Gallic acid-L-leucine (GAL) conjugate protects mice against endotoxic shock via suppressing the production of pro-inflammatory lipid mediators and nitric oxide (NO) in macrophages

Abstract

Aberrant production of pro-inflammatory lipid mediators and nitric oxide (NO) is implicated in the pathology of LPS-induced inflammation and sepsis. We recently synthesized a novel gallic acid-L-leucine (GAL) conjugate and discovered that GAL conjugate could promote macrophage phagocytosis via inducing LTB4DH (Cheng YY et al Mol Immunol. 2016 May 2;74:39-46). The aim of present study was to discover the anti-inflammatory potential of GAL conjugate in the treatment of endotoxic shock. We focused on the effects of GAL conjugate on the generation of the key pro-inflammatory lipid mediators and NO. We not only examined whether GAL could antagonize the effects of liposaccharide (LPS) on the expression of cyclooxygenase-2 (COX-2), 5-lipoxygenase (5-LOX) and inducible NO synthase (iNOS) but also determined the products of these pro-inflammatory enzymes in RAW264.7 cells, primary peritoneal macrophages and mice. Based on Western blot analysis, GAL attenuated LPS-induced expression of COX-2, 5-LOX and iNOS in a concentration-dependent manner. Consistently, the results of ESI-MS based lipidomics profiling verified that GAL suppressed LPS-induced overproduction of prostaglandin E2, prostaglandin F2, leukotriene B4 and thromboxane B2. We further discovered that GAL exhibited anti-inflammatory activities by targeting the following mechanisms: 1) suppressing LPS-induced phosphorylation of MAP kinases such as p38, JNK and ERK1/2; 2) reducing the production of reactive oxygen species and NO; and 3) preventing LPS-induced nuclear translocation of transcription factors NF-κB and AP-1. On the other hand, GAL significantly decreased the plasma levels of pro-inflammatory lipid mediators and the enzyme levels of COX-2 and iNOS expression in LPS-treated mice. Importantly, GAL pretreatment enhanced the survival of mice against LPS-induced endotoxic shock. Collectively, these results suggest that GAL may be a potential anti-inflammatory drug for the treatment of endotoxemia and sepsis.