AhR调控血管微环境对血管平滑肌的影响及其在内皮损伤修复中的作用

Vascular homeostasis depends on the dynamic balance of vessel tissues, and vascular smooth muscle cells (VSMC) maintain homeostasis by phenotypic and functional changes. The applicants suggest that the regulation of VSMC to some extent depends on vascular microenvironment which is regulated by certain inflammation response. Aryl hydrocarbon receptor (AhR) mediates the impact of environmental factors on cerebral vascular disease (CVD), but the mechanism is not clear. We previously shown AHR modulates NLRP3 inflammasome, which can promote exocrine of ASC, we speculate that the latter may be involved in vascular microenvironment. We intend to, using mice carotid endothelial injury model, observe the influence of AHR on NLRP3 and ASC's expression, VSMC differentiation and intimal healing. With ex vivo cell experiments, we are to explore the mechanisms that AhR regulating VSMC's function in intimal repair through NLRP3-ASC- Dock2 signal pathway, and to elucidate the role of environmental factors in intimal injury. We will, from animal, cell to molecular levels, inspect environmental factors’ regulation on vascular microenvironment, through AhR and inflammasome pathway, then affecting on VSMC's phenotype in intimal repair process. So as to verify the hypothesis and mechanism of AhR-mediated environmental factors' inducing CVD, and provide new ideas for prevention and management of CVD.

血管稳态依赖于管壁细胞动态平衡，血管平滑肌细胞（VSMC）通过表型及功能变化，维持保护管壁平衡。申请者认为VSMC的调控在一定程度上依赖于管壁微环境，炎症反应可调节微环境。芳香烃受体（AhR）介导环境因素对脑血管病的影响，但其机制尚未阐明。我们前期证实AhR调控NLRP3炎症小体参与的炎症反应，后者可促进ASC外分泌。我们推测其可能参与血管微环境调控。本项目拟采用小鼠颈动脉内皮损伤模型，观察AhR对管壁中NLRP3、ASC的表达、VSMC表型及损伤修复的影响；并探讨AhR通过NLRP3-ASC-Dock2信号路径调控VSMC并影响内膜修复的作用机制，并就此探讨环境因素在内皮损伤中的作用。从动物、细胞、分子等多层面系统阐明环境因素通过AhR及炎症小体通路调控微环境，进而影响VSMC参与的修复进程。从而验证AhR介导环境因子诱发脑血管病的假设并揭示及其机理，以为脑血管疾病的防治提供新思路。

本研究主要内容有:.1 TCDD 对小鼠颈动脉内皮损伤修复的影响。 方法:建立小鼠颈动脉导丝损伤模型及颈动脉结扎模型; TCDD 慢性给药:分为 0.1μg/kg， 1μg/kg，10μg/kg 三组剂量组。检测CH-223191对TCDD的拮抗作用。建模 2 周后取颈动脉标本进行病理及免疫组化检测，QPCR检测IL-1β 等 mRNA 表达差异。结果:两模型中内膜增生明确。TCDD促进损伤模型的内膜增生，慢性给药内膜增生情况与给药剂量呈正比。TCDD 促进新生内膜中的IL-1β表达。拮抗剂CH-223191减弱TCDD诱导的内膜增生。 .2.TCDD 对人脑血管平滑肌细胞的影响及机制研究。方法:分别以PDGF-BB、IL-1β、TNF-α及 TGF-β及TCDD 对人VSMC进行刺激，检测α-SMA 、calponin及 IL-1β变化;选用 1nM TCDD 和 40ng/ml IL-1β 单独及共同处理后，检测VSMC增殖迁移能力变化;检测 CH-223191 对 TCDD 的拮抗作用;检测 TCDD 对人VSMC分泌 IL-1β 及NLRP3 炎症小体等表达的影响，选用 NF-κB 抑制剂检测TCDD 刺激后 IL-1β 的变化。结果: IL-1β 促使人脑VSMC表型转化，且增强其增殖迁移能力; TCDD促进人VSMC表型转化，同样促使其内 IL-1β表达及分泌增高，NLRP3 水平未随 IL-1β 发生变化。NF-κB 抑制剂可抑制 TCDD 诱导的 IL-1β 的表达增加。.总结: TCDD 可以促进小鼠颈动脉损伤后内膜增生及新生内膜中 IL-1β 表达的增加; TCDD 可促进VSMC表型转化及 IL-1β 的的表达增加，IL-1β 可促进VSMC表型转化及增殖迁移能力的增加，这可能是 TCDD 促进内膜增生的原因; TCDD 促进 IL-1β 的表达增加可能是由 NF-κB 通路介导的，NLRP3 水平未随 IL-1β 发生变化。