Pyroglutamyl
leucine (
pyroGlu-Leu), which is a
peptide isolated from wheat
gluten hydrolysate, has been reported to be a hepatoprotective compound in
acute liver failure. In inflamed liver, proinflammatory
cytokines including
interleukin (IL)-1β and
tumor necrosis factor (TNF)-α stimulate the induction of
inducible nitric oxide synthase (iNOS). Excess production of
nitric oxide (NO) by iNOS is an inflammatory
biomarker in liver injury. We examined proinflammatory
cytokine-stimulated hepatocytes as a simple "in vitro
inflammation model" to determine liver protective effects of
pyroGlu-Leu and its mechanisms of action. We hypothesized that
pyroGlu-Leu inhibits the induction of iNOS gene expression, resulting in the attenuation of hepatic
inflammation. Hepatocytes were isolated from rats by
collagenase perfusion and cultured. Primary cultured cells were treated with IL-1β in the presence or absence of
pyroGlu-Leu. The induction of iNOS and its signaling pathway were analyzed. IL-1β stimulated the enhancement of NO production in hepatocytes and this effect was inhibited by
pyroGlu-Leu.
pyroGlu-Leu decreased the expression of iNOS
protein and its
mRNA. Transfection experiments with iNOS-
luciferase constructs revealed that
pyroGlu-Leu inhibited both of iNOS promoter transactivation and its
mRNA stabilization.
pyroGlu-Leu also decreased the expression of an iNOS gene antisense transcript, which is involved in iNOS mRNA stability. However,
pyroGlu-Leu had no effects on IκB degradation and NF-κB activation. Results demonstrate that
pyroGlu-Leu inhibited the induction of iNOS gene expression at transcriptional and post-transcriptional steps through IκB/NF-κB-independent pathway, leading to the prevention of NO production.
pyroGlu-Leu may have therapeutic potential for liver injury through the suppression of iNOS.