线粒体硫加双氧酶ETHE1介导细胞自噬过度活化在贫铀肾毒性中的作用及其机制研究

Depleted uranium (DU) is widely used in the civil and military activities, and kidney is the main target organ of toxicity of DU. However, the mechanism of DU-induced nephrotoxicity needs to clarify. Our previous research has confirmed that after 24h exposure to DU, the autophagic vacuoles in the human embryonic kidney cells (HEK293) increase, the autophagy marker LC3-Ⅱ increases, and cytotoxicity decreases after using autophagy inhibitor, suggesting that autophagy may play an important role in DU-induced nephrotoxicity. Meanwhile, mitochondrial sulfur dioxygenase ETHE1 decreases as the exposure dose of DU increases, with increasiong autoghagy flux. ETHE1 is a p53 inhibitor, and p53/AMPK axis is an important signal pathway of autophagy. On the basis of the latest research, we speculate that through reducing ETHE1, activation of p53/AMPK pathway and inhibiting mTOR pathways, DU might induce autophagy overactive which affects the nephrotoxicity. To confirm this hypothesis, in vitro and vivo model of DU-induced nephrotoxicity will be set by using HEK293 cells and mice. Molecular biology, siRNA technology and flow cytometry will be used to determine the effect of DU on autophagy and the role of autophage on the nephrotoxicity, and explore the mechanisms of DU-induced autophagy by EHTE1, p53 and AMPK. This study will provide a novel therapeutic target for the prevention and treatment of DU-induced nephrotoxicity.

贫铀被广泛应用于民事和军事活动中，肾脏是其中毒的靶器官，但中毒机制有待于研究。我们前期研究发现人胚肾细胞HEK293暴露于贫铀后24h，胞内自噬体增多，自噬体膜标志蛋白LC3-Ⅱ增高，而使用自噬抑制剂后，贫铀的细胞毒性降低，提示自噬过度活化可能在贫铀肾毒性中发挥了重要作用。同时还发现，随贫铀诱导细胞自噬水平的增高，细胞内线粒体硫加双氧酶ETHE1逐渐降低。结合最新研究进展，ETHE1是p53的抑制蛋白，而p53/AMPK又是自噬调节的关键通路，推测贫铀可能通过下调ETHE1，激活p53/AMPK信号通路，抑制mTOR途径，促进细胞自噬过度活化而导致其肾毒性。为证实这一假说，拟利用本室已建立的体内外贫铀中毒模型，采用分子生物学、基因转染、siRNA干扰、流式细胞术等技术，评价贫铀对肾细胞自噬的影响及其在肾毒性中的作用，探讨ETHE1、p53及AMPK对其调控机制，为防治贫铀中毒提供新靶点。

贫铀被广泛应用于民事和军事活动中，肾脏是其中毒的靶器官，但中毒机制有待于研究。本项目的研究目的是探究自噬在贫铀诱导的肾毒性中的作用并确定其机制。研究发现人胚肾细胞（HEK293）暴露于贫铀4h后，胞内自噬体增多，自噬体膜标志蛋白LC3-Ⅱ增高，而使用自噬抑制剂后，贫铀的细胞毒性降低，提示自噬过度活化可能在贫铀肾毒性中发挥了重要作用。自噬抑制剂通过阻断自噬，进而改善了贫铀诱导的肾毒性。我们还发现HEK293细胞暴露于贫铀后引起细胞核p53的活化和mTOR通路的抑制，同时随着贫铀暴露剂量的增加，细胞自噬水平的增高，细胞内线粒体硫加双氧酶ETHE1逐渐降低。对 HEK293 细胞进行ETHE1基因干扰发现，细胞核p53的表达显著增加，贫铀诱导的肾毒性中细胞自噬过度活化。综上所述，我们的研究结果表明，贫铀通过减少ETHE1，激活p53通路，抑制mTOR通路促进自噬过度活化，从而导致肾毒性。本研究将为预防和治疗贫铀所致的肾毒性提供新的治疗靶点。