Evaluate Increase cAMP through Activating Protein Phosphatase 2A (PP2A) by Zanthoxylum Integrifoliolum in Human Neutrophils and Protect Liver from Ischemia Reperfusion Injury

This study investigates the mechanism underlying the inhibiting effect of a Zanthoxylum integrifoliolum derived compound, N-methylflindersine (ZIL-25) on superoxide anion production and cathepsin G release induced by the chemotactic peptide formly-methionylleucyl- phenylalanine in human neutrophils. ZIL-25 inhibited fMLP-induced superoxide anion production and cathepsin G release in a concentration-dependent manner with the IC50 value of 5.0±0.5 and 8.7±0.8 μM, respectively. We studied the underlying mechanism and found that ZIL-25 suppressed ERK-, Akt-phosphorylation and intracellular calcium mobilization in response to fMLP. In a further study, ZIL-25 rapidly increased intracellular cAMP levels by up to threefold. Inhibition of cAMP-dependent protein kinase A with H-89 abrogated the suppressive effects of ZIL-25, restoring fMLP-induced ERK, Akt phosphorylation, intracellular calcium mobilization and superoxide anion formation. ZIL-25 synergic increased cAMP level induced by PGE1 but not with IBMX, a non-selective phosphodiesterase inhibitor. However, ZIL-25 did not directly inhibit the activity of phosphodiesterase 4 in our study. In another set of experiment, okadaic acid or calyaulin A, two protein phosphatase 2A inhibitors, reversed the ZIL-25-increased cAMP level. This data indicated that protein phosphatase 2A was involved in the cAMP-elevating mechanism of ZIL-25. We found that the activity of protein phosphatase 2A was activated by ZIL-25. Furthermore, we will evaluate the interaction between protein phosphates 2A with phosphodiesterase 4 after ZIL-25-treated human neutrophils. It will be a breakthrough about a new mechanism regulation of cAMP level in human neutrophils. ZIL-25 inhibited fMLP induced neutrophils-platelets interaction in a whole blood system. This data showed us that ZIL-25 overcame the plasma-binding in the whole blood system. Furthermore, we will continue to study the effect of ZIL-25 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 ZIL-25. We already set up this animal model and also test ZIL-25 on this model. According to the histology staining and alanine transaminase （ALT） examination. We found a significantly protect effect of ZIL-25（1 μg/kg）on ischemia-reperfusion injury in mouse liver. Therefore, the more study will carry out in our future study. The detail mechanism for ZIL-25on ischemia-reperfusion injury and the effect of ZIL-25 on this animal model will be carried on. Conclusion: ZIL-25 inhibited fMLP-induced superoxide anion production with a PKA-dependent manner. ZIL-25 could increase cAMP level by activating protein phosphatase 2A and consequently inhibited the activity of phosphodiesterase 4. ZIL-25 also has strong protection from ischemia reperfusion injury in vivo and this offer a good model for development of natural products.

Project ID:PC9807-0301
External Project ID:NSC98-2320-B182-018-MY3