| ObjectiveOur previous study showed that aqueous extract of Whitmania Pigra Whitman(AEW)significantly inhibited deep vein thrombosis(DVT).Therefore,the anti-DVT mechanism of AEW was further investigated.The effect of AEW on blood coagulation,blood fibrinolysis,activity of platelet,whole blood viscosity and Sirtuin 1(SIRT1)modulated inflammation were examined in inferior vena cava(IVC)stenosis induced-DVT rats.Methods(1)Effect of AEW on DVTThe time-effect relationship of AEW on DVT:Rats were randomly divided into three groups:sham,model and AEW(104.2 mg crude W.pigra/kg)groups.IVC stenosis induced-DVT was established in SD rats.The rats in sham and model groups were orally administered with distilled water.Methods 1:AEW was orally administered at 1 h and 24 h after thrombus induction.Methods 2:AEW was orally administered at 1 h,24 h,2 d and 3 d after thrombus induction.Methods 3:AEW was administered once daily for five consecutive days 3 d after thrombus induction.Thrombus weight,histopathological change and leukocytes infiltration were observed.The dose-effect relationship of AEW on DVT:Rats were randomly divided into seven groups:sham group,model group,low,medium and high doses of AEW(34.7,104.2 and 312.5 mg crude W.pigra/kg)groups,heparin(200 U/kg)group and clopidogrel(25 mg/kg)group.IVC stenosis induced-DVT was established in rats.The rats in sham and model groups were orally administered with distilled water.AEW was orally administered at 1 h and 24 h after thrombus induction.Heparin was intravenously administered at 1 h and 24 h after thrombus induction.Clopidogrel was orally administered 2 h prior to and 24 h after thrombus induction.One hour after the final administration,rats were sacrificed,and IVCs were then dissected.Thrombus weight was observed.(2)The effects of AEW on blood coagulation,blood fibrinolysis,activity of platelet and the whole blood viscosityRats were randomly divided into seven groups:sham group,model group,low,medium and high doses of AEW(34.7,104.2 and 312.5 mg crude W.pigra/kg)groups,heparin(200 U/kg)group and clopidogrel(25 mg/kg)group.IVC stenosis induced-DVT was established in SD rats.The rats in sham and model groups were orally administered with distilled water.AEW was orally administered at 1 h and 24 h after thrombus induction.Heparin was intravenously administered at 1 h and 24 h after thrombus induction.Clopidogrel was orally administered 2 h prior to and 24 h after thrombus induction.One hour after the final administration,rats were sacrificed,and IVCs were then dissected.Blood samples were collected to determine blood cell counts,blood viscosity,APTT,PT,TT,Fib,platelet aggregation,plasma levels of tPA and PAI-1.(3)The antiinflammatory effect of AEW is modulated via SIRTI① IVC stenosis induced-down-regulation of SIRT1 promoted inflammation and DVTI.SIRT1 and inflammation were involved in DVTRats were randomly divided into fifteen groups including normal,0.5 h-sham,0.5 h-model,1 h-sham,1 h-model,1 d-sham,1 d-model,3 d-sham,3 d-model,5 d-sham,5 d-model,7 d-sham,7 d-model,14 d-sham and 14 d-model groups.IVC stenosis induced-DVT was established in rats.Thrombus weight,histopathological change and leukocytes infiltration were observed.Protein expression of TF in thrombus and vein wall was assayed by immunohistochemical method.Serum levels of TNF-α and IL-1β was tested by ELISA.Protein expressions of SIRT1,p-p65,p65 and Ace-p65 in thrombus and vein wall were assayed by Western blot.II.Effect of resveratrol(RES,a SIRT1 agonist)on DVTRats were randomly divided into five groups:sham group,model group,low,medium and high doses of RES(25,50 and 75 mg/kg)groups.IVC stenosis induced-DVT was established in rats.The rats in sham and model groups were orally administered with distilled water at 1 h and 24 h after DVT induction.RES was orally administered 1 h prior to and 24 h after thrombus induction.One hour after the final administration,rats were sacrificed and blood samples were collected from carotid artery.Thrombus weight,histopathological change and leukocytes infiltration were observed.Serum levels of TNF-α and IL-1β were tested by ELISA.SIRT1 protein expression was assayed by immunofluorescence.Protein expressions of SIRT1,TF,p65 and Ace-p65 in thrombus and vein wall were examined by Western blot.SIRT1 mRNA expression in thrombus and vein wall were assayed by qRT-PCR.Ⅲ.Effect of EX527(a selective SIRT1 inhibitor)on DVTRats were randomly divided into eight groups which included normal group,sham group,sham+RES(50 mg/kg)group,sham+EX527(10 mg/kg)group,model group,model+RES(50 mg/kg)group,model+RES(50 mg/kg)+EX527(10 mg/kg)group,model+EX527(10 mg/kg)group.IVC stenosis induced-DVT was established in rats.The rats in normal,sham and model groups were administered with distilled water.RES was orally administered at 1 h prior to and 24 h after thrombus induction.EX527 was intraperitoneally injected 20 min prior to thrombus induction.Rats were sacrificed at 25 h after thrombus induction.Morphological changes and thrombus weight were observed.Protein expressions of SIRT1,p-p65,p65 and Ace-p65 in thrombus and vein wall were assayed by Western blot.