| 1 BackgroundAtherosclerosis(As)is a common disease which seriously endangers human health,vascular endothelial injury and dysfunction caused by many risk factors are the key to the development of As.Fluid shear stress(FSS)is the shear stress parallel to the wall of the tube caused by the friction between the blood flow and the wall of the tube,fluid shear stress can protect vascular endothelial function and reduces inflammation of endothelium.Abnormal fluid shear stress such as low or oscillatory,will lead to endothelial dysfunction and induce endothelial inflammation,which can promote the development of of As.The occurrence of abnormal fluid shear stress is closely related to Qixu and Xueyu of TCM.Up to now,there is no very satisfactory drug to prevent the progression of As.At present,statins,surgical and interventional surgery are effective in the treatment of As,but also will sequelae associated with varying degrees of research,pointed out that statins significantly reduce the efficacy of LDL-C,but the effect of reducing LDL-C did not have a linear dose-response relationship,the dose increased 1 times,reduce the effect of the level of LDL-C can only increase 6%,and the adverse reactions such as increased liver toxicity and myopathy.Therefore,it is urgent to find a new method for the treatment of As.The theory of TCM and clinical practice suggest that Qixu Xueyu is the basic etiology and pathogenesis of As.The Chinese medicine of Yiqi,Buxue has the function of expanding the peripheral blood vessels,increasing the blood flow of the organs,and improving the blood concentration,adhesion,coagulation and accumulation.Danggui Buxue Decoction(DBD)has the effect of Buxue,Yiqi.Our previous studies showed that DBD has a good protective effect on endothelial cells,which mainly inhibits endothelial inflammation,promotes endothelial progenitor cell function and strengthens endothelial repair.The endothelial protection of multiple targets in DBD is similar to the anti As effect of physiological FSS.Therefore,the study of the effect of DBD on vascular endothelium in abnormal FSS environment will provide a more reliable experimental basis for the anti As research of DBD. 2 ObjectiveThrough the study of pharmacodynamics of DBD in the abnormal FSS environ-ment protection As vascular endothelial injury,to investigate the DBD on hemorheology,blood lipid,secretion of inflammatory factor,protein expres-sion and mechanism of DBD on the prevention and treatment of As,to provide a scientific basis for DBD in the treatment of As. 3 Methods 3.1 Replication and evaluation of As animal model induced by abnormal FSS30 New Zealand rabbits were reared for 2 weeks after 1 weeks of adaptation to the right carotid artery cannula.Before and 2 weeks after the operation,all the animals were detected by color Doppler ultrasonography of the right common carotid artery and the hemorheology.The FSS value was calculated,and the size of FSS of the right carotid artery before and after operation was compared.The difference of P<0.05 was significant.The abnormal fluid shear stress animal model was successful.30 rabbits with abnormal fluid shear stress were randomly divided into 2 groups according to body weight:the general feeding group(15 cases):fed to the normal diet;the high fat group(15):fed 2% high cholesterol diet.2 times a day,at the end of the 8,16 week,the hemodynamics of right common carotid artery in two groups was detected by ultrasonography,hemorrheology,blood lipid and endothelial function(serum ET-1)were detected,and pathological sections of bilateral common carotid arteries were observed.The vascular endothelial As lesion in the low fluid shear stress area of model animals was used to evaluate the cycle of abnormal FSS induced As. 3.2 Study on the effect and mechanism of DBD on As lesions in animal model of abnormal fluid shear stressAccording to 5:1,3:1,1:1,astragalus and angelica,weigh the compatib-ility proportion of crude drugs,two fried,condensed into water solution containing 1g/mL crude drug.After 1 weeks of adaptive feeding,120 New Zealand rabbits were randomly divided into 8 groups according to their weight,with 15 in each group.After all animals were anesthetized,the sham operation group did not put cannula and common feed,all the animals were given right common carotid artery cannula and fed with high fat for 4 weeks,then the drug is given as follows:A sham operation group:intragastric equal amount of saline;B control group:model animals intragastric equal amount of saline;C TCM group(5 groups):animal models gavage DBD;Qigui ratio 5:1 high dose(6g/kg)group,5:1 medium dose(3g/kg)group,5:1 low dose(1.5g/kg);Qigui ratio 3:1(4g/kg)group;Qigui ratio 1:1(2g/kg)group;D atorvastatin group:animal models gavage atorvastatin 1.7 mg/kg;intragastric dose according to the conversion of human and animal equivalent dose,daily gavage 1 times,administered for 12 weeks,daily feeding diets for 2 times,1.5% of body weight,body weight was measured every week 1 times to adjust the dosage,in 4 weeks,at the end of the 16 detection indicators:the hemodynamics of carotid artery pathological observation;blood rheology,serum ET-1 and NO detection;observation of carotid artery VCAM-1 immunohistochemistry staining,the expression of LOX-1 and NF-K Bp65 protein;identification and function of cultured peripheral blood EPCs rabbit. 