Effects Of Salvia Miltiorrhiza&Crataegus Pinnatifida Herb Couple Combined With Swimming On Atherosclerotic In Rat And Action Mechanism Of Blood Shear Stress In Rabbit Isolated Vessel

1 ObjectiveLow shear stress(SS)in blood vessel is a commen feature in blood stasis syndrome in Chinese medicine,and one of the intension of the therapeutic principle of activating blood circulation to remove blood stasis(ABCRBS)is to elevate shear stress appropriately.Therefore,the functions of endothelial cells are regulated by the elevated SS,and preventive and therapeutic effects could be gained for intervention of atherosclerosis(AS).The project focuses on the interactions among endothelial cells,blood SS and blood.The project consists of two categories of research:Firstly,the dose-response between the factors secreted by endothelial cells of ex vivo vessel which related to atherosclerosis,thrombosis and thrombolysis and the multi-levels of full range physiological shear stress.Secondly,animal models of AS will be employed to investigate the effects of exercise and the herbal pair of ABCRBS(Salvia miltiorrhiza+Crataegus Pinnatifida)individually and jointly.The project focuses on the biomechanopharmacological mechanism and new approach in intervention of AS,and development of the pharmacology of Chinese material medica.2 Methods and Results 2.1 Establishment of the isolated artery perfusion model under different flow conditions of SS and mean normal pressureBy employing ex vivo arterial perfusion system,the rabbit artery perfusion model was established under different flow conditions of SS(1.5~20 dyn/cm2)and mean normal pressure(< 50000 dyn/cm2).And the expression level of COX-1 mRNA and t-PA mRNA was measured by real-time PCR method.When considering the effect of SS on COX-1 mRNA and t-PA mRNA expression separately,the detection results of real-time PCR of COX-1 mRNA and t-PA mRNA expression(y)showed a significant positive correlation with SS(x),respectively,the linear equation was described: y=0.0261x+0.0886(P<0.01)and y=0.2033x+1.2082(P<0.01)accordingly.And we also found that SS(X)and pressure(Y)had a significant combined effect on expression of COX-1 mRNA and t-PA mRNA,the binary quadratic equations were established: COX-1 mRNA = 0.3619-0.0389x+8.8645*10-7y+0.0015x2+1.515*10-6xy-3.1759*10-10y2(P<0.01);t-PA mRNA = 2.9572-0.047x-6.5219*10-5y+0.01x2+2.2973*10-6xy+6.9716*10-10y2(P<0.01).2.2 Study of the joint effects of shear stress and normal pressure on e ndothelial cell function in rabbit isolated vesselBy employing ex vivo arterial perfusion system,the rabbit artery perfusion model was established under different flow conditions of SS(3,8,13,18 dyn/cm2)and mean normal pressure(12,24,36,48 mmHg).The perfusate was collected to determine the level of NO,ET,PGI2,vWF,PAI-1 and t-PA by using radioimmunoassay.And the expression level of COX-1 mRNA,vWF m RNA,PAI-1 mRNA and t-PA mRNA was measured by real-time PCR method.Flow conditions of low shear stress(<4 dyn/cm2)combined with high normal pressure(>40 mmHg),or high shear stress(>18dyn/cm2)combined with low normal pressure(<20 mmHg),can increase the level of PAI-1 in perfusate,and meanwhile can up-regulate the gene expression fo PAI-1;The combination of shear stress(8~18 dyn/cm2)and normal pressure(20~40 mmHg)can increase the level of t-PA in perfusate,and can also up-regulate the gene expression fo t-PA;Flow conditions of shear stress(8~18 dyn/cm2)can increase the level of vWF in perfusate and can up-regulate the gene expression fo vWF;Flow conditions of shear stress(8~18 dyn/cm2)combined with normal pressure(20~40 mmHg)can increase the value of NO/ET in perfusate,thus making blood vessels vasodilation,and can also increase the value of t-PA/PAI-1,being good for thrombolysis.2.3 The joint effects of Salvia Miltiorrhiza&Crataegus Pinnatifida herbal couple and swimming on atherosclerosis in ratsA combined method involving low shear stress caused by partial ligation of left common carotid artery and a high-fat diet was used to establish the atherosclerosis model in rats.Partial ligation of the left common carotid artery was performed,and the rats were then allotted to a 4 × 4 factorial design with two factors(swimming and extract of Salvia Miltiorrhiza&Crataegus Pinnatifida herbal couple)and four levels for each factor,swimming for 0,10 min,20 min and 40 min daily and Salvia Miltiorrhiza&Crataegus Pinnatifida extract administration of 0,DSA-SZA-DSL:42mg/kg-64mg/kg-12.5mg/kg,DSA-SZA-DSL:84 mg/kg-128 mg/kg-25 mg/kg,DSA-SZA-DSL:68mg/kg-256mg/kg-50mg/kg p.o.daily.Next,all of the rats were fed a high-fat diet,and the interventions of swimming and DSA-SZA-DSL were executed for 8 weeks.After the end of the 8th week,blood samples were collected to determine blood lipid profile including total cholesterol(T-CHO),low-density lipoprotein-cholesterol(LDL-C),triglyceride(TG)and high-density lipoprotein-cholesterol(HDL-C),endothelial function cytokines(including nitric oxide(NO),6-keto-prostaglandin(PG)F1?,endothelin(ET),thromboxane(TX)B2,plasminogen activator inhibitor-1(PAI-1),tissue type plasminogen activator enzyme(t-PA),von willebrand factor(vWF)and inflammatory cytokines including interleukin(IL)-1?,IL-6 and IL-10.The common carotid arteries of the rats were harvested to examine pathological changes via Oil red staining and the gene expression of t-PA,PAI-1,vWF and COX-1 was assayed via real-time(RT)quantitative polymerase chain reaction(qPCR).The joint effects of Salvia Miltiorrhiza&Crataegus Pinnatifida extract and swimming showed significant changes in the examined parameters:(1)decreases in serum T-CHO,TG,LDL-C;(2)decreases in serum IL-6,increase in serum IL-10;(3)increase in serum NO and decrease in plasma TX-B2.Swimming alone could increase serum HDL-C and plasma 6-keto-prostaglandin(PG)F1?.Three Dimensional surface plot could been used to describe the joint effects of Salvia Miltiorrhiza&Crataegus Pinnatifida extract and swimming on parameters that could not simply display the tendency of increase or decrease and quadratic equations could described the influences of Salvia Miltiorrhiza&Crataegus Pinnatifida extract and exercise on those factors.The results by the three Dimensional surface fitting method above suggested that serum level and gene expression level of PAI-1,t-PA,v WF and COX-1 were mainly accordant.3 ConclusionThe combined effects of SS and normal pressure should be considered in biomechanopharmacological research.Our result demonstrates that binary quadratic equation is an effective description quantitatively.The combined effects of SS and normal pressure can improve the vascular endothelial function of isolated artery in rabbit.This job demonstrates the combined therapy of Salvia Miltiorrhiza&Crataegus Pinnatifida extract with swimming on inhibiting inflammatory factors,improving blood lipids levels and the vascular endothelial function in rat model of atherosclerosis,and this may be associated with the gene expression of PAI-1,t-PA,COX-1 and vWF.It emphasizes the promising role of the drugs and blood shear stress,the biomechanopharmacological means,for the prevention of atherosclerosis.