miR-17调控TXNIP介导NLRP3炎症小体活化促进动脉粥样硬化的作用及机制

We and other researchers recently demonstrated that the activation of NLRP3 inflammasomes played an important role in the development of atherosclerosis, but the underlying mechanism has not been elucidated. Reactive oxygen species (ROS) is the key activator of NLRP3 inflammasomes. NLRP3 inflammasome is a kind of multiprotein complex consisting of nucleotide-binding oligomerization domain (NOD)-like receptor pyrin domain containing 3 (NLRP3), the adaptor protein apoptosis-associated speck-like protein (ASC), and caspase 1, which functions to switch on the inflammatory process. Thioredoxin-interacting protein (TXNIP) is believed as a critical node in a chain of ROS production. Bioinformatic analysis of the TXNIP 3′-untranslated region (UTR) identified two conserved binding sites for microRNA-17 (miR-17) across multiple species. Our preliminary data suggested that miR-17 was downregulated and TXNIP was upregulated in the aorta under the development of atherosclerosis in apoE deficient mice fed a high fat diet. NLRP3 protein expression was also increased in the aorta of these apoE deficient mice fed a high fat diet. Furthermore, we found that miR-17 inhibitor can induce TXNIP protein expression and miR-17 mimic can inhibit TXNIP protein expression in vitro. These suggested that miR-17 may mediate the expression of TXNIP and increase the production of ROS, which may be involved in the activation of NLRP3 inflammasomes under the development of atherosclerosis. Thus, we hypothesize that miR-17-mediated TXNIP activates NLRP3 inflammasomes and accelerates atherosclerosis. .This project intends to combine animal and cell culture models, to further investigate ①Make sure that miR-17 is downregulated, TXNIP is upregulated and NLRP3 inflammasomes are activated in the development of atherosclerosis. ②Whether there is a causal relationship between miR-17-TXNIP signal and the activation of NLRP3 inflammasomes? If miR-17-TXNIP-mediated activation of NLRP3 inflammasomes are required for vascular inflammation and atherosclerosis? We intend to overexpress miR-17 and block the expression of TXNIP, and observe whether the activation of NLRP3 inflammasomes will be inhibited and atherosclerotic plaque area will be reduced. ③What induces the upregulation of TXNIP in the development of atherosclerosis (translational regulation of miR-17 or/and transcriptional regulation of ChREBP)? And what are the proinflammatory mechanisms of TXNIP? Targeting miR-17-TXNIP and NLRP3 inflammasomes may be a novel strategy to prevent the development of atherosclerosis.

包括我们在内的最新研究提示NLRP3炎症小体活化参与动脉粥样硬化（AS）发生发展，但具体机制不清。活性氧（ROS）是NLRP3炎症小体激活的关键信号，TXNIP是促进ROS产生的关键分子，其基因3′端非翻译区有2个miR-17结合位点。预实验显示AS主动脉miR-17下调、TXNIP上调伴NLRP3表达增强，miR-17体外能调节TXNIP表达，提示miR-17可能调控TXNIP促进ROS产生激活NLRP3炎症小体参与AS。本项目拟进一步明确其作用并探讨机制：①确证AS过程存在miR-17下调、TXNIP上调及NLRP3炎症小体激活；②探讨miR-17-TXNIP通路和NLRP3炎症小体活化之间是否存在因果关系？过表达miR-17、干扰TXNIP，炎症小体激活和AS是否减轻？③阐明AS进程中TXNIP上调（miR-17翻译调控外有无转录调控）及促炎作用的机制。为探讨AS防治新策略提供依据。

NLRP3炎症小体活化参与与动脉粥样硬化（AS）的发生发展，但具体信号机制尚不清楚，本项目提出miR-17调控硫氧还蛋白相互作用蛋白（TXNIP）介导NLRP3炎症小体活化促进AS发生发展的假说。我们在高脂饮食和糖尿病诱导的两种小鼠AS模型中，观察miR-17-5P和TXNIP表达的变化，以及NLRP3炎症小体激活和血管炎症情况。并进一步在糖尿病诱导的AS模型上，利用慢病毒过表达miR-17-5p，以及经典降糖药物二甲双胍，观察对TXNIP表达、NLRP3炎症小体激活、血管炎症反应以及AS的保护作用。研究结果表明：①高脂饮食及糖尿病诱导的AS小鼠模型中，均存在miR-17-5p下调、TXNIP上调及NLRP3炎症小体激活，提示TXNIP-NLRP3可能参与促进AS的发生发展。②miR-17可调控（抑制）TXNIP蛋白表达，miR-17-TXNIP通路和NLRP3炎症小体激活之间存在因果关系，miR-17-5p mimic可以抑制TXNIP表达，进而抑制NLRP3炎症小体活化和细胞焦亡。③高糖通过活性氧（ROS）依赖的信号上调TXNIP表达、下调硫氧还蛋白1（Trx1）表达，促进NLRP3炎症小体激活，诱导炎症反应；经典降糖药物二甲双胍可通过激活AMPK直接抑制TXNIP的表达，降低细胞内ROS水平，抑制NLRP3炎症小体的激活，该作用不依赖于其降糖作用。④二甲双胍可逆转糖尿病诱导的TXNIP表达及NLRP3炎症小体激活，从而减轻血管炎症和AS斑块形成。⑤过表达miR-17-5p可抑制糖尿病小鼠TXNIP表达、NLRP3炎症小体激活及细胞焦亡，并减轻AS。以上结果证实了miR-17-5p-TXNIP-NLRP3在AS发生发展中的重要作用，而干预miR-17-5p-TXNIP信号通路，抑制NLRP3炎症小体的激活，在动物模型上可有效抑制AS斑块的形成，为探索潜在的新的治疗途径提供了基础。