Abdelhamid Ghada, El-Kadi Ayman O S
Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2N8.
Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2N8.
Free Radic Biol Med. 2015 May;82:1-12. doi: 10.1016/j.freeradbiomed.2015.01.005. Epub 2015 Jan 19.
Evidence suggests that upregulation of soluble epoxide hydrolase (sEH) is associated with the development of myocardial infarction, dilated cardiomyopathy, cardiac hypertrophy, and heart failure. However, the upregulation mechanism is still unknown. In this study, we treated H9C2 cells with buthionine sulfoximine (BSO) to explore whether oxidative stress upregulates sEH gene expression and to identify the molecular and cellular mechanisms behind this upregulatory response. Real-time PCR and Western blot analyses were used to measure mRNA and protein expression, respectively. We demonstrated that BSO significantly upregulated sEH at mRNA levels in a concentration- and time-dependent manner, leading to a significant increase in the cellular hypertrophic markers, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP). Furthermore, BSO significantly increased the cytosolic phosphorylated IκB-α and translocation of NF-κB p50 subunits, as measured by Western blot analysis. This level of translocation was paralleled by an increase in the DNA-binding activity of NF-κB P50 subunits. Moreover, our results demonstrated that pretreatment with the NF-κB inhibitor PDTC significantly inhibited BSO-mediated induction of sEH and cellular hypertrophic marker gene expression in a dose-dependent manner. Additionally, mitogen-activated protein kinases (MAPKs) were transiently phosphorylated by BSO treatment. To understand further the role of MAPKs pathway in BSO-mediated induction of sEH mRNA, we examined the role of extracellular signal-regulated kinase (ERK), c-JunN-terminal kinase (JNK), and p38 MAPK. Indeed, treatment with the MEK/ERK signal transduction inhibitor, PD98059, partially blocked the activation of IκB-α and translocation of NF-κB p50 subunits induced by BSO. Moreover, pretreatment with MEK/ERK signal transduction inhibitors, PD98059 and U0126, significantly inhibited BSO-mediated induction of sEH and cellular hypertrophic marker gene expression. These results clearly demonstrated that the NF-κB signaling pathway is involved in BSO-mediated induction of sEH gene expression, and appears to be associated with the activation of the MAPK pathway. Furthermore, our findings provide a strong link between sEH-induced cardiac dysfunction and involvement of NF-κB in the development of cellular hypertrophy.
有证据表明，可溶性环氧化物水解酶（sEH）的上调与心肌梗死、扩张型心肌病、心脏肥大和心力衰竭的发生发展有关。然而，其上调机制仍不清楚。在本研究中，我们用丁硫氨酸亚砜胺（BSO）处理H9C2细胞，以探讨氧化应激是否会上调sEH基因表达，并确定这种上调反应背后的分子和细胞机制。分别使用实时PCR和蛋白质印迹分析来测量mRNA和蛋白质表达。我们证明，BSO以浓度和时间依赖性方式显著上调sEH的mRNA水平，导致细胞肥大标志物心房钠尿肽（ANP）和脑钠尿肽（BNP）显著增加。此外，通过蛋白质印迹分析测量，BSO显著增加细胞质中磷酸化的IκB-α以及NF-κB p50亚基的易位。这种易位水平与NF-κB P50亚基的DNA结合活性增加平行。此外，我们的结果表明，用NF-κB抑制剂PDTC预处理以剂量依赖性方式显著抑制BSO介导的sEH诱导和细胞肥大标志物基因表达。另外，丝裂原活化蛋白激酶（MAPK）经BSO处理后被短暂磷酸化。为了进一步了解MAPK途径在BSO介导的sEH mRNA诱导中的作用，我们研究了细胞外信号调节激酶（ERK）、c-Jun氨基末端激酶（JNK）和p38 MAPK的作用。事实上，用MEK/ERK信号转导抑制剂PD98059处理可部分阻断BSO诱导的IκB-α活化和NF-κB p50亚基的易位。此外，用MEK/ERK信号转导抑制剂PD98059和U0126预处理可显著抑制BSO介导的sEH诱导和细胞肥大标志物基因表达。这些结果清楚地表明，NF-κB信号通路参与了BSO介导的sEH基因表达诱导，并且似乎与MAPK途径的激活有关。此外，我们的研究结果为sEH诱导的心脏功能障碍与NF-κB参与细胞肥大发展之间提供了强有力的联系。
