膜筏介导同型半胱氨酸激活NOX-ROS-NLRP3炎症小体级联效应促进动脉粥样硬化的作用及机制

We recently demonstrated that homocysteine (Hcy) activated NLRP3 inflammasomes through reactive oxygen species (ROS)-dependent model, which played an important role in hyperhomocysteinemia (HHcy)-accelerated atherosclerosis (AS). The mechanism underlying Hcy-promoted ROS generation has not been elucidated. It was reported that Hcy can increase the activity of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX). ROS is mainly derived from NOX in vascular system (including endothelial cell, smooth muscle cell and recruited monocyte-macrophage). Membrane translocation of cytoplasmic subunits and interactions with membrane subunits trigger NOX activity. Membrane rafts are dynamic assemblies of proteins and lipids that harbour many receptors and regulatory molecules and so act as a platform for signal transduction. Ceramide-mediated membrane raft clustering may recruit NOX subunits and then form NOX functional complex. Our preliminary data suggested that inhibition of ceramide synthesis or disruption of membrane rafts can block Hcy-induced ROS generation and NLRP3 inflammasome activation, indicating that both ceramide and membrane rafts are involved in the regulation of Hcy-induced ROS generation and NLRP3 inflammasome activation. Thus, we hypothesize that membrane rafts mediates the cascade activation of NOX-ROS-NLRP3 inflammasome induced by Hcy and accelerates AS. .This project intends to combine animal and cell culture models, to further investigate whether membrane rafts mediates the formation of NOX functional complex induced by Hcy, and the causal relationship with the cascade activation of NOX-ROS-NLRP3 inflammasome in the development of HHcy-accelerated AS; whether ceramide acts as an important messenger of the membrane rafts NOX functional complex induced by Hcy, and what is the underlying mechanism of synthetic regulation of ceramide (hydrolytic synthesis pathway or de novo synthesis). Targeting ceramide and membrane rafts NOX-ROS-NLRP3 inflammasome cascade may be a novel strategy to prevent the development of AS.

我们发现同型半胱氨酸（Hcy）通过活性氧（ROS）模式激活NLRP3炎症小体促进动脉粥样硬化（AS），但其促进ROS生成的机制不清。文献报道Hcy可增强NADPH氧化酶（NOX）活性，而血管ROS主要来源于NOX，其激活需胞浆亚基膜转位与膜亚基结合。神经酰胺介导的膜筏簇聚，可能招募NOX各亚基形成功能复合体。预实验显示抑制神经酰胺合成、破坏膜筏均可阻断Hcy诱导ROS生成及NLRP3炎症小体激活，提示二者参与调控Hcy激活NLRP3炎症小体的作用。由此，本项目率先提出膜筏介导Hcy激活NOX-ROS-NLRP3炎症小体级联效应促进AS发生发展的假说，拟进一步探讨Hcy促进AS进程中：膜筏是否介导NOX功能复合体形成，以及与ROS-NLRP3炎症小体激活等级联效应的因果关系；神经酰胺是否为膜筏NOX功能复合体形成的信使，其合成调控的机制（水解途径？/从头合成？）。为探讨AS防治新策略提供依据

申请人在前期项目资助下，发现同型半胱氨酸（Hcy）通过活性氧（ROS）模式激活NLRP3炎症小体促进动脉粥样硬化（AS）发生发展，然而Hcy促进ROS生成的分子机制尚不清楚。本项目提出Hcy通过脂筏介导还原型辅酶Ⅱ氧化酶（NOX）功能复合体形成，激活NOX-ROS-NLRP3炎症小体及细胞焦亡等级联效应，促进AS发生发展的假说。我们通过喂饲高脂高蛋氨酸饲料构建高同型半胱氨酸血症（HHcy）促进AS的小鼠模型，观察AS斑块面积以及主动脉组织中NLRP3炎症小体激活、细胞焦亡及巨噬细胞浸润情况。并通过脂筏选择性抑制剂干预，评估脂筏在HHcy诱导的NLRP3炎症小体激活、细胞焦亡及AS发生的重要作用。同时在巨噬细胞模型上，观察脂筏簇聚在Hcy诱导NOX-ROS-NLRP3通路激活和细胞焦亡中的作用及机制。研究结果表明：①在HHcy诱导AS小鼠模型上，证实Hcy通过脂筏介导NOX-ROS-NLRP3通路激活，促进血管炎症、诱导巨噬细胞焦亡，促进AS发生发展；脂筏选择性抑制剂可以抑制Hcy诱导的NLRP3炎症小体激活和细胞焦亡，减轻AS。②证实神经酰胺是Hcy促进脂筏NOX功能复合体形成的重要信使，Hcy通过上调酸性鞘磷脂酶（ASMase）的表达促进神经酰胺合成，诱导脂筏簇聚，进而促进NOX膜亚基和胞浆亚基在脂筏区中形成NOX复合体，激活NOX酶活性，增加ROS生成，诱导NLRP3炎症小体激活以及细胞焦亡。.以上结果表明Hcy通过上调ASMase的表达促进神经酰胺合成，诱导脂筏簇聚，进而促进NOX膜亚基和胞浆亚基在脂筏区中形成NOX复合体，激活NOX酶活性，增加ROS生成，诱导NLRP3炎症小体激活以及细胞焦亡，进而促进AS发生发展；而干预脂筏簇聚，抑制NOX激活，可阻断Hcy诱导NLRP3炎症小体激活和细胞焦亡的作用，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。