[논문]Nrf2활성화를 통한 삼출건비탕(蔘朮健脾湯)의 간세포보호효과

김예림; 진효정; 박상미; 변성희; 송창현; 김상찬

doi:10.14374/hfs.2023.31.2.111

Nrf2활성화를 통한 삼출건비탕(蔘朮健脾湯)의 간세포보호효과
Hepatoprotective effect of Samchulgeonbi-tang via Nrf2 Activation 원문보기

大韓韓醫學方劑學會誌 = Herbal formula science, v.31 no.2, 2023년, pp.111 - 124

김예림 (대구한의대학교 한의과대학) , 진효정 (대구한의대학교 한의과대학) , 박상미 (대구한의대학교 한의과대학) , 변성희 (대구한의대학교 한의과대학) , 송창현 (대구한의대학교 한의과대학) , 김상찬 (대구한의대학교 한의과대학)

Abstract ▼ AI-Helper

Objectives : Oxidative stress is an important cause of many diseases including liver injury. Therefore, adequate regulation of oxidative stress plays a pivotal role in maintaining liver function. Until recently, there has been no studies on the hepatoprotective effect of Samchulgeonbi-tang (SCGBT). Therefore, the hepatoprotective effect of SCGBT was investigated in HepG2 cells. In this study, oxidative stress was induced by arachidonic acid (AA) and iron. Methods : To analyze the hepatoprotective effects of SCGBT against oxidative stress induced by AA + iron, the cell viability, apoptosis-related proteins and intracellular ROS, glutathione (GSH), and mitochondrial membrane permeability (MMP) were measured. In addition, nuclear factor erythroid 2-related factor 2 (Nrf2) transcription activation and expressions of Nrf2 target gene were analyzed through immunoblot analysis. Results : SCGBT increased the cell viability from AA + iron - induced cell death and inhibited apoptosis by regulating apoptosis related proteins. SCGBT protected cells by inhibiting ROS production, GSH depletion, and MMP degradation against AA + iron induced oxidative stress. Furthermore, Nrf2 activation was increased by SCGBT, and the Nrf2 target genes were also activated by SCGBT. Conclusions : These results suggest that the SCGBT has a hepatocyte protection effect and antioxidant effect from AA + iron induced oxidative stress.

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그림 Fig. 1. The effect of SCGBT on AA + iron-induced cell death (A) The effect of SCGBT alone on cytotoxicity. HepG2 cells were treated with 0.03-1.0 mg/mL of SCGBT for 24 h. (B) The cytoprotective effect of SCGBT. HepG2 cells were incubated in 0.1-1.0 mg/mL of SCGBT for 1 h prior to the addition of AA (10 μM), and the cells were further incubated for 12 h. Then, the cells were incubated with iron (5 μM) for 1 h. The effect of SCGBT on cell viability was assessed using MTT assays. All data represent the mean ± S.D. of three separated experiments (Significant compared with control; **p < 0.01; Significant compared with AA + iron, ##p < 0.01).
그림 Fig. 2. Inhibitory effect of SCGBT on AA + iron-induced apoptosis (A) Immunoblotting of apoptosis-related proteins. HepG2 cells were treated with 0.3 and 1.0 mg/mL SCGBT. After 1 h, the cells were incubated with AA (10 μM) for the next 12 h, and then subsequently exposed to iron (5 μM) for 1h. Expression of apoptosis-related proteins were observed by immunoblotting. Immunoblot against β-actin was used as loading control. (B) Cleaved PARP, procaspase-9, procaspase-3, and Bcl-2 expression intensities were determined by scanning densitometry. Data represent mean ± S.D. of three separated experiments (Significant compared to control, **p < 0.01; Significant compared to AA + iron, ##p < 0.01)
그림 Fig. 3. Antioxidant effect of SCGBT on AA + iron-induced oxidative stress. (A) Cellular GSH contents. The levels of GSH were measured by commercial kit as mentioned in materials and methods section, and the values were normalized by protein concentration. (B) ROS production. HepG2 cells were treated with 0.3 and 1.0 mg/mL SCGBT. After 1 h, the cells were incubated with AA (10 μM) for the next 12 h, and then subsequently exposed to iron (5 μM) for 1 h. DCFH-DA (20 μM) was added for 1 h after iron treatment. DCF fluorescence was monitored by automated microplate reader. All data represent mean ± S.D. of three separated experiments. (Significant compared to control, **p < 0.01; Significant compared to AA + iron, ##p < 0.01).
$Fig. 4. Effect of SCGBT on AA + iron-induced mitochondrial dysfunction. (A) HepG2 cells were treated with SCGBT (0.3 and 1.0 mg/mL). After 1 h, the cells were incubated with AA (10 μM) for the next 12 h, and then subsequently exposed to iron (5 μM) for 1 h. And the cells were stained with rhodamine123 for 30 minutes. The changes of mitochondrial membrane permeability were analyzed using flow cytometry. (B) Cellular populations with low rho123 intensities (RN1 fraction) are presented as percentages of the l0,000 cells. All data represent mean ± S.D. of three separated experiments. (Significant compared to control, **p < 0.01; Significant compared to AA + iron, ##p < 0.01).$ 그림 Fig. 4. Effect of SCGBT on AA + iron-induced mitochondrial dysfunction. (A) HepG2 cells were treated with SCGBT (0.3 and 1.0 mg/mL). After 1 h, the cells were incubated with AA (10 μM) for the next 12 h, and then subsequently exposed to iron (5 μM) for 1 h. And the cells were stained with rhodamine123 for 30 minutes. The changes of mitochondrial membrane permeability were analyzed using flow cytometry. (B) Cellular populations with low rho123 intensities (RN1 fraction) are presented as percentages of the l0,000 cells. All data represent mean ± S.D. of three separated experiments. (Significant compared to control, **p < 0.01; Significant compared to AA + iron, ##p < 0.01).
그림 Fig. 5. Effect of SCGBT on Nrf2 activation. (A) Reporter gene assays to measure ARE-mediated transcriptional activation. The expressed luciferase activity of recombinant HepG2 cells treated with 0.03-1.0 mg/mL SCGBT for 24 hours was measured. (B) Time-course nuclear Nrf2 expression level. HepG2 cells were treated with 1.0 mg/mL SCGBT for 0.25-6 h. (C) Concentration-dependent nuclear Nrf2 expression changes. HepG2 cells were treated 0.03-1.0 mg/mL SCGBT for 1 h. Lamin A/C was used as control for equal loadings. All data represent mean ± S.D. of three separated experiments. (Significant compared with control, **p < 0.01, *p < 0.05).
그림 Fig. 6. Induction of Nrf2-targeted antioxidant genes by SCGBT. The Sestrin2, NQO1, GCLC were immunoblotted in the HepG2 cells were treated with 1.0 mg/mL SCGBT for 0.5 or 1 h. Equal protein loading was confirmed by β-actin immunoblotting. All Data represent the mean ± S.D. of three separated experiments (Significant compared with control, **p < 0.01, *p < 0.05).

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저자의 다른 논문 :

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용어 설명: PCR은 세균 특이성이 있는 primer를 이용하여 적은 수의 세균이 있을지라도 쉽게 검출할 수 있는 유용한 방법이며, 이를 이용하여 구강 내 치면세균막이나 타액에서 직접 세균을 검출할 수 있게 되었다[8].

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