MAVS在终末期肾病相关的血管内皮损伤中的作用及机制研究

Cardiovascular disease is the most common cause of death in CKD patients. For patients with end-stage renal disease, the endothelial dysfunction resulted from uremia and excessive inflammation was considered as the important pathogenic factor of cardiovascular disease in CKD patients with uncertain mechanisms. MAVS is an important joint protein during the inflammatory stress and its pattern recognition receptor domain contains multiple binding sites of TRAFs. In the preliminary study, we found that uremic toxin indoxyl sulfate (IS) can induce the upregulation of MAVS, TRAF6, and p-NF-κB in mouse aortic endothelial cells (MAECs). More importantly, silencing MAVS attenuated IS-induced cellular apoptosis in MAECs, while overexpression of MAVS led to the aggravation of cell apoptosis induced by IS, suggesting an important role of MAVS in uremic toxin-induced endothelial injury. Meanwhile, bioinformatics analysis predicts 7 binding sites of transcription factor Tcf3 in MAVS promoter region. Furthermore, IS treatment enhanced the mRNA level of Tcf3 in MAECs, suggesting that Tcf3 might be an upstream regulator of MAVS in MAECs in response to the challenge of uremic toxin IS. Thus, we speculated that uremic toxins may enhance the expression of MAVS through transcription factor Tcf3 to activate TRAF6/NF-κB signaling, which further cause the inflammatory injury in endothelial cells contributing to the occurrence of cardiovascular disease in CKD patients. In this proposal, we will fully investigate the role of Tcf3/MAVS/TTAF6/NF-κB signaling pathway in the pathogenesis of uremic toxin IS-induced endothelial injury employing the models of animals and cells, as well as the molecular approach.

心血管并发症是CKD最主要的死亡原因。终末期肾病的尿毒素血症及高炎症状态可能是导致心血管病变的重要因素，但具体机制尚不清楚。MAVS是炎症应激中重要的接头蛋白，其PRR结构域有多个TRAFs结合位点并与NF-κB活化相关。我们发现，尿毒症毒素IS可上调小鼠血管内皮细胞MAVS、TRAF6及p-NF-κB的表达，过表达MAVS可加重IS诱导的血管内皮细胞凋亡，敲低MAVS减轻了细胞损伤。生物信息学预测显示MAVS启动子区存在与转录因子Tcf3结合的位点；IS可上调Tcf3表达，Tcf3siRNA降低了MAVSmRNA水平，提示Tcf3可能是MAVS上游调控分子。由此推测：IS可能通过Tcf3启动MAVS表达，激活TRAF6/NF-κB通路，导致血管内皮受损及血管病变。本项目将在动物、细胞和分子层面深入研究Tcf3/MAVS/TRAF6/NF-κB通路在尿毒症毒素引起的血管内皮损伤中的作用。

心血管并发症是CKD最主要的死亡原因。终末期肾病的尿毒素血症及高炎症状态可能是导致心血管病变的重要因素，但具体机制尚不清楚。MAVS是炎症应激中重要的接头蛋白，其PRR结构域有多个TRAFs结合位点并与NF-κB活化相关。我们发现，尿毒症毒素IS可上调小鼠血管内皮细胞MAVS、TRAF6及p-NF-κB的表达，过表达MAVS可加重IS诱导的血管内皮细胞凋亡，敲低MAVS减轻了细胞损伤。提示MAVS可能参与CKD相关的心血管系统损伤。我们构建了MAVS基因敲除的小鼠，通过体内实验进一步探究MAVS在心血管系统中的作用。我们意外地发现，MAVS-/-小鼠表现出心脏功能受损的表型。这与我们在体外实验中观察到的血管中，MAVS的作用并不一致。我们推测，MAVS在血管系统和心脏组织中可能发挥不同的作用。我们发现MAVS-/-小鼠心脏中ATP含量、血清CK-MB和LDH水平显著低于野生小鼠。基于非靶向的代谢组学OPLS-DA分析结果显示，MAVS-/-小鼠中的一些中/长链脂肪酸的水平显示降低。通过qRT-PCR分析显示，MAVS-/-小鼠的心脏脂肪酸代谢相关基因（ACC1/2、CPT1α、MCAD）下调，CD36和PPARα表达降低，MAVS缺失通过干扰脂质代谢导致能量代谢的紊乱。电子显微镜成像显示，MAVS-/-小鼠的心肌纤维断裂，线粒体解体，线粒体自噬。通过qRT-PCR分析显示，MAVS-/-小鼠心脏中线粒体基因mRNA水平下调。同时，MAVS-/-小鼠心脏中MDA水平升高，2-羟基丁酸水平显着升高，提示，MAVS缺失是通过损害心肌的抗氧化能力和自噬清除能力导致线粒体功能障碍。我们检测了2-3周的新生小鼠的心脏发育状态发现，野生型小鼠和敲除型小鼠之间没有明显的差异，提示MAVS缺乏并不影响心脏的早期发育。利用CRISPR/Cas9技术在体外敲除了H9C2细胞中的MAVS基因后，通过qRT-PCR分析细胞水平中线粒体基因的表达和线粒体形态，观察到线粒体基因mRNA水平均显著下调，H9C2细胞中线粒体受损解体，线粒体自噬。这些结果进一步证实了MAVS的缺失导致H9C2细胞线粒体功能障碍。本研究阐释了MAVS在血管组织和心脏组织中的作用差异，证实了MAVS对心肌细胞及心脏组织的保护作用，为心血管疾病的治疗提供了潜在靶点。