Danhong Huayu Koufuye Prevents Deep Vein Thrombosis Through Antiinflammation Via Sirtuin 1

ObjectiveOur previous study has shown that Danhong Huayu Koufuye(DHK)significantly inhibited deep vein thrombosis(DVT).Therefore,we first optimized conditions for inferior vena cava(IVC)stenosis-induced DVT model in rats.Then we further assayed blood coagulation,blood fibrinolysis,blood cell counts,whole blood viscosity,platelet activity and Sirtuin 1(SIRT1)modulated inflammation to study the anti-DVT mechanism of DHK.Methods1.Optimal conditions for DVT model in rats:Effects of suture,body weight,sex and side branch on IVC stenosis-induced DVT were evaluated.1 d after modeling,thrombus weight was measured.Histopathological change and leukocyte influxes were observed by hematoxylin and eosin(HE)staining.Ly-6G-positive neutrophil was located by immunofluorescence.A multiple linear regression model of thrombus weight was then run by R version.2.The antithrombotic effect of DHK:Rats were randomly divided into 3 groups:sham,model and DHK(3.2 mL/kg/d)groups.IVC stenosis-induced DVT was established in rats.The preventive and therapeutic effects of DHK was evaluated.Thrombus weight,histopathological change and leukocyte influxes were observed.3.The antithrombotic mechanism of DHK:Rats were randomly divided into 8 groups:sham group,model group,low,medium and high dose groups of DHK(1.6,3.2 or 6.4 mL/kg/d),heparin(HEP)group,clopidogrel(CLP)group and resveratrol(RES)group.IVC stenosis-induced DVT was established in rats.DHK,HEP and RES were administered 1 h and 24 h after thrombus induction.CLP was given 2 h before operation and 24 h after operation.IVCs and/or blood were harvested 1.5 h after the final administration.Thrombus weight was measured.Activated partial thromboplastin time(APTT),prothrombin time(PT),thrombin time(TT),fibrinogen(FIB),plasma levels of tissue plasminogen activator(tPA)and plasminogen activator inhibitor(PAI-1),the number of red blood cells(RBC),platelets(PLT)and white blood cells(WBC),whole blood viscosity,and adenosine diphosphate(ADP)-induced platelet aggregation were determined.Histopathological change and leukocyte influxes were observed by HE staining.TNF-α and IL-1β were assayed by ELISA.SIRT1 mRNA expression was measured by quantitative Real Time Polymerase Chain Reaction(qRT-PCR).SIRT1 and tissue factor(TF)protein expressions were tested by immunofluorescence and Western blot.Protein expressions of p-p65,acetylated-p65(Ace-p65)and p65 were assayed by Western blot.Next,DHK combined with SIRT1 inhibitor EX527 were administered to DVT rats.Thrombus weight and protein expressions of SIRT1,p-p65,Ace-p65 and p65 were observed.Results1.Optimal conditions for IVC stenosis-induced DVT model in rats:Silk and monofilament sutures presented no statistic difference in thrombus weight and leukocyte influxes.Thrombus weight of 220-340 g rats was significantly heavier than that of 180-220 g rats after IVC stenosis.Thrombus weight of male rats was significantly heavier than that of females.Although no statistic difference was found in thrombus weight of male and female rats weighing 180-260 g,males weighing 260-300 g formed larger thrombi than weight-matched females.Thrombus weight of rats except 180-220 g females was not impacted by side branch ligation and side branch distance.In the regression model,sex and body weight were the key factors affecting DVT.2.The antithrombotic effect of DHK:Some red blood filaments and leukocyte exudation of vein wall were observed 60 min after stenosing.Thrombi and leukocyte influxes in the thrombosed IVCs were found 1d,3d and 7d after stenosing.Compared to the model group,DHK reduced thrombus weight by 37%,37%and 44%at 1d,3d and 7d after IVC stenosis,respectively.DHK ameliorated leukocyte influxes at 60 min,1d,3d and 7d after IVC stenosis as well.3.The antithrombotic mechanism of DHK:Compared to the model group,DHK had no effects on blood coagulation(APTT,PT,TT and FIB),blood fibrinolysis(tPA and PAI-1),blood cell counts(RBC,PLT,WBC),whole blood viscosity,and platelet activity(ADP-induced maximum platelet aggregation rate).DHK sharply increased SIRT1 mRNA and protein expressions in thrombosed IVCs.Moreover,it decreased leukocyte influxes in the vein wall and thrombus,serum levels of TNF-α and IL-1β,and protein expressions of Ace-p65 and p-p65 in thrombosed IVCs.As compared with DHK group,thrombus weight,and protein expression of Ace-p65 were markedly increased,and SIRT1 protein expression was decreased in DHK+EX527 group.ConclusionMale and female rats weighing 220-260 g are more suitable for establishing a model of DVT induced by stenosing IVC with silk and without side branch ligation.DHK prevents DVT,as well as treats DVT in early and late stages.DHK inhibits DVT by ameliorating inflammation via up-regulation of SIRT1 in the early stage.