Insight into the beneficial immunomodulatory mechanism of the sevoflurane metabolite hexafluoro-2-propanol in a rat model of endotoxaemia

Abstract

Volatile anaesthetics such as sevoflurane attenuate inflammatory processes, thereby impacting patient outcome significantly. Their inhalative administration is, however, strictly limited to controlled environments such as operating theatres, and thus an intravenously injectable immunomodulatory drug would offer distinct advantages. As protective effects of volatile anaesthetics have been associated with the presence of trifluorinated carbon groups in their basic structure, in this study we investigated the water-soluble sevoflurane metabolite hexafluoro-2-propanol (HFIP) as a potential immunomodulatory drug in a rat model of endotoxic shock. Male Wistar rats were subjected to intravenous lipopolysaccharide (LPS) and thereafter were treated with HFIP. Plasma and tissue inflammatory mediators, neutrophil invasion, tissue damage and haemodynamic stability were the dedicated end-points. In an endotoxin-induced endothelial cell injury model, underlying mechanisms were elucidated using gene expression and gene reporter analyses. HFIP reduced the systemic inflammatory response significantly and decreased endotoxin-induced tissue damage. Additionally, the LPS-provoked drop in blood pressure of animals was resolved by HFIP treatment. Pathway analysis revealed that the observed attenuation of the inflammatory process was associated with reduced nuclear factor kappa B (NF-κΒ) activation and suppression of its dependent transcripts. Taken together, intravenous administration of HFIP exerts promising immunomodulatory effects in endotoxaemic rats. The possibility of intravenous administration would overcome limitations of volatile anaesthetics, and thus HFIP might therefore represent an interesting future drug candidate for states of severe inflammation.

Keywords: anaesthesia; fluorinated carbon groups; inflammation; interleukin; sepsis.

Fig. 1

Illustration of experimental setting: after a single injection of lipopolysaccharide (LPS), male Wistar rats were treated either with hexafluoro-2-propanol (HFIP) or sevoflurane. An analysis of inflammatory mediator mRNA and tissue damage markers was performed (kidney, lung, liver, and spleen tissue) 6 h after LPS injection (a). In human lung microvascular endothelial cells, gene expression and pathway analysis was performed after LPS and HFIP exposure (b). MCP-1 = monocyte chemoattractant protein-1; IL-6 = interleukin-6; CINC-1 = cytokine-induced neutrophil chemoattractant protein-1; BALF = bronchoalveolar lavage fluid; AST = aspartate transaminase; BUN = blood urea nitrogen.

Fig. 2

Hexafluoro-2-propanol (HFIP) and sevoflurane treatment attenuates plasma inflammatory mediator secretion in lipopolysaccharide (LPS)-stimulated rats. Monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6) and cytokine-induced neutrophil chemoattractant-1 (CINC-1) protein concentrations were determined hourly (0–6 h, T0–T6) in plasma of LPS-treated rats exposed simultaneously to either hexafluoro-2-propanol (HFIP) or sevoflurane (a,b,c). Values represent mean ± standard deviation (s.d.).

Fig. 3

Monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6) mRNA expression and the amount of neutrophils were determined in kidney and liver tissue of lipopolysaccharide (LPS)-treated rats exposed simultaneously to either hexafluoro-2-propanol (HFIP) or sevoflurane. LPS-induced tissue inflammatory mediator expression and subsequent invasion of effector cells into kidneys and liver was reduced by both HFIP and sevoflurane. Whiskers represent 5% and 95% confidence intervals; *represent 1% and 99% confidence intervals. Sevo = sevoflurane.

Fig. 4

Hexafluoro-2-propanol (HFIP) induces a stabilization of mean arterial pressure (MAP) in endotoxaemic animals. Using invasive blood pressure monitoring, we assessed mean arterial blood pressure every 10 min after injection of lipopolysaccharides (LPS) in male Wistar rats. Hexafluoro-2-propanol (HFIP) or sevoflurane was administered over 30 min (indicated by the blue bar). MAP of control and HFIP-treated animals is shown in Supporting information, Fig. S1.T0–T6 = time-point 0–6 h. Values are mean ± standard deviation (s.d.).

Fig. 5

Hexafluoro-2-propanol (HFIP) reduces lipopolysaccharide-induced nuclear factor kappa B (NF-κB) activity in endothelial cells (HMVEC). Heat map illustrating the effect of lipopolysaccharide (LPS) stimulation on HMVEC mRNA expression in absence or presence of hexafluoro-2-propanol (HFIP) (a). Venn diagrams representing the number of transcripts regulated upon LPS-stimulation and those affected by HFIP (threshold = 1, P < 0·01) (b,c). Summary of significantly different transcripts within the Toll-like receptor 4 (TLR-4)-downstream network (gene symbol and probe name) in LPS-stimulated cells which underwent a treatment with HFIP (d). Gene activity of NF-κB and activator protein 1 (AP-1) in HMVEC measured by firefly luciferase assay (e,f). Whiskers represent 5% and 95% confidence intervals; *represent 1% and 99% confidence intervals. PBS = phosphate-buffered saline.

Fig. 6

Schematic illustration of the Toll-like receptor 4 (TLR-4)-downstream/nuclear factor kappa B (NF-κB) upstream network. Lipopolysaccharide (LPS)-induced inflammatory cytokine release is mediated by the TLR-4 pathway involving activation of interleukin-1 receptor-associated kinase 1/2 (IRAK 1/2), tumour necrosis factor receptor-associated factor 6 (TRAF-6), NF-κB-inducing kinase (NIK, MAP3K14), I kappa B kinases (IKK) and subsequent up-regulation of NF-κB. Tumour necrosis factor (TNF)-α-related cytokine release is mediated through TNF receptor-associated factor 2 (TRAF2), NIK/MAP3K14 and IKK activating NF-κB. Phorbol-12-myristate-13-acetate (PMA) directly provokes cytokine secretion by activation of protein kinase C (PKC) through IKK and subsequent NF-κB activation.

Fig. 7

Attenuation of inflammatory mediators by hexafluoro-2-propanol (HFIP) is mediated at the level of IKK or its downstream effectors. Lipopolysaccharide (LPS)-induced (a,b) and Toll-like receptor 4 (TLR-4)-mediated (c,d) stimulation of interleukin (IL)−6 protein and cytokine-induced neutrophil chemoattractant protein-1 (CINC-1) secretion in human microvascular endothelial cells (HMVEC). Exposure of HMVEC to phorbol-12-myristate-13-acetate (PMA) (e,f). MyD88 = myeloid differentiation primary response 88; TRADD = tumour necrosis factor receptor-1-associated protein.