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目的研究淫羊藿苷(Ica)对H_2O_2诱导的人脐静脉内皮细胞(HUVEC)氧化损伤的保护作用及其机制。方法 H_2O_2诱导氧化损伤模型。药效学研究分为六组:空白组,模型组(750μmol·L-1 H2O2),Ica-L、M、H组(750μmol·L~(-1) H_2O_2+1×10-8、1×10-7、1×10-6mol·L~(-1)Ica)和溶媒组(0.1%DMSO)。Ica给药12 h后加入H_2O_2,Ica作用时间为24、36和48 h。Ica作用机制研究分为五组:空白组、模型组、Ica-H组、LY组(750μmol·L~(-1) H_2O_2+25μmol·L~(-1)LY294002)、Ica-H+LY组。LY294002为PI3K抑制剂,给予LY294002后2 h加入Ica,12 h后加入H_2O_2,Ica作用时间为48 h。MTT法检测细胞活力,ELISA检测细胞培养液中活性氧(ROS)的含量,显微镜观察细胞形态及存活细胞密度,Annexin V-FITC/PI染色荧光显微镜观察细胞凋亡率,Real time RT-PCR检测Bcl-2、Bax mRNA的表达,Western Blot检测p-Akt和Akt蛋白的表达。结果与空白组相比,模型组细胞活力降低(P<0.01),ROS分泌量和凋亡细胞增加(P<0.01);与模型组相比,Ica-L、M、H组均能提高细胞活力(P<0.01),降低细胞培养液中ROS的含量(P<0.05),其抑制率呈现时间和剂量依赖性。同时,Ica能明显降低细胞凋亡率(P<0.01),上调p-Akt蛋白和Bcl-2 m RNA的表达(P<0.05),下调Bax mRNA的表达(P<0.05);Ica的以上作用被LY294002拮抗。结论 Ica(1×10~(-8)-1×10-6 mol·L~(-1))对H_2O_2诱导的HUVEC氧化损伤有保护作用,能缓解氧化应激诱导的HUVEC凋亡,其机制与PI3K/Akt信号通路有关。
Objective To investigate the protective effect and mechanism of icariin (Ica) on the oxidative damage induced by H_2O_2 in human umbilical vein endothelial cells (HUVECs). Methods H 2 O 2 induced oxidative damage model. Pharmacodynamic studies were divided into six groups: blank group, model group (750μmol·L-1 H2O2), Ica-L, M and H group (750μmol·L -1 H 2 O 2 + 1 × 10-8,1 × 10-7,1 × 10-6mol·L -1 Ica) and vehicle group (0.1% DMSO). After 12 h of Ica treatment, H 2 O 2 was added and the Ica action time was 24, 36 and 48 h. The mechanism of Ica was divided into five groups: blank group, model group, Ica-H group, LY group (750μmol·L -1 H 2 O 2 + 25μmol·L -1 LY294002), Ica-H + LY group . LY294002 was a PI3K inhibitor. Ica was added 2 h after LY294002 and H 2 O 2 was added 12 h later. Ica action time was 48 h. Cell viability was measured by MTT assay. ROS content in cell culture medium was detected by ELISA. Cell morphology and viable cell density were observed by microscope. Cell apoptosis rate was observed by Annexin V-FITC / PI staining. Real time RT-PCR Bcl-2, Bax mRNA expression, Western Blot detection of p-Akt and Akt protein expression. Results Compared with the blank group, the viability of the model group decreased (P <0.01) and the amount of ROS secreted and the number of apoptotic cells increased (P <0.01). Compared with the model group, the Ica-L, (P <0.01), and decreased the content of ROS in the cell culture medium (P <0.05), and the inhibition rate showed time-and dose-dependent manner. At the same time, Ica could significantly reduce the apoptosis rate (P <0.01), up-regulate the expression of p-Akt protein and Bcl-2 mRNA (P <0.05) and down-regulate the expression of Bax mRNA Antagonized by LY294002. Conclusions Ica (1 × 10 -8 -1 × 10 -6 mol·L -1) can protect HUVEC from oxidative damage induced by H 2 O 2 and alleviate the oxidative stress-induced apoptosis of HUVEC. The mechanism of Ica And PI3K / Akt signaling pathway.