C/EBP? Promotes LPS-Induced IL-1? Transcription and Secretion in Alveolar Macrophages via NOD2 Signaling

Objective: C/EBP beta, a crucial transcription factor, regulates innate immunity and inflammatory responses. However, the role played by C/EBP beta in alveolar macrophage (AM) inflammatory responses remains unknown. This study aimed to investigate the role and mechanism of C/EBP beta in alveolar macrophages (AMs) from the transcriptional level and to search for natural compounds targeting C/ EBP beta.Methods: Rat AMs were infected with Lv-sh-C/EBP beta and treated with LPS, and the expression levels of iNOS, TNF-alpha, IL-6, and IL-1 beta were measured by RT-qPCR, Western blotting, and ELISA. Mechanistically, transcriptome sequencing (RNA-seq) revealed changes in gene expression patterns in AMs after LPS stimulation and C/EBP beta knockdown. Functional enrichment analyses and rescue experiments identified and validated inflammation-associated cell signaling pathways regulated by C/EBP beta. Furthermore, virtual screening was used to search for natural compounds that inhibit C/EBP beta with the structure of helenalin as a reference.Results: Following stimulation with LPS, AMs exhibited an increased expression of C/EBP beta. C/EBP beta knockdown significantly decreased the expression levels of inflammatory mediators. A total of 374 differentially expressed genes (DEGs) were identified between LPS-stimulated C/EBP beta knockdown and negative control cells. The NOD-like receptor signaling may be a key target for C/ EBP beta, according to functional enrichment analyses of the DEGs. Further experiments showed that the muramyl dipeptide (MDP, NOD2 agonist) reversed the downregulation of inflammatory mediators and the NF-Kappa B pathway caused by the C/EBP beta knockdown. The virtual screening revealed that N-caffeoyltryptophan, orilotimod, and petasiphenone have comparable pharmacological properties to helenalin (a known C/EBP beta inhibitor) and demonstrate a great binding capacity to C/EBP beta.Conclusion: Ablation of C/EBP beta may attenuate LPS-induced inflammatory damage in AMs by inhibiting the NOD2 receptor signaling pathway. Three natural compounds, N-caffeoyltryptophan, orilotimod, and petasiphenone, may be potential C/EBP beta inhibitors.