Spatholobi Caulis Reduces Deep Vein Thrombus Burden Via Sirtuin 1 And Nrf2

ObjectiveDeep vein thrombosis(DVT)is a kind of blood stasis syndrome.Spatholobi Caulis(SC)has been widely used for the treatment of blood stasis syndrome in China,but the underlying mechanism remains poorly understood.The aim of present study was to investigate the anti-DVT mechanism of SC.MethodsThe main compounds in SC formula granules were determined by high performance liquid chromatography(HPLC).A rat model of inferior vena cava(IVC)stenosis-induced DVT was performed.Rats were randomly divided into eight groups:normal group,sham group,model group,low and high doses of SC(0.78 and 1.56 g crude SC/kg/d)groups,heparin(HEP)group and clopidogrel(CLP)group.Rats in low and high doses of SC groups were orally administered with SC once daily for seven consecutive days.IVC stenosis-induced DVT model was operated on the sixth day.Rats in HEP group were intravenous injected with HEP at 1 h and 24 h after thrombus induction.Rats in CLP group was orally given with CLP 2 h before operation and 24 h after operation.All rats were sacrificed 1.5 h after the final administration,then IVCs and/or blood were harvested.Thrombus weight was measured.Blood samples were collected to determine activated partial thromboplastin time(APTT),prothrombin time(PT),blood cell counts,blood viscosity and platelet aggregation.Pathological changes were observed by hematoxylin-eosin staining.Tumor necrosis factor(TNF)-α and interleukin(IL)-1β of serum were analyzed by enzyme-linked immunosorbent assay.C-reactive protein(CRP)was measured with turbidimetric immunoassay.Super oxide dismutase(SOD)activity of serum was measured with colorimetric method.Malondialdehyde(MDA)of thrombosed IVCs was quantified by the thiobarbituric acid assay.Protein expressions in thrombosed IVCs was detected with Western blot.A cell model of oxygen-glucose deprivation(OGD)induced injury in EA.hy926 cell was performed.SC medicated serum was added to OGD-injured cells.Then Cell viability was measured by CCK-8 method,and Western blot was used to detect protein expressions.SIRT1 inhibitor(EX527)or/and Nrf2 inhibitor(ML385)were combined with SC medicated serum,and then Western blot and immunofluorescence were used to detect protein expressions.ResultsThe content of protocatechuic acid,catechin and epicatechi in SC formula granules were 0.23 mg/g,3.32 mg/g,and 2.52 mg/g,respectively.Compared to the model group,SC dramatically decreased thrombus weight.However,SC had no effects on PT,adenosine diphosphate-induced maximum platelets aggregation rate or blood cell counts.SC increased whole blood viscosity and slightly prolonged APTT.SC decreased tissue factor(TF)protein expression.SC reduced inflammatory cells influxes in thrombus and vein wall,and serum levels of TNF-α,IL-1β and CRP.SC enhanced SOD activity and decreased MDA content.Further,SC up-regulated Sirtuin 1(SIRT1)protein expression and down-regulated acetylated-NF-κB p65(Ace-p65)protein expression.Moreover,SC up-regulated nuclear factor-erythroid 2 related factor 2(Nrf2)and heme oxygenase-1(HO-1)protein expressions,and down-regulated phosphorylated-NF-κB p65(p-p65)protein expression.In the OGD cell model,SC medicated serum decreased the protein expression of TF.SC medicated serum enhanced nuclear SIRT1 and reduced nuclear Ace-p65 protein expressions.SC medicated serum up-regulated protein expressions of nuclear Nrf2 and total HO-1,and inhibited nuclear translocation of p65.Furthermore,inhibiting SIRT1 and Nrf2 reversed the effect of SC medicated serum on SIRT1/NF-κB and Nrf2/HO-1/NF-κB signaling pathways in OGD-treated EA.hy926 cells.ConclusionSC prevents DVT through antiinflammation and antioxidation via SIRT1/NF-κB and Nrf2/HO-1/NF-κB signaling pathways.