丹酚酸B通过介导SIRT3/FOXO1信号通路减轻非酒精性脂肪肝的氧化应激反应

doi:10.5246/jcps.2022.09.059

摘要/Abstract

摘要：

丹酚酸B (Salvianolic acid B, Sal B)是一种多酚类抗氧化剂, 已被证明在多种疾病中具有抗脂质积累、抗炎和清除氧自由基的活性。我们旨在研究Sal B是否可以改善非酒精性脂肪性肝病 (Non-alcoholic fatty liver disease, NAFLD)的疾病进展并探索其可能的机制。通过油酸诱导HepG2细胞建立NAFLD模型组细胞, Sal B干预模型细胞建立治疗组细胞, 进行生化分析及油红O染色检测各组细胞内脂质含量, 通过免疫荧光及流式细胞仪检测细胞内活性氧 (reactive oxygen species, ROS)含量; 试剂盒及酶标仪定量细胞内脂氧化物丙二醛 (Malonydialdehyde, MDA)的含量; 通过蛋白质印迹分析、RT-qPCR和免疫沉淀 (Immunoprecipitation, IP)检测参与氧化应激的信号通路蛋白, 包括SIRT3、SOD2和FOXO1通路蛋白。研究发现, 油酸(Oleic acid, OA)可以诱导细胞内脂质、过氧化物及脂氧化物的积累, 降低SIRT3的表达, 并促进FOXO1乙酰化。然而Sal B治疗后显著扭转了这些趋势。进一步通过转染SIRT3质粒及SIRT3 siRNA发现, SIRT3质粒转染后降低了乙酰化FOXO1的表达, 并显著增强了Sal B诱导的SIRT3含量和对FOXO1去乙酰化的调节。SIRT3 siRNA转染后, Sal B诱导的乙酰化FOXO1下调被阻断, 表明Sal B对细胞保护作用是通过SIRT3介导FOXO1去乙酰化所致。SIRT3/FOXO1通路是控制NAFLD氧化应激的关键治疗靶点, Sal B通过SIRT3介导FOXO1去乙酰化能减轻OA诱导的肝脂肪变性和氧化应激反应, 对NAFLD细胞有保护作用。

关键词: 油酸, 氧化应激, 非酒精性脂肪肝, SIRT3, 乙酰化, 丹酚酸B

Abstract:

Salvianolic acid B (Sal B) is a polyphenolic antioxidant that has been shown to have anti-lipid accumulation, anti-inflammatory, and free oxygen radical scavenging activities in various diseases. Here, we aimed to examine whether Sal B could alleviate non-alcoholic fatty liver disease (NAFLD) and explore the possible mechanisms. Signaling pathways involved in oxidative stress, including SIRT3, SOD2, and FOXO1 pathways, were investigated by Western blotting analysis, RT-qPCR, and immunoprecipitation (IP). In the present study, oleic acid (OA) successfully induced lipid and peroxide accumulation, decreased SIRT3 expression, and increased FOXO1 acetylation. However, Sal B significantly reversed these trends. SIRT3 plasmid transfection further reduced the expression of acetylated FOXO1 and considerably enhanced the regulation of SIRT3 and acetylated FOXO1 induced by Sal B. Following SIRT3 siRNA transfection, Sal B-induced down-regulation of acetylated FOXO1 was blocked, suggesting that Sal B-mediated protection occurred through SIRT3-mediated FOXO1 deacetylation. The SIRT3/FOXO1 pathway was a critical therapeutic target for controlling oxidative stress in NAFLD, and Sal B conferred protection against OA-induced hepatic steatosis and oxidative stress through SIRT3-mediated FOXO1 deacetylation.

Key words: Oleic acid, Oxidative stress, Non-alcoholic fatty liver disease, SIRT3, Acetylated, Salvianolic acid B

Supporting:

陈小青, 彭波, 姜红梅, 张昌旭, 李海燕, 李子银. 丹酚酸B通过介导SIRT3/FOXO1信号通路减轻非酒精性脂肪肝的氧化应激反应[J]. 中国药学(英文版), 2022, 31(9): 698-710.

