT cells. To analyze the effect of PLP on mRNA expression of pro-inflammatory cytokine, HaCaT cells were treated with PLP (12.5, 25, 50 and 100 μg/ml) for 1 h and then treated with TNF-α/IFN-γ (each 10 ng/ml) for 24 h. (A) The effect of PLP on the mRNA expression of pro-inflammatory cytokines was measured by RT-PCR. The mRNA expressions of (B) TARC, (C) IL-6, and (D) IL-8 were normalized to GAPDH expression. All experiments were conducted at least 3 times. All data represent the means ± SEM. ^#p＜ 0.05 and ^##p＜0.01 versus nontreated group; *p＜0.05 and **p＜ 0.01 versus only TNF-α/IFN-γ- treated group.
그림 Fig. 4. The effect of PLP on the phosphorylation of MAPKs in HaCaT cell. To analyze the effect of PLP on MAPKs signaling pathway, HaCaT cells were were treated with PLP 100 μg/ml for 1 h and then treated with TNF-α/IFN-γ (each 10 ng/ml) for 5, 15 and 30 min. The phosphorylation of MAPKs were analyzed in whole protein lysates. (A) Total MAPKs (t-ERK, t-JNK and t-p38) and phosphorylated MAPKs (p-ERK, p-JNK and p-p38) were determined by western blot analysis. (B) p-ERK, (C) p-JNK and (D) p-p38 were quantified in each total form. All experiments were conducted at least 3 times. All data represent the means ± SEM. ^#p＜0.05 and ^##p＜ 0.01 versus non-treated group; *p＜ 0.05 versus only TNF-α/IFN-γ- treated group.
그림 Fig. 5. The effect of PLP on NF-κB signaling pathway in HaCaT cell. To analyze the effect of PLP on NF- κB signaling pathway, HaCaT cells were treated with PLP 100 μg/ml for 1 h and then treated with TNF-α/ IFN-γ (each 10 ng/ml) for 5, 15, 30 and 60 min. NF-kB and p-NF-kB were analyzed in nuclear proteins. Iκ Bα and p-IκBα were analyzed in cytoplasmic proteins. (A) The effect of PLP on NF-κB signaling pathway was investigated by western blot analysis. The expressions of (B) NF- κB and (C) p-NF-κB were quantified with lamin B. The expression of (D) IκBα and (E) p-IκBα was quantified with actin. All experiments were conducted at least 3 times. All data represent the means ± SEM. ^#p＜0.05 and ^##p＜0.01 versus non-treated group; *p＜0.05 versus only TNF-α/IFN-γ-treated group.

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