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Published on: February 9, 2014
The macrophage DAG/PKCα/ROS axis exacerbates sepsis by inducing endothelial dysfunction through activation of the p38
Wen Chen1, Xiangye Bo1, Qianqian Wang2
1Department of General Practice, Affiliated Hangzhou First People's Hospital, Westlake University School of Medicine, Hangzhou, China.
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.
Insights
The macrophage DAG/PKCα/ROS axis drives sepsis-induced endothelial injury by activating p38 MAPK. Inhibiting this axis improves survival and reduces organ damage in sepsis models.
Area of Science:
- Immunology
- Cell Biology
- Pathophysiology
Background:
- Sepsis can cause endothelial injury, a critical factor in disease severity.
- Macrophage activation plays a key role in sepsis pathogenesis.
- The specific mechanisms linking macrophages to endothelial dysfunction in sepsis require further elucidation.
Purpose of the Study:
- To investigate the role of the macrophage diacylglycerol (DAG)/protein kinase C-alpha (PKCα)/reactive oxygen species (ROS) axis in sepsis-induced endothelial injury.
- To elucidate the underlying molecular mechanisms, including the involvement of the p38 mitogen-activated protein kinase (MAPK) pathway.
Main Methods:
- In vitro studies using RAW264.7 macrophages and pulmonary microvascular endothelial cells co-cultured in a Transwell system.
- In vivo studies utilizing cecal ligation and puncture (CLP) mouse models of sepsis.
- Pharmacological inhibition of PKCα using Go6976 and activation of p38 MAPK using anisomycin.
Main Results:
- Activation of the macrophage DAG/PKCα/ROS axis was linked to M1 polarization and inflammation, which was mitigated by Go6976.
- Go6976 treatment improved endothelial cell viability, reduced apoptosis, and restored barrier function in vitro.
- In vivo, Go6976 administration or macrophage depletion improved survival rates and reduced organ damage in septic mice, effects reversed by anisomycin.
Conclusions:
- The macrophage DAG/PKCα/ROS axis is a significant contributor to sepsis-induced endothelial dysfunction.
- This axis exacerbates sepsis by activating the p38 MAPK pathway, leading to increased inflammation and organ injury.
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