Sun Yilei, Liu Dan, Liu Yanchen, Chi Lijun
Department of Neurology, The First Affiliated Hospital of Harbin Medical University, Harbin, China.
Mol Neurobiol. 2025 Jun 19. doi: 10.1007/s12035-025-05106-x.
Cerebral ischemia-reperfusion injury (CIRI) induces significant microglial inflammation. V-type immunoglobulin domain-containing suppressor of T cell activation (VISTA), a novel inhibitory immune checkpoint, participates in myeloid cell metabolism. This study aims to investigate the molecular mechanisms of VISTA's protective effects on CIRI by modulating microglial metabolism. In this study, differentially expressed genes (DEGs) were extracted from GSE77986 to identify hub gene VISTA. Transient middle cerebral artery occlusion (tMCAO) and oxygen-glucose deprivation and reoxygenation (OGD/R) were conducted to mimic CIRI. AAVMG1.2-VSIR was injected intracerebroventricularly into Cx3cr1 mice, while over-expression plasmids were transfected into BV2 to intervene VISTA. The mice underwent LONGA scoring, H&E, Nissl, and TTC staining. Western blot and qRT-PCR were conducted for VISTA, IL-6, TNFα, IL-1β, and IL-10. Microglial proliferation was assessed by Edu staining and CCK8. RNA-sequencing (RNA-seq) analysis was used to investigate downstream pathways. Tricarboxylic acid (TCA) cycle intermediates were measured using ELISA. ACOD1/IκBα/NF-κB pathway was validated by Western blot. Eight DE-ICGs were identified through differential analysis, with VSIR exhibiting the highest expression. Additionally, VISTA was found decreased in microglia around the infarction site. Compared with CIRI group, VISTA reduced the infarct volume, improved neurological deficit, and decreased IL-6, TNFα, and IL-1β, while increasing IL-10, and suppressing microglia proliferation. RNA-seq showed that the DEGs primarily participated in microglial glucose metabolism and the IκBa/NF-κB pathway. VISTA promoted ACOD1 expression and itaconate (ITA). The protective function on CIRI and inhibitory effect on IκBa/NF-κB of VISTA were abrogated by ACOD1 knockdown.
脑缺血再灌注损伤（CIRI）会引发显著的小胶质细胞炎症。含V型免疫球蛋白结构域的T细胞活化抑制因子（VISTA）是一种新型抑制性免疫检查点，参与髓系细胞代谢。本研究旨在探讨VISTA通过调节小胶质细胞代谢对CIRI产生保护作用的分子机制。在本研究中，从GSE77986中提取差异表达基因（DEG）以鉴定核心基因VISTA。进行短暂性大脑中动脉闭塞（tMCAO）以及氧糖剥夺和复氧（OGD/R）以模拟CIRI。将腺相关病毒载体MG1.2-VSIR脑室内注射到Cx3cr1小鼠体内，同时将过表达质粒转染到BV2中以干预VISTA。对小鼠进行LONGA评分、苏木精-伊红（H&E）染色、尼氏染色和TTC染色。对VISTA、白细胞介素-6（IL-6）、肿瘤坏死因子α（TNFα）、白细胞介素-1β（IL-1β）和白细胞介素-10（IL-10）进行蛋白质免疫印迹法（Western blot）和实时定量聚合酶链反应（qRT-PCR）检测。通过Edu染色和CCK8评估小胶质细胞增殖。采用RNA测序（RNA-seq）分析来研究下游通路。使用酶联免疫吸附测定（ELISA）法检测三羧酸（TCA）循环中间体。通过蛋白质免疫印迹法验证ACOD1/IκBα/核因子κB（NF-κB）通路。通过差异分析鉴定出8个差异表达的炎症相关基因（DE-ICG），其中VSIR表达最高。此外，发现梗死灶周围小胶质细胞中的VISTA表达降低。与CIRI组相比，VISTA减小了梗死体积，改善了神经功能缺损，降低了IL-6、TNFα和IL-1β水平，同时增加了IL-10水平，并抑制了小胶质细胞增殖。RNA-seq显示，DEG主要参与小胶质细胞葡萄糖代谢和IκBa/NF-κB通路。VISTA促进了ACOD1表达和衣康酸（ITA）生成。敲低ACOD1可消除VISTA对CIRI的保护作用及其对IκBa/NF-κB的抑制作用。
