时钟基因DEC2调控TLR3信号通路介导狼疮性肾炎肾小球内皮细胞损伤的机制研究

Glomerular endothelial cell injury plays an essential role in the pathogenesis of lupus nephritis (LN). Differentiated embryonic chondrocyte gene 2(DEC2), as a member of clock gene that has been highlight reported to be associated with inflammatory response in recent years. However, whether DEC2 participating in the pathogenesis of glomerular endothelial cell injury in lupus nephritis has not been well illuminated yet. In our previous study, we confirmed the contribution of TLR3 signaling pathway in human glomerular endothelial cell injury. On the base of our previous study, we sought to investigate whether DEC2 regulates TLR3-mediated human glomerular endothelial cell injury in lupus nephritis. First, we will analyze the correlation between the expression of DEC2 in renal biopsy tissue and systemic lupus erythematosus disease activity index (SLEDAI) as well as renal tissue activity index in patients with LN, which suggests DEC2 may be a potent candidate for disease activity of LN patients. Second, we will detect that DEC2 and TLR3 are extensively expressed in glomeruli of MRLlpr/lpr mice by using dual-labeling immunofluorescence staining. The correlation between the expression of DEC2 and TLR3, ICAM-1, CCL2 as well as CXCL10 in glomeruli will be analyzed. Last, we will explore the functional site for DEC2 and elucidate the molecular mechanisms underlying the involvement of DEC2 in TLR3-mediated human glomerular endothelial cell injury. Our study will give insight to theoretical and experimental basis for revealing the pathogenesis of LN.

肾小球内皮细胞损伤是狼疮性肾炎（LN）发病机制的重要环节。近年来时钟基因分化型胚胎软骨基因2（DEC2）调控炎症反应受到广泛关注，但DEC2是否介导LN肾小球内皮细胞损伤尚未见报道。本研究拟在前期结果证明TLR3信号通路参与人肾小球内皮细胞损伤的基础上，探讨DEC2是否通过调控TLR3信号通路介导人肾小球内皮细胞损伤，参与LN的发生发展。分析LN患者肾组织DEC2表达与SLEDAI评分及肾脏病理活动性指数(AI)的相关性，提示DEC2可能作为LN患者病情活动指标。免疫荧光双染定位DEC2在狼疮性肾炎MRLlpr/lpr小鼠肾小球内皮细胞中表达，分析DEC2与TLR3信号通路粘附因子ICAM-1、趋化因子CCL2及CXCL10的相关性，鉴定DEC2在人肾小球内皮细胞TLR3信号通路的作用位点，明确DEC2调控TLR3信号通路参与LN肾小球内皮细胞损伤，为揭示LN发病机制提供理论和实验依据。

本研究在狼疮性肾炎MRL/lpr及Fcgr2b-/-小鼠和人肾小球内皮细胞及小鼠肾小球系膜细胞MES-13中探讨miR-183、miR-16、DEC2、TLR3及TLR4在狼疮性肾炎肾脏损伤中的作用机制。分析LN小鼠肾组织miRNAs表达变化，筛选与LN发病密切相关的miRNAs及下游作用靶点，探讨DEC2及TLR3、TLR4在LN肾脏损伤及炎症中的作用机制。结果发现LN小鼠肾组织中miR-183及miR-16存在差异表达。通过外源性给予miRNAs类似物可减轻LN小鼠蛋白尿，肾脏损伤，炎症细胞浸润及纤维化。通过生信分析预测miR-183下游靶向基因。体内和体外研究证明miR-183可通过靶向抑制Tgfbr1表达，下调TGF-β/Smad/TLR3，抑制炎症因子IL-6, IL-8及TNF-α，纤维化相关因子α-SMA及Vimentin 表达，从而减轻LN肾脏损伤及纤维化。通过生信分析预测miR-16下游靶向基因为DEC2。通过双荧光素酶试验验证miR-16与DEC2结合位点。进一步在体内及体外研究发现DEC2可促进下游TLR4表达。外源给予miR-16类似物，在体内及体外研究中均发现miR-16可通过抑制DEC2表达，下调下游TLR4表达，从而抑制促炎因子IL-1β，IL-6，TNF-α及MCP-1释放，抑制细胞增殖，促进细胞凋亡，凋亡相关蛋白Bax及Cleaved Caspase-3表达增多，LN肾脏损伤及纤维化减轻。本项目在深入研究miRNAs在LN发生过程中作用机制的同时，阐明了miRNA下游作用靶点，通过增加相应miRNAs表达或干预靶点下游基因，可减轻LN肾脏损伤的治疗作用，为LN的预防和记住你提供了新的治疗策略。