Abstract

Danshensu (DSS) is a water-soluble phenolic compound in Danshen (Salvia Miltiorrhiza Radix et Rhizoma). Although various pharmacological activities have been recognized, little is known regarding its anti-inflammatory effect and related molecular mode of action. In the current study, bone marrow-derived macrophages (BMMs) were activated by a Toll-like receptor 2 (TLR2) agonist Pam3CSK4 with or without DSS intervention. Production of pro-inflammatory cytokines interleukin-6 (IL-6) and interleukin-12 (IL-12) was detected by both enzyme-linked immunosorbent assay (ELISA) and real-time quantitative PCR (RT-qPCR). Activation of signaling pathways involving nuclear factor kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) was assessed by Western blot. Additionally, RNA sequencing (RNA-seq) combined with bioinformatics analyses was applied to investigate the molecular mechanisms of DSS. Emphasis was placed on the construction of the protein–protein interaction (PPI) network and transcription factor (TF) enrichment analysis of data including co-regulated differentially expressed genes (DEGs) in the Pam3CSK4 vs. control and DSS vs. Pam3CSK4 groups. The RT-qPCR and ELISA results showed that DSS effectively inhibited the expressions of IL-6 and IL-12, indicating a significant anti-inflammatory effect. Western blot verified that DSS suppressed the phosphorylation of p65, which was in accordance with the results of the TF enrichment analysis. Additionally, the PPI network analysis showed several key molecules, including lactoferrin (Ltf), CC-chemokine receptor 7 (Ccr7), interferon-gamma (IFN-γ) and C-X-C motif chemokine ligand 9 (Cxcl9), to be regulatory genes that responded to DSS treatment. Overall, our study revealed that DSS has a pronounced anti-inflammatory effect involving TLR2 and macrophages through the NF-κB signaling pathway, which supports the novel application of DSS in the treatment of relevant diseases including atherosclerosis and ischemic or is