Study On The Mechanism Of Xueshuantong Injection In Preventing Platelet Aggregation And Improving Blood Flow

ObjectiveTo explore the biopharmaceutical mechanism of anti-platelet aggregation of Xueshuantong(XST)for injection.BackgroundIt is widely accepted that platelets play a pivotal role in the development of cardiovascular disease.Anti-platelet aggregation has become an important way of preventing and treating cardiovascular and cerebrovascular diseases.XST,as a traditional Chinese medicine for promoting blood circulation and removing blood stasis,is mainly composed of panax notoginseng saponins.Our previous data have demonstrated that the efficacy and mechanism of XST on platelets activation and adhesion were influenced by different shear stress conditions in vitro.XST inhibits platelet activation and adhesion to injured ECs under controlled shear stress in vitro,and the effects of XST are not only dose-dependent,but also shear stress-influenced Therefore,it is speculated that Panax notoginseng saponins may exert the overall effect of promoting blood circulation and removing blood stasis by inhibiting platelet aggregation and improving the hemodynamic environment of the organismThrombosis is a complex process involving the interaction of blood vessels,blood,and the mechanical environment.The previous studies have found that platelets are a device that can sense mechanical stimuli.Abnormal blood flow shear forces can induce platelet activation and aggregation,which in turn affects the development of thrombus It is known that platelet adhesion to immobilized von Willebrand factor(vWF)and subsequent formation of platelet-derived micro-particles are mediated by glycoprotein Iba(GPIb?)under high shear stress Further research found that there is a mechanical ion channel Piezol protein on the surface of the platelet membrane,which can sense the shear stress of the blood flow and induce the Ca2+entry.thereby affecting the activation of platelets.In this study,we take XST as the research object,explore the effect of XST on ?-carrageenan induced thrombosis and blood flow from the aspects of interactions among blood flow,vascular endothelium and platelets;We used the Bioflux1000z controlled shear stress microfluidic culture system to simulate different levels of hydrodynamic conditions in the body.To explore the effect of XST on platelet adhesion and rolling mediated by vWF under high shear stress To investigate the effects of XST on platelet aggregation under different flow conditions.Then,the effect of XST on Ca2+fluorescence intensity under flow conditions was determined with a fluorescence microscope.At last,and the effect of XST on platelet mechanics-related protein expression under different conditions was investigated by using western blot.To investigate the effects of XST on thrombosis and blood flow status,and the biological pharmacological mechanism of XST anti-shear-induced platelet aggregation.Research content1.Effects of XST on thrombosis formation and blood flow in ratsFifty male Sprague-Dawley rats were randomized into five groups:control group.model group,heparin sodium(1000 U·kg-1),low-dose and high-dose XST(50,150 mg·kg-1).Rats were intraperitoneally injected with corresponding drugs and saline for 10 days.One hour after drugs were administered intraperitoneally on the 7th days.each rat was injected with carrageenan(Type ?,1 mg·kg-1)which was dissolved in physiological saline by intravenous administration in the tail.And the experiments were performed at 18±1?.Rats were observed for the formation of thrombosis and thrombus lengths were measured and photographed at 2 h,6 h.24 h and 48 h.The blood flow was calculated with the average velocity and the vessel diameter was measured b)Visual Sonics Vevo 2100 ultrasound imaging system on the 8th days.Meanwhile.the blood flow perfusion in the thigh surface and caudal microcirculation of rats were detected by laser speckle blood flow imaging system.Platelet aggregometry was used to quantify rat platelet aggregation with the maximum aggregation rate ex vivo Histological examination in tail was performed through hematoxylin-eosin staining and light microscopy and Western blot was used to detect the protein content of platelet Piezo1.2.Platelets adhesion and rolling assay under high shear stressThe vWF-mediated platelet adhesion and rolling model was established under high shear flow conditions.The High Shear BioFlux 48-well plates were utilized.The microfluidic channels were coated with 70 ?g·mL-1 vWF for 1 h and then blocked with 0.5%(v/v)BSA in PBS for 10 min at room temperature.The sodium citrate anti-coagulated whole blood was treated with XST(0.15 g·L-1 or 0.6 