miRNA-141通过调节TLR4信号通路抑制大鼠血栓形成

miR-141 inhibits thrombosis in vascular pathways through the TLR4 signaling pathway

目的研究微小RNA(miR)-141对血栓模型大鼠血栓形成、血小板聚集及血浆中一氧化氮(NO)、环磷酸鸟苷(cGMP)和前列环素(PGI2)的影响。方法90只SD雄性大鼠随机进行分组,10只为正常对照组;模型组使用5 g/L的CMC-Na建立大鼠动静脉旁路血栓模型(20只);治疗组在CMC-Na基础上分别静脉注射100 mg/kg阿司匹林(Asp)作为阳性对照(10只)、miR-141的拟似物mimic(20只)、mimic阴性对照mimic-NC(10只)、mimic联合Toll样受体4(TLR4)过表达重组质粒pcDNA3.1-TLR4(mimic+pcDNA3.1-TLR4)(20只),连续4周。观察各组血栓形成的影响,比浊法观察ADP诱导的血小板聚集变化,试剂盒法检测各组血浆中NO,cGMP,PGI2的水平;蛋白免疫印迹法检测TLR4、NF-κB、Rac1、IL-1β的蛋白水平。结果miR-141-mimic可显著抑制血栓形成和血小板聚集率(P<0.05),升高血浆中NO(P<0.05)、cGMP(P<0.05)、PGI2(P<0.05)浓度,miR-141-mimic组效果优于阿司匹林组(P<0.05)。miR-141-mimic可显著抑制NF-κB(P<0.05),Rac1(P<0.05),IL-1β(P<0.05)的蛋白水平。mimic+pcDNA3.1-TLR4组的逆转miR-141-mimic组的效果(P<0.05)。结论miR-141通过抑制TLR4信号通路发挥抗血栓作用。

Objective To study the effects of microRNA(miR)-141 on thrombosis,platelet aggregation,and plasma nitric oxide(NO),cyclic guanosine monophosphate(cGMP),and prostacyclin(PGI2)in thrombus model rats.Methods Ninety SD male rats were randomly divided into groups.10 of them are normal control group.Five g/L CMC-Na was used to establish rat arteriovenous bypass thrombosis models(20 rats).In the treatment group,100 mg/kg aspirin(ASP)was intravenously injected on the basis of CMC-Na as positive controls(n=10).miR-141 mimics(n=20),mimic negative control mimic-NC group(n=10),mimic combined with Toll-like receptor 4(TLR4)recombinant plasmid pcDNA3.1-TLR4(mimic+pcDNA3.1-TLR4)(n=20)were respectively injected in rats for 4 weeks.The effects of thrombosis in each group and the changes of platelet aggregation induced by ADP with turbidimetry were observed,and the levels of NO,cGMP,PGI2 in plasma of each group were measured by kit method.Western blot was used to detect TLR4,NF-κB,Rac1,IL-1βprotein levels.Results miR-141-mimic significantly inhibited thrombosis and platelet aggregation rate(P<0.05),increased plasma NO(P<0.05),cGMP(P<0.05),PGI2(P<0.05)concentrations.The effect of 141-mimic group was better than that of aspirin group(P<0.05).MiR-141-mimic significantly inhibited the protein levels of NF-κB(P<0.05),Rac1(P<0.05),and IL-1β(P<0.05).The mimic+pcDNA3.1-TLR4 group reversed the effect of the miR-141-mimic group(P<0.05).Conclusion miR-141 exerts antithrombotic effect through TLR4 signaling pathway.