Anti-thrombosis Effects And Mechanisms By Xueshuantong Capsule

ObjectiveXueshuantong capsule(XST)is a patented traditional Chinese medicine used for the prevention and treatment of thrombosis.The molecular mechanism of XST anti-thrombosis effect through the cross-talk among the platelets/leucocytes,endothelial cells and flow shear stress was investigated.BackgroundBlood flow is typically characterized by shear stress,which is the force per unit area applied between adjacent layers of fluid.Shear stress is one of the most important factors that influence the formation of thrombus,and it can influence the structure and function of endothelial cells(EC)and modulate pathophysiologically relevant genes and proteins expressions.Typical physiological range of wall shear stresses in artery is about 1-7 Pa,and in susceptible regions shear stress decreased by 0.4 Pa.Abnormal shear stress may influence the function of endothelium,leading to various of thrombotic diseases.Thrombotic events occurring in either arteries or veins are the primary causes of fatal perioperative cardiovascular events.Blood composition is the most well studied component of the triad.Interactions of shear stress,endothelium and platelets/leucocytes may play important roles in thrombotic disease.Endothelium mediates the pathological consequences and protective responses in acute and chronic inflammation,cardiovascular disorders.Platelets and leukocytes adhesion to the activated endothelial cells lining the blood vessels is the first step to perform tasks related to injury and inflammation.It has been widely proposed that the anti-adhesion therapy is a rational and effective approach for the treatment of thrombotic disease.In this study,we focus on the molecular mechanism of XST anti-thrombosis effect through the cross-talk among the blood cells,blood vessel and blood flow,as well as by the method of biomechanopharmacology.We used ADP to induce platelet activation,the influence of XST was determined by flow cytometry.To investigate the effects of XST on platelets or leukocyte adhesion,TNF-? was used to induce endothelial cell injury,so that the endothelium injury model was established.Then,we used western blot to explore the effects of XST on VCAM-1,VE-cadherin and Cx43 expression under different conditions.At last,the effects of XST on the secretion of TXA2 and PGI2 by HUVECs were determined by radioimmunoassay.To explore the possible mechanism of XST in the treatment of thrombosis,this can provide a scientific experimental basis for further study on the molecular pharmacology mechanism of XST.Research content1.Inhibitory effects of XST on platelets activation under different flow conditionsWashed platelets were pretreated with varying concentrations of XST(0.15 mg·mL-1,0.3 mg·mL-1,and 0.6 mg·mL-1)or aspirin(1 mmol·L-1)under 0.1 or 0.9 Pa flow condition.Adenosine diphosphate(ADP)(10 ?mol·L-1)was used to activate platelets.Then,platelets were incubated with antibodies against CD61(FITI-conjugated)and CD62p(PE-conjugated).Platelets were again centrifugated and resuspended in Tyrode buffer and flow cytometric analysis was subsequently performed to detect activation of platelets.2.Inhibitory effects of XST on platelets and leukocyte adhesion under different flow conditionsThe influences of XST at different concentrations on HUVECs viability were examined by MTT assay.The safe dose range of XST was determined,and the dosage of XST(0.3 mg mL-1 and 0.6 mg mL-1)was used for the following experiments.HUVECs were cultured under flow conditions using the Bioflux 1000 flow system.In brief,HUVECs were incubated in the Bioflux 48-well plates,and were treated with TNF-a(20 ng·mL-1)in the absence or presence(12-hours preincubation)of XST(0.3 mg mL-1)or aspirin(1 mmol·L-1).Platelets or THP-1 cells were introduced into the viewing channels.Then,the platelets or THP-1 cell suspension was continued flowing under controlled flow conditions,and images were captured by time-lapse microscopy.Platelets or THP-1 cell adhesion numbers were measured by Bioflux Montage software.3.Study of XST on the expression of VC AM-1,VE-cadherin,and Cx43 under different