NLRP3炎性小体介导同型半胱氨酸诱导动脉粥样硬化炎症反应的作用及机制

Hyperhomocysteinemia (HHcy) is an independent risk factor for cardiovascular disease. Recently studies have shown that homocysteine (Hcy) can induce vascular inflammation and accelerate atherosclerosis, but the mechanism underlying Hcy-induced inflammation has not been elucidated. Our previous study showed Hcy can induce endothelial dysfunction and damage, and promote the generation of reactive oxygen species (ROS) in endothelial cells. ROS was reported as the key factor of NLRP3 inflammasomes activation. The activation of NLRP3 inflammasomes can trigger pro-inflammatory programmed cell death, namely pyroptosis, thereby recruit monocytes and T cells and induce the inflammatory response. 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. Can Hcy activate NLRP3 inflammasomes? Our preliminary data suggested that Hcy can induce NLRP3 protein expression and activate caspase-1 in endothelial cells. Furthermore, cell swelling, chromatin condensation (nucleus remains intact and karyorrhexis does not occur) and osmotic lysis were observed in endothelial cell in the aortic wall by electron microscopic examination. Which meets the characteristics of pyroptosis, suggesting the presence of pyroptosis. Thus, we hypothesize that Hcy induces inflammatory pyroptosis via formation and activation of NLRP3 inflammasomes and accelerates atherosclerosis. This project intends to combine animal and cell culture models, to further investigate ①Make sure that Hcy can activate NLRP3 inflammasomes and trigger pro-inflammatory programmed cell death pyroptosis. ②If NLRP3 inflammasomes are required for Hcy-induced vascular inflammation in atherosclerotic murine model? We intend to block the expression and activation of NLRP3 inflammasomes by using NLRP3 siRNA and caspases-1 inhibitor, and observe if Hcy-induced inflammatory response will disappear. ③How did Hcy activate NLRP3 inflammasomes (ROS/K+ channel/cathepsin)? Targeting NLRP3 inflammasomes at the stage of its assembling or activation may be a novel strategy to prevent the development of HHcy-induced AS.

同型半胱氨酸（Hcy）可诱导血管炎症促进动脉粥样硬化（AS），但其诱导炎症机制尚未阐明。我们既往研究表明Hcy能导致血管内皮损伤并促进内皮细胞活性氧（ROS）生成，而ROS可激活NLRP3炎性小体；作为炎症反应的核心，炎性小体可介导pyroptosis死亡模式，促进炎症反应发展。预实验显示Hcy可诱导内皮细胞NLRP3蛋白表达，并且产生符合pyroptosis特征的细胞形态改变，提示Hcy诱导的炎症反应可能与NLRP3炎性小体相关。本项目拟进一步明确其作用并探讨相关机制：①确证Hcy能激活NLRP3炎性小体并介导pyroptosis；②NLRP3炎性小体是否为Hcy诱导AS炎症反应所必需？干扰NLRP3表达阻断pyroptosis发生，Hcy诱导炎症反应的作用是否消失？③揭示Hcy激活NLRP3炎性小体的具体机制（ROS/K+通道/组织蛋白酶？）。为探讨AS防治的新策略提供理论依据。

高同型半胱氨酸血症（HHcy）可促进动脉粥样硬化（AS），但其机制尚未完全阐明。本项目提出同型半胱氨酸（Hcy）激活NLRP3炎症小体诱导血管炎症促进AS的假说。我们在apoE-/-小鼠AS模型上，观察HHcy对NLRP3炎症小体（炎性小体）的激活作用，以及对血管炎症反应和AS斑块面积的影响；并利用NLRP3 siRNA干扰技术，阐明NLRP3-caspase-1通路在Hcy致AS中的作用。研究随机分为对照组、高脂组、HHcy组、HHcy+NLRP3 shRNA组、HHcy+scramble shRNA组。对照组喂食普通饲料，高脂组喂饲高脂饲料，其它三组喂饲高脂高蛋氨酸饲料，共10周。研究结果发现，HHcy小鼠血浆IL-1β、IL-18水平显著升高，粥样斑块里巨噬细胞的浸润增加，AS病变加重，NLRP3炎症小体成分表达及共定位增加。NLRP3 shRNA干扰后血浆促炎细胞因子IL-1β和IL-18水平降低，粥样斑块里巨噬细胞的浸润减少，而且HHcy诱导的动脉粥样硬化减轻，HHcy诱导的NLRP3炎症小体成分表达及共定位减少。以上结果提示Hcy能通过激活NLRP3炎症小体促进AS的发生发展。为探讨Hcy激活NLRP3炎症小体的具体机制，我们采用Hcy诱导巨噬细胞NLRP3炎症小体激活，并观察到活性氧（ROS）清除剂N-乙酰基-L-半胱氨酸（NAC）可以阻断该作用，我们进一步在apoE-/-小鼠AS模型验证了NAC治疗抑制Hcy激活NLRP3炎症小体的作用。该结果表明Hcy通过ROS依赖的途径激活NLRP3炎症小体，诱导血管炎症反应，促进AS发生发展。研究结果为揭示Hcy 诱导炎症反应促进AS 机理提供新的依据，而干预NLRP3 炎性小体的生成、活化，阻断pyroptosis 促炎作用的恶性循环，可望为AS 防治提供新的思路，并有利于探索潜在的新的治疗途径。