锌指蛋白A20调节泛素羧基末端水解酶-L1表达在狼疮性肾炎足细胞损伤中的作用研究

Dysfunction of ubiquitin-proteasome system (UPS) is a key process in podocyte injury of lupus nephritis (LN). Recent studies have demonstrated that the activation of NF-κB could up-regulate the expression of ubiquitin carboxy-terminal hydrolase 1 (UCH-L1), which disturbed the proteasome activity of UPS, followed by the accumulation of target proteins and ubiquitin, and leading to the injury of podocytes. Zinc finger protein A20 is a negative regulatory feedback inhibitor of NF-κB activation. Our preliminary experiment observed that the diffuse podocyte foot-process effacement emerged in the early onset of LN in the MRL/lpr mice, accompanying uneven thickened glomerular basement membrane. The expression of A20 was down-regulated with UCH-L1 up-regulated significantly in the podocytes of LN patients, the phenomenon indicated that A20 and UCH-L1 might involve in the pathogenesis of podocyte injury, while the actual mechanisms need to be investigated. In this study, we intended to disclose that A20 regulates the expression of UCH-L1 through NF-κB pathway, which plays important roles in changing the structure and function of podocytes, and leads to UPS impairment and apoptosis in vitro and in vivo. This study might provide new theoretical and experimental basis for clinical management of LN.

泛素-蛋白酶体（UPS）途径异常是狼疮性肾炎（LN）足细胞损伤的重要机制。近期研究证实，NF-κB活化可上调泛素羧基末端水解酶-L1（UCH-L1）表达，UCH-L1影响UPS途径，造成靶蛋白及泛素蓄积，导致足细胞损伤。锌指蛋白A20是NF-κB信号通路的负向调节因子，我们预实验中发现，LN小鼠肾脏病早期即出现明显足突融合；LN患者肾小球足细胞A20蛋白表达明显下调伴UCH-L1表达明显上调，提示A20及UCH-L1可能参与LN足细胞损伤的发生，但确切机制仍不清楚。本研究拟通过动物及体外细胞培养实验，利用真核表达载体转染、siRNA等多种干预和检测手段，探讨A20蛋白是否通过NF-κB信号通路调节UCH-L1表达，及上述通路对足细胞结构、功能、UPS途径和细胞凋亡的影响，以阐明LN足细胞损伤的新机制，为临床防治LN提供新的理论依据。

狼疮性肾炎（LN）是系统性红斑狼疮（SLE）最常见且严重的并发症之一，NF-ƙB信号通路激活是LN发病的重要机制，锌指蛋白A20是NF-ƙB通路的负反馈调节因子，泛素羧基末端水解酶-L1（UCH-L1）是NF-ƙB通路的下游靶蛋白。然而，它们在LN发病机制中的作用仍不清楚。本研究选择MRL/lpr小鼠为LN动物模型，随周龄增长，MRL/lpr小鼠出现蛋白尿，肾功能减退，循环抗dsDNA抗体、TNF-α和IL-1β水平增加，肾脏病理可见肾小球细胞增殖、新月体和间质炎性细胞浸润，电镜显示LN肾脏足细胞病变早于肾小球内免疫复合物沉积，因此，该小鼠是成功的LN动物模型。本课题组研究发现，LN病程中，MRL/lpr小鼠肾脏A20蛋白存在表达缺陷，无法限制NF-ƙB信号通路活化，从而导致UCH-L1过度表达，影响泛素蛋白酶体活性，造成泛素蓄积和足细胞损伤，糖皮质激素治疗可通过减轻A20缺陷来延缓LN进展。通过尾静脉注射A20过表达腺病毒载体，上调MRL/lpr小鼠的肾脏A20蛋白表达，可抑制NF-ƙB通路活化，减少UCH-L1表达和泛素积累、从而改善足细胞损伤，延缓LN病情进展。使用PDTC抑制NF-ƙB活性或LDN57444抑制UCH-L1活性，可达到与上调A20蛋白表达类似的效果。进一步体外细胞实验证实，A20蛋白通过NF-ƙB信号通路调节UCH-L1表达，进而参与调节足细胞损伤。本研究通过动物及体外细胞培养实验，首次系统地揭示了锌指蛋白A20通过NF-κB信号通路调节UCH-L1表达的机制，及该A20/NF-ƙB/UCH-L1通路在LN发生发展和足细胞损伤中的作用，阐述了A20蛋白表达缺陷可能是LN发病的始动环节和重要因素，A20有望成为干预LN进展的新靶点，为临床防治LN提供了新的理论和实验依据。