组织因子途径抑制剂对血管紧张素II诱导的血管平滑肌细胞焦亡的影响。

Effect of tissue factor pathway inhibitor on the pyroptosis of vascular smooth muscle cells induced by angiotensin II.

作者信息

Dai Yue, Xu Rui, Yang Kelaier, Jiang Tingting, Wei Yongkang, Liu Yue, Chen Wenjia, Fu Yu, Zhao Yong

机构信息

Department of Cardiology, The First Affiliated Hospital of Harbin Medical University, Harbin, China.

Department of Endocrinology, Shenzhen University General Hospital, Shenzhen, China.

出版信息

Cardiovasc Diagn Ther. 2024 Feb 15;14(1):72-83. doi: 10.21037/cdt-23-355. Epub 2024 Jan 19.

DOI:10.21037/cdt-23-355

PMID:38434568

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC10904294/

Abstract

BACKGROUND

In recent years, a mass of studies have shown that pyroptosis plays an important role in the proliferation of vascular smooth muscle cells (VSMCs). We investigated whether angiotensin II (Ang II) induces the pyroptosis of rat aortic VSMCs and the role of NOD-like receptor family pyrin domain containing 3 (NLRP3) in this process. Additionally, we explored the effect and related mechanism of recombinant tissue factor pathway inhibitor (rTFPI) in Ang II-induced VSMC pyroptosis.

METHODS

Cultured VSMCs were divided into five groups: control group, Ang II group (1×10 mol/L), MCC950 group (NLRP3 inhibitor, 15 nmol/L), Ang II + MCC950 group and Ang II + rTFPI (50 µg/L) group. Cell viability was measured by cell counting kit-8 (CCK8) assays and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assays. Propidium iodide (PI) staining and immunofluorescence were performed to determine the pyroptosis of VSMCs. Changes in VSMC ultrastructure were evaluated through transmission electron microscopy. The expression levels of NLRP3, pro-caspase-1, gasdermin D-N (GSDMD-N), and interleukin-1β (IL-1β) were determined by western blot analysis.

RESULTS

The cell viability, the positive rate of PI staining, and the expression level of GSDMD detected by immunofluorescence in the Ang II group were higher than that in the control group, whereas they all decreased in Ang II + MCC950 group and Ang II + rTFPI group compared with Ang II group (P<0.05). Electron microscopy analysis revealed less extracellular matrix, increased myofilaments, and decreased endoplasmic reticulum, Golgi complex, and mitochondria in Ang II + rTFPI-treated VSMCs than in Ang II-treated VSMCs. The protein expression levels of the pyroptosis-related molecules NLRP3, pro-caspase-1, GSDMD-N, and IL-1β in Ang II group showed an increasing trend compared with those in control group (P<0.05); however, these expression levels in Ang II + MCC950 and Ang II + rTFPI groups were significantly lower than those in Ang II group (P<0.05).

CONCLUSIONS

Ang II may induce pyroptosis in VSMCs by activating NLRP3. rTFPI can inhibit Ang II-induced VSMC pyroptosis. Furthermore, rTFPI might exert this effect by inhibiting the NLRP3 pathway and therefore play an important role in the treatment of vascular remodeling induced by hypertension.

摘要

背景

近年来，大量研究表明，细胞焦亡在血管平滑肌细胞（VSMCs）增殖中起重要作用。我们研究了血管紧张素 II（Ang II）是否诱导大鼠主动脉 VSMCs 发生细胞焦亡以及含 NOD 样受体家族 pyrin 结构域 3（NLRP3）在此过程中的作用。此外，我们探讨了重组组织因子途径抑制剂（rTFPI）在 Ang II 诱导的 VSMC 细胞焦亡中的作用及相关机制。

方法

将培养的 VSMCs 分为五组：对照组、Ang II 组（1×10⁻⁶ mol/L）、MCC950 组（NLRP3 抑制剂，15 nmol/L）、Ang II + MCC950 组和 Ang II + rTFPI（50 μg/L）组。通过细胞计数试剂盒 -8（CCK8）检测法和 3 -（4,5 - 二甲基 -2 - 噻唑基）-2,5 - 二苯基 -2H - 四氮唑溴盐（MTT）检测法测定细胞活力。进行碘化丙啶（PI）染色和免疫荧光检测以确定 VSMCs 的细胞焦亡情况。通过透射电子显微镜评估 VSMC 超微结构的变化。采用蛋白质免疫印迹分析测定 NLRP3、前半胱天冬酶 -1、gasdermin D - N（GSDMD - N）和白细胞介素 -1β（IL -1β）的表达水平。

结果

Ang II 组的细胞活力、PI 染色阳性率以及免疫荧光检测的 GSDMD 表达水平均高于对照组，而与 Ang II 组相比，Ang II + MCC950 组和 Ang II + rTFPI 组的上述指标均降低（P<0.05）。电子显微镜分析显示，与 Ang II 处理的 VSMCs 相比，Ang II + rTFPI 处理的 VSMCs 细胞外基质减少、肌丝增加、内质网、高尔基体复合体和线粒体减少。与对照组相比，Ang II 组中细胞焦亡相关分子 NLRP3、前半胱天冬酶 -1、GSDMD - N 和 IL -1β 的蛋白表达水平呈上升趋势（P<0.05）；然而，Ang II + MCC950 组和 Ang II + rTFPI 组的这些表达水平显著低于 Ang II 组（P<0.05）。