Ding Yuanyuan, Sun Yu, Wang Hongyan, Zhao Hongqin, Yin Ruihua, Zhang Meng, Pan Xudong, Zhu Xiaoyan
Department of Neurology, The Affiliated Hospital of Qingdao University, Qingdao, Shandong Province, China.
Qingdao Cadre Health Care Service Center, The Affiliated Hospital of Qingdao University, Qingdao, Shandong Province, China.
Neural Regen Res. 2024 Nov 1;19(11):2488-2498. doi: 10.4103/NRR.NRR-D-23-01355. Epub 2024 Mar 8.
JOURNAL/nrgr/04.03/01300535-202419110-00029/figure1/v/2024-03-08T184507Z/r/image-tiff Our previous study has demonstrated that lnc_000048 is upregulated in large-artery atherosclerotic stroke and promotes atherosclerosis in ApoE-/- mice. However, little is known about the role of lnc_000048 in classically activated macrophage (M1) polarization. In this study, we established THP-1-derived testing state macrophages (M0), M1 macrophages, and alternately activated macrophages (M2). Real-time fluorescence quantitative PCR was used to verify the expression of marker genes and the expression of lnc_000048 in macrophages. Flow cytometry was used to detect phenotypic proteins (CD11b, CD38, CD80). We generated cell lines with lentivirus-mediated upregulation or downregulation of lnc_000048. Flow cytometry, western blot, and real-time fluorescence quantitative PCR results showed that down-regulation of lnc_000048 reduced M1 macrophage polarization and the inflammation response, while over-expression of lnc_000048 led to the opposite effect. Western blot results indicated that lnc_000048 enhanced the activation of the STAT1 pathway and mediated the M1 macrophage polarization. Moreover, catRAPID prediction, RNA-pull down, and mass spectrometry were used to identify and screen the protein kinase RNA-activated (PKR), then catRAPID and RPIseq were used to predict the binding ability of lnc_000048 to PKR. Immunofluorescence (IF)-RNA fluorescence in situ hybridization (FISH) double labeling was performed to verify the subcellular colocalization of lnc_000048 and PKR in the cytoplasm of M1 macrophage. We speculate that lnc_000048 may form stem-loop structure-specific binding and activate PKR by inducing its phosphorylation, leading to activation of STAT1 phosphorylation and thereby enhancing STAT1 pathway-mediated polarization of THP-1 macrophages to M1 and inflammatory factor expression. Taken together, these results reveal that the lnc_000048/PKR/STAT1 axis plays a crucial role in the polarization of M1 macrophages and may be a novel therapeutic target for atherosclerosis alleviation in stroke.
《期刊》/nrgr/04.03/01300535 - 202419110 - 00029/图1/v/2024 - 03 - 08T184507Z/图像 - tiff 我们之前的研究表明，lnc_000048在大动脉粥样硬化性卒中中上调，并在载脂蛋白E基因敲除（ApoE-/-）小鼠中促进动脉粥样硬化。然而，关于lnc_000048在经典活化巨噬细胞（M1）极化中的作用知之甚少。在本研究中，我们建立了THP - 来源的测试状态巨噬细胞（M0）、M1巨噬细胞和交替活化巨噬细胞（M2）。采用实时荧光定量PCR验证巨噬细胞中标记基因的表达以及lnc_000048的表达。使用流式细胞术检测表型蛋白（CD11b、CD38、CD80）。我们构建了慢病毒介导的lnc_000048上调或下调的细胞系。流式细胞术、蛋白质免疫印迹法和实时荧光定量PCR结果表明，lnc_000048的下调减少了M1巨噬细胞极化和炎症反应，而lnc_000048的过表达则产生相反的效果。蛋白质免疫印迹结果表明，lnc_000048增强了信号转导和转录激活因子1（STAT1）通路的激活，并介导了M1巨噬细胞极化。此外，使用catRAPID预测、RNA下拉和质谱法鉴定并筛选蛋白激酶RNA激活剂（PKR），然后使用catRAPID和RPIseq预测lnc_000048与PKR的结合能力。进行免疫荧光（IF） - RNA荧光原位杂交（FISH）双重标记以验证lnc_000048和PKR在M1巨噬细胞胞质中的亚细胞共定位。我们推测lnc_000048可能形成茎环结构特异性结合并通过诱导其磷酸化激活PKR，导致STAT1磷酸化激活，从而增强STAT1通路介导的THP - 1巨噬细胞向M1极化和炎症因子表达。综上所述，这些结果揭示lnc_000048/PKR/STAT1轴在M1巨噬细胞极化中起关键作用，可能是缓解卒中后动脉粥样硬化的新治疗靶点。