g·L-1)or vehicle(saline)for 10 min at 37? prior to perfusion at a shear rate of 3000 s-1 for 10 min.Subsequently,the channels were washed for 5 min at a shear rate of 1000 s-1 with HBSS(with calcium and magnesium)until all red and white blood cells were clear of the channels.Only platelets were loaded on the surface of channels coating with vWF protein.The rolling of platelets under a shear rate of 6000 s-1 with HBSS containing saline or XST(0.15 g·L-1 or 0.6 g·L-1)was recorded by time-lapse imaging every second for 30 s.The platelet adhesion and rolling speed were measured using BioFlux Montage software.Measurements of distance/time were taken for 10 rolling platelets in each field.3.Inhibitory effects of XST on shear induced platelets aggregationPRP was prepared after taking blood from the abdominal aorta of normal rats,and PRP was adjusted to 0.5×108/mL using Tyrode-platelet buffer.Then incubate PRP with calcein-AM fluorescent stain for 1 h at room temperature in the dark.Calcein-AM labelled platelets were pretreated with XST(0.15 g·L-1,0.6 g·L-1).GsMTx-4(2.5?mol·L-1),Yoda1(25?mol·L-1),Yoda1(25 ?mol·L-1)plus XST(0.15 g·L-1,0.6 g·L-1)or vehicle(saline)for 5 min at 37? in the dark.Pretreated platelets suspensions were perfused to the microfluidic channels with incremental pressures of 0.13,0.33,0.66,1.32,2.64 psi for 5 min each resulted in wall shear stresses of 400 s-1,000 s-1,2000 s-1,4000 s-1 and 8000 s-1 respectively,and the whole shear flow duration continued for 25 min.Fluorescent micrographs of platelets adhesion and aggregation were captured by time-lapse inverted microscopy(wavelength FITC;exposure time 400ms).The BioFlux1000z system was used to control the flow shear rate and image acquisition settings.The platelets aggregation rates were determined by fluoresce intensity module of BioFlux Montage software.4.Ca2+ Imaging in platelets assay under shear stressThe 24-well microfluidic channels were coated with 40 ?g·mL-1 collagen followed by incubation at room temperature for 1 h and blocked with 0.5%BSA/PBS.The washed platelets suspension were initially incubated with fluo3 AM(5 ?mol·L-1)for 45min at 37? in the dark.Then the washed platelets were adjusted to 3 × 108/mL with Tyrode buffer containing 1 mmol·L-1 calcium.All inlet A wells were filled with fluo3 AM labeled platelets.At the same time in separate channels,inlet B wells were added with 1mL of XST(0.15 g·L-1),or GsMTx-4(2.5?mol·L-1)an inhibitor of Piezol channel,or Yoda1(25 ?mol·L-1)a mediator of Piezo1 channel,or Yoda1(25 ?mol·L-1)combined with XST or saline vehicle respectively.Flow was initiated from inlet A wells at shear stress of 200 s-1 for 2 min to load platelets on the surface of channels,sequentially perfusion from inlet B wells started automatically and continued for 10 min at shear stress of 1000 s"1 to observe the instant effects of XST on calcium influx under flow.During the 12 minutes,Calcium fluorescent images recorded time-lapse every 30 s.The fluorescent signals were back-ground-corrected,and fluorescence levels(F)were normalized against before administration fluorescence level(F0)to yield F/Fo values.5.Effects of XST on platelet Piezol and related-protein under different conditionsFor the static condition,washed platelets from rats were incubated with saline,XST(0.15 g·L-1,0.6 g·L-1),GsMTx-4(2.5 ?mol·L-1),or Yoda1(25 ?mol·L-1)for 10 min.and stimulated with 10 ?M ADP for 30 min at 37? except control group.Total platelet proteins were extracted and the expressions of platelet Piezo1 protein were detected by western blot.For the shear flow conditions,washed platelets were incubated with saline.XST(0.15 g·L-1,0.6 g·L-1),GsMTx-4(2.5?mol·L-1),Yoda1(25 ?mol·L-1),or Yoda1(25 ?mol·L-1)plus XST(0.15 g·L-1.0.6 g·L-1)for 10 min,after which the pretreated platelets were added into BioFlux 48 well Plates.Then the platelets suspension continued flowing in the microfluidic channels at shear stress of 1000 s-1?4000 s-1 or 10000 s-1 for 30 min.after which the platelets were followed by lysis in RIPA buffer containing 1 mM PMSF.Total platelet proteins were extracted and the expressions of platelet Piezo1 protein.Calpain-2 protein,Talinl protein and Vinculin protein were detected by western blot Results1.Inhibitory effects of XST on platelets activation under different flow conditionsAccording to the results,XST could inhibit the rat tail arterial thrombosis and significantly reduce the length of black tail(P<0.05).The blood flow of common carotid artery in XST low doses could significantly higher than in model group(P<0.05).XST high dose group can prominently increase the blood flow perfusion of the tail microcirculation in rats contrast with the model