Fang Haiqin, Cong Liangzi, Zhi Yuan, Xu Haibin, Jia Xudong, Peng Shuangqing
Evaluation and Research Centre for Toxicology, Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing 100071, China; Key Laboratory of Food Safety Risk Assessment of Ministry of Health, China National Center for Food Safety Risk Assessment, Beijing 100021, China.
Huaiyin District Center for Disease Control and Prevention, Jinan, Shandong, China.
Toxicol Lett. 2016 Sep 6;258:259-266. doi: 10.1016/j.toxlet.2016.06.2103. Epub 2016 Jun 27.
To investigate the effect of T-2 toxin on murine embryonic stem cells (ESCs) cardiac differentiation and mitochondrial biogenesis in vitro.
Cardiac differentiation of the mouse ESCs was initiated by embryoid bodies (EBs) formation in hanging drops. EBs were exposed to 0.5ng/ml T-2 toxin for 24, 72 and 120h. Cultures were observed daily for the appearance of contracting clusters, and cardiac-specific protein (α-actiniin) were measured by Western blot and immunocytochemistry. Mitochondrial ultrastructure was observed by confocal laser scanning microscopy and transmission EM photography. Reactive oxygen species (ROS) was monitored by H2-dichlorofluorescein-diacetate (H2DCF-DA). The phosphorylation of the p38 (p-p38) and p38 mitogen-activated protein kinase (MAPK) and the expression of mitochondrial biogenesis proteins, including peroxisome proliferator activated receptor coactivator-1 alpha (PGC-1α), nuclear respiratory factor 1 (NRF-1), mitochondrial transcription factor A (mtTFA), and mitochondrial respiratory chain complex IV (COXIV) were analyzed using Western blot. In some experiments, mESCs were pre-treated with the antioxidant Trolox (200μM) for 30min, then exposed to Trolox (200μM) and T-2 toxin (0.5ng/ml) for 72h.
Contracting clusters were observed under the microscope light and cardiac-specific protein (α-actinin) expressed positively indicated mESCs directly differentiated in cardiomyocytes. However, the cardiac differentiation was inhibited by T-2 toxin treatment 72 and 120h. ROS accumulated in murine ES cells in a time-dependent manner. The expression of p-p38 significantly increased in 24h group and decrease in 72 and 120h groups. The decrease of mitochondrial number and the mitochondrial biogenesis-related proteins expression, including PGC-1α, NRF-1, mtTFA, and COXIV decreased in a time-dependent manner with T-2 toxin treatment. However, the inhibition of mitochondrial biogenesis by T-2 toxin in differentiated mESCs was recovered significantly in the presence of the antioxidant Trolox.
Taken together, T-2 toxin decreased the expression of PGC-1α, NRF-1, and mtTFA, inhibited mitochondrial biogenesis, and then inhibited the cardiac differentiation of murine ES cells, and the effect was partly responsible for the p38 MAPK mediated by ROS.
研究T-2毒素对小鼠胚胎干细胞（ESCs）体外心脏分化及线粒体生物合成的影响。
通过悬滴法形成拟胚体（EBs）启动小鼠ESCs的心脏分化。将EBs暴露于0.5ng/ml T-2毒素中24、72和120小时。每天观察培养物中收缩簇的出现情况，并通过蛋白质免疫印迹法和免疫细胞化学法检测心脏特异性蛋白（α-肌动蛋白）。通过共聚焦激光扫描显微镜和透射电子显微镜观察线粒体超微结构。用二氯荧光素二乙酸酯（H2DCF-DA）监测活性氧（ROS）。使用蛋白质免疫印迹法分析p38丝裂原活化蛋白激酶（p-p38）和p38的磷酸化以及线粒体生物合成相关蛋白的表达，包括过氧化物酶体增殖物激活受体γ共激活因子-1α（PGC-1α）、核呼吸因子1（NRF-1）、线粒体转录因子A（mtTFA）和线粒体呼吸链复合物IV（COXIV）。在一些实验中，mESCs先用抗氧化剂曲克芦丁（200μM）预处理30分钟，然后暴露于曲克芦丁（200μM）和T-2毒素（0.5ng/ml）中72小时。
在显微镜下观察到收缩簇，心脏特异性蛋白（α-肌动蛋白）阳性表达表明mESCs直接分化为心肌细胞。然而，T-2毒素处理72和120小时后心脏分化受到抑制。ROS在小鼠ES细胞中呈时间依赖性积累。p-p38的表达在24小时组显著增加，在72和120小时组降低。随着T-2毒素处理，线粒体数量减少以及线粒体生物合成相关蛋白表达降低，包括PGC-1α、NRF-1、mtTFA和COXIV呈时间依赖性下降。然而，在抗氧化剂曲克芦丁存在下，T-2毒素对分化的mESCs中线粒体生物合成的抑制作用显著恢复。
综上所述，T-2毒素降低了PGC-1α、NRF-1和mtTFA的表达，抑制了线粒体生物合成，进而抑制了小鼠ES细胞的心脏分化，且该效应部分由ROS介导的p38丝裂原活化蛋白激酶所致。