②AEW prevents DVT by antiinflammation modulated via SIRT1Ⅰ.Effects of AEW on SIRT1/NF-κB signaling pathwayRats were randomly divided into five groups:sham group,model group,low,medium and high doses of AEW(34.7,104.2 and 312.5 mg crude W.pigra/kg)groups.IVC stenosis induced-DVT was established in rats.The rats in sham and model groups were orally administered with distilled water.AEW was orally administered at 1 h and 24 h after thrombus induction.Rats were sacrificed,and blood was collected from carotid artery at 25 h after thrombus induction.Thrombus weight,histopathological change and leukocytes infiltration were observed.SIRT1 protein expression was tested by immunofluorescence and Western blot.Protein expressions of p-p65,p65 and Aceep65 were assayed by Western blot.Ⅱ.SIRT1 inhibitor was used to clarify that AEW prevent DVT via SIRT1Rats were randomly divided into six groups which included:normal group,sham group,model group,model+EX527 group(10 mg/kg/d),model+AEW(104.2 mg crude W.pigra/kg/d)group,and model+EX527(10 mg/kg/d)+AEW(104.2 mg crude W.Pigra/kg/d)group.IVC stenosis induced-DVT was established in SD rats.AEW was orally administered at 1 h and 24 h after thrombus induction.EX527 was intraperitoneally injected 20 min before surgery.Rats were sacrificed at 25 h after thrombus induction.Thrombus weight was assayed.Protein expressions of SIRT1,p-p65,p65 and Ace-p65 in thrombus and vein wall were observed by Western blot.Results(1)Effect of AEW on DVTAEW with three methods of administration significantly decreased thrombus weight and leukocytes recruitment in thrombus and vein wall.AEW at middle and high doses signifieantly decreased thrombus weight.(2)The effect of AEW on blood coagulation,blood fibrinolysis,blood cell counts,activity of platelet and the whole blood viscosityHaparin significantly increased APTT,PT and TT in DVT rats.Clopidogrel significantly reduced adenosine diphosphate(ADP)-induced platelet aggregation.AEW had no effects on blood coagulation(APTT,PT,TT and Fib.),plasma tPA and PAI-1 levels,blood cell counts(RBC,PLT,WBC,NEUT,LYMPH and MONO),maximum platelet aggregation rate induced by ADP,and whole blood viscosity.(3)The antiinflammatory effect of AEW is modulated via SIRT1① IVC-stenosis induced-down-regulation of SIRT1 promoted inflammation and DVTⅠ.Inflammation and SIRT1 are involved in DVTNo thrombus was observed in sham groups.A red blood filament was observed at 1 h after stenosis,and a fragile thrombus was formed at 1 d after stenosis.The thrombus grew into a hard one with time.New vessels was observed at 7 d and 14 d after stenosis.There was no differences of leukocytes infiltration in lumen and vein wall among normal and various sham groups.Numbers of neutrophils reached a maximum at 1 h and 1 d in the vein wall,while at 1 d in the thrombus.Monocytes in thrombus and vein wall reached a maximum at 5 d.TF was little expressed in normal and 0.5 h groups,while it was highly expressed in active monocytes,neutrophils and endothelium 1 h-14 d after stenosis.Serum levels of IL-1β and TNF-α were markedly elevated at 1 d after stenosis.SIRT1 protein expression was down-regulated by 75.4%at 1 d after stenosis.In contrast,p-p65 and Ace-p65 protein expressions were up-regulated by 185.4%and 92.4%at 1 d after surgery,respectively.The protein expressions of SIRT1 and Ace-p65 restored towards normal level roughly 3-14 d after stenosis,while p-p65 protein expression recovered towards normal level 14 d after stenosis.Ⅱ.Effects of RES on DVT formationRES inhibited thrombus formation and neutrophils and monocytes infiltration in the thrombi and exudation in the vein wall.Moreover,RES restored serum IL-1βand TNF-α towards normal level.RES increased SIRT1 protein and mRNA expressions.In contrast,RES restored Ace-p65 and TF protein expressions towards normal level.Ⅲ.Effect of EX527 on DVTThere were no differences in thrombus weight and protein expressions of SIRT1,Ace-p65 and p-p65 between normal and sham groups.RES and EX527 had no influence in sham-operated rats.Moreover,EX527 alone had no influence on above parameters in stenosis-treated rats.As compared with model+RES group,thrombus weight,Ace-p65 and p-p65 protein expressions were markedly increased,and SIRT1 protein expression in vein wall and thrombus was decreased in the model+RES+EX527 group.② AEW prevents DVT by antiinflammation modulated via SIRT1Ⅰ.Effect of AEW on SIRT1/NF-κB signaling pathwayAEW sharply increased SIRT1 protein expression in vein wall and thrombus.Moreover,it decreased serum levels of IL-1β and TNF-α,and protein expressions of Ace-p65 and p-p65 in the vein wall and thrombus.Ⅱ.SIRT1 inhibitor was used to clarify that AEW prevents DVT via SIRT1As compared with AEW group,thrombus weight,protein expressions of Ace-p65 and p-p65 were markedly increased,and SIRT1 protein expression in vein wall and thrombus was decreased in AEW+EX527 group.Conclusion:AEW significantly prevents DVT.But it has no effect on blood coagulation,blood fibrinolysis,blood cell counts,platelet activity and whole blood viscosity.DVT is regulated by inflammation modulated via SIRT1/NF-κB.And AEW inhibits DVT regulated by ameliorating inflammation via SIRT1. |
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