Okada Muneyoshi, Suzuki Atsushi, Yamawaki Hideyuki, Hara Yukio
Laboratory of Veterinary Pharmacology, School of Veterinary Medicine, Kitasato University, Higashi 23 Bancho 35-1, Towada, Aomori 034-8628, Japan.
Eur J Pharmacol. 2013 Oct 15;718(1-3):332-9. doi: 10.1016/j.ejphar.2013.08.013. Epub 2013 Sep 3.
Cell migration and matrix metalloproteinases (MMPs) secretion in cardiac fibroblasts are important processes in the cardiac remodeling during the development of cardiac diseases and are regulated by proinflammatory cytokines. Although levosimendan, a novel inotropic agent, is expected to have some beneficial influences on preventing cardiac remodeling, its effects on proinflammatory cytokines-induced functional changes in cardiac fibroblasts have not been clarified. Therefore, we investigated the effects of levosimendan on interleukin (IL)-1β-induced MMP-9 secretion and migration in adult rat cardiac fibroblasts. Primary cardiac fibroblasts were isolated from adult male Wistar rats. MMP-9 secretion in culture medium and extracellular signal-regulated kinase (ERK) phosphorylation in cell lysate were measured by using Western blotting. Gelatin zymography was performed to measure activity of secreted MMP-9. MMP-9 mRNA expression in the cell was measured by using reverse transcription polymerase chain reaction. Boyden chamber assay was performed for detection of migration. Levosimendan (3-100 μM) concentration-dependently inhibited IL-1β (4 ng/ml)-induced MMP-9 secretion, activity and mRNA expression. Levosimendan inhibited IL-1β (4 ng/ml)-induced ERK phosphorylation. Levosimendan (10 and 100 μM) inhibited IL-1β-induced migration, and CTTHWGFTLC peptide (10 μM), an MMP inhibitor, or PD98059 (50 μM), an ERK inhibitor, also suppressed it. The present study for the first time demonstrated in adult rat cardiac fibroblasts that levosimendan inhibits IL-1β-induced migration at least partly through the inhibition of MMP-9 secretion via suppressing ERK phosphorylation.
在心脏疾病发展过程中，心脏成纤维细胞的细胞迁移和基质金属蛋白酶（MMPs）分泌是心脏重塑中的重要过程，且受促炎细胞因子调控。尽管新型正性肌力药物左西孟旦有望对预防心脏重塑产生一些有益影响，但其对促炎细胞因子诱导的心脏成纤维细胞功能变化的影响尚未阐明。因此，我们研究了左西孟旦对白细胞介素（IL）-1β诱导的成年大鼠心脏成纤维细胞中MMP-9分泌和迁移的影响。从成年雄性Wistar大鼠中分离出原代心脏成纤维细胞。通过蛋白质免疫印迹法检测培养基中MMP-9的分泌以及细胞裂解物中细胞外信号调节激酶（ERK）的磷酸化。进行明胶酶谱分析以测量分泌的MMP-9的活性。通过逆转录聚合酶链反应测量细胞中MMP-9 mRNA的表达。采用Boyden小室试验检测迁移情况。左西孟旦（3 - 100 μM）浓度依赖性地抑制IL-1β（4 ng/ml）诱导的MMP-9分泌、活性和mRNA表达。左西孟旦抑制IL-1β（4 ng/ml）诱导的ERK磷酸化。左西孟旦（10和100 μM）抑制IL-1β诱导的迁移，MMP抑制剂CTTHWGFTLC肽（10 μM）或ERK抑制剂PD98059（50 μM）也能抑制迁移。本研究首次在成年大鼠心脏成纤维细胞中证明，左西孟旦至少部分通过抑制ERK磷酸化从而抑制MMP-9分泌，进而抑制IL-1β诱导的迁移。
