Metabolomic analysis of spontaneous neutrophil apoptosis reveals the potential involvement of glutathione depletion.

Spontaneous apoptosis of neutrophils plays a key role in maintaining immune homeostasis and resolving inflammation. However, the mechanism triggering this apoptosis remains obscure. In the present study, we performed a global metabolomics analysis of neutrophils undergoing spontaneous apoptosis by using hydrophilic interaction chromatography ultra-high-performance liquid chromatography-tandem quadrupole/time-of-flight mass spectrometry and found 23 metabolites and 42 related pathways that were altered in these cells. Among them, glutathione, which is known to be involved in apoptosis, was particularly interesting. We found that L-pyroglutamic acid, glutamate, and their glutathione-mediated embolic pathways were all changed. Our findings confirmed the glutathione levels decreased in apoptotic neutrophils. Exogenous glutathione and LPS treatment delayed neutrophil apoptosis and decreased the levels of pro-apoptotic protein caspase-3. γ-glutamylcyclotransferase, 5-oxoprolinase, and ChaC1, which participated in glutathione degradation, were all activated. At the same time, the down-regulation of ATP production suggested the activity of glutathione biosynthesis may be attenuated even if glutamate-cysteine ligase and glutathione synthase, which are two ATP-dependent enzymes participating in glutathione biosynthesis, were enhanced. To our knowledge, this is the first report highlighting a global metabolomics analysis using hydrophilic interaction chromatography ultra-high-performance liquid chromatography-tandem quadrupole/time-of-flight mass spectrometry and the potential involvement of glutathione depletion in spontaneous apoptosis of neutrophils demonstrating that LPS could delay this process.

Asiatic acid attenuates lipopolysaccharide-induced injury by suppressing activation of the Notch signaling pathway.

Sepsis is a severe multisystem disease with high mortality rates and limited treatment options. However, advances during the last decade have opened opportunities to develop novel therapeutic strategies. The Notch signaling pathway plays a critical role in inflammation, and its inhibition offers an avenue to treat inflammatory diseases, such as sepsis. Asiatic acid (AA), a triterpenoid isolated from Centella asiatica, reportedly exerts anti-oxidant, anti-tumor, and anti-inflammatory effects, but its mechanisms remain unclear. In our study, we found that AA decreased levels of interleukin-1ß (IL-1ß), IL-6, alanine aminotransferase and blood urea nitrogen in serum; attenuated liver, lung and kidney damage; and improved the survival among mice with experimental sepsis. AA also reduced lipopolysaccharide-stimulated expression of proinflammatory mediators, including nitric oxide, IL-1ß and IL-6 in RAW 264.7 macrophages. Notably, we demonstrated for the first time that AA is a novel small molecule inhibitor of the Notch signaling pathway. Its effects include upregulation of Notch receptor (Notch3) and delta-like ligand (DLL4), inhibition of Notch3 binding to the IL-6 promoter and regulation of mitochondrial function. These novel effects of AA may provide new approaches and strategies for the treatment of inflammatory disorders.