Ethyl Pyruvate Reduces Systemic Leukocyte Activation via Caspase-1 and NF-κB After Blunt Chest Trauma and Haemorrhagic Shock

Abstract

Background: Blunt chest (thoracic) trauma (TxT) and haemorrhagic shock with subsequent resuscitation (H/R) induce strong systemic and local inflammatory response, which is closely associated with apoptotic cell loss and subsequently impaired organ function. The underlying mechanisms are not completely understood, therefore, the treatment of patients suffering from TxT+H/R is challenging. In our recent studies, we have demonstrated local anti-inflammatory effects of ethyl pyruvate (EtP) in lung and liver after TxT+H/R. Here, the therapeutic potential of a reperfusion regime with EtP on the early post-traumatic systemic inflammatory response and apoptotic changes after TxT followed by H/R were investigated. Methods: Female Lewis rats underwent TxT followed by haemorrhagic shock (60 min). Resuscitation was performed with own blood transfusion and either lactated Ringers solution (LR) or LR supplemented with EtP (50 mg/kg). Sham group underwent the surgical procedures. After 2 h blood as well as lung and liver tissues were obtained for analyses. Systemic activation of neutrophils (expression of CD11b and CD62L), leukocyte phagocytosis, apoptosis (caspase-3/7 activation), pyroptosis (caspase-1 activation) and NF-κB p65 activity were assessed. p < 0.05 was considered significant. Results: TxT+H/R-induced systemic activation of neutrophils (increased CD11b and reduced CD62L expression) was significantly reduced by EtP. Trauma-induced delayed neutrophil apoptosis was further reduced by EtP reperfusion but remained unaltered in monocytes. Reperfusion with EtP significantly increased the phagocytizing capacity of granulocytes. Trauma-induced inflammasome activation, which was observed in monocytes and not in neutrophils, was significantly reduced by EtP in both cell entities. NF-κB p65 activation, which was increased in neutrophils and monocytes was significantly decreased in monocytes. Conclusion: TxT+H/R-induced systemic activation of both neutrophils and monocytes concomitant with increased systemic inflammation was reduced by a reperfusion with EtP and was associated with a down-regulation of NF-κB p65 activation.

Keywords: NF-κB; ethyl pyruvate; inflammation; leukocytes; trauma.

Figure 1

Flow cytometric analysis of cellular adhesion molecule CD11b and CD62L on circulating neutrophils after blunt thoracic trauma (TxT) followed by haemorrhagic shock and resuscitation (H/R). Two hours after resuscitation flow cytometric analysis of CD11b or CD62L expression on circulating granulocytes (A-F) was performed. Sham operated animals underwent all surgical procedures without induction of TxT and H/R. TxT+H/R_LR animals received lactated Ringers solution (LR) and TxT+H/R_EtP animals were resuscitated with LR supplemented with ethyl pyruvate (EtP). (A) Gating strategy for CD11b positive granulocyte. (B) Gating strategy for CD62L positive granulocytes. (C) Percentage of CD11b⁺ cells out of all gran⁺ granulocytes. (D) Mean fluorescence units (MFU) of CD11b⁺ on gran⁺ granulocytes. (E) Percentage of CD62L⁺ cells out of all gran⁺ granulocytes. (F) MFU of CD62L⁺ on gran⁺ granulocytes. Data are given as mean ± standard error of the mean, *p < 0.05 vs. indicated, n = 10.

Figure 2

Flow cytometric analysis of granulocyte and monocyte phagocytosis after blunt thoracic trauma (TxT) followed by haemorrhagic shock and resuscitation (H/R). Two hours after resuscitation flow cytometric analysis of phagocytosis in circulating granulocytes and monocytes was performed. Sham operated animals underwent all surgical procedures without induction of TxT and H/R. TxT+H/R_LR animals received lactated Ringers solution (LR) and TxT+H/R_EtP animals were resuscitated with LR supplemented with ethyl pyruvate (EtP). (A) Percentage of phagocytizing granulocytes in absolute cell numbers. (B) Mean fluorescence units (MFU) of phagocytizing granulocytes. (C) Percentage of phagocytizing monocytes in absolute cell numbers. (D) MFU of phagocytizing monocytes. Data are given as mean ± standard error of the mean, *p < 0.05 vs. indicated, n = 10.

Figure 3

Caspase-3/7 activation after blunt thoracic trauma (TxT) followed by haemorrhagic shock and resuscitation (H/R) in granulocytes and monocytes. Two hours after resuscitation flow cytometric analysis of apoptosis (caspase-3/7 activation) in circulating granulocytes (A) and monocytes (B) was performed. Sham operated animals underwent all surgical procedures without induction of TxT and H/R. TxT+H/R_LR animals received lactated Ringers solution (LR) and TxT+H/R_EtP animals were resuscitated with LR supplemented with ethyl pyruvate (EtP). (A) Mean fluorescence units (MFU) of caspase-3/7 positive granulocytes and (B) monocytes. Data are given as mean ± standard error of the mean, *p < 0.05 vs. indicated, n = 10.

Figure 4

Caspase-1 activation after blunt thoracic trauma (TxT) followed by haemorrhagic shock and resuscitation (H/R) in granulocytes and monocytes. Two hours after resuscitation flow cytometric analysis of pyroptosis (caspase-1 activation) in circulating granulocytes (A) and monocytes (B) was performed. Sham operated animals underwent all surgical procedures without induction of TxT and H/R. TxT+H/R_LR animals received lactated Ringers solution (LR) and TxT+H/R_EtP animals were resuscitated with LR supplemented with ethyl pyruvate (EtP). (A) Mean fluorescence units (MFU) of caspase-1 positive granulocytes and (B) monocytes. Data are given as mean ± standard error of the mean, *p < 0.05 vs. indicated, n = 10.

Figure 5

Flow cytometric analysis of NF-κB p65 phosphorylation after blunt thoracic trauma (TxT) followed by haemorrhagic shock and resuscitation (H/R). Two hours after resuscitation flow cytometric analysis of phosphorylated NF-κB p65 in circulating granulocytes (A) and monocytes (B) was performed. Sham operated animals underwent all surgical procedures without induction of TxT and H/R. TxT+H/R_LR animals received lactated Ringers solution (LR) and TxT+H/R_EtP animals were resuscitated with LR supplemented with ethyl pyruvate (EtP). The ratio of phosphorylated to total NF-κB protein in granulocytes (A,B) monocytes is given. Data are given as mean ± standard error of the mean, *p < 0.05 vs. indicated, n = 10.