Department of Cardiology, Shuguang Hospital of Shanghai University of Traditional Chinese Medicine, Shanghai, 201203, China.
Chin J Integr Med. 2022 Sep;28(9):785-793. doi: 10.1007/s11655-022-2891-6. Epub 2022 Jul 15.
To investigate the regulatory roles of Shexiang Baoxin Pill (SXBXW) in neointimal formation and vascular smooth muscle cells (VSMCs) invasion and apoptosis as well as the potential molecular mechanisms using cultured VSMCs model of vascular injury (platelet-derived growth factor (PDGF)-BB-stimulated) in vitro.
VSMCs were randomly assigned to 5 groups: blank, PDGF-BB (20 ng/mL+ 0.1% DMSO), SXBXW-L (PDGF-BB 20 ng/mL + SXBXW low dose 0.625 g/L), SXBXW-M (PDGF-BB 20 ng/mL + SXBXW medium dose 1.25 g/L) and SXBXW-H (PDGF-BB 20 ng/mL+ SXBXW high dose 2.5 g/L) group. Cell proliferation was assessed using cell counting kit-8 (CCK-8) assay and bromodeoxyuridine (BrdU) incorporation assay, the migration effects were detected by Transwell assay, cell apoptosis rate was measured by the Annexin V/propidium iodide (PI) apoptosis kit. The markers of contractile phenotype of VSMCs were detected with immunofluorescent staining. To validate the effects of miR-451 in regulating proliferation, migration and apoptosis treated with SXBXW, miR-451 overexpression experiments were performed, the VSMCs were exposed to PDGF-BB 20 ng/mL + 0.1% DMSO and later divided into 4 groups: mimic-NC (multiplicity of infection, MOI=50), SXBXW (1.25 g/L) + mimic-NC, mimic-miR451 (MOI=50), and SXBXW (1.25 g/L) + mimic-miR451, and alterations of proteins related to the miR-451 pathway were analyzed using Western blot.
PDGF-BB induced VSMCs injury causes acceleration of proliferation and migration. SXBXW inhibited phenotypic switching, proliferation and migration and promoted cell apoptosis in PDGF-BB-induced VSMCs. In addition, miR-451 was shown to be down-regulated in the VSMCs following PDGF-BB stimulation. SXBXW treatment enhanced the expression of miR-451 in PDGF-BB-induced VSMCs (P<0.05). Compared with SXBXW + mimic-NC and mimic-miR451 groups, the expression of tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (Ywhaz) and p53 was further reduced in SXBXW + mimic-miR451 group, while activating transcription factor 2 (ATF2) was increased in VSMCs (P<0.05).
SXBXW regulated proliferation, migration and apoptosis via activation of miR-451 through ATF2, p53 and Ywhaz in PDGF-BB-stimulated VSMCs.
通过体外培养的血管损伤模型（血小板衍生生长因子（PDGF）-BB 刺激）中的血管平滑肌细胞（VSMCs），探讨麝香保心丸（SXBXW）在新生内膜形成和血管平滑肌细胞（VSMCs）浸润和凋亡中的调节作用及其潜在的分子机制。
将 VSMCs 随机分为 5 组：空白组、PDGF-BB（20ng/ml+0.1%DMSO）组、SXBXW-L 组（PDGF-BB 20ng/ml+SXBXW 低剂量 0.625g/L）、SXBXW-M 组（PDGF-BB 20ng/ml+SXBXW 中剂量 1.25g/L）和 SXBXW-H 组（PDGF-BB 20ng/ml+SXBXW 高剂量 2.5g/L）。用细胞计数试剂盒-8（CCK-8）检测细胞增殖，用溴脱氧尿苷（BrdU）掺入法检测细胞迁移，用 Transwell 检测细胞迁移，用 Annexin V/碘化丙啶（PI）凋亡试剂盒检测细胞凋亡率。用免疫荧光染色法检测 VSMCs 收缩表型标志物。为了验证 SXBXW 对增殖、迁移和凋亡的调节作用，进行了 miR-451 的过表达实验，将 VSMCs 暴露于 PDGF-BB 20ng/ml+0.1%DMSO 中，然后分为 4 组：mimic-NC（感染复数，MOI=50）、SXBXW（1.25g/L）+mimic-NC、mimic-miR451（MOI=50）和 SXBXW（1.25g/L）+mimic-miR451，并用 Western blot 分析与 miR-451 通路相关的蛋白变化。
PDGF-BB 诱导的 VSMCs 损伤导致增殖和迁移加速。SXBXW 抑制 PDGF-BB 诱导的 VSMCs 表型转换、增殖和迁移，并促进细胞凋亡。此外，在 PDGF-BB 刺激后，miR-451 在 VSMCs 中表达下调。SXBXW 处理增强了 PDGF-BB 诱导的 VSMCs 中 miR-451 的表达（P<0.05）。与 SXBXW+mimic-NC 和 mimic-miR451 组相比，SXBXW+mimic-miR451 组中酪氨酸 3-单加氧酶/色氨酸 5-单加氧酶激活蛋白 zeta（Ywhaz）和 p53 的表达进一步降低，而激活转录因子 2（ATF2）在 VSMCs 中增加（P<0.05）。
SXBXW 通过激活 ATF2、p53 和 Ywhaz 调节 miR-451 的表达，从而调节 PDGF-BB 刺激的 VSMCs 中的增殖、迁移和凋亡。