group(P<0.05).Adenosine diphosphate(ADP)of 2.5 ?M mediated platelet aggregation was obviously inhibited after intraperitoneal injection of high doses of XST for 10 days in rats.And the low doses of XST could increase the expression of Piezo1 protein from the platelet.2.Effects of XST on platelets interaction with vWF under high shear stressUnder high shear conditions,platelet adhesion increased with increasing vWF concentration.The vWF concentration of 70 ?mol·L-1 was used to evaluate the effect of XST on platelet adhesion and rolling.The results showed that compared with the vehicle group,XST had no significant difference in vWF-mediated platelet adhesion and rolling3.Inhibitory effects of XST on shear induced platelets aggregationPlatelets suspension with XST(0.15 g·L-1,0.6 g·L-1),GsMTx-4(2.5 ?M),Yodal(25?M),mixture of Yodal and XST,or vehicle(saline)was perfused over the microfluidic channels and platelet adhesion and aggregation was measured.Observation of the inhibitory effects of XST under fluorescent microscopy indicated that XST reduced the number and size of the aggregates formed significantly at physiologic shear stresses(1000 s·-1 and 2000 s-1)and pathological high-shear stresses(4000 s-1 and 8000 s-1)such as observed in arteries and stenosis arteries respectively.Compared with the vehicle group,XST significantly inhibited shear-induced platelet aggregation,and showed a dose-dependent relationship.GsMTx-4.the selective inhibitor of Piezol protein,also inhibited shear stress induced platelet aggregation compared with the vehicle group(P<0.05 or P<0.01);Yodal promoted platelet aggregation only under pathological high shear flow;mixture of XSTL and XSTH group can significantly inhibit platelet aggregation,with an average inhibition rate of 66.37%and 78.63%,respectively(P<0.01)4.Effect on platelet Ca2+ entry under flow shear stressTo determine how XST influences platelets aggregation under shear stress,we measured the Ca2+ entry into platelets before and after XST perfusion.The Ca2+fluorescence intensity of each group increased with time at shear rate of 1000 s-1.Treatment with XST decreased the elevation of intracellular calcium in a concentration-dependent manner.Arterial shear 1000 s-1 induced a sustained increase in Ca2+ influx by 90%in platelets.These Ca2+increases were abrogated by the MS channel inhibitor GsMTx-4 or potentiated by the Piezo1 agonist Yodal by 150%(P<0.05).Both GsMTx-4 and XST significantly reduced Ca2+ fluorescence intensity compared with the model group(P<0.05 or P<0.01);Yodal combine XST group obviously decreased platelet Ca2+ entry compared with the Yoda1 group(P<0.05),which implied that XST acted on the Piezo 1 channel.5.Effects of XST on platelet Piezo1 and related-protein under different conditionsUnder static conditions,XST had no significant effect on the expression of platelet Piezo1 protein.Under 1000 s-1,4000 s-1,or 10000 s-1 shear rate conditions,Piezo1 protein expression were measured.Compared with the static group.the Piezo1 protein level was significantly enhanced after exposure to shear(1000 s-1.4000 s-1 or10000 s-1).which may contribute to shear-induced platelet aggregation.Treatment with GsMTx-4.XSTL or XSTH was no effects on the Piezol protein under high shear rate.Talin1 was cleaved into 230kDa and 190kDa molecular weight under high shear flow conditions.After treatment with Yoda1.the platelet 230kDa Talin1 protein level was significantly reduced by 32%(P<0.01);the 190kDa Talin1 level was significantly increased by 64%(P<0.01).XST(0.15 g·L-1)combined with Yodal group significantly reduced the expression of 230kDa Talin1(P<0.01),XST(0.6 g·L-1)combined with Yoda1 treatment significantly reduced the expression of 190kDa Talin1((P<0.05)Conclusion1.XST could play an effect in fighting against thrombosis induced by ?-carrageenan in rats,which may be related to significantly inhibiting platelet aggregation,improving body's blood flow state,maintaining normal hemodynamic environment and affecting mechanical ion channel protein piezol;2.XST has no significant effect on vWF-mediated platelet adhesion and rolling under high shear conditions.The above-mentioned findings indicated that XST may not act on the GPIb?-vWF bond to influence shear-induced platelet aggregation.3.XST could significantly inhibit shear-induced platelet aggregation,which may be related to inhibition the function of Piezo1,thereby inhibiting Ca2+ entry.4.XST inhibits shear-induced platelet aggregation via Piezo1-mediated Ca2+ signalling and subsequent calpain-2 activation and talinl cleavage as downstream signal pathways,which is recruited to form a complex with the GP?b?a protein to induce activation of platelets.