Myostatin介导塑化剂DEHP母体暴露对子代肌肉生长抑制的分子机制

Phthalic acid esters are a category of global environmental organic pollutants, classified as priority controlled pollutants by the U.S. Environmental Protection Agency and the China Environmental Monitoring Station as priority pollutants. We and others have reported that maternal exposure of phthalates (DEHP as a typical representative) reduced offspring birth weight and lean muscle mass, mainly due to the inhibitory effect of DEHP on the development and growth of skeletal muscle, however, the mechanisms leading to this responses are unclear. Studies have shown that myostatin (Abbreviation as the MSTN) can strongly inhibit muscle growth and development. Our preliminary studies showed that the expression of MSTN is significantly increased in mouse muscle treated with DEHP compared with control group. We propose that DEHP exposure inhibits muscle development and weight loss via stimulating MSTN expression. In this project, we will employ the Cre-loxP technology to create a mouse model with muscle-specific MSTN gene knockout and investigate the mechanisms by which DEHP influences muscle growth and development in progeny. We also will setup an in vitro model with small RNA interfering technology (siRNA) to identify the mechanisms how DEHP stimulates MSTN expression. We believe that the results of this project will provide a new strategy for prevention and treatment of DEHP-caused health problems especially the muscle growth defects/muscle wasting.

邻苯二甲酸酯是一类全球性环境有机污染物，被美国环保署和我国环保部列为优先控制污染物。申请人前期研究及文献均显示，邻苯二甲酸酯(DEHP是典型代表)母体暴露会降低子代出生体重及肌肉量，骨骼肌发育受到抑制，但具体机制尚不清楚。研究显示，Myostatin（简称MSTN）能强烈抑制肌肉的生长发育。我们前期实验发现，当母体暴露DEHP后，仔鼠肌肉中MSTN表达较对照组显著升高，因此我们推测：DEHP通过刺激MSTN升高从而引起肌肉发育抑制、体重减轻。本项目拟采用Cre-loxP技术，特异性敲除仔鼠骨骼肌细胞中MSTN基因，研究DEHP母体暴露对子代肌肉生长发育的影响及分子机制。同时采用siRNA技术构建细胞模型，阐明DEHP引起MSTN升高的作用机制。本项目研究结果可为塑化剂DEHP的健康危害尤其是肌肉发育抑制/肌肉萎缩的临床防治提供一种新的思路，具有较好的社会价值和临床应用前景。

塑化剂，又称邻苯二甲酸酯类化合物，是一种环境内分泌干扰物，对生殖、肝肾功能、胚胎发育等多方面均有潜在的危害作用。本课题在前期研究基础上，进一步利用Cre-loxP技术培育了骨骼肌中MSTN基因特异性敲除小鼠模型，利用该模型研究了MSTN基因在母体暴露塑化剂DEHP引起子代肌肉发育抑制中的作用及可能的相关分子机制。具体结果如下：（1）与对照组相比，MSTN基因敲除后仔鼠体重及骨骼肌重量均显著增加，尤以第7和21天最为明显；免疫荧光结果显示MSTN基因敲除组（KO组）仔鼠的肌纤维横截面积较对照组（WT组）增大；RT-qPCR结果提示KO组肌肉降解相关分子Atrogin-1和MuRF-1的mRNA表达水平较WT组下降。（2）在特定时间点（0、7、14、21天）分别检测4组仔鼠的体重及骨骼肌重量，结果显示WT-DEHP组仔鼠的体重与骨骼肌重量较WT-Con组明显降低，而KO-DEHP组与KO-Con组相比则降低不明显。（3）RT-qPCR检测4组仔鼠相关基因表达情况，结果显示WT-DEHP组仔鼠的肌肉降解因子Atrogin-1和MuRF-1 mRNA表达水平较WT-Con组升高，肌肉合成因子MyoD和Myogenin表达下降，而KO-DEHP组仔鼠较KO-Con组的Atrogin-1和MuRF-1并没有明显升高，MyoD和Myogenin也没有明显下调。（4）用DEHP活性产物MEHP处理骨骼肌细胞C2C12，结果显示，随着MEHP浓度的增加以及暴露时间的延长，C2C12细胞的增殖活性显著降低；荧光素酶标记结果显示MEHP组的荧光相对表达量均较对照组高，说明MEHP激活了MSTN的启动子；RT-qPCR结果显示C2C12细胞经MEHP处理后，MSTN、Atrogin-1和MuRF-1的mRNA水平表达均升高，而MyoD和Myogenin的表达均下调。（5）利用siRNA技术干扰C2C12细胞中C/EBPδ的表达，再经MEHP处理，结果发现干扰C/EBPδ不仅明显抑制了MEHP对MSTN以及Atrogin-1、MuRF-1等肌肉降解因子表达的上调，同时也抑制了MEHP对MyoD、Myogenin等肌肉合成因子表达的下调。本研究结果为认清塑化剂DEHP的健康危害提供一种新的思路，也为肌肉发育抑制相关疾病的防治提供一定的理论依据。