Regulatory role of PI3K-protein kinase B on the release of interleukin-1ß in peritoneal macrophages from the ascites of cirrhotic patients.

Great effort has been paid to identify novel targets for pharmaceutical intervention to control inflammation associated with different diseases. We have studied the effect of signalling inhibitors in the secretion of the proinflammatory and profibrogenic cytokine interleukin (IL)-1ß in monocyte-derived macrophages (M-DM) obtained from the ascites of cirrhotic patients and compared with those obtained from the blood of healthy donors. Peritoneal M-DM were isolated from non-infected ascites of cirrhotic patients and stimulated in vitro with lipopolysaccharide (LPS) and heat-killed Candida albicans in the presence or absence of inhibitors for c-Jun N-terminal kinase (JNK), mitogen-activated protein kinase kinase 1 (MEK1), p38 mitogen-activated protein kinase (MAPK) and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K). The IL1B and CASP1 gene expression were evaluated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The expression of IL-1ß and caspase-1 were determined by Western blot. IL-1ß was also assayed by enzyme-linked immunosorbent assay (ELISA) in cell culture supernatants. Results revealed that MEK1 and JNK inhibition significantly reduced the basal and stimulated IL-1ß secretion, while the p38 MAPK inhibitor had no effect on IL-1ß levels. On the contrary, inhibition of PI3K increased the secretion of IL-1ß from stimulated M-DM. The activating effect of PI3K inhibitor on IL-1ß release was mediated mainly by the enhancement of the intracellular IL-1ß and caspase-1 content release to the extracellular medium and not by increasing the corresponding mRNA and protein expression levels. These data point towards the role of MEK1 and JNK inhibitors, in contrast to the PI3K-protein kinase B inhibitors, as potential therapeutic tools for pharmaceutical intervention to diminish hepatic damage by reducing the inflammatory response mediated by IL-1ß associated with liver failure.

Characterization of a circulating N-extended form of the thyrotropin-releasing hormone-like peptide pGlu-Glu-Pro amide in human plasma.

The TRH-like peptides pGlu-Glu-Pro amide, pGlu-Phe-Pro amide, and pGlu-Gln-Pro amide were isolated and identified some years ago, and these peptides have been proven to be present in many tissues and fluids. The presence of TRH-like immunoreactivity distinct from TRH in blood has been observed previously. In the present study, the presence of N-extended forms of TRH-like peptides in plasma has been investigated. Peripheral blood samples of human, rat, and rabbit were obtained and plasma was extracted. The peptides were separated in several steps of chromatography, including gel filtration, cation and anion exchange, and HPLC. The concentrations of the TRH-like peptides in the column fractions were measured by RIA with TRH antibody. The N-extended forms of TRH-like peptides were determined by RIA after trypsin digestion. In human plasma it was observed an N-extended form of TRH-like peptides in substantial concentration. After trypsin and heating, the N-extended forms of TRH-like peptides were rechromatographed on Sephadex G-50. This showed that the TRH-like peptides released have a similar size to TRH. The peptides were then separated by cation exchange chromatography, and the major fraction was unretained, indicating a neutral or acidic nature. Part of the unretained fraction was then chromatographed on anion exchange column in which the major fraction was retained, demonstrating the acidic nature of the peptides. Similar results have been observed in rat and rabbit. The other part of the unretained fraction from cation exchange chromatography of human plasma was purified on HPLC. The results demonstrated that the major component observed by HPLC cochromatographed with synthetic pGlu-Glu-Pro amide. This study represents the first demonstration of a circulating N-extended form of any TRH-like peptide.