基于AMPK通路的马度米星调控内质网应激致骨骼肌毒性分子机理研究

Maduramicin, a high potent coccidiostat, presents serious toxicity to skeletal muscle in animals and humans. As an ionophore, maduramicn possesses the ability to carry Na+ into cells, which may thus alter intracellular Ca2+ and ATP concentrations. Endoplasmic reticulum is an important organelle, whose function is affected by Ca2+ and ATP levels. Endoplasmic reticulum stress is associated with cytotoxicity and is regulated by AMPK. ATP and Ca2+ can regulate AMPK. Our preliminary work showed that maduramicin induces endoplasmic reticulum stress and activates AMPK in C2C12 cells. However, the mechanism of maduramicin-regulated endoplasmic reticulum stress in skeletal muscle cells remains largely unknown. Using C2C12, L6, mouse primary skeletal muscle cells, broiler skeletal muscle tissue as experimental models and RNA interference, Western Blot, immunohistochemistry as methods, this project intends to uncover the importance of endoplasmic reticulum stress in maduramicin-caused skeletal muscle toxicity, to disclose the relationship between maduramicin-activated AMPK and -induced endoplasmic reticulum stress, and to demonstrate that maduramicin induces endoplasmic reticulum stress through AMPK by regulating Na+-dependent intracellular ATP and Ca2+ concentrations in skeletal muscle. This study will deeply reveal the molecular mechanism of maduramicin-induced endoplasmic reticulum stress in skeletal muscle cells, thereby providing the new idea and theoretical basis for the prevention and treatment of maduramicin-caused skeletal muscle toxicity.

马度米星是一种高效抗球虫药，但对动物和人有很强的骨骼肌毒性。其作为离子载体可携带Na+内流入胞，可能因此改变胞内Ca2+和ATP浓度。内质网是重要的细胞器，功能受Ca2+和ATP水平影响。内质网应激与细胞毒性相关，会被AMPK调节。ATP和Ca2+则能调控AMPK。申请人前期发现马度米星诱导C2C12细胞内质网应激并激活AMPK，而马度米星调控骨骼肌细胞内质网应激的机制未明。本项目拟以C2C12、L6、小鼠原代骨骼肌细胞和肉鸡骨骼肌组织为实验模型，用RNA干扰、Western Blot和免疫组化等方法，确认内质网应激在马度米星骨骼肌毒性中的重要性，阐明马度米星调控AMPK和其诱导内质网应激的相互关系，论证马度米星调控Na+依赖的胞内ATP和Ca2+浓度经AMPK诱导骨骼肌细胞内质网应激。本研究将深入揭示马度米星诱导骨骼肌细胞内质网应激分子机理，为防治马度米星骨骼肌毒性提供新思路和理论依据。

马度米星作为一种高效抗球虫药被广泛添加于动物饲料中，但其存在毒性大、安全范围窄等问题，常因使用不当造成动物中毒，还可经动物源食品和环境进入人体，从而威胁人类健康。因此，对于其毒性机理的研究具有重要意义。马度米星主要损伤骨骼肌，但内质网应激相关马度米星骨骼肌毒性分子机制还未阐明。本项目用马度米星处理L6和C2C12细胞，喂养小鼠、大鼠和肉鸡，运用细胞培养、流式细胞术、RNA干扰、Western blot、转录组学等技术开展研究。结果显示，在C2C12细胞中观察到差异基因2450个；在L6和C2C12细胞中，马度米星影响细胞正常功能，如诱导细胞空泡化、抑制细胞生长、降低线粒体膜电位、升高钠离子、钙离子、ATP浓度和pH值；在细胞和骨骼肌组织中，发现马度米星升高钠钾ATP酶蛋白表达；升高细胞和组织LC3-II、p62蛋白水平，表明其阻断自噬流；升高eIF2α磷酸化和ATF4、CHOP蛋白水平，表明其诱导内质网应激，特别是激活PERK-eIF2α通路；该通路抑制剂IRSIB联合处理可削弱马度米星对ATF4和自噬流的作用，回升细胞活性，表明此通路介导马度米星对自噬流的调控和骨骼肌毒性；升高AMPK磷酸化水平，表明其激活AMPK通路；沉默AMPK可减弱马度米星对eIF2α-ATF4通路和自噬流的影响，表明AMPK蛋白活性在马度米星诱导内质网应激和调控自噬流中发挥重要作用。以上结果说明马度米星的骨骼肌毒性是由于其激活AMPK通路，从而诱导内质网应激，最终阻断自噬流。本研究深化了对马度米星中毒机制的认识，提示干预AMPK、内质网应激、自噬可有效缓解其骨骼肌毒性，为下一步防治马度米星中毒奠定了重要基础。