Ninj1通过TLR4/NF-κB/STAT1/3通路调控巨噬细胞极化促进糖尿病AS斑块形成的作用机制研究

Diabetes accelerates the formation of atherosclerotic plaque, in which macrophage (Mø) sub-phenotypes play an important regulatory role. Ninj1, a membrane protein that is significantly upregulated in high glucose environment, has not been clarified whether it promotes the formation of diabetic atherosclerotic plaques by regulating Mø phenotypical polarization. Our previous study found that: (1) Ninj1 overexpression in diabetic AS plaques was associated with notable infiltration of Mø and increased amount of pro-inflammatory M1 Mø; (2) Overexpression of NINJ1 gene in vitro promoted Mø polarization to M1, silencing of NINJ1 inhibited high glucose-induced M1 polarization, and STAT1/3 activity was also regulated. Accordingly, we hypotheized that Ninj1 promotes the formation of diabetic atherosclerotic plaques by regulating the polarization of Mø to M1 and aggravating the inflammatory response. This project intends to further investigate the effect of Ninj1 on the migration, recruitment and polarization of Mø by using cells and Mø NINJ1 conditional knockout ApoE-/- mice at the level of molecular, protein,cellular and tissue, revealing the regulatory role of TLR4/NF-κB/STAT1/3 signaling pathway and elucidating the mechanism of Ninj1 in regulating the formation of diabetic atherosclerotic plaque and finally provide a new idea for the prevention and treatment of diabetes-accelerated atherosclerosis.

糖尿病加快动脉粥样硬化（AS）斑块的形成，巨噬细胞（Mø）亚型在其中起重要调节作用。神经损伤诱导蛋白（Ninj1）是一种高糖环境下显著上调的促炎膜蛋白，其是否通过调控Mø极化促进糖尿病AS斑块的形成尚未明确。我们前期发现：①糖尿病患者AS斑块内Ninj1高表达伴随Mø大量浸润，且促炎的M1Mø数量显著增多；②体外过表达NINJ1促进Mø向M1极化;沉默NINJ1可抑制高糖诱导的M1极化，且STAT1/3活性受其调节。据此提出假说：Ninj1通过调控Mø向M1极化，加重炎症反应，促进糖尿病AS斑块的形成。本项目拟在前期基础上，应用Mø条件性敲除NINJ1的ApoE小鼠，从分子、蛋白、细胞及组织水平进一步研究Ninj1对Mø迁移、吞噬和极化的影响，揭示TLR4/NF-κB/STAT1/3通路在其中的调控作用，阐明Ninj1在糖尿病AS斑块形成中的作用机制，为糖尿病AS的防治提供新思路。

糖尿病加快动脉粥样硬化（AS）斑块的形成，巨噬细胞（Mø）亚型在其中起重要调节作用。神经损伤诱导蛋白（Ninj1）是一种高糖环境下显著上调的促炎膜蛋白，与巨噬细胞功能和心血管事件关系紧密。 我们统计分析了Ninj1表达与糖尿病动脉粥样硬化疾病的相关性。利用条件性敲基因小鼠 NINJ1Mø-CKO ApoE-/-条件性敲基因小鼠及NINJ1flox ApoE-/-对照小鼠的培养规模，获取敲基因小鼠的骨髓来源巨噬细胞，同过有参转录组测序技术分析两者巨噬细胞基因表达变化，并通过Western diet构建条件性敲基因小鼠 NINJ1Mø-CKO ApoE-/-条件性敲基因小鼠及NINJ1flox ApoE-/-对照小鼠糖尿病动脉粥样硬化模型，组织学染色分析动脉斑块形成比例、斑块大小、脂质核心区范围、钙化情况、血管内皮完整性、巨噬细胞浸润量、以及炎症蛋白表达情况等，检测糖尿病动脉粥样硬化斑块内巨噬细胞活化及炎症情况。构建Raw264.7-NINJ1-kd细胞系，检测Raw264.7和Raw264.7-NINJ1-kd在低糖和高糖环境下迁移能力和炎症反应的水平，最终确认Ninj1水平对巨噬细胞迁移和高糖下炎症响应能力的调节作用，并证明此作用可最终引起效应细胞的功能改变。