丹酚酸A激活AMPK及SIRT1减轻棕榈酸诱导的肝细胞脂毒性

doi:10.12092/j.issn.1009-2501.2021.03.001

中国临床药理学与治疗学 ›› 2021, Vol. 26 ›› Issue (3): 241-249.doi: 10.12092/j.issn.1009-2501.2021.03.001

• 基础研究 • 下一篇

丹酚酸A激活AMPK及SIRT1减轻棕榈酸诱导的肝细胞脂毒性

赵芳卿¹，杨雯雯¹，殷玉杰¹，张斌²，王邦才²，窦晓兵^1,3，李松涛^3,4，朱林文思⁵

¹浙江中医药大学生命科学学院，杭州 310053，浙江；²宁波市中医院消化内科，宁波 310000，浙江；³浙江中医药大学分子医学研究所，杭州 310053，浙江；⁴浙江中医药大学基础医学院、公共卫生学院，杭州 310053，浙江；⁵浙江中医药大学附属第一医院消化内科，杭州 310003，浙江

收稿日期:2020-10-26 修回日期:2020-12-05 出版日期:2021-03-26 发布日期:2021-04-06
通讯作者: 朱林文思，通信作者，女，医学博士，主治医师，研究方向：消化系统疾病的分子机制和防治策略。 Tel: 0571-87068001 E-mail: zhulinwensi@163.com
作者简介:赵芳卿，女，硕士研究生，研究方向：营养相关代谢性疾病的分子机制及防治。 Tel: 18958084811 E-mail: zhaofangqing0316@163.com
基金资助:
国家自然科学基金（81973041，81773981）；浙江省杰出青年基金（LR20H260001）；浙江中医药大学2020年校级科研基金重点项目（2020ZZ09）

Salvianolic acid A activates AMPK and SIRT1 to reduce palmitic acid-induced lipotoxicity in hepatocyte

ZHAO Fangqing ¹, YANG Wenwen ¹, YIN Yujie ¹, ZHANG Bin ², WANG Bangcai², DOU Xiaobing ^1,3, LI Songtao ^3,4, ZHU Linwensi ⁵

¹School of Life Sciences, Zhejiang University of Traditional Chinese Medicine, Hangzhou 310053, Zhejiang, China

Received:2020-10-26 Revised:2020-12-05 Online:2021-03-26 Published:2021-04-06

摘要/Abstract

摘要： 目的：研究丹酚酸A对棕榈酸诱导的AML12肝细胞脂毒性的保护作用并初步探究其分子机制。方法：采用棕榈酸诱导AML12细胞建立脂毒性模型并给予丹酚酸A进行干预，采用乳酸脱氢酶（LDH）法检测细胞损伤，采用酶法检测细胞内甘油三酯含量，采用Bodipy染色法观察胞内的脂滴，采用甲基偶氮唑盐微量酶反应比色法（MTT）检测细胞存活率，采用荧光细胞通透性染料罗丹明123和荧光显微镜检测线粒体膜电位。采用2'，7'-二氯荧光黄双乙酸盐（DCFH-DA）和荧光显微镜检测细胞内活性氧（ROS）水平。采用Western blot技术检测沉默信息调节因子相关酶Ⅰ（silent information regulator 1, SIRT1）及腺苷酸活化蛋白激酶（AMP-activated protein kinase, AMPK）蛋白表达。结果：棕榈酸作为肝细胞脂毒性诱导剂可显著降低AML12细胞存活率，加重细胞内脂质沉积，而丹酚酸A干预可改善棕榈酸诱导的肝细胞脂毒性损伤（P<0.05）。其次，丹酚酸A干预可改善棕榈酸诱导的细胞线粒体膜电位降低（P<0.01）及细胞内ROS水平升高（P<0.01）。此外，Western blot结果显示棕榈酸可显著抑制AMPK及SIRT1蛋白表达（P<0.05），而经过丹酚酸A处理可显著升高棕榈酸抑制的SIRT1及AMPK磷酸化水平（P<0.05）。结论：丹酚酸A可有效改善脂毒性诱导的肝细胞损伤，该保护作用可能与其激活AMPK及SIRT1蛋白有关。

关键词: 丹酚酸A, 脂毒性, 腺苷酸活化蛋白激酶, 沉默信息调节因子相关酶Ⅰ

Abstract: AIM: To investigate the protective affect of salvianolic acid A on palmitic acid-induced lipotoxicity in hepatocyte and its potential molecular mechanism. METHODS: The lipotoxicity model of AML12 hepatocytes induced by PA was established. Different concentrations of Sal A (20, 40, 80, 120 μmol/L) were intervened. The hepatocyte injury was detected by the Lactate dehydrogenase (LDH) method, the intracellular triglyceride (TG) content was detected by enzyme assay and the lipid droplets were observed by Bodipy staining, cell viability was detected by MTT, Intracellular reactive oxygen species (ROS) were detected by 2'eci'- dichlorofluorescein diacetate (DCFH-DA) and fluorescence microscope. Mitochondrial membrane potential was detected by rhodamine 123 and fluorescence microscope. The expression of phosphorylation of AMP-activated protein kinase (AMPK) protein and silent information regulator 1 (SIRT1) protein were observed by Western blot. RESULTS: Model of hepatocyte lipotoxicity was established after intervented for 12 h in vitro with PA (0.5 mmol/L). Different concentrations of Sal A could significantly reduce the lipid deposition and hepatocytes injury induced by PA (P<0.05), and the protective effect was dose-dependent. Secondly, Sal A could significantly improve cell mitochondrial membrane potential (P<0.01) and abate the ROS level of hepatocytes induced by PA (P<0.01). In addition, PA could significantly inhibit AMPK and SIRT1 protein expression (P<0.05). Salvianolic acid A can significantly up-regulate SIRT1 and AMPK protein expression (P<0.05). CONCLUSION: Sal A improves PA induced lipotoxicity in hepatocyte, AMPK and SIRT1 may be a potential molecular target.

Key words: salvianolic acid A, lipotoxicity, AMP-activated protein kinase, silent information regulator 1

中图分类号:

R965.2

赵芳卿, 杨雯雯, 殷玉杰, 张斌, 王邦才, 窦晓兵, 李松涛, 朱林文思. 丹酚酸A激活AMPK及SIRT1减轻棕榈酸诱导的肝细胞脂毒性[J]. 中国临床药理学与治疗学, 2021, 26(3): 241-249.

ZHAO Fangqing, YANG Wenwen, YIN Yujie, ZHANG Bin, WANG Bangcai, DOU Xiaobing, LI Songtao, ZHU Linwensi. Salvianolic acid A activates AMPK and SIRT1 to reduce palmitic acid-induced lipotoxicity in hepatocyte[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2021, 26(3): 241-249.

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丹酚酸A激活AMPK及SIRT1减轻棕榈酸诱导的肝细胞脂毒性

Salvianolic acid A activates AMPK and SIRT1 to reduce palmitic acid-induced lipotoxicity in hepatocyte

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