Xiaoqing Chen, Bo Peng, Hongmei Jiang, Changxu Zhang, Haiyan Li, Ziyin Li. Salvianolic acid B alleviates oxidative stress in non-alcoholic fatty liver disease by mediating the SIRT3/FOXO1 signaling pathway[J]. Journal of Chinese Pharmaceutical Sciences, 2022, 31(9): 698-710.

图/表 5

Figure 1. Sal B eliminates OA-induced HepG2 cell injury and hepatic steatosis. (A) Supernatant levels of ALT and AST. (B) Intracellular levels of TC and TG. The results are mean ± SD compared with the control group. *P < 0.01 and **P <0.01 compared with the model group. (C) Oil red O staining of HepG2 cells in the experimental group: a) cells in the control group were cultured in the complete medium for 24 h; b) cells in the model group were treated with 1 mM OA for 24 h; c) cells in the intervention group were pretreated with 8 μM Sal B for 3 h and then exposed to 1 mM OA for 24 h. Oil red O-stained sections were photographed at 400× magnification. The lipid droplets were dissolved with 60% isopropanol for 10 min, and the OD at a wavelength of 510 nm was measured in a 96-well plate reader.

Figure 2. Sal B alleviates the OA-induced accumulation of peroxide in HepG2 cells. (A) MDA content in HepG2 cells treated with OA and Sal B is shown. After HepG2 cells were treated, 10 μM H2DCFH was added to the cells for 60 min, and the ROS level was observed by fluorescence microscopy. (B) The cells were then collected to detect the ROS content (C) By flow cytometry.

Figure 3. Effects of OA and Sal B on the expressions of SIRT3, SOD2, and acetylated FOXO1 at the mRNA and protein levels. mRNA levels are expressed as the fold change relative to untreated control cells and are expressed as mean ± SD. Western blotting results of SIRT3 and SOD2 protein levels are expressed as acetylated protein/total protein ratio, and β-tubulin was used as an internal reference protein. (A) SIRT3 mRNA levels and (B) SOD2 mRNA levels in HepG2 cells. *P < 0.01 compared with the control group, and **P < 0.01 compared with the model group. Western blotting analysis of (C) SIRT3 and (D) SOD2 protein levels in HepG2 cells. *P < 0.01 compared with the control group, and **P < 0.01 compared with the model group. (E) IP analysis of acetylated FOXO1 protein levels in HepG2 cells. *P < 0.01 compared with the control group, and **P < 0.01 compared with the model group.

Figure 4. Efficiency screening of SIRT3-siRNA and SIRT3 overexpression plasmids transfected into HepG2 cells. HepG2 cells were plated in 24-well plates. When the cell density reached approximately 30%–50%, Lipo2000 was used as the transfection medium. The transfection conditions were observed under a fluorescence microscope (100×) after transfection for 6 h. A–C: The ratio of transfection reagent to siRNA was 1 μL: 20 pmol, 1 μL: 30 pmol, and 1 μL: 40 pmol, respectively. D–F: The ratio of transfection reagent to plasmid was 2 μL: 1 μg, 3 μL: 1 μg, and 4 μL: 1 μg. According to the screening results, the selected transfection conditions were the transfection reagent to siRNA ratio of 1 μL: 40 pmol and the transfection reagent and plasmid ratio of 3 μL: 1 μg.

Figure 5. Sal B-mediated protection is dependent on the SIRT3-induced inhibition of acetylated FOXO1 expression. HepG2 cells were transfected with control siRNA, SIRT3-siRNA, control plasmid, or SIRT3 plasmid and were subsequently exposed to 8 μM Sal B for 3 h. The expression of SIRT3 at the protein level was detected by Western blotting analysis. The FOXO1 protein in whole-cell lysates was immunoprecipitated with an anti-FOXO1 antibody and immunoblotted with an anti-acetyl antibody. *P < 0.01 compared with the control-siRNA group or the control plasmid group, and **P < 0.05 compared with the control-siRNA group or the control plasmid group.

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