S100a6通过β-catenin/TCF1依赖性转录抑制调控PARP9/STAT1改善冠状动脉微循环障碍

刘春; 杨清媛; 邓忠芳; 蔡筱诗; 赖晓玥; 陈铭; 万小勤; 汪紫阳; 郑攀; 邓芳; 张志辉

doi:10.16016/j.2097-0927.202602088

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S100a6通过β-catenin/TCF1依赖性转录抑制调控PARP9/STAT1改善冠状动脉微循环障碍

基础医学 | 更新时间：2026-05-30

- S100a6通过β-catenin/TCF1依赖性转录抑制调控PARP9/STAT1改善冠状动脉微循环障碍
- S100a6 ameliorates coronary microcirculatory dysfunction by modulating PARP9/STAT1 via β-catenin/TCF1-dependent transcriptional repression
- 陆军军医大学学报 2026年48卷第10期页码：1368-1382
- 作者机构：
  
  1.陆军军医大学（第三军医大学）第一附属医院心血管内科，代谢生物钟与心血管疾病中心，老年心脑血管病教育部重点实验室，重庆
  2.陆军军医大学（第三军医大学）第二附属医院超声医学科，重庆
  3.陆军军医大学（第三军医大学）高原军事医学系病理生理学教研室，重庆
- 作者简介：
  
  邓芳，E-mail： celldf@tmmu.edu.cn
  张志辉，E-mail： xyzpj@tmmu.edu.cn
- 基金信息：
  
  国家自然科学基金面上项目(82570409);重庆市技术创新与应用发展专项重点项目(CSTB2023TIAD-KPX0061-2)
- DOI：10.16016/j.2097-0927.202602088
  中图分类号： R341;R394.3;R543.3
- 收稿：2026-02-24，
  
  修回：2026-04-28，
  
  录用：2026-04-30，
  
  纸质出版：2026-05-30
- 稿件说明：
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刘春，杨清媛，邓忠芳，等.S100a6通过β-catenin/TCF1依赖性转录抑制调控PARP9/STAT1改善冠状动脉微循环障碍［J］.陆军军医大学学报，2026，48（10）：1368-1382.DOI：10.16016/j.2097-0927.202602088.

LIU C，YANG Q Y，DENG Z F，et al.S100a6 ameliorates coronary microcirculatory dysfunction by modulating PARP9/STAT1 via β-catenin/TCF1-dependent transcriptional repression［J］.J Army Med Univ，2026，48（10）：1368-1382.DOI：10.16016/j.2097-0927.202602088.
刘春，杨清媛，邓忠芳，等.S100a6通过β-catenin/TCF1依赖性转录抑制调控PARP9/STAT1改善冠状动脉微循环障碍［J］.陆军军医大学学报，2026，48（10）：1368-1382.DOI：10.16016/j.2097-0927.202602088. DOI：

LIU C，YANG Q Y，DENG Z F，et al.S100a6 ameliorates coronary microcirculatory dysfunction by modulating PARP9/STAT1 via β-catenin/TCF1-dependent transcriptional repression［J］.J Army Med Univ，2026，48（10）：1368-1382.DOI：10.16016/j.2097-0927.202602088. DOI：

摘要

目的

冠状动脉微循环障碍（coronary microvascular dysfunction， CMD）在冠心病群体中患病率高，且作为独立危险因素大幅增加心血管事件风险，但缺乏靶向干预措施。钙周期蛋白（S100a6）具有调节内皮细胞周期的作用，但其在CMD中的表达特征及保护机制尚不明确。本研究旨在明确S100a6在CMD中的内源性保护作用，阐明其通过稳定β-catenin/TCF1复合体抑制PARP9/STAT1信号轴活化改善CMD的分子机制。

