Journal
PLATELETS
Volume 30, Issue 8, Pages 1047-1052Publisher
TAYLOR & FRANCIS INC
DOI: 10.1080/09537104.2019.1588241
Keywords
Apocynin; infliximab; NADPH oxidase; R-7050; sepsis
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Funding
- Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)
- Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES)
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Increased reactive oxygen species (ROS) production leads to tissue damage observed in sepsis and lipopolysaccharide (LPS)-exposed animals. LPS stimulates cytokines releasing, including tumor necrosis factor alpha (TNF-alpha), that is important to ROS production. Platelets, considered inflammatory cells, generate ROS when exposed to LPS in vivo, but not when they are incubated in vitro with this compound. Therefore, we investigated the role of TNF-alpha on the increased intraplatelet ROS levels after LPS treatment. Mice were injected with LPS (1 mg/kg) or TNF-alpha (10 ng/kg), and blood was collected to prepare the washed platelets. Animals were treated with infliximab (anti-TNF-alpha antibody), R-7050 (non-selective TNF-alpha receptor antagonist) or apocynin (NADPH oxidase inhibitor). At 48 h after LPS or TNF-alpha injection, the ROS levels in ADP (25 mu M)-activated platelets were evaluated by flow cytometry. Our data showed that injection of mice with LPS increased by 4-fold the ROS production (p < 0.05), which was significantly reduced by the treatments with infliximab, R-7050 or apocynin. Injection of mice with TNF-alpha markedly elevated the ROS formation in platelets (p < 0.05) that was reduced by infliximab, R-7050 or apocynin treatments. In separate experiments, platelets from saline-injected mice were incubated with TNF-alpha (30 to 3000 pg/mL) in absence or presence of infliximab, R-7050, apocynin or GKT137831 (NOX1/NOX4 inhibitor) before ROS measurements. TNF-alpha in vitro markedly increased the ROS levels, an effect significantly reduced by all treatments. Therefore, platelets are involved in the oxidative stress induced by LPS through TNF-alpha action, and NADPH oxidase takes part in this effect.
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