3.3 Statistical analysisThe experimental data were processed with SPSS22.0 statistical software,and the data were expressed with mean standard deviation(X±s).The normal distribution data were tested by single factor variance analysis(ANOVA).If the variance was homogeneous and LSD was used,and the Tamhane ’s T2 method was used when the variance was not homogeneous.If Mann-Whitney U rank sum test is used for non normal distribution data,P<0.05 is considered to be statistically significant.4 Results 4.1 Replication and evaluation of As animal model induced by abnormal FSS 4.1.1 The hemorheological changes of the two groupsCompared with the general feeding group,the whole blood viscosity of the high-fat group increased significantly,the plasma viscosity increased,the erythrocyte deformability index decreased,the erythrocyte deformability was poor,and the blood viscosity increased. 4.1.2 The change of serum ET-1 content in two groupsThe serum ET-1 of rabbits in high fat group was significantly higher than that of the high fat group. 4.1.3 The changes of blood lipid content in two groupsCompared with the general feeding group,the serum level of TC,TG,LDL increased significantly(P<0.05)and HDL decreased in the high fat group(P<0.05). 4.2 Study on the effect and mechanism of DBD on As lesions in animal model of abnormal fluid shear stress 4.2.1 The change of FSSThe model group and the sham operation group,the end diastolic diameter increased,systolic peak velocity and diastolic blood flow velocity decreased significantly(P<0.05).There was no significant change in the end diastolic diameter of the treatment group compared with the model group,but the peak systolic velocity or the diastolic blood flow speed increased significantly(P<0.05). 4.2.2 The results of HE staining in the region of the low shear stress of the total arteryCompared with sham operation group,model group and proximal vascular cannula can see significant As plaques,plaque fibrous cap formation,which shows a large number of foam cells and lymphocytes infiltration,deep fibrous cap has a large number of cholesterol crystal deposition and necrosis,description model success;compared with the model group,treatment group 3:1 and group 1:1 only form a small part of AS plaque,AVT intimal thickening,partial endometrium is not smooth,the endothelial cells do not complete off phenomenon,while 5:1 treatment group without plaque formation,and membrane integrity. 4.2.3 Hemorheological resultsCompared with the sham operation group,the whole blood viscosity of the model group increased obviously,the plasma viscosity increased and the erythro-cyte deformability index decreased.Compared with model group,treatment group whole blood viscosity;plasma viscosity was decreased in different degree;which Qigui high dose of 5:1 group and AVT group decreased the most obvious effect of Qigui 5:1 dose group,erythrocyte deformation index only Qigui middle dose of 5:1 group and AVT group significantly increased(P<0.05). 4.2.4 Changes of blood lipid contentCompared with the sham operation group,the model group TC,TG,LDL-C were significantly increased,and HDL-C decreased significantly(P<0.05).Compared with the model group,TC,TG and LDL-C decreased to a certain extent in the treatment group compared with the model group.The TC content in the 5:1 group was the lowest,and the high-dose group was the most significant.HDL-C level was the most significant in the middle dose group.In addition,the content of LDL-C in the 5:1 group was significantly lower than that in the model group(P<0.05),but the decrease in the 3:1 group was not statistically significant compared with that in the AVT group. 4.2.5 Serum ET-1 contentCompared with the sham operation group,the ET-1 content in the model group increased significantly(P<0.05).Compared with the model group,the content of ET-1 in the 5:1 group and the AVT group decreased significantly compared with the model group,of which the high dose group decreased most obviously(P<0.05).Although the levels of serum ET-1 decreased in the 3:1 and 1:1 groups,the level of the decrease was not statistically significant. 4.2.6 Serum NO contentStatistical analysis showed that the results of serum NO in each group were similar,without statistical significance. 4.2.7 ICAM-1 detection of vascular tissueCompared with sham operation group,the ICAM-1 content in the model group increased significantly,and the difference between them was statistically significant(P<0.05).Compared with the model group,the content of ICAM-1 in the vascular tissue was significantly lower than that in the model group,and the difference was statistically significant(P<0.05). 4.2.8 Expression of VCAM-1 protein in blood vesselThe model group compared with the sham operation group,model group,vascular tissue VCAM-1 fluorescence intensity,high expression of VCAM-1 in plaque;the treated group compared with the model group,the fluorescence intens-ity of the vascular tissue VCAM-1 was significantly lower than model group;in treatment group in Qigui ratio of 5:1 group and AVT group was the strength is weak,the high dose group of 5:1 fluorescence intensity is the weakest,proved the expression of VCAM-1 at least,and the dose of 1:1 fluorescence in the treatment group in the strongest. 