Studies Of Resveratrol Regulates Endothelial Cells Apoptosis And Expression Of Pro-thrombosis Molecules P-Selectin And VWF

DVT is a common clinical complication in orthopedic surgery.It is one of the important causes of perioperative death in orthopedic surgery patients.It is complicated and involving endothelial cells,platelets,white blood cells and other factors in the pathogenesis of DVT.In recent years,studies have shown that vascular endothelial cell injury is one of the most important causes of venous thrombosis,endothelial cell injury can release a variety of adhesion molecules,platelet activating factor,tissue factor,enhance coagulation,promote DVT formation;However,Oxidative stress,inflammatory response is caused by intravenous endothelial cell damage in the common causes of oxidative stress,inflammation can cause the body reactive oxygen species,increased inflammatory factors,cell lipid peroxidation,NO reduction,causing endothelial cell damage,withered And studies have shown that NF-?B can increase the expression of tissue factor and promote coagulation,which affects DVT formation;many studies have shown that resveratrol has antioxidant,anti-inflammatory,protective endothelial cell damage Function,but in the formation of DVT is rarely reported.Therefore,the purpose of this study is to investigate:Inhibitory effect of resveratrol on oxidative damage and apoptosis of venous endothelial cells,and regulate the molecular mechanism of pro-thrombotic molecular P-Selectin and vWF expression of after venous endothelial cell injury.Objective1.To investigate the effect of resveratrol on oxidative damage and apoptosis of endothelial cells.2.To investigate the effect and molecular mechanism of resveratrol on the expression of pro-thrombotic molecular P-Selectin and vWF after venous endothelial cell injury.MethodsThe study is divided into two parts:1.The first part,the experimental part is divided into three groups:(1)blank control group;(2)H2O2 group:200?mol/L H2O2 treatment of human umbilical vein endothelial cells 24 hours to establish a model of endothelial cell injury;(3)RES+H2O2 group:HUVECs were pretreated with 30?mol/L resveratrol for 2 hours and then treated with 200?mol/L H2O2 for 24 hours.Cell viability was measured by MTT assay to investigate the protective effect of resveratrol on H2O2-induced HUVECs injury;Intracellular ROS content were measured by Fluorescent probe DCFH-DA capture method and laser confocal microscopy,to clarify the effect of resveratrol on the oxidative stress of endothelial cells;Hoechst 33258 staining,fluorescence microscopy,Annexin V-FITC/PI and flow cytometry were used to detect cell apoptosis,to aim at the effect of resveratrol on H2O2-induced apoptosis of HUVECs.2.The second part,the experimental part is divided into four groups:(1)blank control group;(2)H2O2 group:this group is the same as the first part;(3)RES+H2O2 group:this group is the same as the first part;(4)BAY 11-7082+H2O2 group:Cells were pretreated with 5 ?mol/L NF-?B specific inhibitor BAY 11-7082 for 4 hours and then incubated with 200 ?mol/L H2O2 for 24 hours.PCR and Western blot were used to detect NF-?B,P-selectin and vWF gene and protein expression changes,to verify the effect and molecular mechanism of resveratrol on the expression of pro-thrombotic molecular P-Selectin and vWF after endothelial cell injury.Results1.The first part of the experimental results:HUVECs were treated with 200 ?mol/L H2O2 for 24 hours,is H2O2 group,compared with the control group,the cell viability was significantly decreased(P<0.01),and the intracellular ROS content was significantly increased(P<0.01),and the apoptotic rate was significantly increased(P<0.01)(P<0.01);However,the cells were pretreated 30?mol/L resveratrol for 2 hours,and then cultured with 200?mol/L H2O2 for 24 hours,is RES+H2O2 group,compared with the H2O2 group,the cell viability was significantly higher(P<0.01),The intracellular ROS content was significantly decreased(P<0.01),and the apoptotic rate was significantly decreased(P<0.01).2.The second part of the experimental results:(1)The mRNA and protein expression of NF-?B,P-Selectin and vWF in HUVECs treated with 200(pmol/L H2O2 for 24 hours were significantly higher than the control group(P<0.01).(2)However,after pretreatment with NF-?B-specific inhibitor 5?mol/L BAY 11-7082 for 4 hours,then treated with 200?mol/L H2O2 for 24 hours,is BAY 11-7082+H2O2 group,compared with H2O2 group,the protein and mRNA expression of NF-?B was down-regulated(P<0.01),and the protein and mRNA expression of P-Selectin,vWF also were down-regulated(P<0.05).(3)Whereas,after treatment with 30?mol/L resveratrol for 2 hours,the cells were pretreated with 30p?mol/L resveratrol for 2 hours and then treated with 200?mol/L H2O2 for 24 hours,is RES+H2O2 group,compared with H2O2 group,the protein and mRNA expression of NF-?B was down-regulated(P<0.01),and the protein and mRNA expression of P-Selectin,vWF also were down-regulated(P<0.05).(4)Compared with RES+H2O2 group,the protein and mRNA expression of NF-?B and vWF of BAY 11-7082+HH2O2 group were reduced more obvious(P<0.01).Conclusion1.Resveratrol can reduce endothelial cell damage and apoptosis.2.Resveratrol can reduce ROS production after endothelial cells injury.3.NF-?B is involved in the expression of Pro-thrombosis Molecules P-Selectin and vWF after endothelial cells injury.4.Resveratrol can inhibit Pro-thrombosis Molecules P-Selectin,vWF activates after endothelial cells injury,and may by NF-?B signaling pathway.Suggesting resveratrol has a certain prevention and treatment effect on DVT formation.