Aqueous Extract Of Whitmania Pigra Whitman Has Antithrombotic Effect And Inhibits Venous Thrombosis Through Activation Of Nrf2 Signaling

ObjectiveThe aims of the study were to compare the antithrombotic effect of colon delayed-release pellets（CDP）and regular pellets（RP）of aqueous extract of Whitmania pigra Whitman（AW）and to explore anti-venous thrombotic mechanism of AW basing on nuclear factor E2-related factor 2（Nrf2）.Methods1.Antithrombotic effects of RP and CDP（1）Time-effect relationship of anticoagulationRabbits were randomly divided into three groups:normal group（distilled water,50 m L）,RP group（43.7mg/kg/d）and CDP group（64mg/kg/d）.Capillary tube method was applied to detect coagulation time of New Zealand rabbits after oral administration coresponding test article.（2）Dose-effect relationship of anticoagulationRabbits were randomly divided into sixteen groups:normal group（distilled water,50 m L）,seven dosages of RP groups（10.9,21.8,32.8,43.7,65.6,87.4 and131.1mg/kg/d）,seven dosages of CDP groups（16,32,48,64,96,128 and 192mg/kg/d）and aspirin group（2.88mg/kg/d）.Capillary tube method was applied to detect coagulation time in New Zealand rabbits after oral administration coresponding test article.（3）Ligation-induced inferior vena cava（IVC）thrombosis in ratsRats were randomly divided into six groups:model group（distilled water,10m L/kg）,two dosages of RP groups（59.4,118.8mg/kg/d）,two dosages of CDP groups（86.8,173.6mg/kg/d）and aspirin group（7.8mg/kg/d）.The wet weight and inhibition rate of venous thrombosis were measured after oral administration coresponding test article.（4）Arteriovenous shunt-induced thrombosis in ratsRats were randomly divided into six groups:model group（distilled water,10m L/kg）,two dosages of RP groups（59.4,118.8mg/kg/d）,two dosages of CDP groups（86.8,173.6mg/kg/d）and aspirin group（7.8mg/kg/d）.All rats were orally administered coresponding test article.The wet weight and inhibition rate of thrombosis were then measured.（5）FeCl₃-induced arterial thrombosis in ratsRats were randomly divided into seven groups:sham group（distilled water,10m L/kg）,model group（distilled water,10 m L/kg）,two dosages of RP groups（59.4,118.8mg/kg/d）,two dosages of CDP groups（86.8,173.6mg/kg/d）and clopidogrel group（30mg/kg/d）.All rats were orally administered coresponding test article.The arterial thrombosis were evaluated by histological analysis and calculated the percentage and inhibition rate of thrombus formation.2.The anti-venous thrombosis（VT）mechanism study of AW in rats（1）Establishment of FeCl₃-induced IVC thrombus model in rats（1）Effect of the concentration of FeCl₃ on IVC thrombosis in rats:Rats were randomly divided into four groups:sham group,three concentration of FeCl₃ groups（5%,10%,20%w/v）.Thrombosis of the IVC was induced by applying a piece of filter paper saturated with 5%,10%or 20%FeCl₃ solution for 5min,then the filter paper was removed and the times of stabilization of thrombosis was 30min.The wet weight of venous thrombosis was measured and the tissue specimens were performed routine histopathological evaluation.（2）Effect of rinse on FeCl₃-induced IVC thrombosis in rats:Rats were randomly divided into two groups:rinse group and no rinse group.Thrombosis of the IVC was induced by applying a piece of filter paper saturated with10%FeCl₃ solution for 5min,then the filter paper was removed and the vein was wiped with a cotton swab dipped with saline.The times of stabilization of thrombosis was 30min.The wet weight of venous thrombosis was measured.（3）Effect of the time of thrombosis on FeCl₃-induced IVC thrombosis in rats:Rats were randomly divided into six groups:three model groups（20,30 and 40min）,three warfarin groups（20,30 and 40min）.All rats were orally administered with distilled water（10 m L/kg/d）or warfarin（0.2mg/kg/d）once daily,and consecutively for 5 days.Rats were carried out operation 1 h post-administration.Thrombosis of the IVC was induced by applying a piece of filter paper saturated with10%FeCl₃ solution for 5min,then the filter paper was removed.Three different stabilization times of thrombosis were tested after induction of 5min with FeCl₃:20,30 and 40min.The wet weight of venous thrombosis was measured.