方法

选用8周龄、体质量38~42 g的雄性ob/ob小鼠共12只，采用随机数字表法将小鼠分为CMD组与CMD-S100a6KD组（

=6）。其中，CMD组小鼠经尾静脉注射scramble对照病毒，CMD-S100a6KD组小鼠经尾静脉注射内皮特异性的腺病毒以敲低S100a6的表达。另选取6只同周龄的雄性C57BL/6J野生型小鼠（体质量18~22 g）作为健康对照组（Control组），常规饲养不做任何模型构建及药物或基因干预。通过尾静脉注射内皮特异性Tie启动子驱动的AAV9-shS100a6实现S100a6内皮特异性敲低，20周后通过心脏超声检测小鼠冠状动脉血流储备（coronary flow reserve， CFR）及左心室舒张功能（E/e'），取小鼠心脏组织行免疫荧光染色检测心肌微血管密度及相关蛋白表达与定位。体外培养人冠状动脉微血管内皮细胞（human coronary microvascular endothelial cells， HCMECs），以高糖（25 mmol/L）联合游离脂肪酸（0.5 mmol/L）处理48 h构建CMD细胞模型，采用siRNA及质粒进行基因干预。根据不同实验目的分为：基线对照组［正常培养的对照组（Control组）和CMD模型组（HG组）］、S100a6功能缺失组（HG+siS100a6和HG+siNC组）及下游机制挽救组（HG+siS100a6+siPARP9及其HG+siS100a6+siNC组、HG+siS100a6+OE β-catenin及其HG+siS100a6+OE NC组）。通过单细胞 RNA测序分析心脏组织S100a6的细胞亚群表达，并通过批量RNA测序筛选差异基因与富集通路；以Western blotting、RT-qPCR检测S100a6、β-catenin、TCF1、PARP9、STAT1及p-STAT1的蛋白相对表达量与mRNA表达水平；采用免疫共沉淀方法验证β-catenin与TCF1相互作用，通过染色质免疫共沉淀方法检测TCF1与PARP9启动子结合能力，明确相关分子机制。通过EdU增殖实验、划痕愈合实验及成管实验评估内皮细胞功能。

结果

与CMD组比较，CMD-S100a6KD组小鼠CFR显著降低（

0.05），E/e'比值显著升高（

0.05），心肌微血管密度显著降低（

0.05）。单细胞RNA测序结果显示，CMD组小鼠心脏组织中S100a6整体表达显著升高，且在心脏内皮细胞亚群中显著升高（

0.05）；免疫荧光共定位分析显示S100a6与CD31的共定位信号明显增强（

0.000 1）。体外实验中，高糖高脂刺激使HCMECs S100a6蛋白表达水平显著高于正常培养的对照组（

0.05）。S100a6敲低导致HCMECs成管分支数显著减少（

0.05）、划痕愈合率显著降低（

0.05）及EdU阳性率显著降低（

0.05）。批量RNA测序结果显示S100a6敲低后

PARP9

基因表达显著上调，基因富集分析显示STAT1信号通路显著富集；在敲低S100a6的同时敲低PARP9，STAT1磷酸化水平较单纯S100a6敲低组显著降低（

0.000 1）；功能学实验证实沉默PARP9显著改善了S100a6敲低引起的HCMECs成管功能障碍（

0.05）、迁移能力受损（

0.05）及EdU阳性率降低（

0.05）。分子对接实验显示S100a6结合在TCF1和β-catenin的结合界面缝隙中；敲低S100a6使β-catenin蛋白水平显著降低（

0.05），β-catenin与TCF1的相互作用显著减弱（

0.05），TCF1与

PARP9

启动子区域的结合显著减少（

0.05）。在敲低S100a6的同时过表达β-ca

tenin使PARP9蛋白表达水平显著降低（

0.05），磷酸化STAT1水平显著下降（

0.05）；功能学层面，β-catenin过表达显著改善了S100a6缺失导致的HCMECs成管分支数减少（

0.05）、划痕愈合率降低（

0.05）及EdU阳性率降低（

0.05）。

结论

S100a6是冠状动脉微循环障碍的内源性保护因子，其通过β-catenin/TCF1复合体依赖性转录抑制调控PARP9/STAT1信号通路的活化，维持内皮细胞稳态，进而改善冠状动脉微循环障碍。

Abstract

Objective

Coronary microvascular dysfunction (CMD) is highly prevalent among patients with coronary artery disease and

as an independent risk factor

increases the risk of cardiovascular events

yet there is a lack of targeted interventions. S100a6 has been shown to regulate endothelial cell cycle progression

but its expression pattern and protective mechanism in CMD remain unclear. This study aimed to determine the endogenous protective role of S100a6 in CMD and to elucidate the molecular mechanism by which it ameliorates CMD through stabilizing the β-catenin/TCF1 complex and suppressing the activation of the PARP9/STAT1 signaling axis.