4.2.9 Expression of LOX-1 protein in blood vesselThe model group compared with the sham operation group,vascular tissue LOX-1 fluorescence intensity,high expression of LOX-1 in plaque;the treated group compared with the model group,the fluorescence intensity of the vascular tissue LOX-1 was significantly lower than model group;in treatment group in 1 groups of Qigui ratio of 5:1 fluorescence intensity decreased significantly the high dose group of 5:1 fluorescence intensity is the weakest,proved the expression of LOX-1 and 1:1 at least;dose group in treatment group in the strongest fluorescence intensity of AVT. 4.2.10 Expression of NF-kappa Bp65 protein in blood vesselThe model group compared with the sham group,NF-kappa Bp65 protein fluorescence intensity group vascular tissue model significantly,the expression of NF-Bp65 protein was highly expressed in plaque;the treated group compared with the model group,the fluorescence intensity of NF-kappa Bp65 protein in vascular tissue was lower than that in the model group;treatm-ent in the group of Qigui the proportion of 5:1 group and AVT group,the fluorescence intensity is weak,the fluorescence intensity of group AVT was that the expression of NF-K Bp65 protein was the least,and the dose of 1:1 fluorescence in the treatment group in the strongest. 4.2.11 Identification of EPCs by fluorescent stainingThe sixteenth weekend,rabbit peripheral blood was extracted,and EPCs was isolated and cultured for 5-7 days,we used FITC-UEA-I,DiI-acLDL and DAPI respectively on the isolated cells were stained with fluorescence microscopy,showed green fluorescence for UEA-I positive cells,red fluorescence for DiI-acLDL positive cells,blue fluorescence the three is the nucleus staining cells are differentiated EPCs. 4.2.12 Effect of DBD on EPCs function in peripheral blood of rabbitsIn the model group compared with normal group EPCs colony number,migration and tubule decreased(P < 0.05);treatment group and model group except 1:1 group,other groups of EPCs colonies increased,and EPCs migration and small tube were enhanced in different degrees;the most obvious high dose of 5:1 group and AVT group and 1:1 treatment group effect,the worst. 5 Conclusion and discussion 5.1 Replication and evaluation of As animal model induced by abnormal FSSIn this study,an abnormal FSS animal model was established by unilateral common carotid artery cannula.That special fed high fat diet for 8 weeks.After 16 weeks,the general feeding group and high fat group right carotid artery atherosclerotic plaque formation,and in the proximal end and low shear force should be the most obvious side plaque,and aggravated over time,but did not appear in the left common carotid artery atherosclerotic plaque.Because the experiment was made on the left side of the control,with strong comparability,in addition to shear stress in different conditions,other high fat group on both sides of the common carotid artery(including hyperlipidemia in arteries)are the same,so we can take the right carotid As attributed to abnormal shear stress and the pathological changes.The results suggest that the proximal end and low shear stress should be the most obvious side of the plaque,and other scholars’ research reports are consistent,we can draw the following conclusions:abnormal shear stress can produce atherosclerotic les-ions,including hyperlipidemia and low shear atherosclerotic plaque formation is the most obvious.The study also found that fed high fat diet after 16 weeks and 4 lipid indicators in rabbits is significantly increased,shear stress decreased significantly compared with ordinary feeding group,high fat diet increased blood viscosity,increased serum ET-1 levels,suggesting that As hyperlipidemia on abnormal shear stress model to promote and predict effect. 5.2 Study on the effect and mechanism of DBD on As lesions in animal model of abnormal fluid shear stressIn this study,we established the abnormal FSS animal model of atheroscle-rotic lesions and used different Qigui ratios of DBD to carry out drug intervention.It was found that DBD could effectively improve the formation of atherosclerotic plaques induced by low shear stress.DBD can effectively regulate blood lipids,improve hemorheological changes,reduce ET-1 production and increase blood flow velocity,thereby restoring the shear stress in low shear stress area.In addition,DBD effectively inhibited the expression of ICAM-1,VCAM-1,LOX-1 and NF-kappa B protein in the vascular tissue of low shear stress region.We also found that DBD could effectively improve the function of EPCs by separating and extracting the peripheral blood endothelial progenitor cells of the rabbit.The results showed that the effect of Qigui ratio 5:1 group was the most obvious,and the effect decreased with the decrease of Qigui ratio.These results demonstrate that DBD functions in the abnormal shear stress environment and anti As function may regulate blood lipids,improve blood rheology changes,improve blood flow,closely related to the protection of EPCs and anti-inflammatory effects,and the effect of different Qigui ratio has obvious difference,the ratio of Qigui in 5:1 group the most obvious effect. 6 Innovation pointThe protective effect of DBD on vascular endothelial function in abnor-mal FSS environment was first discussed,and a new idea of biomechanics combined with TCM against As was developed. 7 Deficiencies and prospectsNow with the rapid development of various disciplines,the modern study of DBD in the treatment of As is constantly learn new ideas and techniques,because of my limited ability,in addition effect of DBD has multi-target multi way,may play a role in the treatment of As by other means,needs further study. |
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