Paper Title
RABDOSIARUBESCENS EXTRACTSATTENUATES PLATELET ACTIVATION THROUGH THE INHIBITION OF AKT, ERKAND PLCΓ2-PKC PATHWAYS

Abstract
Abstract:- Introduction - Blood platelets are recognized for their crucial role in hemostasis, yet they are implicated in cardiovascular diseases like heart attacks and strokes. Medicinal herbs, including those traditionally used, have gained increased significance in Western countries. Additionally, herbs and natural products have been pivotal in discovering drugs for human diseases. Extracts from Rabdosiarubescens demonstrate various biological activities, including anti-inflammatory and antimicrobial effects. However, the potential antiplatelet and antithrombotic effects of Rabdosiarubescens, particularly its compound oridonin, are not clearly understood. Consequently, our objective is to explore the intricate mechanism of oridonin in platelet activation and thrombus formation. Methods: In the in vitro study, platelet aggregation, flow cytometry, and immunoblotting were employed to assess the antiplatelet effect of oridonin. Approval for this study was obtained from the Taipei Medical University—Joint Institutional Review Board, and the research adhered to the principles of the Declaration of Helsinki. Written informed consent was obtained from all participants. For animal studies, pulmonary thrombosis and thrombus formation in mesenteric vessels in mice were investigated to evaluate the antithrombotic effect of oridonin. Additionally, a zebrafish assay for developmental toxicity was conducted to assess the safety of oridonin. All procedures were conducted in accordance with the Animal Use Protocol of Taipei Medical University, following the guidelines outlined in the Guide for the Care and Use of Laboratory Animals (Eighth Edition, 2011), and The Zebrafish Book (5th Edition, 2007). Data were analyzed using analysis of variance, and post hoc analysis was performed using the Newman–Keuls test. Results are presented as the mean ± standard error of the mean (SEM), with statistical significance considered at P < 0.05. Results:The data indicated that oridonin hindered platelet aggregation induced by collagen (1 μg/ml) but not by thrombin (0.02 U/ml) or U46619 (1 μM). This suggests that oridonin possesses the ability to specifically impede collagen-induced platelet activation. Oridonin demonstrated a reduction in ATP release and calcium mobilization and mitigated the phosphorylation of PLCγ2, PKC, Akt, and ERK. Additionally, oridonin prevented platelet activation-related events, such as granule release and GPIIbIIIa activation. Furthermore, the data revealed that oridonin did not impact the survival rate, body length, or eyeball diameter and area of zebrafishes. This suggests that oridonin does not exhibit direct developmental toxicity under these concentrations. Moreover, oridonin attenuated pulmonary thrombosis and delayed platelet thrombus formation in mesenteric microvessels without any significant influence on hemostasis. Conclusion - These findings provide support for the notion that the oridonin can impede platelet activation by affecting the Akt, ERK and PLCγ2-PKC pathways. This, in turn, leads to the inhibition of platelet aggregation and subsequent thrombus formation. As a result, oridonin holds therapeutic potential for thromboembolic disorders, including heart attacks and strokes. Keywords - Akt/ERK, PLC2/PKC,oridonin, platelet activation