RESUMO
BACKGROUND: Sepsis is a systemic inflammatory response which is frequently associated with acute lung injury (ALI). Activating transcription factor 3 (ATF3) promotes M2 polarization, however, the biological effects of ATF3 on macrophage polarization in sepsis remain undefined. METHODS: LPS-stimulated macrophages and a mouse model of cecal ligation and puncture (CLP)-induced sepsis were generated as in vitro and in vivo models, respectively. qRT-PCR and western blot were used to detect the expression of ATF3, ILF3, NEAT1 and other markers. The phenotypes of macrophages were monitored by flow cytometry, and cytokine secretion was measured by ELISA assay. The association between ILF3 and NEAT1 was validated by RIP and RNA pull-down assays. RNA stability assay was employed to assess NEAT1 stability. Bioinformatic analysis, luciferase reporter and ChIP assays were used to study the interaction between ATF3 and ILF3 promoter. Histological changes of lung tissues were assessed by H&E and IHC analysis. Apoptosis in lungs was monitored by TUNEL assay. RESULTS: ATF3 was downregulated, but ILF3 and NEAT1 were upregulated in PBMCs of septic patients, as well as in LPS-stimulated RAW264.7 cells. Overexpression of ATF3 or silencing of ILF3 promoted M2 polarization of RAW264.7 cells via regulating NEAT1. Mechanistically, ILF3 was required for the stabilization of NEAT1 through direct interaction, and ATF3 was a transcriptional repressor of ILF3. ATF3 facilitated M2 polarization in LPS-stimulated macrophages and CLP-induced septic lung injury via ILF3/NEAT1 axis. CONCLUSION: ATF3 triggers M2 macrophage polarization to protect against the inflammatory injury of sepsis through ILF3/NEAT1 axis.
Assuntos
Fator 3 Ativador da Transcrição , Macrófagos , RNA Longo não Codificante , Sepse , Animais , Humanos , Camundongos , Fator 3 Ativador da Transcrição/genética , Fator 3 Ativador da Transcrição/metabolismo , Lipopolissacarídeos , Macrófagos/metabolismo , Proteínas do Fator Nuclear 90/genética , Proteínas do Fator Nuclear 90/metabolismo , Células RAW 264.7 , Sepse/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismoRESUMO
BACKGROUND: Patients with sepsis resulting in acute lung injury (ALI) usually have increased mortality. Ferroptosis is a vital regulator in sepsis-induced ALI. Exploring the association of ferroptosis and sepsis-induced ALI is crucial for the management of sepsis-induced ALI. METHODS: Whole blood was collected from sepsis patients. Mice were treated with cecal ligation and puncture (CLP) to model sepsis. Primary murine pulmonary microvascular endothelial cells were treated with lipopolysaccharide as a cell model. Ferroptosis was evaluated by analyzing levels of iron, malonaldehyde, glutathione, nonheme iron, ferroportin, ferritin, and GPX4. Hematoxylin and eosin and Masson's trichrome staining were applied to examine lung injury and collagen deposition. Cell apoptosis was analyzed by caspase-3 activity and TUNEL assays. Gene regulatory relationship was verified using RNA pull-down and immunoprecipitation assays. RESULTS: CircEXOC5 was highly expressed in sepsis patients and CLP-treated mice, in which knockdown alleviated CLP-induced pulmonary inflammation and injury, and ferroptosis. CircEXOC5 recruited IGF2BP2 to degrade ATF3 mRNA. The demethylase ALKBH5 was responsible for circEXOC5 upregulation through demethylation. CircEXOC5 silencing significantly improved sepsis-induced ALI and survival rate of mice by downregulating ATF3. CONCLUSIONS: ALKBH5-mediated upregulation of circEXOC5 exacerbates sepsis-induced ALI by facilitating ferroptosis through IGF2BP2 recruitment to degrade ATF3 mRNA.
