{"title":"动脉粥样硬化相关lnc_000048激活PKR,增强STAT1介导的THP-1巨噬细胞极化至M1表型。","authors":"Yuanyuan Ding, Yu Sun, Hongyan Wang, Hongqin Zhao, Ruihua Yin, Meng Zhang, Xudong Pan, Xiaoyan Zhu","doi":"10.4103/NRR.NRR-D-23-01355","DOIUrl":null,"url":null,"abstract":"<p><p>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.</p>","PeriodicalId":19113,"journal":{"name":"Neural Regeneration Research","volume":null,"pages":null},"PeriodicalIF":5.9000,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11090429/pdf/","citationCount":"0","resultStr":"{\"title\":\"Atherosis-associated lnc_000048 activates PKR to enhance STAT1-mediated polarization of THP-1 macrophages to M1 phenotype.\",\"authors\":\"Yuanyuan Ding, Yu Sun, Hongyan Wang, Hongqin Zhao, Ruihua Yin, Meng Zhang, Xudong Pan, Xiaoyan Zhu\",\"doi\":\"10.4103/NRR.NRR-D-23-01355\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>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.</p>\",\"PeriodicalId\":19113,\"journal\":{\"name\":\"Neural Regeneration Research\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":5.9000,\"publicationDate\":\"2024-11-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11090429/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Neural Regeneration Research\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://doi.org/10.4103/NRR.NRR-D-23-01355\",\"RegionNum\":2,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2024/3/8 0:00:00\",\"PubModel\":\"Epub\",\"JCR\":\"Q2\",\"JCRName\":\"CELL BIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Neural Regeneration Research","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.4103/NRR.NRR-D-23-01355","RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2024/3/8 0:00:00","PubModel":"Epub","JCR":"Q2","JCRName":"CELL BIOLOGY","Score":null,"Total":0}
Atherosis-associated lnc_000048 activates PKR to enhance STAT1-mediated polarization of THP-1 macrophages to M1 phenotype.
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.
期刊介绍:
Neural Regeneration Research (NRR) is the Open Access journal specializing in neural regeneration and indexed by SCI-E and PubMed. The journal is committed to publishing articles on basic pathobiology of injury, repair and protection to the nervous system, while considering preclinical and clinical trials targeted at improving traumatically injuried patients and patients with neurodegenerative diseases.