The Decoupling Of Circulating Microparticles And ENOS In The Early Mechanism Of AS And The Intervention Study Of Qiyu Jiedu Tongluo Decoction

BackgroundAtherosclerosis is the basic disease of various clinical diseases.Lipid lowering drugs do not block the development and outcome of atherosclerosis.The incidence of cardiovascular and cerebrovascular diseases,based on atherosclerosis,is increasing.Therefore,it is of great practical value and scientific significance to explore new pathological mechanism of atherosclerosis and effective intervention.Circulating microparticles have been neglected in the past as cell garbage.In recent years,it has been found to play an important role in the early stage of atherosclerotic lesions and has the potential to be a new marker for predicting the development of cardiovascular diseases.Endothelial dysfunction plays a key role in the pathogenesis of atherosclerosis.The endothelial nitric oxide synthase(eNOS)decoupling leads to the barrier of protective nitric oxide(NO)synthesis and oxidative stress,which is the fuse of endothelial dysfunction,which further promotes the development of atherosclerosis.The most important factor of eNOS decoupling is four hydrogen biopterin(BH4)deficiency or increased oxidation.In recent years,studies have shown that circulating microparticles can promote endothelial dysfunction through eNOS related pathways.However,it is not reported whether circulating microparticles can promote AS through BH4 dependent eNOS decoupling.Huo Tan Jie Du Tong Luo Yin Decoction is an effective prescription for the treatment of atherosclerosis,and it can improve the function of endothelium.Whether this prescription can improve the mechanism of atherosclerosis by regulating circulating particles needs further study.Objective1 To screen recycling particles which is related with the pathological progress of AS in early carotid atherosclerosis(CAS),evaluate its diagnostic value and provides the evidence as a new biological marker for the evaluation of the AS disease process.2 To estimate the endothelial function of early CAS population,analyze the correlation between endothelial nitric oxide synthase(eNOS),tetrahydrobiopterin(BH4)and blood lipid and cardiovascular risk correlative blood count indicators.3 To investigate the effect of recycling particles on the coupling of eNOS and the mechanism of early AS formation.4 To build up the early AS model and estimate it with ultrasound and pathology,then use this model to study the Huotanjiedutongluo decoction on improvement of early AS and the intervention effect on recycling microparticles/BH4/eNOS uncoupling/ROS pathways.Method1 All the volunteers are from physical examination center of Beijing University Of Chinese Medicine.According to include-exclusion standard,we select 23 early CAS patients and 22 healthy subjects,all of them volunteer for the experiment after informed consent.We collect patients peripheral blood 3ml since morning on an empty stomach and then centrifuge it gradiently to extract recycling particles.Use BD FACS Canto Ⅱ flow cytometry instrument to detect five kinds of recycling particles,including the expression of white blood cells derived particles(LMP)CD45,CD11a and CDlla+/CD45+,platelet particles(PMP)CD31+/CD42b+ and endothelial particles(EMP)CD31+ CD42b-.Carotid ultrasound is used to measure the thickness(CIMT)and the maximum patch area.We carry out the correlation analysis and diagnostic test of the carotid artery ultrasound index and five kinds of particles.The diagnostic sensitivity and specificity of the evaluation of the AS progress indicators were evaluated.2 The subjects are the same as above.We use nitrate reduction method,enzymatic method,ELISA in peripheral blood to detect the expression of nitric oxide(NO)levels,TG CHOHDLLDL in early carotid atherosclerosis patients and endothelin 1(ET 1),eNOS,BH4.Automatic blood cell analyzer is used to measure blood count level.3 According to assessment of particles numbers,we choose the Kunming mouse(20-22g,Male,36)and randomly divide them into four groups,including CAS particles,healthy particles,saline,BH4(ig 3d,10mg/kg),each group of nine.The mice are sacrificed when particles are injected through the tail vein of them 24h later.HE staining observes vascular pathological structure,western-blot detects the protein level of eNOS dimer/monomer,GTPCH-1,ETRA,ETRB