KMUP-3藉由誘導細胞自噬作用降低氧化壓力引起心肌纖維母細胞之細胞凋亡

KMUP-3 Attenuated Oxidative Stress-Induced Apoptosis in Cardiac Fibroblasts by Activating Adaptive Autophagy

王菱誼(Ling-Yi Wang)

指導教授 ： 葉竹來

高雄醫學大學/醫學院/藥理學科研究所/碩士(2014年)

摘要

細胞自噬 (Autophagy) 對於正常狀況下胞器低基礎水平的轉換和在受到缺血/再灌流及心臟衰竭等心臟疾病壓力下的上調是重要的。細胞凋亡 (Apoptosis) 在許多疾病的發病機制中也扮演了重要的生物角色。由心肌梗塞而引起之氧化壓力是心臟衰竭的主要因子。在我們先前的研究中發現，化學合成的黃嘌呤 (Xanthine) 衍生物KMUP-3於心肌纖維母細胞 (Cardiac fibroblast) 中能誘導細胞自噬。本研究目的為探討KMUP-3如何調節H2O2 (Hydrogen peroxide) 對於心肌纖維母細胞所引起之細胞凋亡以及闡述細胞和分子機制。
　　在本研究中，細胞存活率分析顯示H2O2所引起之心肌纖維母細胞死亡，藉由KMUP-3的前處理可以減少細胞死亡。以流式細胞儀分析後，發現前處理KMUP-3可防止H2O2引起之細胞凋亡。在西方墨點法的分析結果顯示KMUP-3在H2O2存在下能增加磷酸化内皮型一氧化氮合成酶(phosphorylation of endothelial nitric oxide synthase, p-eNOS)、内皮型一氧化氮合成酶 (Endothelial nitric oxide synthase, eNOS)、蛋白激酶G (protein kinase G, PKG)、細胞自噬指標蛋白 (microtubule-associated protein light chain 3-II, LC3-II)、細胞自噬相關蛋7 ( autophagy-related gene 7, Atg7) 之形成且增加Bcl-2/Bax蛋白比值。此外，KMUP-3降低H2O2引起之MMP-2、MMP-9和caspase-3裂解的蛋白表現。這些作用會因給予非專一性和專一性的NOS抑制劑而被阻斷，指出eNOS在KMUP-3調節H2O2引起之細胞凋亡作用中所扮演的角色。然而，當心肌纖維母細胞以小干擾RNA Atg7處理後，會阻斷細胞自噬進而增加細胞凋亡。
　　整體來說，KMUP-3能促進細胞自噬而降低氧化壓力在心肌纖維母細胞中引起的細胞凋亡。同時透過調控細胞自噬和細胞凋亡在心臟疾病中具有心臟保護作用。這些心臟保護作用可能是透過eNOS增加及cGMP/PKG-依賴訊息傳遞路徑。

關鍵字

氧化壓力 ； 細胞凋亡 ； 細胞自噬 ； 心肌纖維母細胞

並列摘要

Autophagy is important for the turnover of organelles at low basal levels under normal conditions and it is upregulated in response to stresses such as ischemia/reperfusion and in cardiovascular diseases such as heart failure. Apoptosis also plays important biological roles in the pathogenesis of many diseases. Oxidative stress induced by myocardial infarction is one of the major factor of heart failures. In our previous studies, 7-[2-[4-(4-nitrobenzene)piperazinyl]ethyl]-1,3-dimethylxanthine (KMUP-3) is a chemically synthetic xanthine-based derivative. It has been shown to induce autophagy in cardiac fibroblasts. The aim of this study was to investigate how KMUP-3 modulates hydrogen peroxide (H2O2)-induced apoptosis in neonatal rat cardiac fibroblasts and to elucidate the cellular and molecular mechanism.
　　In this study, MTT assay showed that H2O2 induced cardiac fibroblasts death, and pre-treat KMUP-3 block it. Flow cytometry analysis H2O2 induced cardiac fibroblasts death through apoptosis, and pre-treat KMUP-3 prevent cell death. Western blot showed that KMUP-3 enhanced p-eNOS, eNOS, PKG, LC3-II, Atg7 formation, and increased Bcl-2/Bax ratio in H2O2-treated neonatal rat cardiac fibroblasts. Moreover, KMUP-3 attenuated H2O2-induced MMP-2, MMP-9 and cleaved caspase-3 protein expressions. These effects were blocked by both the L-NAME and L-NIO, indicating that eNOS plays a role in the modulation of KMUP-3 in H2O2-induced apoptosis. However, when cardiac fibroblasts treated with Atg7 siRNA, which blocked the autophagy in the cells and resulted in a further increase in cell apoptosis.
　　These results showed that KMUP-3 may promote autophagy to decrease oxidative stress-induced apoptosis in cardiac fibroblasts. KMUP-3 might exert cardioprotective effects in heart diseases through regulation of autophagy and apoptosis. These cardioprotective effects are possibly mediated through e-NOS enhancing and cGMP/ PKG-dependent signalling pathways.

並列關鍵字

oxidative stress ； apoptosis ； autophagy ； cardiac fibroblast

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