Objective: Abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) are essential for vascular remodeling. Natural compounds with diterpene chinone or phenolic acid structure from Salvia miltiorrhiza, an eminent medicinal herb widely used to treat cardiovascular diseases in China, can effectively attenuate vascular remodeling induced by vascular injury. However, it remains unknown whether Salvia miltiorrhiza-derived miRNAs can protect VSMCs from injury by environmental stimuli. Here, we explored the role and underlying mechanisms of Salvia miltiorrhiza-derived Sal-miR-1 and 3 in the regulation of VSMC migration and monocyte adhesion to VSMCs induced by thrombin./nMethods: A mouse model for intimal hyperplasia was established by the ligation of carotid artery and the injured carotid arteries were in situ-transfected with Sal-miR-1 and 3 using F-127 pluronic gel. The vascular protective effects of Sal-miR-1 and 3 were assessed via analysis of intimal hyperplasia with pathological morphology. VSMC migration and adhesion were analyzed by the wound healing, transwell membrane assays, and time-lapse imaging experiment. Using loss- and gain-of-function approaches, Sal-miR-1 and 3 regulation of OTUD7B/KLF4/NMHC IIA axis was investigated by using luciferase assay, co-immunoprecipitation, chromatin immunoprecipitation, western blotting, etc./nResults: Salvia miltiorrhiza-derived Sal-miR-1 and 3 can enter the mouse body after intragastric administration, and significantly suppress intimal hyperplasia induced by carotid artery ligation. In cultured VSMCs, these two miRNAs inhibit thrombin-induced the migration of VSMCs and monocyte adhesion to VSMCs. Mechanistically, Sal-miR-1 and 3 abrogate OTUD7B upregulation by thrombin via binding to the different sites of the OTUD7B 3'UTR. Most importantly, OTUD7B downregulation by Sal-miR-1 and 3 attenuates KLF4 protein levels via decreasing its deubiquitylation, whereas decreased KLF4 relieves its repression of transcription of NMHC IIA gene and thus increases NMHC IIA expression levels. Further, increased NMHC IIA represses VSMC migration and monocyte adhesion to VSMCs via maintaining the contractile phenotype of VSMCs./nConclusions: Our studies not only found the novel bioactive components from Salvia miltiorrhiza but also clarified the molecular mechanism underlying Sal-miR-1 and 3 inhibition of VSMC migration and monocyte adhesion to VSMCs. These results add important knowledge to the pharmacological actions and bioactive components of Salvia miltiorrhiza. Sal-miR-1 and 3-regulated OTUD7B/KLF4/NMHC IIA axis may represent a therapeutic target for vascular remodeling.

中文翻译：

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丹参衍生的 miRNA 通过调节 OTUD7B/KLF4/NMHC IIA 轴来抑制血管重塑。

目的：血管平滑肌细胞（ VSMCs）的异常增殖和迁移对血管重塑至关重要。丹参是我国广泛用于治疗心血管疾病的名药，具有二萜或酚酸结构的天然化合物可有效减轻血管损伤引起的血管重构。然而，尚不清楚丹参衍生的 miRNAs 是否可以保护 VSMCs 免受环境刺激的伤害。在这里，我们探讨了丹参衍生的 Sal-miR-1 和 3 在调节凝血酶诱导的 VSMC 迁移和单核细胞与 VSMC 粘附中的作用和潜在机制。/n方法：通过结扎颈动脉建立小鼠内膜增生模型，并用F-127 pluronic凝胶原位转染损伤的颈动脉。Sal-miR-1和3的血管保护作用通过分析内膜增生与病理形态学进行评估。通过伤口愈合、transwell 膜测定和延时成像实验分析 VSMC 迁移和粘附。使用功能丧失和获得的方法，通过使用荧光素酶测定、免疫共沉淀、染色质免疫沉淀、蛋白质印迹等研究了 Sal-miR-1 和 3 对 OTUD7B/KLF4/NMHC IIA 轴的调节。/n结果： 丹参——衍生的Sal-miR-1和3可在灌胃后进入小鼠体内，显着抑制颈动脉结扎引起的内膜增生。在培养的 VSMC 中，这两种 miRNA 抑制凝血酶诱导的 VSMC 迁移和单核细胞与 VSMC 的粘附。从机制上讲，Sal-miR-1 和 3 通过与 OTUD7B 3'UTR 的不同位点结合来消除凝血酶对 OTUD7B 的上调。最重要的是，Sal-miR-1 和 3 对 OTUD7B 的下调通过降低其去泛素化来减弱 KLF4 蛋白水平，而降低的 KLF4 解除其对 NMHC IIA 基因转录的抑制，从而增加 NMHC IIA 表达水平。此外，增加的 NMHC IIA 通过维持 VSMC 的收缩表型来抑制 VSMC 迁移和单核细胞与 VSMC 的粘附。/n结论：我们的研究不仅发现了丹参中的新型生物活性成分，而且阐明了 Sal-miR-1 和 3 抑制 VSMC 迁移和单核细胞粘附于 VSMC 的分子机制。这些结果为丹参的药理作用和生物活性成分增加了重要知识。Sal-miR-1 和 3 调节的 OTUD7B/KLF4/NMHC IIA 轴可能代表血管重塑的治疗靶点。