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

目的考察五味子木脂素缓解血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)诱导的人脐静脉内皮细胞(human umbilical vein endothelialcells,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AngⅡ诱导氧化应激损伤。结果 Sch B(50μmol·L^(-1))和Sch C(26μmol·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H_(2)O_(2)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。

OBJECTIVE To investigate schizandrin lignan relieving angiotensin Ⅱ(AngⅡ) 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 AngⅡ-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 AngⅡ, 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 AngⅡ. CONCLUSION Sch B and Sch C can relieve AngⅡ-induced oxidative stress damage in HUVEC by regulating the Nrf-2/Keap-1 antioxidant pathway.