Liu Jie, Pan Defeng, Luo Yuanyuan, Wu Wanling, Jiang Tingbo
Department of Cardiology, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu, China.
Department of Cardiology, The Affiliated Hospital of Xuzhou Medical University, Xuzhou, Jiangsu, China.
Anatol J Cardiol. 2024 Dec 20;29(2):73-82. doi: 10.14744/AnatolJCardiol.2024.4605.
Cardiac fibrosis, a key contributor to heart failure, is driven by the activation of cardiac fibroblasts (CFs), often induced by angiotensin II (Ang II). Relaxin, a peptide hormone, has been reported to counteract fibrotic processes. This study aims to investigate the antifibrotic effects of relaxin on Ang II-induced CF activation, with a focus on the involvement of the nitric oxide/cyclic guanosine monophosphate (NO/cGMP) signaling pathway.
Primary CFs were isolated and treated with Ang II to induce fibrotic activation. Relaxin was used to assess its antifibrotic effects. Inhibitors of the NO/cGMP pathway, NG-nitro-L-arginine methyl ester (L-NAME) (a nitric oxide synthase inhibitor) and 1H-(1 ,2,4) -Oxadiazolo-(4, 3-a) quinoxalin-1-one (ODQ) (a guanylyl cyclase inhibitor), were co-administered to examine their effects on relaxin-mediated inhibition. Proliferation and migration were assessed using 5-Ethynyl-2'-de oxyur idine incorporation and Transwell assays. Western blot analysis was conducted to measure the expression of alpha-smooth muscle actin (α-SMA), collagen I, and collagen III, key markers of fibroblast activation. Nitric oxide, cGMP, total nitric oxide synthase (TNOS), and inducible nitric oxide synthase (iNOS) levels were measured in the culture media.
Ang II significantly increased CF proliferation, migration, and the expression of fibrosis markers α-SMA, collagen I, and collagen III. Relaxin treatment markedly reduced these effects. Inhibition of the NO/cGMP pathway by L-NAME or ODQ partially reversed relaxin's suppressive effects on CF proliferation and migration. Relaxin restored Ang II-induced reductions in NO, cGMP, and TNOS levels, while iNOS levels remained largely unchanged, except for a reduction in the L-NAME group.
Relaxin attenuates Ang II-induced cardiac fibroblast activation and fibrosis primarily through the NO/cGMP signaling pathway.
心脏纤维化是心力衰竭的关键促成因素，由心脏成纤维细胞（CFs）的激活驱动，而这通常由血管紧张素II（Ang II）诱导。松弛素是一种肽类激素，据报道可对抗纤维化过程。本研究旨在探讨松弛素对Ang II诱导的CF激活的抗纤维化作用，重点关注一氧化氮/环磷酸鸟苷（NO/cGMP）信号通路的参与情况。
分离原代CFs并用Ang II处理以诱导纤维化激活。使用松弛素评估其抗纤维化作用。联合使用NO/cGMP途径抑制剂Nω-硝基-L-精氨酸甲酯（L-NAME）（一种一氧化氮合酶抑制剂）和1H-（1,2,4）-恶二唑并-（4,3-a）喹喔啉-1-酮（ODQ）（一种鸟苷酸环化酶抑制剂），以检查它们对松弛素介导的抑制作用的影响。使用5-乙炔基-2'-脱氧尿苷掺入法和Transwell测定法评估增殖和迁移。进行蛋白质免疫印迹分析以测量α-平滑肌肌动蛋白（α-SMA）、I型胶原蛋白和III型胶原蛋白的表达，这些是成纤维细胞激活的关键标志物。在培养基中测量一氧化氮、cGMP、总一氧化氮合酶（TNOS）和诱导型一氧化氮合酶（iNOS）水平。
Ang II显著增加CF增殖、迁移以及纤维化标志物α-SMA、I型胶原蛋白和III型胶原蛋白的表达。松弛素处理显著降低了这些作用。L-NAME或ODQ对NO/cGMP途径的抑制部分逆转了松弛素对CF增殖和迁移的抑制作用。松弛素恢复了Ang II诱导的NO、cGMP和TNOS水平的降低，而iNOS水平在很大程度上保持不变，除了L-NAME组有所降低。
松弛素主要通过NO/cGMP信号通路减弱Ang II诱导的心脏成纤维细胞激活和纤维化。
