Data_Sheet_1_Andrographolide Promotes Interaction Between Endothelin-Dependent EDNRA/EDNRB and Myocardin-SRF to Regulate Pathological Vascular Remodeling.docx

IntroductionPathological vascular remodeling is a hallmark of various vascular diseases. Smooth muscle cell (SMC) phenotypic switching plays a pivotal role during pathological vascular remodeling. The mechanism of how to regulate SMC phenotypic switching still needs to be defined. This study aims to investigate the effect of Andrographolide, a key principle isolated from Andrographis paniculate, on pathological vascular remodeling and its underlying mechanism.
MethodsA C57/BL6 mouse left carotid artery complete ligation model and rat SMCs were used to determine whether Andrographolide is critical in regulating SMC phenotypic switching. Quantitative real-time PCR, a CCK8 cell proliferation assay, BRDU incorporation assay, Boyden chamber migration assay, and spheroid sprouting assay were performed to evaluate whether Andrographolide suppresses SMC proliferation and migration. Immunohistochemistry staining, immunofluorescence staining, and protein co-immunoprecipitation were used to observe the interaction between EDNRA, EDNRB, and Myocardin-SRF.
ResultsAndrographolide inhibits neointimal hyperplasia in the left carotid artery complete ligation model. Andrographolide regulates SMC phenotypic switching characterized by suppressing proliferation and migration. Andrographolide activates the endothelin signaling pathway exhibited by dramatically inducing EDNRA and EDNRB expression. The interaction between EDNRA/EDNRB and Myocardin-SRF resulted in promoting SMC differentiation marker gene expression.
ConclusionAndrographolide plays a critical role in regulating pathological vascular remodeling.

引言
病理性血管重构是多种血管疾病的标志性病理特征。平滑肌细胞（Smooth Muscle Cell, SMC）表型转换在病理性血管重构过程中发挥关键作用。目前，调控平滑肌细胞表型转换的具体分子机制仍有待阐明。本研究旨在探讨从穿心莲中提取的关键活性成分穿心莲内酯（Andrographolide）对病理性血管重构的影响及其潜在作用机制。
方法
本研究采用C57/BL6小鼠左侧颈总动脉完全结扎模型及大鼠平滑肌细胞，以验证穿心莲内酯是否可有效调控平滑肌细胞表型转换。通过定量实时荧光定量PCR（qRT-PCR）、CCK8细胞增殖实验、溴脱氧尿苷（BrdU）掺入实验、Boyden小室迁移实验及球体出芽实验，评估穿心莲内酯对平滑肌细胞增殖与迁移的抑制作用。采用免疫组织化学染色、免疫荧光染色及蛋白质免疫共沉淀技术，观察内皮素受体A（Endothelin Receptor A, EDNRA）、内皮素受体B（Endothelin Receptor B, EDNRB）与心肌素-血清反应因子（Myocardin-SRF）复合物之间的相互作用。
结果
穿心莲内酯可抑制小鼠左侧颈总动脉完全结扎模型中的内膜增生。穿心莲内酯可调控平滑肌细胞表型转换，具体表现为抑制细胞增殖与迁移。穿心莲内酯可激活内皮素信号通路，该效应通过显著上调EDNRA与EDNRB的表达得以体现。EDNRA/EDNRB与Myocardin-SRF复合物的相互作用可促进平滑肌细胞分化标记基因的表达。
结论
穿心莲内酯在调控病理性血管重构过程中发挥关键作用。