The Effects Of Fruitflow Water Soluble Tomato Extract On Platelet Function

Objective To investigate the effects of Fruitflow water soluble tomato extract on platelet function and its molecular mechanism,and provide a new molecular mechanism and experimental basis for the effects on platelet function by Fruitflow.Materials and Methods In vitro study,we collected blood from healthy donors and separated platelet rich plasma（PRP）by centrifugation.Then PRP was incubated with Fruitflow,platelet aggregation was stimulated by ADP or Collagen.In vivo study was conducted with subjects over 50 years old.The subjects were randomly divided into four groups:placebo group,Fruitflow group,acetylsalicylic acid（ASA）group,and Fruitflow+ASA groups.All subjects received corresponding interventions for 7 days.1.In vitro study to evaluate the effects of Fruitflow on platelet function.（1）Measurement of platelet aggregation:PRP was incubated with Fruitflow and platelet aggregation was stimulated by ADP or Collagen.The platelet aggregation rate was measured by Chrono-Log platelet aggregator.（2）Detection of platelets P-selectin（CD62P）expression by flow cytometry:the purified platelets were labeled with fluorescent-labeled CD61 and CD62P antibodies,and the effect of Fruitflow on the expression of P-selectin of platelets was analyzed by flow cytometry.（3）ELISA assay:ELISA kits were used to analyze the effects of Fruitflow on the release of TXB₂,6-Keto-PGF_1αand PF4 of platelets.（4）Detection of platelet spreading:Immunofluorescence assay was used to analyze the effects of Fruitflow on platelet spreading.（5）Western blot analysis:western blot was used to analyze Akt,GSK-3βand p38MAPK phosphorylation of platelet stimulated by Collagen.2.In vivo study to evaluate the effects of Fruitflow intervention on platelet function.（1）General examination:biochemical indexes,blood routine,coagulation function and fecal occult blood of the subjects were checked before and after the intervention.（2）In vivo study was used to evaluate the effects of Fruitflow on platelet aggregation:blood was collected before and after the intervention to separate PRP.ADP or Collagen was used to stimulate platelet aggregation,and the platelet aggregation rate was measured by Chrono-log aggregator.（3）ELISA assay:the phosphorylation of Akt,GSK-3βand p38 MAPK in Collagen stimulated platelets were analyzed by specific antibody.Results1.In vitro study results:the analysis of platelet aggregation showed that ADP and Collagen can stimulate platelet activation.Platelets were incubated with different concentrations of Fruitflow,our results suggested Fruitflow can decrease the platelet aggregation rate stimulated by ADP and Collagen in a dose-dependent manner.The inhibitory rate of 100μg/ml Fruitflow on platelet aggregation stimulated by ADP was88.6%,and the inhibitory rate of 100μg/ml Fruitflow on platelet aggregation stimulated by Collagen was 89.5%.The results of flow cytometry showed that the P-selectin expression of platelets increased by 1.9 folds induced by Collagen compared with the control group（P<0.001）.The P-selectin expression of platelets treated with 100μg/ml Fruitflow significantly decreased compared with the Collagen group（P<0.01）.Our results showed that 100μg/ml Fruitflow could completely inhibit the expression of P-selectin in platelets stimulated by Collagen.ELISA results showed that the production of TXB₂in platelets stimulated by ADP increased by 4.2 folds compared with the baseline level,and the production of TXB₂was completely inhibited by 100μg/ml Fruitflow,the difference was statistically significant（P<0.001）.TXB₂level was increased 19.5 folds after Collagen stimulation,and decreased 48.7%after Fruitflow treatment,the difference was statistically significant（P<0.001）.Analysis of 6-keto-PGF_1αshowed that the Fruitflow reduced the production of 6-keto-PGF_1α,and the level of 6-keto-PGF_1αin the Fruitflow group was 39%lower than the ADP-stimulated group,and the difference was statistically significant（P<0.001）.The Fruitflow also significantly reduced the production of 6-keto-PGF_1αinduced by Collagen,and the level of6-keto-PGF_1αin the Fruitflow group decreased by 46.3%compared with the Collagen group,the difference was statistically significant（P<0.01）.The analysis of PF4showed that ADP-stimulated platelet PF4 secretion increased 1.8 folds compared with the control group,and Fruitflow effectively inhibited ADP-stimulated PF4 production,the difference was statistically significant（P<0.05）.Compared with the control group,platelet PF4 increased 2.3 folds(stimulated by Collagen.Fruitflow effectively inhibited the release of PF4 stimulated by Collagen,and the difference was statistically significant（P<0.01）.The platelet spreading experiments showed morphologic