To evaluate the ingredient of Rhaphiolepis indica manipulation of immunity responses by modulate PI3K activity and the study these ingredients on liver ischemia re-perfusion injury in mice

The first part of this study investigates the mechanism underlying the inhibiting effect of a Rhaphiolepis indica derived compound, (4-Hydroxy-3-Methoxyphenyl)-3,7-Dimethoxy- 4H-Chromen-4-one (MSF-2), on superoxide anion production and cathepsin G release induced by the chemotactic peptide formly-methionyl-leucyl-phenylalanine (fMLP) in human neutrophils. The second part of this study investigates the mechanism underlying the manipulating effect of another compound, 2-(1'-geranyloxy)-4,6-dihydroxyacetophenone (MSF-4), stimulated neutrophil activating effect. Briefly, MSF-2 inhibited fMLP-induced superoxide anion production and cathepsin G release in a concentration-dependent manner with the IC50 value of 22.8±4.8 and 14.2±1.7 μM, respectively. MSF-2 inhibited fMLP-induced neutrophil superoxide anion production, cathepsin G release and migration in human neutrophils isolated from healthy volunteers, reflecting inhibition of phosphatidylinositol 3-kinase (PI3K) activation. Specifically, PI3K/AKT activation results in migration, degranulation and superoxide anion production in neutrophils. MSF-2 suppresses PI3K activation and phosphatidylinositol (3,4,5)-trisphosphate (PIP3) production, and consequently inhibits downstream activation of PDK1 and AKT. Further, PI3K also stimulates respiratory burst via PLC-dependent elevation of intracellular calcium. MSF-2 reduces fMLP-mediated PLCγ2 activation and intracellular calcium accumulation notably through extracellular calcium influx in a PI3K and PLC-dependent manner. However, MSF-2 is not a competitive or allosteric antagonist of fMLP. In conclusion, MSF-2 opposes fMLP-mediated neutrophil activation and inflammation by inhibiting PI3K activation and subsequent activation of AKT and PLCγ2. Additionally, in an in vivo study, MSF-2 prevents fMLP-induced neutrophil infiltration and inflammation in mice. 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 MSF-2. We already set up this animal model and also test MSF-2 on this model. According to the histology staining and alanine transaminase （ALT） examination. We found a significantly protect effect of MSF-2 on ischemia-reperfusion injury in mouse liver. Therefore, the more studies will be carried out in our future study. The detail mechanism for MSF-2 on ischemia-reperfusion injury and the effect of MSF-2 on this animal model will be carried on. In another set of experiment, MSF-4 increases superoxide anion with a concentration dependent manner. MSF-4 at 30 μM has a maximum effect on superoxide anion production. In order to evaluate the possible effect of MSF-4 on increasing superoxide anion, several pharmacological inhibitors are used in our study. According to preliminary studies, we suggest PLD, PKC and intracellular calcium are involved in the mechanisms for MSF-4 induced free radical production. Further investigations are needed to evaluation of the rational of these second messengers.

Project ID:PC10202-0640
External Project ID:NSC101-2320-B182-019-MY3