Methods

A total of 12 male ob/ob mice

aged 8 weeks and weighing 38 to 42 g

were randomly assigned into 2 groups (

=6 per group) using a random number table: the CMD group and the CMD-S100a6KD group. Mice in the CMD group received injection

via

tail vein of scrambled control virus

whereas those in the CMD-S100a6KD group received endothelium-specific adenovirus to knock down S100a6 expression. Additionally

six male C57BL/6J wild-type mice of the same age

weighing 18 to 22 g

were used as a healthy control group (Control group) and were fed routinely without any model construction or drug/gene intervention. Endothelial-specific knockdown of S100a6 was achieved by tail vein injection of AAV9-shS100a6 driven by an endothelium-specific Tie promoter. After 20 weeks

coronary flow reserve (CFR) and left ventricular diastolic function (E/e') were assesse

d by echocardiography; heart tissues were collected for immunofluorescence staining to evaluate myocardial microvessel density and related protein expression and localization. Human coronary microvascular endothelial cells (HCMECs) were cultured in vitro and treated with high glucose (25 mmol/L) combined with free fatty acids (0.5 mmol/L) for 48 h to establish a CMD cell model

followed by gene intervention using siRNA and plasmids. Experimental groups were designed according to different objectives: baseline control groups [the normal culture control group (Control group) and the CMD model group (HG group)

]

S100a6 loss-of-function groups (HG+siS100a6 and its siNC control)

and downstream mechanism rescue groups (HG+siS100a6+siPARP9 and its siNC control; HG+siS100a6+OE-β-catenin and its empty vector control). Single-cell RNA sequencing was used to analyze the cell-subset expression of S100a6 in heart tissues

and bulk RNA sequencing was performed to identify differentially expressed genes and enriched pathways. Western blotting and qRT-PCR were used to measure the relative protein and mRNA expression levels of S100a6

β-catenin

TCF1

PARP9

STAT1

and p-STAT1. Co-immunoprecipitation was performed to validate the interaction between β-catenin and TCF1

and chromatin immunoprecipitation was used to detect the binding of TCF1 to the PARP9 promoter

thereby clarifying the molecular mechanism. Endothelial cell function was assessed by EdU proliferation assay

scratch wound healing assay

and tube formation assay.

Results

Compared with the CMD group

mice in the CMD-S100a6KD group showed a significantly reduced CFR (

0.05)

a significantly increased E/e’ ratio (

0.05)

and a significantly decreased myocardial microvessel density (

0.05). Single-cell RNA sequencing revealed that S100a6 expression was markedly elevated overall in the heart tissues of CMD group mice

and it is also significantly upregulated in the subsets of c

ardiac endothelial cells (

0.05); immunofluorescence co-localization analysis showed significantly enhanced co-localization signals of S100a6 and CD31 (

0.000 1).

In vitro

high glucose and high lipid stimulation significantly increased S100a6 protein expression levels in HCMECs compared with normally cultured controls (

0.05). Knockdown of S100a6 led to a significant reduction in the number of tube branches (

0.05)

a significant decrease in scratch wound healing rate (

0.05)

and a significant decrease in EdU-positive rate (

0.05) in HCMECs. Bulk RNA sequencing showed that PARP9 gene expression was significantly upregulated after S100a6 knockdown

and gene enrichment analysis revealed significant enrichment of the STAT1 signaling pathway. When S100a6 and PARP9 were knocked down simultaneously

STAT1 phosphorylation levels were significantly reduced compared with the S100a6 knockdown alone group (

0.000 1). Functional assays confirmed that PARP9 silencing significantly ameliorated the S100a6 knockdown-induced impairments in tube formation (

0.05)

migration capacity (

0.05)

and EdU-positive rate (

0.05) in HCMECs. Molecular docking experiments showed that S100a6 binds within the crevice of the TCF1/β-catenin interaction interface. Knockdown of S100a6 significantly reduced β-catenin protein levels (

0.05)

significantly weakened the interaction between β-catenin and TCF1 (

0.05)

and significantly decreased the binding of TCF1 to the PARP9 promoter region (

0.05). Overexpression of β-catenin in parallel with S100a6 knockdown significantly reduced PARP9 protein expression levels (

0.05) and reduced phosphorylated STAT1 levels (

0.05). Functionally

β-catenin overexpression significantly

rescued the S100a6 deficiency-induced reductions in tube branch number (

0.05)

scratch wound healing rate (

0.05)

and EdU-positive rate (

0.05) in HCMECs.

Conclusion

S100a6 is an endogenous protective factor for coronary microcirculatory dysfunction. It regulates the activation of the PARP9/STAT1 signaling pathway through β-catenin/TCF1 complex-dependent transcriptional repression

maintains endothelial cell homeostasis

and thereby ameliorates coronary microcirculatory dysfunction.

关键词

Keywords

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