Okada Muneyoshi, Yamawaki Hideyuki
Laboratory of Veterinary Pharmacology, School of Veterinary Medicine, Kitasato University, Higashi 23 Bancho 35-1, Towada city, Aomori 034-8628, Japan.
Laboratory of Veterinary Pharmacology, School of Veterinary Medicine, Kitasato University, Higashi 23 Bancho 35-1, Towada city, Aomori 034-8628, Japan.
Eur J Pharmacol. 2015 Dec 15;769:86-92. doi: 10.1016/j.ejphar.2015.10.056. Epub 2015 Oct 31.
Levosimendan, a calcium sensitizer, known as an inotropic agent has a cytoprotective effect against apoptosis in cardiomyocytes. However, the cytoprotective effect of levosimendan on cardiac fibroblasts has not been clarified. The aim of this study was to examine whether levosimendan modulates interleukin (IL)-1β-induced apoptosis in adult rat cardiac fibroblast. Cardiac fibroblasts were isolated from adult male Wistar rats. Apoptosis of cardiac fibroblasts was evaluated by 4',6-diamidino-2-phenylindole staining and TdT-mediated d-UTP nick end labeling (TUNEL)-based staining. Expression of inducible nitric oxide (NO) synthase (iNOS) and phosphorylation of Akt (Ser473) were determined by Western blotting. Assessment of NO production in the culture medium was performed by using 4,5-diaminofluorescein as a fluorescent probe. Immunofluorescence staining was performed to examine cytochrome-c translocation. Levosimendan (3-100μM) concentration-dependently inhibited IL-1β (4ng/ml, 24h)-induced apoptotic changes in cardiac fibroblasts, such as cellular shrinkage, nuclear condensation and the increase of TUNEL-positive cells. Levosimendan inhibited IL-1β-induced iNOS expression and subsequent NO production. 1400W, an iNOS inhibitor, suppressed the IL-1β-induced apoptosis. Levosimendan alone-treatment concentration-dependently increased phosphorylation of Akt (Ser473). LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, reversed the protective effect of levosimendan on IL-1β-induced apoptosis in TUNEL assay. In addition, levosimendan and 1400W inhibited the IL-1β-induced cytosolic translocation of cytochrome-c. LY294002 reversed the suppressive effects of levosimendan. The present study for the first time demonstrated that levosimendan inhibits IL-1β-induced apoptosis via the activation of PI3K/Akt pathway and the inhibition of iNOS expression in adult rat cardiac fibroblasts.
左西孟旦是一种钙增敏剂，作为一种正性肌力药物，对心肌细胞凋亡具有细胞保护作用。然而，左西孟旦对心脏成纤维细胞的细胞保护作用尚未阐明。本研究的目的是检测左西孟旦是否能调节白细胞介素（IL）-1β诱导的成年大鼠心脏成纤维细胞凋亡。从成年雄性Wistar大鼠中分离出心脏成纤维细胞。通过4',6-二脒基-2-苯基吲哚染色和基于末端脱氧核苷酸转移酶介导的dUTP缺口末端标记（TUNEL）染色评估心脏成纤维细胞的凋亡情况。通过蛋白质印迹法测定诱导型一氧化氮（NO）合酶（iNOS）的表达和Akt（Ser473）的磷酸化。使用4,5-二氨基荧光素作为荧光探针评估培养基中NO的产生。进行免疫荧光染色以检测细胞色素c的转位。左西孟旦（3-100μM）浓度依赖性地抑制IL-1β（4ng/ml，24小时）诱导的心脏成纤维细胞凋亡变化，如细胞收缩、核浓缩和TUNEL阳性细胞增加。左西孟旦抑制IL-1β诱导的iNOS表达和随后的NO产生。iNOS抑制剂1400W可抑制IL-1β诱导的凋亡。单独使用左西孟旦处理可浓度依赖性地增加Akt（Ser473）的磷酸化。磷脂酰肌醇3激酶（PI3K）抑制剂LY294002在TUNEL试验中逆转了左西孟旦对IL-1β诱导凋亡的保护作用。此外，左西孟旦和1400W抑制IL-1β诱导的细胞色素c胞质转位。LY294002逆转了左西孟旦的抑制作用。本研究首次证明，左西孟旦通过激活PI3K/Akt途径和抑制成年大鼠心脏成纤维细胞中iNOS的表达来抑制IL-1β诱导的凋亡。
