Gastrin-releasing peptide receptor antagonist induces a protection from lethal sepsis: involvement of toll-like receptor 4 signaling

Introduction

In lethal polymicrobial sepsis, toll-like receptor 4 (TLR-4) mediates a critical role in impeding the migration of neutrophils to infectious foci, thereby favoring increased bacteremia and ultimately leading to mortality. We have previously shown that the selective gastrin-releasing peptide receptor antagonist RC-3095 can reduce organ dysfunction in experimental sepsis. Thus the aim of the present study is to report a novel link between GRPR and TLR-4 signaling and its relationship with inflammatory parameters in in vitro and in vivo experimental models as well as in sepsis patients.

Methods

For the in vitro experiment, RAW 264.7 macrophages were stimulated with LPS and treated with RC-3095 for RT-PCR analyses of TLR-4 mRNA, immunoblotting of pERK1/2, pJNK, pAkt, and EMSA of NF-κB and activator protein 1 (AP-1). In the in vivo studies, male Wistar rats were divided and submitted, into sham surgery, cecal ligation and puncture (CLP) surgery, and CLP plus RC-3095. Six hours after, all rats were anesthetized and sacrificed by cardiac puncture. Blood was collected for bacterial count and cytokine analyses; bronchoalveolar lavage fluid for cell count, levels of TLR-4 and cytokines; peritoneal lavages for bacterial count; and lung tissue for levels of TLR-4 and RT-PCR analyses of TLR-4 mRNA. In a human study 12 patients, admitted to an adult medical ICU with a clinical diagnosis of septic shock, received a continuous infusion with RC-3095 over a period of 12 hours and concentrations of IL-6 and IL-10 in plasma were determined. Results are expressed as means ± SD. Differences between groups were determined by ANOVA, followed by Tukey's post hoc test. Differences between two groups were determined by t test.

Results

RC-3095 inhibited expression of TLR-4 and reduced phosphorylation of extracellular signal-regulated kinase (ERK-1/2), c-Jun NH2-terminal kinase (JNK), and Akt, leading to decreased activation of NF-κB and AP-1 in macrophages. In a rat model of sepsis, RC-3095 treatment decreased lung TLR-4 content, reduced the migration of inflammatory cells to the lung, reduced systemic cytokine levels, and attenuated bacterial dissemination. Continuous infusion with RC-3095 for 12 hours decreased IL-6 plasma levels in septic patients, but did not significantly affect IL-10 plasma levels.

Conclusions

These findings demonstrate the beneficial action of GRPR antagonists in controlling the inflammatory response in sepsis through a mechanism involving inhibition of TLR-4 signaling.