The macrophage DAG/PKCα/ROS axis exacerbates sepsis by inducing endothelial dysfunction through activation of the p38 MAPK pathway.

Abstract

BACKGROUND: This study aimed to investigate the role and mechanism of macrophage DAG/PKCα/ROS axis in sepsis‑induced endothelial injury. METHODS: In vitro, RAW264.7 macrophages were polarized to the M1 phenotype using lipopolysaccharide/interferon‑γ and treated with Go6976 (PKCα inhibitor). The following parameters were assessed: DAG and ROS levels, PKCα phosphorylation, M1/M2 phenotype markers and inflammatory cytokine levels. In a Transwell system, pulmonary microvascular endothelial cells were co-cultured with macrophages to evaluate endothelial cellular viability, apoptosis, inflammatory cytokine levels, barrier function, the expressions of tight junction proteins and adhesion junction proteins, and p38 MAPK phosphorylation. In vivo, mouse sepsis models were established by cecal ligation and puncture (CLP) surgery and treated with Go6976 and the p38 MAPK agonist, anisomycin. The 24 h survival rate was recorded, macrophage depletion was verified, inflammatory cytokine levels were measured, and histopathology of lung, liver and kidney was assessed. Additionally, peritoneal macrophages were isolated from mice and examined for DAG and ROS levels, inflammatory cytokine levels, M1/M2 phenotype markers, the expressions of tight junction proteins and adhesion junction proteins, and p38 MAPK phosphorylation. RESULTS: Lipopolysaccharide/interferon‑γ activated the macrophage DAG/PKCα/ROS axis, promoted M1 polarization and inflammation, which was reversed by Go6976. LPS impaired endothelial viability, apoptosis and barrier function, which were alleviated by Go6976. In vivo, macrophage depletion or transfer of Go6976‑pretreated macrophages improved survival, reduced inflammation/organ injury, and restored junctional proteins in CLP‑induced septic mice, these effects were abolished by anisomycin. CONCLUSIONS: The macrophage DAG/PKCα/ROS axis exacerbates sepsis by inducing endothelial dysfunction through activation of the p38 MAPK pathway.