changes during platelet activation.Our results showed that the platelets adhered to the fibrinogen coating in the control group without spreading.The platelets adhered to the fibrinogen coating in the Collagen group with obvious spreading.The platelet spreading stimulated by Collagen was obviously inhibited by Fruitflow（100μg/ml）.Our study suggested that Akt、GSK-3βand p38 MAPK phosphorylation of platelets stimulated by Collagen increased by 2.7 folds（P<0.001）,3.2 folds（P<0.01）and 3.0 folds（P<0.001）respectively compared with the control group.The Akt、GSK-3βand p38 MAPK phosphorylation of platelets stimulated by Collagen were decreased by Fruitflow in a dose-dependent manner compared.These results suggested that the inhibition of platelet function by Fruitflow was related to the inhibition of phosphorylation of these protein kinases.2.In vivo study to investigate the effects of Fruitflow on platelet function,our results showed no changes in biochemical indexes,coagulation function and fecal occult blood before and after the intervention.The platelet aggregation rate induced by ADP was decreased by 7.7%after 7 days intervention with Fruitflow（P<0.05）,and decreased by 9.4%after 7 days intervention with acetylsalicylic acid（P<0.05）.Fruitflow intervention reduced the Collagen stimulated platelet aggregation rate decreased by 10.2%（P<0.01）.Acetylsalicylic acid intervention reduced the Collagen stimulated platelet aggregation rate by 39%（P<0.001）.The platelet aggregation induced by ADP and Collagen decreased by 9.3%（P<0.01）and 38.3%（P<0.001）respectively after the combined intervention of Fruitflow and acetylsalicylic acid.Intergroup analysis showed that there was no statistical difference in the platelets aggregation rate stimulated by ADP and Collagen before intervention（P>0.05）.The platelets aggregation rates were statistically significant differences in the Fruitflow group,acetylsalicylic acid group,Fruitflow+acetylsalicylic acid group compared with placebo group after intervention（P<0.05）.There was no difference in platelet aggregation rate between acetylsalicylic acid group and Fruitflow and acetylsalicylic acid combined group（P>0.05）.We also evaluated the effect of Fruitflow on TXB₂,6-keto-PGF_1αand PF4.The plasma level of TXB₂decreased significantly after 7 days Fruitflow intervention,and the difference was statistically significant compared with before the intervention（P<0.01）.The level of 6-keto-PGF_1αof plasma significantly decreased after 7 days Fruitflow intervention,and the difference was statistically significant compared with before intervention（P<0.001）.The level of PF4 was also reduced after Fruitflow intervention,and the difference between before and after intervention was statistically significant（P<0.001）.The similar trend was also seen in the acetylsalicylic acid group and in the combination of Fruitflow and acetylsalicylic acid group.Intergroup analysis showed that there were no significant differences in TXB₂,6-keto-PGF_1αand PF4 levels before intervention（P>0.05）.The levels of TXB₂,6-keto-PGF_1αand PF4 were significant difference in Fruitflow group,acetylsalicylic acid group,Fruitflow+acetylsalicylic acid group compared with placebo group after intervention（P<0.05）.There was no difference in TXB₂,6-keto-PGF_1αand PF4 levels between acetylsalicylic acid group and Fruitflow and acetylsalicylic acid combined group（P>0.05）.Conclusions1.Fruitflow can inhibit platelet aggregation,reduce the production of TXB₂,6-keto-PGF_1αand the release of PF4 stimulated by ADP and Collagen.Fruitflow also can inhibit platelet spreading and the expression of P-selectin induced by Collagen.The Collagen stimulated phosphorylation of Akt,GSK-3βand p38 MAPK was effectively inhibited by the Fuitflow,suggesting that the molecular mechanism of the inhibition of platelet activation was related to the inhibition of the phosphorylation of Akt,GSK-3βand p38 MAPK.Our study provided a new theoretical and experimental basis for the inhibition of platelet function by Fruitflow.2.In vivo study showed that Fruitflow can inhibit platelet aggregation and decrease the levels of TXB₂,6-keto-PGF_1αand PF4 in aging human,suggesting that Fruitflow intervention could improve platelet function.There was no adverse effect on blood coagulation after short-term intervention of Fruitflow.These results suggested that Fruitflow can reduce the risk of cardiovascular disease by inhibiting platelet function and provide health benefits for the aging human.3.Fruitflow belongs to natural food extract with good safety characteristics,which can be used as an effective strategy to prevent or improve cardiovascular disease.Fruitflow has certain application prospects and important scientific significance in the prevention of cardiovascular disease research.