氧化应激相关蛋白p66Shc和GDF1在砷介导的心脏毒性中的作用机制研究

p66shc, a member of Shc family,contributes to mitochondrial ROS metabolism and regulating the mitochontrial apoptosis pathway. The expression of Growth/differentiation factor 1(GDF1),a transforming growth factor-βmember, is downregulated via ROS produced in mitochontria. Both p66shc and GDF1 play an important role in embryo development and cardiac remodeling. Arsenic contamination is a major worldwide public health problem and associated with abnormal embryo development and cardiotoxicity. Our data showed that arsenic increased intracellular ROS level,upregulated the expression of p66shc and stimulated its translocation into the mitochondrial intermembrane space, which mediated arsenic-induced embryonic retardation. On the other hand, arsenic impaired embryo development via down-regulating GDF1 expression. Furthermore, we found that antioxidant folic acid decreased the expression upregulation of p66shc and ROS level caused by arsenic, markedly rescued the expression of GDF1, and antagonized arsenic-mediated cytoxicity. However, the molecular mechanism underlying the expression change of both p66shc and GDF1 and their role in cardiotoxicity mediated by arsenic need to be further investigated. Based on these findings,we will use both mice and cardiomyocytes to examine the effect of p66shc on arsenic-mediated ROS production, clarify the molecular mechanism of the expression change of both p66shc and GDF1 with arsenic treatment and the relationship between p66shc,ROS and GDF1,confirm the role of p66shc-ROS-GDF1 in arsenic-mediated cardiotoxicity. These findings will provide important evidences for the prevention and treatments of arsenic-mediated cardiotoxicity.

Shc家族成员p66Shc和TGF-β家族成员GDF1都与氧化应激相关，在胚胎发育和心脏功能中扮演重要作用。砷是一种广泛的环境污染物，具有胚胎发育和心脏毒性，我们前期研究表明砷通过上调p66shc和抑制GDF1的表达引起胚胎发育毒性，抗氧化剂叶酸能拮抗砷的细胞毒性，降低砷介导的p66shc表达和ROS升高，显著恢复GDF1的表达。然而砷调控p66shc和GDF1表达的机制以及其是否介导砷的心脏毒性效应尚需进一步研究。本项目拟以小鼠和细胞为模型，应用报告基因、染色质免疫沉淀、siRNA等技术，探讨p66shc对砷介导ROS 产生的调控作用，阐明砷对p66shc和GDF1的表达调控机制以及p66shc/ROS在砷抑制GDF1中的作用，明确p66shc/ROS和GDF1通路在砷介导心肌细胞毒性和心脏损伤中的作用，为探讨砷所致心脏毒性的发生提供新机制，为寻找有效防护措施提供新思路。

砷是一种广泛存在的环境污染物，砷暴露增加了心肌毒性及心血管疾病发病风险，氧化应激是其主要毒性机制之一。然而，砷调控活性氧（ROS）产生的机制及氧化应激响应蛋白p66shc和Dvr1/GDF1在砷心脏毒性中的调控和作用机制尚不清楚。.本项目围绕这些科学问题开展了如下研究：研究了长期砷暴露对小鼠心脏功能的影响，分析p66shc在砷诱导ROS中的作用，阐明砷调控p66shc和Dvr1/GDF1表达的分子机制及在心肌毒性中的作用，探索了营养保护等拮抗途径。主要取得了如下重要成果：发现长期砷暴露引起心肌氧化应激以及凋亡和自噬，导致心脏功能损伤，抗氧化剂NAC和叶酸能拮抗这些毒性效应，发现砷上调p66shc的表达和线粒体转位，过表达野生型p66shc促进砷诱导的ROS产生，而过表达显性负效应突变体p66shcS36A和敲低p66shc都显著抑制ROS产生。前期我们发现砷通过抑制斑马鱼Dvr1/GDF1的表达诱导心脏环化异常和发育毒性，在此基础上我们发现叶酸能拮抗砷的心脏发育毒性，显著逆转砷对Dvr1/GDF1的抑制。报告基因和染色质免疫沉淀等实验证明转录因子Sp1能转录激活GDF1的表达。文献报道组蛋白去乙酰化酶Sirt1是Sp1的下游基因，而Sirt1能负调控p66shc的表达。我们发现砷引起细胞Sp1、GDF1和Sirt1转录及蛋白水平剂量依赖性降低，而p66shc相应增加，进一步发现过表达Sp1逆转了砷对GDF1、Sirt1的抑制和对p66shc的上调，而敲低Sp1增强了砷对这些基因的调控效应。敲低Sirt1的表达能促进砷对p66shc表达上调。NAC和叶酸能显著拮抗砷对这些基因的调控。过表达野生型p66shc增强了砷对Sp1、GDF1和Sirt1表达的抑制，而过表达p66shcS36A和敲低p66shc显著逆转了砷对这些基因表达调节。基于体内外研究，我们结果表明砷暴露引起心脏发育异常和功能损伤，砷通过上调p66shc表达引起氧化应激，进而下调转录因子Sp1及下游基因GDF1和Sirt1的表达，而Sirt1又进一步负调控p66shc的表达，抗氧化剂NAC和叶酸能拮抗砷对这些基因的调控和心肌毒性，发挥心肌保护作用，本研究阐明了砷的毒性效应及新颖的分子机制，为环境污染物砷的风险评估和心肌保护途径提供新视角。