RESUMO
Sepsis remains a significant health care burden, with high morbidities and mortalities. Patients with sepsis often require general anesthesia for procedures and imaging studies. Knowing that anesthetic drugs can pose immunomodulatory effects, it would be critical to understand the impact of anesthetics on sepsis pathophysiology. The volatile anesthetic sevoflurane is a common general anesthetic derived from ether as a prototype. Using a murine sepsis model induced by cecal ligation and puncture surgery, we examined the impact of sevoflurane on sepsis outcome. Different from volatile anesthetic isoflurane, sevoflurane exposure significantly improved the outcome of septic mice. This was associated with less apoptosis in the spleen. Because splenic apoptosis was largely attributed to the apoptosis of neutrophils, we examined the effect of sevoflurane on FasL-induced neutrophil apoptosis. Sevoflurane exposure significantly attenuated apoptosis. Sevoflurane did not affect the binding of FasL to the extracellular domain of Fas receptor. Instead, in silico analysis suggested that sevoflurane would bind to the interphase between Fas death domain (DD) and Fas-associated DD (FADD). The effect of sevoflurane on Fas DD-FADD interaction was examined using fluorescence resonance energy transfer (FRET). Sevoflurane attenuated FRET efficiency, indicating that sevoflurane hindered the interaction between Fas DD and FADD. The predicted sevoflurane binding site is known to play a significant role in Fas DD-FADD interaction, supporting our in vitro and in vivo apoptosis results.-Koutsogiannaki, S., Hou, L., Babazada, H., Okuno, T., Blazon-Brown, N., Soriano, S. G., Yokomizo, T., Yuki, K. The volatile anesthetic sevoflurane reduces neutrophil apoptosis via Fas death domain-Fas-associated death domain interaction.
Assuntos
Apoptose/efeitos dos fármacos , Proteína de Domínio de Morte Associada a Fas , Neutrófilos/metabolismo , Receptor fas , Animais , Sítios de Ligação , Proteína de Domínio de Morte Associada a Fas/química , Proteína de Domínio de Morte Associada a Fas/metabolismo , Camundongos , Neutrófilos/citologia , Sevoflurano/química , Sevoflurano/farmacologia , Receptor fas/química , Receptor fas/metabolismoRESUMO
BACKGROUND: Perioperative infections, particularly surgical site infections pose significant morbidity and mortality. Phagocytosis is a critical step for microbial eradication. We examined the effect of commonly used anesthetics on macrophage phagocytosis and its mechanism. METHODS: The effect of anesthetics (isoflurane, sevoflurane, propofol) on macrophage phagocytosis was tested using RAW264.7 mouse cells, mouse peritoneal macrophages, and THP-1 human cells. Either opsonized sheep erythrocytes or fluorescent labeled Escherichia coli were used as phagocytic objects. The activation of Rap1, a critical protein in phagocytosis was assessed using the active Rap1 pull-down and detection kit. To examine anesthetic binding site(s) on Rap1, photolabeling experiments were performed using azi-isoflurane and azi-sevoflurane. The alanine scanning mutagenesis of Rap1 was performed to assess the role of anesthetic binding site in Rap1 activation and phagocytosis. RESULTS: Macrophage phagocytosis was significantly attenuated by the exposure of isoflurane (50% reduction by 1% isoflurane) and sevoflurane (50% reduction by 1.5% sevoflurane), but not by propofol. Photolabeling experiments showed that sevoflurane directly bound to Rap1. Mutagenesis analysis demonstrated that the sevoflurane binding site affected Rap1 activation and macrophage phagocytosis. CONCLUSIONS: We showed that isoflurane and sevoflurane attenuated macrophage phagocytosis, but propofol did not. Our study showed for the first time that sevoflurane served as a novel small GTPase Rap1 inhibitor. The finding will further enrich our understanding of yet-to-be determined mechanism of volatile anesthetics and their off-target effects. The sevoflurane binding site was located outside the known Rap1 functional sites, indicating the discovery of a new functional site on Rap1 and this site would serve as a pocket for the development of novel Rap1 inhibitors.