Espitia-Corredor Jenaro Antonio, Boza Pía, Espinoza-Pérez Claudio, Lillo José Miguel, Rimassa-Taré Constanza, Machuca Víctor, Osorio-Sandoval José Miguel, Vivar Raúl, Bolivar Samir, Pardo-Jiménez Viviana, Sánchez-Ferrer Carlos Félix, Peiró Concepción, Díaz-Araya Guillermo
Laboratory of Molecular Pharmacology, Faculty of Chemical and Pharmaceutical Sciences, Department of Pharmacological & Toxicological Chemistry, University of Chile, Santiago, Chile.
Faculty of Medicine, Department of Pharmacology, Universidad Autónoma de Madrid, Madrid, Spain.
Inflammation. 2022 Dec;45(6):2498-2512. doi: 10.1007/s10753-022-01707-z. Epub 2022 Jul 22.
Angiotensin II (Ang-II) is a widely studied hypertensive, profibrotic, and pro-inflammatory peptide. In the heart, cardiac fibroblasts (CF) express type 1 angiotensin II receptors (AT1R), Toll-like receptor-4 (TLR4), and the NLRP3 inflammasome complex, which play important roles in pro-inflammatory processes. When activated, the NLRP3 inflammasome triggers proteolytic cleavage of pro-IL-1, resulting in its activation. However, in CF the mechanism by which Ang-II assembles and activates the NLRP3 inflammasome remains not fully known. To elucidate this important point, we stimulated TLR4 receptors in CF and evaluated the signaling pathways by which Ang-II triggers the assembly and activity. In cultured rat CF, pro-IL-1β levels, NLRP3, ASC, and caspase-1 expression levels were determined by Western blot. NLRP3 inflammasome complex assembly was analyzed by immunocytochemistry, whereas by ELISA, we analyzed NLRP3 inflammasome activity and [Formula: see text] release. In CF, Ang-II triggered NLRP3 inflammasome assembly and caspase-1 activity; and in LPS-pretreated CF, Ang-II also triggered [Formula: see text] secretion. These effects were blocked by losartan (AT1R antagonist), U73221 (PLC inhibitor), 2-APB (IP3R antagonist), and BAPTA-AM (Ca chelator) indicating that the AT1R/PLC/IP3R/Ca pathway is involved. Finally, bafilomycin A1 prevented Ang-II-induced [Formula: see text] secretion, indicating that a non-classical protein secretion mechanism is involved. These findings suggest that in CF, Ang-II by a Ca-dependent mechanism triggers NLRP3 inflammasome assembly and activation leading to [Formula: see text] secretion through a non-conventional protein secretion mechanism.
血管紧张素II（Ang-II）是一种经过广泛研究的具有升高血压、促纤维化和促炎作用的肽。在心脏中，心脏成纤维细胞（CF）表达1型血管紧张素II受体（AT1R）、Toll样受体4（TLR4）和NLRP3炎性小体复合物，它们在促炎过程中发挥重要作用。激活后，NLRP3炎性小体触发前白细胞介素-1（pro-IL-1）的蛋白水解切割，使其活化。然而，在心脏成纤维细胞中，Ang-II组装和激活NLRP3炎性小体的机制仍不完全清楚。为阐明这一要点，我们刺激心脏成纤维细胞中的TLR4受体，并评估Ang-II触发组装和活性的信号通路。在培养的大鼠心脏成纤维细胞中，通过蛋白质印迹法测定pro-IL-1β水平、NLRP3、凋亡相关斑点样蛋白（ASC）和半胱天冬酶-1（caspase-1）的表达水平。通过免疫细胞化学分析NLRP3炎性小体复合物的组装，而通过酶联免疫吸附测定法（ELISA），我们分析NLRP3炎性小体活性和白细胞介素-1β（IL-1β）释放。在心脏成纤维细胞中，Ang-II触发NLRP3炎性小体组装和caspase-1活性；在经脂多糖（LPS）预处理的心脏成纤维细胞中，Ang-II也触发IL-1β分泌。这些效应被氯沙坦（AT1R拮抗剂）、U73221（磷脂酶C（PLC）抑制剂）、2-氨基乙氧基二苯硼酸（2-APB，肌醇1,4,5-三磷酸受体（IP3R）拮抗剂）和1,2-双（2-氨基苯氧基）乙烷-N,N,N',N'-四乙酸四乙酰甲酯（BAPTA-AM，钙螯合剂）阻断，表明AT1R/PLC/IP3R/Ca途径参与其中。最后，巴弗洛霉素A1阻止了Ang-II诱导的IL-1β分泌，表明涉及一种非经典的蛋白质分泌机制。这些发现表明，在心脏成纤维细胞中，Ang-II通过一种钙依赖性机制触发NLRP3炎性小体的组装和激活，导致IL-1β通过一种非传统的蛋白质分泌机制分泌。
