From Study Inhibitroy Effect of Piper Philippinum on Human Neutrophils to Evaluate the Role of Neutrophils on Ischemia Refperfusion Injury and the Therapeutic Mechanisms of Natural Products

The purpose of this study is to evaluate the roles of neutrophils and platelets on ischemia-reperfusion injury by evaluated the anti-inflammatory effect of two natural products, 4』-O,O-demethylenehinokinin (PP-4) and (＋)-bornyl caffeate (PP-12) which were extracted from Piper Philippinum. In in vitro experimental model for evaluation of action mechanisms of PP-4 and PP-12 on neutrophils and neutrophils-platelets interaction, furthermore, a liver ischemia-reperfusion injury animal model for study the effect of PP-4 and PP-12. This is the first report for the anti-inflammatory effect of Piper Philippinum, therefore, we evaluate the anti-inflammatory effect of PP-4 or PP-12. Briefly, PP-4 specific and concentration-dependent inhibited fMLP-induced free radical production, the IC50 value for PP-4 on fMLP-induced free radical was 0.3±0.1 μM. On the other hand, PP-12 inhibited free radical production that induced by fMLP, PMA or LTB4, the IC50 values for PP-12 on fMLP, PMA, LTB4 induced free radical production were 0.6±0.1, 3.6±0.2 or 5.3 ±0.2 μM, respectively. PP-4 specifically inhibited fMLP induced intracellular signaling, such as intracellular calcium mobilization, protein phosphorylation (ERK). However, PP-12 did not affect the intracellular signaling caused by fMLP, PMA and LTB4. Moreover, PP-4 inhibited FLPEP (FITC-fMLP) binding to fMLP receptor on human neutrophil. The IC50 value for PP-4 on FLPEP binding was 0.8±0.2 μM. In another set of experiment, PP-4 specific inhibited fMLP-induced MAC-1 expression. Furthermore, a high concentration of PP-4 （10 μM）had no inhibitory effect on superoxide anion production induced by NaF, a direct activator of G-protein, that target of the inhibitory action of PP-4 appears to be a component of signal transduction pathway upstream of G-protein. This data showed us that PP-4 could be a fMLP-receptor antagonist. The detail action mechanism of PP-4 will carry out in the future. These studies also show the different mechanisms of PP-4 and PP-12. Neutrophils-platelets interaction play an important role ischemia-reperfusion, we evaluated the effect of PP-4 and PP-12 on neutrophil-platelets interaction. PP-4 and PP-12 inhibited fMLP induced neutrophils-platelets interaction in a whole blood system. This data showed us that PP-4 and PP-12 overcome the plasma-binding in the whole blood system. Furthermore, we will continue to study the effect of PP-4 and PP-12 on the adhesion molecular (MAC-1) expression on human neutrophils and platelets. The intracellular signaling molecules which regulate adhesion molecular, such as cdc42, cyclic nucleotides, protein phosphorylation, will be studied in the future. Finally, we will perform an ischemia-reperfusion animal model in mouse liver. According to this model, we can evaluate the roles of neutrophils and platelets in reperfusion injury and we also evaluate the in vivo effect of PP-4 and PP-12. We already set up this animal model and also test PP-4 on this model. According to the histology staining and alanine transaminase （ALT） examination. We found a significantly protect effect of PP-4 （1 μg/kg）on ischemia-reperfusion injury in mouse liver. Therefore, the more study will carry out in our future study. The detail protect mechanism for PP-4 on ischemia-reperfusion injury and the effect of PP-12 on this animal model will be carried on.

Project ID:PC9706-0482
External Project ID:NSC96-2320-B182-022-MY2