The Mechanism Of Salvianolic Acid B Actions In Inhibiting Proliferation Of Vascular Smooth Muscle Cells

Vascular remodeling is a series of adaptive functional and structural compensatory reactions in which the blood vessels undergo changes to the internal environment,including pathological dysfunction,proliferation,migration,phenotypic transformation,imbalances of extracellulax matrix(ECM)synthesis and degradation and other pathological processes.The formation of neointima is one of the main pathological features of pathological vascular remodeling,and its main pathological features are the abnormal proliferation and migration of vascular smooth muscle cells and the infiltration of local inflammatory cells.Therefore,the study of its mechanism and effective prevention can provide new strategies for the prevention and treatment of cardiovascular diseases and other related diseases.Angiotensin II,a major peptide hormone of the renin-angiotensinaldosterone system(RAAS),induces the proliferation and migration of VSMC.MicroRNA(miRNA),which regulate gene expression at the posttranscriptional level by binding to target mRNAs,are involved in the pathophysiological processes of many kinds of diseases,which are closely related to the occurrence and development of cardiovascular diseases.miR-146 a is a member of the miR-146 family,which is abundant in vivo.Studies have shown that miR-146 a is involved in many biological processes such as cell proliferation,differentiation,apoptosis,and migration.Sal B(Salvianolic acid B,Sal B)is a water-soluble active ingredient extracted from the roots and rhizomes of the traditional Chinese medicine Salvia miltiorrhiza,which belongs to the class of phenolic acids and has the effects of reducing myocardial ischemia-reperfusion injury,anti-myocardial cell injury,protection of vascular endothelial cells,prevention and treatment of atherosclerosis,and anti-tumor and other broad-spectrum role.However,there are few reports on the effect of salvianolic acid B on Vascular smooth muscle proliferation and its underlying mechanism.Objective: In this paper,we established mouse vascular smooth muscle cell proliferation model induced by Ang II.Salvianolic acid B(Sal B)was used to explore the molecular mechanism whereby Sal B inhibits Ang II-induced proliferation of vascular smooth muscle cells.The aim of this study provided a theoretical basis for promoting blood circulation to dissipate blood stasis and preventing vascular intima hyperplasia.Methods:1 Ang II induced proliferation model of VSMC.Quantitative real time PCR(qRT-PCR)was used to detect the expression of miR-146 a.Western blot and q RT-PCR were used to detect the expression of PCNA,cyclin D1 and KLF5.MTS and cell-counting assays were performed to examine the proliferation of VSMC.2 VSMC was transfected with miR-146 a mimic or miR-146 a inhibitor,and then was treated with Ang II,Western blot and q RT-PCR were used to detect the expression of PCNA,cyclin D1 and KLF5.MTS and cell-counting assays were performed to examine the proliferation of VSMC.3 C57BL/6 mice were randomly divided into three groups: unligated group,ligated+miR-146 a antagomir group and ligated+control antagomir group.Morphology of neointima hyperplasia was assessed by hematoxylin/eosin staining.4 Ang II stimulation was performed after pre-incubation of mouse VSMC with Sal B,cell counts and MTS assay were used to detect cell proliferation,and Western blot and qRT-PCR were used to analyze PCNA,cyclin D1 and KLF5 expression.qRT-PCR analysis detected miR-146 a expression.5 C57BL/6 mice were randomly divided into three groups: control group,NaCl+ligated group and Sal B+ligated group,and morphology of neointima hyperplasia was assessed by hematoxylin/eosin staining.Results:1.miR-146 a mediates Ang II-induced proliferation of VSMC.Ang II stimulation upregulated the expression of miR-146 a in a time-dependent manner compared with the control group.Simultaneously,the expression of cell nuclear antigen(PCNA),cell cycle protein(cyclin D1)and KLF5 was significantly increased.MTS and cell-counting also showed an increased cell proliferation.2.Overexpression and knockdown of mir-146 a,respectively,promotes or inhibits the proliferation of VSMC in vitro.Western blot and qRT-PCR showed that overexpression of miR-146 a significantly promoted the expression of PCNA,cyclin D1 and KLF5.MTS and cell-counting showed an increase in cell proliferation.Knockdown of miR-146 a expression strongly reduced PCNA,cyclin D1 and KLF5 expression compared with the control group.MTS and cell-counting assays yielded similar results.3.miR-146 a antagomir inhibits neointimal formation.In order to further observe the role of miR-146 a in cell proliferation and intimal hyperplasia,miR-146 a was knocked down by transfection of miR-146 a antagomir after carotid artery ligation.HE staining showed that miR-146 a antagonist significantly reduced intimal hyperplasia,intima/media ratio and intima area,indicating that miR-146 a plays an important role in neointima formation after carotid artery ligation.4.Sal B inhibits the proliferation of Ang II-induced VSMC by inhibiting the expression of miR-146 a.Pretreating VSMC with Sal B markedly decreased Ang II-induced,cell proliferation.Western blot and qRT-PCR analysis showed that the expression of PCNA,cyclin D1 and KLF5 was significantly down-regulated after pre-incubation of VSMC with Sal B compared with the control group.Sal B also significantly reduced the expression of miR-146 a in Ang II-induced VSMC.Moreover,Sal B obviously inhibited Ang II-induced proliferation of vascular smooth muscle cells by inhibiting the expression of miR-146 a.5.Sal B reduced the intimal hyperplasia caused by carotid artery ligation.HE staining showed that intraperitoneal injection of Sal B could inhibit the intimal hyperplasia of the blood vessels.Compared with the unligatedgroup,ligation of carotid artery resulted in a significant increase in I/M ratio and neointimal area.Administration of Sal B significantly reduced I/M ratio and neointimal area.The above results indicate that Sal B can inhibit the intimal hyperplasia caused by ligation.Conclusion:1.Ang II promotes VSMC proliferation and expression of miR-146 a.2.miR-146 a promotes the proliferation of VSMC through up-regulating the expression of cyclin D1 and KLF5.3.miR-146 a mediates Ang II-induced VSMC proliferation.4.miR-146 a antagomir suppresses carotid intimal hyperplasia induced by ligated.5.Sal B reduces Ang II-induced VSMC proliferation and intimal hyperplasia by inhibiting the expression of miR-146 a.