Department of Orthopaedics, The First Affiliated Hospital of Kunming Medical University, Kunming, Yunnan 650032, P.R. China.
Department of Ultrasound, The First Affiliated Hospital of Kunming Medical University, Kunming, Yunnan 650032, P.R. China.
Mol Med Rep. 2018 Feb;17(2):2479-2487. doi: 10.3892/mmr.2017.8124. Epub 2017 Nov 21.
Reactive oxygen species (ROS) are implicated in the pathogenesis of thrombosis. Studies have reported that resveratrol exhibits antioxidative activities, however, the effect and underlying mechanisms of resveratrol on venous thrombosis remain largely unknown. To investigate the effect of resveratrol on venous thrombosis and the underlying mechanisms, the present study investigated the effects of resveratrol on cell viability, apoptosis, ROS generation and the expression of thrombosis‑associated markers in human umbilical vein endothelial cells (HUVECs). HUVECs were pretreated with resveratrol for 2 h and incubated with hydrogen peroxide (H2O2) for 24 h prior to the evaluation of cell viability, ROS generation, apoptosis and thrombosis‑associated marker expression by performing MTT assays, 2',7'‑dichlorofluorescin diacetate reagent, flow cytometry, and reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) and western blot analysis, respectively. Subsequently, to validate whether resve-ratrol functions via mitogen‑activated protein kinase (MAPK) pathways, the expression of thrombosis‑associated markers was detected by western blot analysis and RT‑qPCR following treatment of cells with resveratrol and the MAPK pathway activators anisomycin and curcumin. The results demonstrated that cell viability was markedly reduced by H2O2, and resveratrol treatment reversed the reductions in cell viability in a dose‑dependent manner. In addition, the levels of cell apoptosis and ROS generation were significantly increased by H2O2 alone, and resveratrol also reduced these effects in a dose‑dependent manner. Furthermore, the mRNA and protein expression of caspase‑3, P‑selectin glycoprotein ligand‑1 and von Willebrand factor was upregulated by H2O2 treatment in HUVECs. However, resveratrol decreased the protein expression these proteins in a dose‑dependent manner. Resveratrol also significantly inhibited the induction of phosphorylated (p)‑p38, P‑c‑Jun N‑terminal kinase and P‑extracellular signal‑regulated kinase by H2O2, and these effects were attenuated by the MAPK pathway activators anisomycin and curcumin. In conclusion, these results indicate that resveratrol protected HUVECs against oxidative stress and apoptosis. Furthermore, to the best of our knowledge, the present study is the first to demonstrate that resveratrol attenuates the expression of thrombosis‑associated markers induced by H2O2, which may occur through the suppression of the MAPK signalling pathways, indicating a potential novel therapeutic approach to prevent venous thrombosis.
活性氧（ROS）参与血栓形成的发病机制。研究表明白藜芦醇具有抗氧化活性，然而，白藜芦醇对静脉血栓形成的作用及其潜在机制仍知之甚少。为了研究白藜芦醇对静脉血栓形成的影响及其潜在机制，本研究探讨了白藜芦醇对人脐静脉内皮细胞（HUVEC）活力、细胞凋亡、ROS 生成和血栓相关标志物表达的影响。HUVEC 用白藜芦醇预处理 2 h，然后用过氧化氢（H2O2）孵育 24 h，通过 MTT 检测、2'，7'-二氯二乙酸荧光素试剂、流式细胞术和逆转录-定量聚合酶链反应（RT-qPCR）和 Western blot 分析分别评估细胞活力、ROS 生成、细胞凋亡和血栓相关标志物的表达。随后，为了验证白藜芦醇是否通过丝裂原活化蛋白激酶（MAPK）通路发挥作用，用白藜芦醇和 MAPK 通路激活剂anisomycin 和 curcumin 处理细胞后，通过 Western blot 分析和 RT-qPCR 检测血栓相关标志物的表达。结果表明，H2O2 显著降低细胞活力，白藜芦醇处理呈剂量依赖性逆转细胞活力降低。此外，H2O2 单独作用可显著增加细胞凋亡和 ROS 生成水平，白藜芦醇也呈剂量依赖性降低这些作用。此外，HUVEC 中 H2O2 处理可上调 caspase-3、P-选择素糖蛋白配体-1 和血管性血友病因子的 mRNA 和蛋白表达，而白藜芦醇呈剂量依赖性降低这些蛋白的蛋白表达。白藜芦醇还显著抑制 H2O2 诱导的磷酸化（p）-p38、P-c-Jun N-末端激酶和 P-细胞外信号调节激酶的诱导，而 MAPK 通路激活剂 anisomycin 和 curcumin 可减弱这些作用。总之，这些结果表明白藜芦醇可防止 HUVEC 发生氧化应激和凋亡。此外，据我们所知，本研究首次证明白藜芦醇可减弱 H2O2 诱导的血栓相关标志物的表达，这可能是通过抑制 MAPK 信号通路实现的，表明预防静脉血栓形成的一种潜在新治疗方法。
