Cao Qian, Jiang Yan, Shi Jin, Xu Changlu, Liu Xiaoxia, Yang Tiangui, Fu Peng, Niu Tiesheng
Department of Cardiology, Shengjing Hospital of China Medical University, Shenyang, People's Republic of China.
Department of Emergency Medicine, Shengjing Hospital of China Medical University, Shenyang, People's Republic of China.
J Surg Res. 2015 Apr;194(2):667-678. doi: 10.1016/j.jss.2014.12.013. Epub 2014 Dec 11.
Atherosclerosis is an inflammatory disease with the most common pathologic process leading to cardiovascular diseases. The aim of this study was to evaluate the effect of artemisinin (ART) on the proliferation, migration, and inflammation induced by tumor necrosis factor-α (TNF-α) of rat vascular smooth muscle cells (VSMCs).
Primary rat VSMCs were pretreated with ART and then co-incubated with TNF-α. Cell proliferation was evaluated by MTT assay. Cell migration was assessed by transwell assay. Reactive oxygen species (ROS) production was measured by flow cytometry after staining with dichloro-dihydro-fluorescein diacetate. Inflammation factors of nitric oxide and prostaglandin E2 (PGE2) were measured by responding assay kits. Expression levels of nuclear factor kappa B (NF-κB) subunit NF-κB p65 and the regulator inhibitor of nuclear factor kappa-B kinase-alpha (IκBα) were tested by Western blot, meanwhile, the activation of NF-κB was observed by immunofluorescence assay.
The proliferation, migration, and inflammation of VSMCs induced by TNF-α were significantly inhibited by ART treatment in a dose-dependent manner. Treatment with 100 μM ART for 2 h significantly reduced the expression of proliferating cell nuclear antigen and migration-related proteins matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9). On the other hand, the same treatment decreased the inflammation factors production of nitric oxide and PGE2. Fluorescence-activated cell sorting analysis revealed that ART suppressed the ROS production induced by TNF-α. Western blot analysis showed that both inflammation mediators inducible nitric oxide synthase and cyclooxygenase and the NF-κB pathway subunit NF-κB p65 were downregulated by ART.
The results suggest that ART can effectively inhibit the proliferation, migration, and inflammation of VSMCs induced by TNF-α through ROS-mediated NF-κB signal pathway.
动脉粥样硬化是一种炎症性疾病，是导致心血管疾病最常见的病理过程。本研究旨在评估青蒿素（ART）对大鼠血管平滑肌细胞（VSMC）增殖、迁移以及肿瘤坏死因子-α（TNF-α）诱导的炎症的影响。
原代大鼠VSMC先用ART预处理，然后与TNF-α共同孵育。通过MTT法评估细胞增殖。通过Transwell法评估细胞迁移。用二氯二氢荧光素二乙酸酯染色后，通过流式细胞术测量活性氧（ROS）的产生。通过相应的检测试剂盒测量一氧化氮和前列腺素E2（PGE2）等炎症因子。通过蛋白质免疫印迹法检测核因子κB（NF-κB）亚基NF-κB p65和核因子κB激酶α调节抑制剂（IκBα）的表达水平，同时通过免疫荧光法观察NF-κB的激活情况。
ART处理以剂量依赖性方式显著抑制了TNF-α诱导的VSMC增殖、迁移和炎症。用100μM ART处理2小时可显著降低增殖细胞核抗原以及迁移相关蛋白基质金属蛋白酶-2（MMP-2）和基质金属蛋白酶-9（MMP-9）的表达。另一方面，相同处理降低了一氧化氮和PGE2等炎症因子的产生。荧光激活细胞分选分析显示，ART抑制了TNF-α诱导的ROS产生。蛋白质免疫印迹分析表明，ART下调了炎症介质诱导型一氧化氮合酶和环氧化酶以及NF-κB信号通路亚基NF-κB p65。
结果表明，ART可通过ROS介导的NF-κB信号通路有效抑制TNF-α诱导的VSMC增殖、迁移和炎症。
