The Effect of Vi Tis Thunbergii Sieb, &Amp; Zucc. Var. Thunbergii on the Interaction between Human Platelets and Neutrophils---Natural Products Inhibit Neutrophil Extracellular Traps (Net) Caused by Activated Platelets

This study investigates the mechanism underlying the inhibiting effect of two natural products from Vitis thunbergii Sieb. & Zucc. var. thunbergii, VT-1 and VT-6, on the neutrophil (neutrophil extracellular traps; NET), and on the interaction between human platelets and neutrophils. VT-1 and VT-6 were found to inhibit fMLP induced neutrophil-platelet aggregation in the whole blood system. VT-1 and VT-6 were also found to inhibit neutrophil NET formation caused by LPS or thrombin-activated platelets. However, VT-1 and VT-6 did not affect the platelet function. Briefly, VT-1 inhibited fMLP-induced superoxide anion production in a concentration-dependent manner with the IC50 value of 21.88±2.59 μM. VT-1 was found to specifically inhibit fMLP-induced ERK, AKT phosphorylation. Moreover, VT-1 was found to compete the binding of FLPEP (FITC-fMLP) on fMLP receptor and this data indicated VT-1 could be a fMLP receptor antagonist. There is addition inhibitory effect on fMLP-induced free radical production and receptor binding study when VT-1 combines with a FPRL1 (N-formyl peptide receptor-like 1:a subtype of fMLP receptor) antagonist (WRW4). This information indicated that VT-1 could be a FPR (N-formyl peptide receptor) antagonist. In another set of experiment, VT-6 specific inhibits fMLP-induced superoxide anion and cathepsin G release in a concentration dependent manner. According to the data from flow cytometer analysis, we exclude that VT-6 is a fMLP receptor antagonist. VT-6 was found to inhibit fMLP induced Src kinase activation, however, VT-6 did not affect the Src kinase activity. There is synergic inhibitory effect on fMLP induced ROS when VT-6 combine with PP2. β-arrestin was found to endocytosis FPRL1 and activate Src. According to this data, we speculate that VT-6 could inhibit the β-arrestin translocation and subsequently affect the Src activation. Moreover, we have to figure out how VT-6 affects β-arrestin and Src tyrosine kinase. In conclusion, VT-1 and VT-6 opposes fMLP-mediated neutrophil activation and inflammation by antagonizing FPR receptor or interring intracellular signaling. According to this information, the inhibitory effect of VT-1 and VT-6 on neutrophil NET which caused by activated-platelets will be studied. Finally, we will perform an ischemia-reperfusion animal model in mouse liver and a thrombosis animal model in mouse mesentery. Therefore, the more studies will be carried out in our future study. The detail mechanism for VT-1 or VT-6 on these animal models will be carried on and evaluate the applications of natural products in the future.

Project ID:PC10601-0585
External Project ID:MOST105-2320-B182-004-MY3