miR-186-5p对冠心病大鼠血管内皮细胞损伤及FGF2/FGFR1信号通路的影响

Effects of miR-186-5p on vascular endothelial cell injury and FGF2/FGFR1 signaling pathway in rat model of coronary heart disease

目的探究微小RNA-186-5p(miR-186-5p)对冠心病(CHD)大鼠血管内皮细胞损伤的影响,以及对成纤维细胞生长因子2(FGF2)/成纤维细胞生长因子受体1(FGFR1)信号通路的作用。方法48只雄性SD大鼠均分成对照组、模型组、miR-186-5p抑制剂组和miR-186-5p抑制剂NC组。除对照组外,其余组大鼠均构建CHD模型,并给予相应干预4周。测定大鼠左心室射血分数(LVEF)、左心室缩短分数(LVFS)以及血清中总胆固醇(TC)、三酰甘油(TG)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)、白细胞介素(IL)-1β、IL-6和肿瘤坏死因子(TNF)-α、一氧化氮(NO)和内皮素(ET)-1水平;苏木素-伊红(HE)染色观察各组大鼠冠状动脉组织的病理学变化;Western blot法检测大鼠冠状动脉组织中FGF2、FGFR1蛋白相对表达水平;双荧光素酶验证miR-186-5p与FGF2的靶向关系。结果对照组大鼠冠状动脉血管管壁形态均匀,无炎性细胞浸润;模型组和miR-186-5p抑制剂NC组血管内皮细胞排列紊乱,炎性细胞浸润明显;miR-186-5p抑制剂组较模型组病理变化程度减轻,炎性细胞浸润减少。与对照组比较,模型组LVEF、LVFS、HDL-C、NO水平及FGF2、FGFR1蛋白相对表达水平降低,而TC、TG、LDL-C、IL-1β、IL-6、TNF-α、ET-1水平升高(P<0.05)。与模型组和miR-186-5p抑制剂NC组比较,miR-186-5p抑制剂组大鼠LVEF、LVFS、HDL-C、NO水平及FGF2、FGFR1蛋白相对表达水平升高,而TC、TG、LDL-C、IL-1β、IL-6、TNF-α、ET-1水平降低(P<0.05)。模型组和miR-186-5p抑制剂NC组各指标差异无统计学意义(P>0.05)。双荧光素酶报告实验结果证实,miR-186-5p与FGF2存在靶向结合位点。结论miR-186-5p的下调可减轻CHD大鼠的炎症反应和血管内皮细胞损伤,该作用与激活FGF2/FGFR1信号通路有关。

Objective To investigate the effect of microRNA-186-5 p(miR-186-5 p)on vascular endothelial cell injury and fibroblast growth factor 2(FGF2)/fibroblast growth factor receptor 1(FGFR1)signaling pathway in model rats with coronary heart disease(CHD).Methods A total of 48 male SD rats were divided into the control group,the model group,the miR-186-5 p inhibitor group and the miR-186-5 p inhibitor NC group(n=12).Except for the control group,rats in the other groups were all constructed CHD models and given corresponding interventions for 4 weeks.The serum levels of left ventricular ejection fraction(LVEF),left ventricular shortening fraction(LVFS),total cholesterol(TC),triglyceride(TG),high density lipoprotein cholesterol(HDL-C)and low density lipoprotein cholesterol(LDL-C),interleukin-1β(IL-1β),interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),nitric oxide(NO)and endothelin-1(ET-1)were measured in the four groups of rats.HE staining was used to observe the pathological changes of coronary artery in each group.Western blot assay was used to detect the expression levels of FGF2 and FGFR1 proteins in rat coronary artery.The double luciferase was used to verify the target relationship between miR-186-5 p and FGF2.Results In the control group,the vascular wall of the coronary arteries of rats was uniform without inflammatory cell infiltration.In the model group and the miR-186-5 p inhibitor NC group,vascular endothelial cells arranged disorderly and inflammatory cells infiltrated obviously.Compared with the model group,less pathological changes and less inflammatory cell infiltration were found in the miR-186-5 p inhibitor group.Compared with the control group,the contents of LVEF,LVFS,HDL-C,NO,the protein expression levels of FGF2 and FGFR1-were significantly decreased in the model group,and the levels of TC,TG,LDL-C,IL-1β,IL-6,TNF-αand ET-1 were significantly increased(P<0.05).Compared with the model group and the miR-186-5 p inhibitor NC group,the contents of LVEF,LVFS,HDL-C,NO,the protein expression levels of FGF2 and FGFR1 were significantly increased,and the levels of TC,TG,LDL-C,IL-1β,IL-6,TNF-αand ET-1-were significantly decreased in the miR-186-5 p inhibitor group(P<0.05).There were no significant differences in the above indicators between the model group and the miR-186-5 p inhibitor NC group(P>0.05).The results of double luciferase assay confirmed that miR-186-5 p had a targeted binding site with FGF2.Conclusion The down-regulation of miR-186-5 p can reduce inflammatory reaction and vascular endothelial cell injury in CHD rats,which is related to the activation of FGF2/FGFR1 signaling pathway.