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目的:研究血瘀证相关的micorRNA-520b(miR-520b)对白介素-8(IL-8)的影响及丹参酮ⅡA(TanⅡA)的干预作用。方法:采用脂质体介导的转染方法将microRNA-520b模拟物(50 nmol·L-1)或抑制物(100 nmol·L-1)转染进入含1.6×104cells/well人脐静脉血管内皮细胞(HUVECs)的6孔板中24,36,48 h,采用5,10,20,40 mg·L-1TanⅡA干预24,48 h。荧光显微镜观察转染情况,MTT法检测细胞活性(n=5),硝酸还原酶法、酶联免疫法(ELISA)测定培养液中一氧化氮(NO),内皮素(ET),蛋白C受体(EPCR),血管内假性血友病因子(v WF)和血栓调节蛋白(TM)含量(n=3)。半定量反转录-聚合酶链反应(RT-PCR),蛋白质免疫印迹(Western blotting)检测细胞IL-8信使核糖核酸(mRNA)和蛋白表达水平(n=3)。结果:与对照组比较,模型组(miR-520b组)细胞有荧光,48 h荧光最强。细胞活性降低(P<0.01)且48 h最显著,NO含量下降(P<0.01),EPCR,v WF,ET含量升高(P<0.01),转染48 h后IL-8 mRNA和蛋白质表达下调(P<0.01)。与模型(miR-520b)组比较,给药组(TanⅡA组)细胞活性升高(P<0.01),TanⅡA(10 mg·L-1,48 h)干预效果最佳;NO含量升高(P<0.01);EPCR,v WF,TM,ET含量降低(P<0.01),IL-8 mRNA和蛋白质表达上调(P<0.01)。结论:miR-520b调节IL-8在血瘀证中发挥作用,可能为TanⅡA作用靶点。
Objective: To investigate the effect of micorRNA-520b (miR-520b) on interleukin-8 (IL-8) and the effect of TanⅡA on blood stasis syndrome. Methods: Liposome-mediated transfection method was used to transfect microRNA-520b mimics (50 nmol·L-1) or inhibitor (100 nmol·L-1) into human umbilical vein with 1.6 × 104cells / Endothelial cells (HUVECs) in 6-well plates 24,36,48 h, 5,10,20,40 mg · L-1Tan Ⅱ A intervention 24,48 h. The transfection conditions were observed by fluorescence microscopy. The cell viability was detected by MTT assay (n = 5), nitric oxide reductase and enzyme-linked immunosorbent assay (ELISA) were used to determine the nitric oxide (NO), endothelin (ET) (EPCR), von Willebrand factor (v WF) and thrombomodulin (TM) content (n = 3). Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting were used to detect IL-8 messenger RNA (mRNA) and protein expression (n = 3). Results: Compared with the control group, the cells in the model group (miR-520b group) had fluorescence and the fluorescence peaked at 48 hours. (P <0.01), the content of NO was decreased (P <0.01), the contents of EPCR, vWF and ET were increased at 48 h (P <0.01), and the mRNA and protein expression of IL- Down (P <0.01). Compared with the model group (miR-520b), the cell viability of Tan Ⅱ A group was significantly increased (P <0.01), TanⅡA (10 mg · L -1,48 h) <0.01). The contents of EPCs, vWF, TM and ET were decreased (P <0.01), and the mRNA and protein expressions of IL-8 were up-regulated (P <0.01). Conclusion: The regulation of IL-8 by miR-520b may play a role in the pathogenesis of blood stasis, which may be the target of TanⅡA.