Effects And Mechanisms Of Supplementing Qi And Activating Blood Chinese Herbs Combined With Dual Antiplatelet Drugs On Platelet Activation

Part 1 Effects of supplementing qi and activating blood herbs combined with dual antiplatelet drugs on platelet adhesion induced by HUVEC injuryObjective:To observe the effects of Panax quinquefolium saponin (PQS)+DA, Panax notoginseng saponin (PNS)+DA and PQS+PNS+DA on inhibiting platelet adhesion to injured endothelial cells (ECs) and to explore the possible mechanisms focusing on PI3K/AKT, COX-2/6-keto-PGF1α, and COX-1/TXB2 pathways.Methods:Human umbilical vein endothelial cells (HUVECs) injured by oxidized low-density lipoprotein (ox-LDL) were randomly allocated into control, model, DA, PQS+DA, PNS+DA, PQS+PNS+DA, and LY294002+PQS+DA groups. HUVEC apoptosis, platelet adhesion to injured HUVECs, and platelet CD62p expression were assessed by fluorescence activated cell sorting (FACS). The concentrations of 6-keto-PGF1α and TXB2 in the supernatant were measured by radioimmunoassay. Protein expression of phosphorylated-PI3K, PI3K, phosphorylated-AKT, AKT, COX-1, and COX-2 in both platelets and HUVECs was evaluated by western blot.Results:When compared with control, HUVEC apoptosis, platelet adhesion, platelet CD62p expression and concentration of 6-keto-PGF1α and TXB2 in model group were all increased (P<0.05). HUVEC p-Akt pretein expression in model group was decreased. HUVEC COX-2 protein expression and platelet p-Akt protein expression in model group were increased (P<0.05). When compared with model group, DA group showed decreased platelet adhesion, platelet CD62p expression and supernatant concentration of 6-keto-PGF1α and TXB2. Compared to DA group, both PQS+DA and PNS+DA reduced platelet adhesion and HUVEC apoptosis, increased the concentration of supernatant 6-keto-PGF1α, of which PQS+DA up-regulated p-AKT protein expression in HUVECs, and PNS+DA down-regulated COX-1 protein expression in platelets. LY294002 mitigated the effects of PQS on HUVEC apoptosis and platelet adhesion. PQS+PNS+DA showed more potent effects on HUVEC apoptosis and platelet adhesion when compared with PQS+DA or PNS+DA.Conclusions:Both PQS+DA and PNS+DA reduce EC apoptosis and platelet adhesion induced by EC injury. PQS+PNS+DA showed more potent effects than either of the two and the underlying mechanisms involve up-regulation of PI3K/Akt and COX-2/PGI2 pathways in ECs and down-regulation of PI3K/Akt and COX-1/TXA2 pathways in platelets.Part 2 Effects of Panax notoginseng saponin on platelet adhesion induced by HUVEC injuryObjective:This study was designed to investigate the effect of PNS on platelet adhesion to injured ECs and platelet activation induced by injured ECs, and to explore its underlying mechanisms.Methods:HUVECs pretreated with ASA (15 μg/mL) or PNS (160 μg/mL), or neither, were exposed to ox-LDL(80 mg/L) for 16h. Platelets were then added and co-cultured with HUVECs for 5min. Platelet adhesion to HUVECs, platelet CD62p expression, and HUVEC apoptosis were assessed by fluorescence activated cell sorting (FACS). Supernatant concentration of 6-keto-PGFla and thromboxane 2 (TXB2) were measured by radioimmunoassay. Cyclooxygenase-1 (COX-1) and COX-2 protein expression were measured by western blotting.Results:The inhibitory effect of PNS on platelet activation was similar to ASA, but the inhibitory effect of PNS on platelet adhesion to ECs was superior to ASA. PNS up-regulated COX-2 expression, and increased 6-keto-PGF1α concentration in HUVECs, while down-regulated COX-1 expression and decreased supernatant TXB2 concentration in platelets.Conclusions:PNS is superior to ASA in protecting ECs and in inhibiting platelet adhesion to injured ECs, and the regulation of COX pathway in both ECs and platelets might be the underlying mechanisms of PNS.Part 3 Effects of Panax notoginseng saponin on gastric mucosal epithelial cell injury induced by dual antiplatelet drugsObjective:This study was designed to investigate the effects of PNS on gastric mucosal epithelial cells injury induced by dual antiplatelet drugs, and to explore its underlying mechanisms.Methods:Human gastric mucosal epithelial cells (GES-1) were allocated into control group, DA group, PNS+DA group and LY294002+PNS+DA group. GES-1 apoptosis was assessed by FACS. GES-1 monolayer permeability was assessed using Transwell. Supernatant concentration of 6-keto-PGF1α, PGE2 and VEGF were measured by E-lisa. p-PI3K/PI3K, p-Akt/Akt, COX-1, COX-2, GSK-3p, GTP-RhoA/RhoA protein expression were measured by western blotting.Results:When compared with control, DA increased the GES-1 apoptosis and GES-1 permeability, decreased the supernatant concentration of PGE2,6-keto-PGF1α and VEGF. In addition, DA also decreased the protein expression of COX-1, p-PI3K and p-Akt, and increased the protein expression of COX-2, GTP-RhoA and GSK-3β. When compared to DA, PNS+DA decreased GES-1 apoptosis and GES-1 permeability, increased the supernatant concentration of PGE2,6-keto-PGFla and VEGF and down-regulated the protein expression of COX-2, GTP-RhoA and GSK-3p. LY294002 mitigated the effects of PNS on GES-1 apoptosis and permeability, increased the protein expression of GTP-RhoA and GSK-3β.Conclusions:PNS attenuates the inhibition of COX pathways in gastric mucosal epithelial cells by DA, and attenuates DA induced gastric mucosal epithelial cell injury by modulating PI3K/Akt/GSK-3(3-VEGF-RhoA pathway.