从内质网应激探讨清除肌红蛋白对挤压综合征肾小管上皮细胞凋亡的干预机制

Endoplasmic reticulum stress (ERS) plays a key role in the induction of apoptosis in renal tubular epithelia, which is a critical process in acute kidney injury (AKI)caused by crush syndrome. However, the mechanisms of AKI in crush syndrome remain unclear. Our previous study found that continuous veno-venous hemofiltration (CVVH) had extraordinary advantages on the treatment of crush syndrome by effective clearance of myoglobin. Our preliminary studies also detected that renal tubular cell apoptosis and ERS activation occurred in both goat and mouse crush syndrome models. Therefore, we propose our hypothesis as follows: CVVH can remove myoglobin, prohibiting the apoptosis process of renal tubular epithelia caused by oxidative stress. The purpose of our research is to create goat crush syndrome model, commit CVVH, and observe the dynamic change of plasma myoglobin level and the correlation between ERS pathway and renal tubular epithelia apoptosis. In addition, we aim to establish mouse crush syndrome models, suppress oxidative stress, and detect the apoptosis level of renal tubular epithelia. Crush syndrome will also be induced in CHOP knockout mice to investigate the level of ERS and renal tubular epithelia apoptosis. Besides, in vitro study is designed to elucidate the exquisite pathway of ERS leading to renal tubular apoptosis by using SiRNA techniques. In this proposal, we combine in vivo and in vitro studies to verify the treatment mechanism of CVVH for crush syndrome, which may provide novel theoretical evidence for this treatment protocol.

内质网应激（ERS）在肾小管上皮细胞凋亡中发挥着关键作用。肌红蛋白是挤压综合征致AKI的关键致病蛋白，但其致病机制尚不明确。我们的前期研究发现挤压综合征山羊及小鼠模型中肾小管上皮细胞明显的ERS激活及凋亡征象, 而连续性静-静脉血液滤过（CVVH）治疗挤压综合征疗效显著，具有快速清除肌红蛋白的优势。因此我们建立如下假说，在挤压综合征，肌红蛋白可能通过ERS诱导肾小管上皮细胞调亡，而CVVH可能通过阻断这一机制而起治疗作用。本研究将从整体及细胞两个层次验证这一假说。在体内，我们拟通过CVVH干预羊挤压综合征模型，观察肌红蛋白的动态变化与肾组织中ERS通路和肾小管上皮细胞凋亡的相关关系；通过建立CHOP敲除小鼠挤压综合征模型及阻断氧化应激等干预方式，分析肌红蛋白通过ERS介导肾小管上皮细胞凋亡的机制。在体外，我们拟通过siRNA基因沉默技术，研究肌红蛋白致肾小管上皮细胞凋亡的ERS信号传导通路

挤压综合征（crush syndrome, CS）是指四肢或躯干肌肉丰富部位受重物长时间挤压或长期固定体位而造成肌肉组织的缺血性坏死，从而出现的以肢体肿胀、高钾血症、肌红蛋白尿及急性肾损伤（AKI）等为特点的临床综合征。在各种重大灾难（地震、战争、塌方等）的幸存者中，因挤压综合征及其并发症是导致灾后伤亡的主要原因，死亡率可高达50%以上。挤压综合征致AKI的确切机制尚未完全阐明，主要的病理生理改变是肌肉受到长期挤压后所导致的横纹肌溶解，其中肌红蛋白是致病的关键蛋白，而肌红蛋白与肾脏损伤过程中内质网应激和氧化应激诱发的凋亡是本课题研究的侧重点。本课题采用体内、体外实验探究横纹肌溶解致AKI的可能治病机制。体内实验采用了抗氧化剂N-乙酰半胱氨酸(NAC)阻断内质网应激和氧化应激。与对照组相比，甘油造模组(50%, 10ml/kg, IM)在功能学和组织学方面均表现出更严重的肾脏损伤，凋亡和内质网应激相关蛋白如caspase-12, cleaved caspase-3, BAX, GRP78 和CHOP的表达也较高。甘油造模组血清SOD和MDA水平下降，而NAC预处理组在功能学和组织学方面的改变均呈现出部分逆转趋势。此外，体外实验中用200mM的肌红蛋白处理锦缎小管上皮细胞(PTECs)，观察到细胞凋亡水平、凋亡和内质网应激相关蛋白（GRP78, CHOP, caspase-4, cleaved caspase-3, BAX）、活性氧族(ROS)水平均有所上升，提示PTECs参与了内质网应激和氧化应激的激活。上述研究成果阐明了横纹肌溶解诱导急性肾损伤过程中，肌红蛋白与内质网应激和氧化应激诱发的凋亡的作用机制，对挤压综合征的临床治疗具有重要的指导意义。