conditionsHUVECs were incubated with TNF-a(20 ng·mL-1)in the absence or presence(12-hour preincubation)of XST(0.3 mg-mL-1 and 0.6 mg·mL-1)or aspirin(1 mmol·L-1),under static,0.1 or 0.9 Pa flow conditions.After washed three times with cold PBS,cell lysis(RIPA buffer containing 1 mmol · L-1 PMSF)was performed.And the protein concentration in the supernatant was determined by BCA assay.The expression of VCAM-1,VE-cadherin,and Cx43 was determined by western blot assay.4.Effects of XST on secretion of TXA2 and PGI2 in HUVECs under flow conditionsHUVECs were cultured under flow conditions using the Bioflux 1000 flow system.In brief,HUVECs were seeded in the Bioflux 48-well plates,and were treated with XST(0.3 mg·mL-1)or aspirin(1 mmol·L-1)under 0.1 or 0.9 Pa.Then,TNF-a(20 ng·mL-1)was used to injure the HUVECs.After 2 hours treatment,the supernatant was collected,and the concentration of TXB2 and 6-keto-PGF1?(metabolite of TXA2 and PGI2)were determined by radioimmunoassay.Results1.Inhibitory effects of XST on platelets activation under different flow conditionsXST inhibited the platelets activation in a dose-dependent manner under 0.1 Pa flow condition,the inhibition ratio was 37.8%,51.1%,and 55.6%,respectively,with XST concentrations of 0.15 mg·mL-1,0.30 mg·mL-1 and 0.60 mg·mL-1.Under 0.9 Pa flow condition,low dose XST of 0.15 mg·mL-1 reduced the platelets activation significantly by 29.5%,however,0.30 mg·mL-1 and 0.60 mg·mL-1 of XST showed no significant reduction.XST showed higher efficacy of anti-ADP induced platelets activation than aspirin under controlled shear stress.2.Inhibitory effects of XST on platelets and leukocyte adhesion under different flow conditionsUnder 0.1 Pa flow condition,XST pre-treatment demonstrated an obvious reduction in adhesion platelets compared with those in TNF-a activated HUVECs,the inhibition ratio was 34.1%.Under 0.9 Pa flow condition,XST pre-treatment reduced platelets adhesion,with the inhibitory ratio of 15.0%.Under 0.1 and 0.9 Pa flow conditions,XST showed similar effects on THP-1 adhhesion.And the inhibitory ratios were 30.2%and 28.3%respectively.3.Study of XST on the expression of VCAM-1,VE-cadherin,and Cx43 under different conditionsUnder static,XST(0.3,0.6 mg·mL-1)treatment decreased the expression of the VCAM-1,VE-cadherin and Cx43 significantly.XST at concentrations of 0.30 mg·mL-1 and 0.60 mg mL-1 showed inhibitory effect on the expression of these membrane proteins.Treatment with 0.30 mg·mL-1 XST decreased VCAM-1,VE-cadherin and Cx43 expression by 15.9%,23.5%and 31.9%,respectively;0.60 mg·mL-1 XST reduced their expression by 21.9%,16.6%and 24.0%accordingly.Under 0.1 Pa flow condition,XST pre-treatment(0.3 mg mL-1)decreased the expression of VCAM-1 by 24.6%,with ?-actin served as a loading control(P<0.05).However,the effects of XST on the VE-cadherin and Cx43 expression were not obvious.Under 0.9 Pa flow condition,treatment with 0.3 mg·mL-1 XST significantly decreased the expression of the VE-cadherin and Cx43(P<0.05),and the inhibition ratios were 30.6%and 16.0%respectively.While the effect of XST on VCAM-1 expression was not evident,but aspirin showed an apparent inhibitory effect.4.Effects of XST on secretion of TXA2 and PGI2 in HUVECs under flow conditionsUnder flow conditions,TNF-a(20 ng·mL-1)induced the TXB2 secretion from HUVECs,while XST and aspirin inhibited the secretion of TXB2(P<0.01),and the inhibitory ratios were 49.9%and 65.4%.Furthermore,XST had the trend of increasing 6-keto-PGF1?/TXB2(0.47±0.08 vs 0.70±0.01).Conclusion1.Shear stress influence the effects of XST on platelets activation,platelets and THP-1 adhesion;2.XST inhibited ADP induced platelet activation significantly.Under low(0.1 Pa)flow condition,XST showed stronger inhibitory effect of platelet activation in a dose-dependent manner.The anti-thrombosis effect might be related to inhibit platelets activation;3.The inhibition on platelets and THP-1 adhesion by XST may be through decreasing the VCAM-1,VE-cadherin,and Cx43 expression of HUVECs.4.XST might inhibit the formation of thrombosis by inhibiting the secretion of TXB2 in HUVECs and increasing the ratio of 6-keto-PGF1?/TXB2.