马度米星铵调控Ras和Rac1蛋白介导鸡心肌细胞methuosis的机制研究

Maduramicin, one of the most commonly used anti-coccidial drugs in poultry industry, often induces intoxication of broiler chicken and human due to its narrow safety range and high toxicity, which results in potential risks of animal health and food safety for human. Studies show that chicken myocardium is the major target tissue induced by maduramicin intoxication. Our previous studies demonstrated that maduramicin induces a novel cell death type ‘methuosis’ in primary chicken myocardial cells along with the regulation of Ras and Rac1. However, the molecular mechanism of maduramicin-induced methuosis in chicken myocardial cells is still unclear. Thus, in this study, we aim to understand the expression pattern of Ras and Rac1 in chicken myocardial cells and H9c2 cells, as well as the important role of Ras and Rac1 in the process of maduramicin-induced methuosis by using RNA interference, protein overexpression, Co-Immunoprecipitation assay and other techniques. Furthermore, we will identify the key upstream and downstream signal molecules of Ras and Rac1 and will reveal its underlying mechanism induced by maduramicin in myocardial cells. This study will shed new light on the mechanism of methuosis-mediated cardiotoxicity of maduramicin, and also will provide scientific basis for the detoxification of maduramicin in order to guarantee broiler chicken and human health.

马度米星铵是肉鸡养殖中最常用的抗球虫药之一，但其安全范围窄、毒性大，易引起肉鸡和人的中毒，给动物健康和人类食品安全带来潜在风险。研究显示心肌是马度米星铵毒性损伤的主要靶组织，申请者研究发现新型细胞死亡方式methuosis介导马度米星铵引发的鸡心肌毒性，且Ras和Rac1蛋白与心肌细胞methuosis的发生存在相关性，但其分子机制尚未阐明。本项目拟以鸡心肌细胞和心肌细胞（H9c2）为模型，采用RNA干扰、蛋白过表达及免疫共沉淀等技术，研究Ras和Rac1在心肌细胞发生methuosis时的表达规律，阐释Ras和Rac1调控心肌细胞methuosis的重要作用，鉴定Ras和Rac1的上下游关键信号分子，揭示其调控心肌细胞methuosis介导马度米星铵心肌毒性的发生机制。本项目为认识马度米星铵的心肌毒性机制提供新思路，为解除马度米星铵的毒性以保障肉鸡和人类健康提供科学依据。

马度米星铵作为兽医临床最常用的治疗肉鸡球虫病药物之一，因其抗球虫谱广、效价高、成本低且球虫不易产生耐药性在临床广泛应用，但其安全范围极窄、毒性大，常引起肉鸡中毒和药物残留，严重限制了其在兽医临床的应用且对动物健康和人类健康具有潜在风险。早期研究显示心脏是马度米星铵毒性损伤的靶器官，细胞凋亡和非凋亡依赖性细胞死亡是马度米星铵引起心肌损伤的重要原因。本研究前期发现一种新型细胞死亡methuosis介导马度米星铵诱发鸡心肌损伤，且Ras和Rac1与methuosis的发生高度相关，然而其调控机制尚不清楚。本研究以鸡原代心肌细胞和H9c2心肌细胞为体外模型，探究了Ras和Rac1在心肌细胞methuosis时的表达规律，解析了Rac1参与调控马度米星铵诱导心肌细胞毒性的重要作用，阐明了Ras-Rac1下游信号分子MAPK介导马度米星铵引起心肌细胞methuosis的作用与机制。研究发现细胞过度巨胞饮和内体肿胀共同导致马度米星铵引起心肌细胞methuosis，马度米星铵上调Ras和Rac1基因和蛋白水平的表达，采用siRNA干扰和化学性抑制Rac1可显著抑制马度米星铵引起的心肌细胞死亡但无法抑制其空泡化，马度米星铵激活心肌细胞中Rac1下游MAPK的P38表达，化学性抑制P38可缓解马度米星铵引起的心肌细胞毒性但无法抑制其空泡化。本研究结果为靶向抑制methuosis缓解马度米星铵引起的肉鸡心脏毒性奠定理论依据，也丰富了兽医毒理学基础理论。