艳山姜挥发油通过Nrf2/Notch1信号通路抑制高糖诱导的内皮间质转分化  

Inhibitory Effect of Essential Oil from Fructus Alpinia zerumbet on Endothelial-to-mesenchymal Transition Induced by High Glucose via Nrf2/Notch1 Signaling Pathway

作　　者：赵爽 何丽 黄梅 张嫩玲 姜丰 沈祥春 李玲 张彦燕 ZHAO Shuang;HE Li;HUANG Mei;ZHANG Nen-ling;JIANG Feng;SHEN Xiang-chun;LI Ling;ZHANG Yan-yan(School of Basic Medical Sciences,School of Pharmaceutical Sciences,Guizhou Medical University,Guiyang 550025,China)

机构地区：[1]贵州医科大学基础医学院,药学院,贵阳550025

出　　处：《中国实验方剂学杂志》2020年第23期99-105,共7页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(81560811,U1812403-4-4);贵州省科技支撑计划项目(黔科合[2020]1Z069)。

摘　　要：目的:分析艳山姜挥发油(EOFAZ)抑制高糖(HG)诱导的内皮间质转分化(EndMT)的作用及其机制。方法:体外培养人脐静脉内皮细胞(HUVECs),分析EOFAZ对HG诱导EndMT及氧化应激损伤的药效学作用,设定空白组,EOFAZ低、高剂量组(1,4μg·L-1),高糖组(35 mmol·L-1),EOFAZ预孵育2 h后加入HG共同孵育72 h复制EndMT细胞模型。采用蛋白免疫印迹法(Western blot)检测波形蛋白(vimentin)和血小板-内皮细胞黏附分子(CD31)的蛋白表达水平,血管生成实验检测细胞迁移能力以分析EOFAZ对EndMT的影响;采用2’,7’-二氯二氢荧光素二乙酸酯(DCFH-DA)荧光探针检测活性氧(ROS)水平的变化以及试剂盒法检测细胞内丙二醛(MDA),超氧化物歧化酶(SOD),过氧化氢酶(CAT)的含量,以分析EOFAZ对氧化应激的影响;采用Western blot检测核转录因子E2相关因子2(Nrf2)与Notch1的蛋白表达水平。通过腺病毒(AD)转染实现Nrf2的过表达,进一步分析EOFAZ抑制EndMT的作用机制,设定空白组,AD-Nrf2+EOFAZ组(4μg·L-1),AD-Nrf2组,高糖组(35 mmol·L-1),EOFAZ组(4μg·L-1)。Nrf2基因重组腺病毒过表达质粒感染细胞6 h,更换为正常培养基24 h,EOFAZ预孵育2 h后加入HG共同孵育72 h诱导EndMT。通过Western blot检测Nrf2,CD31,vimentin,Notch1和Snail的蛋白表达。结果:与高糖组比较,EOFAZ干预给药后明显上调HG诱导的CD31蛋白表达水平和下调vimentin蛋白表达水平(P<0.05,P<0.01),降低细胞迁移能力(P<0.01),同时降低ROS和MDA的水平(P<0.05,P<0.01),提高CAT和SOD的水平(P<0.01)。此外,EOFAZ干预给药能够显著上调抗氧化信号Nrf2的蛋白表达水平(P<0.01),下调Notch1的蛋白表达水平(P<0.01)。通过稳定的腺病毒转染HUVECs可实现Nrf2的高表达,Western blot结果显示,与高糖组比较,各给药处理组显著提高Nrf2和CD31的蛋白表达水平(P<0.01),显著下调vimentin,Notch1和Snail蛋白表达水平(P<0.01);与AD-Nrf2组相比,AD-Nrf2+EOFAZ组能够进一步上调Nrf2和CD31的�Objective: To investigate the inhibitory effect and the possible mechanism of essential oil from fructus Alpinia zerumbet(EOFAZ)on endothelial-to-mesenchymal transition(EndMT)induced by high glucose(HG). Method:Human umbilical vein endothelial cells(HUVECs)was cultured in vitro to analyze the pharmacodynamic effects of EOFAZ on EndMT and oxidative stress damage induced by HG. The experiment was set the blank group,HG group(35 mmol·L-1),EOFAZ low dose group(1 μg·L-1)and EOFAZ high dose group(4 μg·L-1). After EOFAZ intervention for 2 h,HG was added to incubate for 72 h in order to establish EndMT cell model. Western blot was used to detect the protein expression of vimentin and platelet endothelial cell adhesion molecule(CD31). Angiogenesis experiment was used to detect the ability of cell migration ability in order to analyze the effect of EOFAZ on EndMT. The changes of reactive oxygen species(ROS)levels were detected by 2’,7’-dichlorodihydrofluorescein diacetate(DCFH-DA)fluorescence probe and the contents of malondialdehyde(MDA),superoxide dismutase(SOD)and catalase(CAT)in cells were detected by the kit method to analyze the effect of EOFAZ on oxidative stress. Western blot was used to detect the protein expression levels of nuclear transcription factor E2 related factor 2(Nrf2)and Notch1. The overexpression of Nrf2 was achieved by adenovirus(AD) transfection and the mechanism of EOFAZ inhibiting EndMT was further analyzed. The experiment was set the blank group,HG group(35 mmol·L-1),AD-Nrf2 group,EOFAZ group(4 μg·L-1),AD-Nrf2+EOFAZ group(4 μg·L-1). The cells were infected with recombinant adenovirus overexpression plasmid of Nrf2 gene for 6 h,then replaced with normal medium for 24 h. After EOFAZ intervention for 2 h,HG was added to co-incubate for 72 h to induce EndMT. Western blot was used to detect the protein expressions of Nrf2,CD31,vimentin,Notch1 and Snail. Result: Compared with the HG group,after treatment with EOFAZ,the protein expression of CD31 was significantly up-regulated(P<0.05),the

关 键 词：艳山姜 挥发油 氧化应激 内皮间质转分化(EndMT) 核转录因子E2相关因子2(Nrf2) NOTCH信号 蛋白免疫印迹法 

分 类 号：R22[医药卫生—中医基础理论] R285[医药卫生—中医学]