（2）The mechanism study of AW in rats of FeCl₃-induced IVC thrombosisRats were randomly divided into five groups:sham group（distilled water,10m L/kg）,model group（distilled water,10 m L/kg）,two dosages of AW group（52,104mg/kg/d）and warfarin group（0.2mg/kg/d）.All rats were orally administered coresponding test article once daily and consecutively for 5 days.Blood samples were collected for detecting activated partial thromboplastin time（APTT）,prothrombin time（PT）,thrombin time（TT）,blood cell count and whole blood viscosity.The wet weight and inhibition rate of venous thrombosis were measured.Serum activities of superoxide dismutase（SOD）,catalase（CAT）and T-AOC,serum levels of total glutathione（T-GSH）,oxidized glutathione（GSSG）and glutathione（GSH）and level of malondialdehyde（MDA）in vascular tissue were detected by commercial kits.Enzyme linked immunosorbent assay was used to evaluate plasma level of plasminogen activator inhibitor-1（PAI-1）and tissue plasminogen activator（tPA）.reactive oxygen species（ROS）in vascular tissue was evaluated by DCFH-DA probe.Protein expression of Nrf2 in vascular tissue was evaluated using immunofluorescence.Protein expressions of phosphoinositide 3-kinase（PI3K）,Akt,p-Akt,Nrf2 and heme oxygenase 1（HO-1）were determined by Western blot.Results1.Antithrombotic effects of RP and CDP（1）RP and CDP significantly prolonged coagulation time at 0.5 h and 4 h after single administration（P≤0.05 vs.normal group）,respectively.And their anticoagulant effects were peak at 1.5 h and 6 h,and maintained for 1 h and 4 h,respectively.（2）RP and CDP dose-dependently prolonged coagulation time（P≤0.05 vs.normal group）.The anticoagulability effect of CDP was better than RP.（3）RP and CDP dose-dependently inhibited ligation-induced IVC thrombosis（P≤0.05 vs.model group）.The antithrombotic effect of CDP was better than RP.（4）RP and CDP dose-dependently inhibited AV shunt-induced thrombosis（P≤0.05 vs.model group）,and the antithrombotic effect of CDP was better than RP.（5）RP and CDP significantly inhibited FeCl₃-induced arterial thrombosis（P≤0.05 vs.model group）.2.The anti-VT mechanism study of AW in rats（1）Establishment of FeCl₃-induced IVC thrombus model in rats（1）Effect of the concentration of FeCl₃ on IVC thrombosis in rats:Wet weight of thrombus were significantly increased in three concentration of FeCl₃ groups（P≤0.05 vs.sham group）.As compared with 5%FeCl₃ group,wet weight of thrombus was significantly increased in 10%FeCl₃ group（P≤0.05）.（2）Effect of rinse on FeCl₃-induced IVC thrombosis in rats:There was no significant difference in the formation of thrombosis between rinse group and no rinse group.（3）Effect of the time of thrombosis on FeCl₃-induced IVC thrombosis in rats:Thrombosis of warfarin group（30min）was significantly reduced（P≤0.05 vs.30min model group）.（2）The mechanism study of AW in rats of FeCl₃-induced IVC thrombosisAs compared with sham group,wet weight of thrombus was significantly increased in model group（P≤0.01）.Activities of SOD（P≤0.05）,CAT（P≤0.01）and T-AOC（P≤0.05）,the levels of T-GSH（P≤0.01）and tPA（P≤0.01）,the ratio of GSH/GSSG（P≤0.01）,fluorescence intensity of Nrf2,and protein expressions of Nrf2（P≤0.01）and HO-1（P≤0.01）were marked decreased.While the levels of ROS,MDA（P≤0.01）,whole blood viscosity（P≤0.05）,PAI-1（P≤0.05）,protein expression of PI3K（P≤0.05）and p-Akt（P≤0.01）were significantly increased in model rats.As compared with model group,AW dose-dependently inhibited FeCl₃-induced thrombosis（P≤0.05）.But AW had no effect on APTT,PT,TT,blood cell count,whole blood viscosity,PAI-1 and tPA.As compared with model group,high-dose of AW reduced the levels of ROS,MDA（P≤0.01）and down-regulated protein expression of PI3K（P≤0.05）and p-Akt（P≤0.05）,increased fluorescence intensity of protein of Nrf2,strengthened the activities of SOD（P≤0.01）,CAT（P≤0.01）and T-AOC（P≤0.05）,increased the serum level of T-GSH（P≤0.05）and the ratio of GSH/GSSG（P≤0.05）.As compared with model group,high-dose of AW significantly down-regulated protein expressions of PI3K（P≤0.05）and p-Akt（P≤0.01）,marked up-regulated protein expressions of Nrf2（P≤0.05）and HO-1（P≤0.05）.ConclusionBoth RP and CDP have anticoagulant and antithrombotic effects,but CDP is obviously more effective than RP.Moreover,CDP has better inhibitory effect on VT than arterial thrombosis.The optimal model of FeCl₃-induced IVC thrombosis in rats was induced by applying a piece of filter paper saturated with 10%FeCl₃ solution for5min,then the filter paper was removed and the times of stabilization of thrombus was 30min.The antioxidant activity regulated by activation of Nrf2 signaling may contribute to the anti-VT effect of AW.