Ding Ling, Liang Xing-Guang, Hu Ying, Zhu Dan-Yan, Lou Yi-Jia
Institute of Pharmacology-Toxicology and Biochemical Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China.
Stem Cells Dev. 2008 Aug;17(4):751-60. doi: 10.1089/scd.2007.0206.
We previously reported that treatment of icariin could significantly induce cardiomyocyte differentiation of murine embryonic stem (ES) cells in vitro. In the present study, the exact activity initiated by icariin was further confirmed and the underlying molecular mechanism was investigated. We found that cardiomyocyte differentiation was efficiently stimulated only if icariin was administrated between days 5 and 8 in differentiation course, which indicated with elevated percentage of embryoid bodies (EB) and with beating areas and up- regulated expression of alpha-actinin and troponin T. Exposure of icariin triggered intracellular reactive oxygen species (ROS) generation of EBs in 3 h, which was abolished in the presence of either NADPH oxidase inhibitor DPI or antioxidant Trolox. Meanwhile, expression of NOX4, a membrane combined enzyme responsible for ROS generation, was promoted by icariin in a dose-dependent manner. Although p38MAPK (mitogen-activated protein kinase), extracellular signal-regulated kinase (ERK), and c-Jun N-terminal protein kinase (JNK) were spontaneously activated in early differentiation, only the phosphorylation of p38MAPK was enhanced and prolonged when icariin was present, whereas both ERK and JNK showed no response to icariin treatment. Moreover, the inducible effect of icariin was blunted by SB203580, a specific inhibitor of p38MAPK. On the contrary, neither UO126 nor SP600125, the specific inhibitor of ERK and JNK, could abolish icariin-stimulated differentiation. Nuclear location of MEF2C, which played a critical role in cardiomyocyte differentiation and could be activated by p38MAPK, was stimulated after icariin exposure. Taken together, these results suggest that ROS generation and the subsequent activation of p38MAPK are essential for the inducible function of icariin on cardiomyocyte differentiation of murine embryonic stem cells in vitro.
我们之前报道过，淫羊藿苷处理可在体外显著诱导小鼠胚胎干细胞向心肌细胞分化。在本研究中，进一步证实了淫羊藿苷引发的确切活性，并对其潜在分子机制进行了研究。我们发现，仅在分化过程的第5至8天给予淫羊藿苷时，心肌细胞分化才会受到有效刺激，这表现为胚状体（EB）百分比升高、出现跳动区域以及α-肌动蛋白和肌钙蛋白T的表达上调。淫羊藿苷处理3小时后可触发EB细胞内活性氧（ROS）生成，在存在NADPH氧化酶抑制剂DPI或抗氧化剂Trolox的情况下，这种现象会被消除。同时，淫羊藿苷以剂量依赖的方式促进了负责ROS生成的膜结合酶NOX4的表达。尽管p38丝裂原活化蛋白激酶（MAPK）、细胞外信号调节激酶（ERK）和c-Jun氨基末端蛋白激酶（JNK）在早期分化过程中会自发激活，但当存在淫羊藿苷时，只有p38MAPK的磷酸化增强且持续时间延长，而ERK和JNK对淫羊藿苷处理无反应。此外，p38MAPK的特异性抑制剂SB203580可减弱淫羊藿苷的诱导作用。相反，ERK和JNK的特异性抑制剂UO126和SP600125均不能消除淫羊藿苷刺激的分化。淫羊藿苷处理后，在心肌细胞分化中起关键作用且可被p38MAPK激活的MEF2C的核定位受到刺激。综上所述，这些结果表明，ROS生成以及随后p38MAPK的激活对于淫羊藿苷在体外诱导小鼠胚胎干细胞向心肌细胞分化的功能至关重要。
