Wang Lei, Yan Weijun, Wang Jing
Department of Cardiology, The First People's Hospital of Shangqiu, Shangqiu, Henan, People's Republic of China.
Emergency Department, Yan'an People's Hospital, Yan'an, Shannxi, People's Republic of China.
J Cell Physiol. 2019 Sep;234(9):15206-15214. doi: 10.1002/jcp.28162. Epub 2019 Feb 3.
MEKK3 is a member of MAP3K, which plays a pivotal role in cardiac diseases. In this study, we aimed to investigate the effects and potential mechanisms of MEKK3 on hypoxia/reoxygenation (H/R) injury of cardiomyocytes. After exposing H9C2 cells to H/R insult, real-time polymerase chain reaction and western blot analysis showed that MEKK3 was highly expressed. Cell viability, cell apoptosis, caspase 3/7 activity, and cleaved-caspase 3 expression were tested using a CCK-8 assay, Cell Death Detection PLUS ELISA, Caspase-Glo 3/7 Assay Kit and western blot analysis, respectively. Mitochondrial membrane potential, cytochrome C expression, adenosine triphosphate (ATP), and reactive oxygen species also were measured using JC-1 staining, western blot analysis, an ATP Assay Kit, and DCFH -DA staining, respectively. The messenger RNA (mRNA) levels and secretions of TNF-α, IL-6, and IL-1β were evaluated. The results revealed that MEKK3 silencing promoted cell survival and attenuated lactate dehydrogenase leakage, cell apoptosis, caspase 3/7 activity, and the protein level of cleaved-caspase 3. Moreover, knockdown of MEKK3 blocked mitochondrial impairment by inhibiting the loss of mitochondrial membrane potential and cytochrome C expression as well as promoting ATP synthesis. MEKK3 deficiency led to a decrease in reactive oxygen species and malondialdehyde (MDA) generation and an increase in superoxide dismutase (SOD) activity. Deletion of MEKK3 led to reduced inflammatory cytokines in mRNA level and secretion. MEKK3 suppression activated the sonic hedgehog (Shh) signaling pathway in H9C2 cells. After blocking the Shh signaling pathway with a specific inhibitor, cyclopamine, the cardioprotective functions of MEKK3 downregulation were partly abolished. In conclusion, downregulation of MEKK3 prevented apoptosis and inflammation in H9C2 cells via the Shh signaling pathway.
MEKK3是丝裂原活化蛋白激酶激酶激酶（MAP3K）家族的成员，在心脏疾病中起关键作用。在本研究中，我们旨在探究MEKK3对心肌细胞缺氧/复氧（H/R）损伤的影响及潜在机制。将H9C2细胞暴露于H/R损伤后，实时聚合酶链反应和蛋白质印迹分析显示MEKK3高表达。分别使用CCK-8法、细胞死亡检测PLUS ELISA试剂盒、Caspase-Glo 3/7检测试剂盒和蛋白质印迹分析检测细胞活力、细胞凋亡、半胱天冬酶3/7活性及裂解的半胱天冬酶3表达。分别使用JC-1染色、蛋白质印迹分析、ATP检测试剂盒和DCFH-DA染色检测线粒体膜电位、细胞色素C表达、三磷酸腺苷（ATP）和活性氧。评估肿瘤坏死因子-α（TNF-α）、白细胞介素-6（IL-6）和白细胞介素-1β（IL-1β）的信使核糖核酸（mRNA）水平及分泌情况。结果显示，沉默MEKK3可促进细胞存活，减轻乳酸脱氢酶泄漏、细胞凋亡、半胱天冬酶3/7活性及裂解的半胱天冬酶3蛋白水平。此外，敲低MEKK3可通过抑制线粒体膜电位丧失和细胞色素C表达以及促进ATP合成来阻止线粒体损伤。MEKK3缺失导致活性氧和丙二醛（MDA）生成减少，超氧化物歧化酶（SOD）活性增加。删除MEKK3导致炎症细胞因子的mRNA水平和分泌减少。抑制MEKK3可激活H9C2细胞中的音猬因子（Shh）信号通路。用特异性抑制剂环杷明阻断Shh信号通路后，MEKK3下调的心脏保护功能部分被消除。总之，MEKK3的下调通过Shh信号通路预防H9C2细胞凋亡和炎症。
