五味子木脂素缓解AngⅡ诱导人脐静脉内皮细胞氧化应激损伤的研究

Study on Schisandra Lignan Relieves Ang II-induced Oxidative Stress Damage of Human Umbilical Vein Endothelial Cells

关键词:  五味子木脂素  氧化应激  核因子E2相关因子/Kelch样环氧氯丙烷相关蛋白-1  人脐静脉内皮细胞

Key words:  schisandra lignans  oxidative stress  Nrf-2/Keap-1  human umbilical vein endothelial cells (HUVEC)

摘要:

目的 考察五味子木脂素缓解血管紧张素Ⅱ（angiotensin Ⅱ，AngⅡ）诱导的人脐静脉内皮细胞（human umbilical veinendothelial cells，HUVEC）氧化应激损伤，并探究其内在机制。方法 HUVEC分别于一定浓度梯度的五味子乙素（schisandrin B，Sch B）、五味子丙素（schisandrin C，Sch C）共孵育24 h，利用CCK8确定药物作用浓度； Annexin V-FITC/PI双染法检测细胞凋亡变化情况； DCFH-DA和DHE探针检测胞内活性氧表达情况；采用RT-qPCR检测Bax、Bcl-2 mRNA水平，抗氧化蛋白核因子E2相关因子（Nrf-2）、血红素氧合酶-1（HO-1）和醌氧化还原酶（NQO-1）的mRNA表达情况；采用Western blotting检测Bax、Bcl-2、Nrf-2和Kelch样环氧氯丙烷相关蛋白-1（Keap-1）的表达情况；利用特异性的siRNA构建低表达Nrf-2的HUVEC，探究五味子木脂素是否靶向Nrf-2缓解Ang II诱导氧化应激损伤。结果 Sch B（50 μmol·L^-1）和Sch C（26 μmol·L^-1）作用于AngⅡ诱导的HUVEC损伤模型中，发现Sch B、Sch C可通过调控抗氧化轴Nrf-2/Keap-1，抑制细胞凋亡（P < 0.01或P < 0.001）和ROS水平（P < 0.05），促进HO-1（P < 0.05）、NQO-1（P < 0.05）等抗氧化酶的表达，并有效缓解H₂O₂诱导的氧化应激损伤。在特异性沉默Nrf-2的HUVEC中，发现Sch B、Sch C可靶向Nrf-2激活下游抗氧化酶（NQO-1、HO-1）的表达，缓解AngⅡ诱导的氧化应激损伤。结论 Sch B、Sch C可通过调控Nrf-2/Keap-1抗氧化通路缓解AngⅡ诱导的HUVEC氧化应激损伤。

Abstract:

OBJECTIVE To investigate schizandrin lignan relieving angiotensin II(Ang II) induced oxidative stress damage of human umbilical vein endothelial cells(HUVEC) and explore its internal mechanism. METHODS HUVEC were incubated with a certain concentration gradient of schisandrin B(Sch B) and schisandrin C(Sch C) for 24 h. Annexin V-FITC/PI double staining method was used to detect changes in cell apoptosis. DCFH-DA and DHE probes was used to detect the expression of intracellular reactive oxygen species. RT-qPCR was used to detect the levels of Bax and Bcl-2 mRNA, the antioxidant protein nuclear factor E2 related factor(Nrf-2), heme oxygenase-1(HO-1) and quinone oxidoreductase(NQO-1) mRNA expression. Western blotting was used to detect the expression of Bax, Bcl-2, Nrf-2 and Kelch-like epichlorohydrinrelated protein-1(Keap-1). Used specific siRNA to construct HUVEC with low expression of Nrf-2, and explore whether Schisandra lignans target Nrf-2 to alleviate Ang II-induced oxidative stress damage. RESULTS Using Sch B(50 μmol·L^-1) and Sch C(26 μmol·L^-1) in the HUVEC injury model induced by Ang II, it was found that Sch B and Sch C could inhibit cell apoptosis(P < 0.01 or P < 0.001) and ROS level(P < 0.05) by regulating the antioxidant axis Nrf-2/Keap-1, promoting the expression of HO-1(P < 0.05), NQO-1(P < 0.05) and other antioxidant enzymes, and effectively relieved H₂O₂-induced oxidation stress injury. In HUVEC that specifically silenced Nrf-2, it was found that Sch B, Sch C can target Nrf-2 to activate the expression of downstream antioxidant enzymes(NQO-1, HO-1) and relieve the oxidative stress damage induced by Ang Ⅱ. CONCLUSION Sch B and Sch C can relieve Ang II-induced oxidative stress damage in HUVEC by regulating the Nrf-2/Keap-1 antioxidant pathway.