Ninj1通过调控单核/巨噬细胞迁移和炎性活化促进腹主动脉瘤形成的实验研究

Abdominal aortic aneurysm (AAA) is a dangerous arterial dilatation disease that has a very high mortality rate. Macrophage (Mø) infiltration of arterial wall-mediated chronic inflammatory injury plays an important role in the course of AAA, but its mechanism has not been fully elucidated. Previous studies have confirmed that Ninj1 can regulate monocyte/macrophage (Mo/Mø) migration, and we have previously found that Ninj1 also promotes Mø inflammatory activation. We also found that Ninj1 expression was up-regulated in clinical or animal AAA tissues with pro-inflammatory Mø infiltration; whereas in Ninj1 knockout mice, Mø infiltration was significantly reduced in vascular wall and AAA formation rate was also significantly reduced. Thus, we hypothesize that Ninj1 can promote the migration of Mo/Mø to the arterial wall, then Ninj1 induces the inflammatory activation of Mø, and ultimately leads to the formation and progression of AAA. To test this hypothesis based on ApoE-/-Ninj1-/- mice, we intend to use siRNA and signaling pathway inhibitors, etc. to illustrate the regulatory role of Ninj1 and its downstream signaling in promoting Mo/Mø migration and inflammatory activation from molecular, cellular and animal levels, thus clarify the mechanism by which Ninj1 promotes AAA formation and provide new ideas and theoretical basis for AAA non-surgical intervention strategies.

腹主动脉瘤（AAA）是一种凶险的动脉扩张性疾病，一旦破裂死亡率极高。巨噬细胞（Mø）浸润动脉壁介导的慢性炎症损伤在AAA病程中发挥重要促进作用，但其机制尚未完全阐明。既往研究证实Ninj1可调控单核/巨噬细胞（Mo/Mø）迁移，而我们前期发现Ninj1还能促进Mø炎性活化。我们还发现，临床或动物AAA组织中Ninj1表达上调，伴随促炎型Mø浸润；而Ninj1敲基因小鼠建模后瘤壁中Mø浸润量明显减少，AAA形成率显著降低。由此假设：Ninj1可趋化Mo/Mø迁移至动脉管壁，进而诱导其向促炎型Mø活化，最终促进AAA的形成及进展。因此，本项目拟基于ApoE-/-Ninj1-/-小鼠，利用siRNA、信号通路抑制剂等从分子、细胞及动物水平验证Ninj1及下游信号在促进Mo/Mø迁移及炎性活化中的调控作用，阐明Ninj1促进AAA形成的机制，从而为AAA的非手术干预策略提供新思路和理论依据。

腹主动脉瘤（AAA）是一种凶险的动脉扩张性疾病，一旦破裂死亡率极高。巨噬细胞浸润动脉壁介导的慢性炎症损伤在AAA病程中发挥重要促进作用，但其机制尚未完全阐明。本研究旨在探明AAA中神经损伤诱导蛋白1（NINJ1）表达水平的变化，探索NINJ1调控巨噬细胞炎性活化及迁移的作用与具体分子机制。收集临床主动脉组织、外周血血清以及小鼠主动脉组织样本，通过免疫荧光、Western Blot、ELISA等方法分析AAA组织及血液中NINJ1表达水平变化。构建Ninj1基因条件性敲除小鼠，应用血管紧张素II诱导AAA形成，评估成瘤率及AAA严重程度，通过免疫荧光和免疫组化评估主动脉管壁病理学变化、巨噬细胞浸润及活化情况。构建Ninj1基因过表达及敲低巨噬细胞系，通过RT-qPCR、Western Blot、Transwell、双荧光素酶报告基因检测等实验分析NINJ1对巨噬细胞活化、粘附和迁移表型的影响和具体信号通路，明确其分子机制。AAA患者及小鼠主动脉组织及外周血血清中NINJ1表达水平显著上调，伴主动脉管壁巨噬细胞浸润显著增多，且NINJ1与巨噬细胞存在共定位。与野生型小鼠相比，髓系特异性Ninj1基因敲除小鼠AAA形成率、严重程度及死亡率显著下调，主动脉管腔扩张、管壁结构紊乱显著减轻，伴炎性巨噬细胞浸润显著减少。敲低Ninj1后，巨噬细胞促炎标记物基因表达下调，抗炎标记物基因表达上调，且细胞粘附、迁移能力减弱；而过表达Ninj1后，巨噬细胞粘附、迁移显著上调。Western Blot结果表明Ninj1可能通过激活TLR4/MyD88/NF-κB信号通路促进巨噬细胞黏附及迁移，双荧光素酶报告基因实验结果表明转录因子NF-κB直接调控CCR2基因表达。基因过表达与敲低实验证实CCR2能够促进巨噬细胞黏附及迁移。AAA使主动脉组织及血液中NINJ1水平显著升高。Ninj1基因条件性敲除小鼠AAA形成率及严重程度显著降低，伴巨噬细胞浸润和促炎性表型显著下调。NINJ1可能通过上调TLR4/MyD88/NF-κB/CCR2信号通路促进巨噬细胞黏附及迁移至主动脉管壁并介导炎症反应，从而促进AAA形成。本研究明确了NINJ1调控巨噬细胞炎性活化及迁移在AAA中的关键作用和调控机制，为AAA的精准治疗提供了新思路。