in mice’s circulatory organizations,immunohistochemical tests the expression of ETRA,ETRB,a-actin,PCNA,nitrate reduction method detects the expression of NO in mice’s circulatory organizations and ELISA tests ROS,BH2,iNOS of supernatant of cardiovascular tissues which are homogenated.4 The early AS model is made with Japanese big ear white rabbit(1.6～1.8kg,male).We feed rabbits with high fat and operate with endometrial nitrogen drying and carotid balloon strain,then use ultrasound and pathology to evaluate the model.According to pressure level of balloon and weights,we randomly divide rabbits into four groups,including model,huotanjiedutongluo decoction,atorvastatin,sham-operated.Oil red staining is used to evaluate the aortaventrali’s lipid deposition,peroxidase method for TC,TG,HDL,LDL,elisa for serum of ox-LDL and BH4,HE staining for the morphological indexes of vascular pathologic changes and histological internal diameter,flow cytometry for recycling particles,biochemical technology for serum of ROS,western-blot for eNOS dimer/monomer of circulatory organizations.Result1 The number and%parant of white blood cells derived particles CD45+LMP/μl,CD 11 a+LMP/μl and CD 11 a +/CD45 +LMP/μl,platelet particles CD31 +/CD42b +PMP/μl in early CAS group is significantly higher than healthy group(p<0.01),but there is no statistically significant difference between two groups of CD31 +/CD42b-EMP/μl and%parant.In early CAS group,the number of CD45 +LMP/μl,CD11a+LMP/μl,CD11a +/CD45 +LMP/μl and CD31 +/CD42b +PMP/μl have no linear correlation with age,gender,complications(p>0.05)while its have positive correlation with maximum plaque area.Moreover CIMT have positive correlation with the number of CD 11 a+ LMP/μl,CD11a+/CD45+MP/μl.Areas under the ROC curve of CD45+LMP/μl,CD11a+ MP/μl,CDlla+/CD45+MP/μl and CD31+/CD42b+ MP/μl are respectively 0.689(95%CI,0.531-0.847,p = 0.030),0.747(95%CI,0.604-0.890,p=0.005),0.741(95%CI,0.596-0.886,p =0.006)and 0.701(95%CI,0.545-0.856,p=0.021),and the diagnostic points are 4.07,2.73,2.15 and 7.64.The sensitivities are 61%,57%,57%,61%and the specificities are 77%,86%,86%and 82%.2 The expressions of eNOS and BH4 in early CAS group are significantly lower than healthy group and the difference is statistically significant(p<0.05).The expressions of total cholesterol,LDL,TC,WBC,PLT in early CAS group are significantly higher than healthy group and the difference is statistically significant(p<0.05).The expressions of eNOS have negative correlation with total cholesterol,LDL,WBC,PLT,PLR,NEUT in early CAS group.The expressions of BH4 have positive correlation with RBC in early CAS group.3 The eNOS monomers in the circulation tissues(heart and blood vessels)of saline mice are rarely expressed,however in the other three groups it increased.Injecting with recycling particles of CAS and healthy groups can significantly induce the eNOS coupling of the cardiovascular system in mice.The performance of eNOS dimer/monomer is significantly reduced(Comparison with saline control group,p<0.01,p<0.05).Compared with healthy group,the ratio of eNOS dimer/monomer is significantly reduced in early CAS group which explain that recycling particles in early CAS group can cause a greater degree of eNOS decoupling.Feeding with BH4 can significantly reverse the uncoupling of eNOS in cardiovascular system caused by the recycling particles in the early CAS patients(p<0.05).There is no statistical difference in GTPCH-1 expression in the four groups of mice.(p>0.05).Compared with saline group,the expression of ROS in mice organization is highly increased in early CAS group and healthy group(p=0.006,p=0.000).The expression of iNOS in early CAS group,healthy group and BH4 group is more than in saline group(p=0.045,p=0.000,p=0.000).In addition,the expression of iNOS in mice with normal microparticles group was significantly higher than that in the group of mice with early CAS(p=0.019).The expression of iNOS in mice with BH4 group was significantly higher than that in the group of mice with early CAS and normal microparticles group(p=0.000,p=0.000).The content of NO in early CAS,healthy and BH4 group is highly increased,compared with saline group(p=0.001,p=0.000,p=0.001).The expression of BH2 is remarkably increased in Healthy and BH4 group(p=0.005,p=0.001).However,recycling particles have little effect on ETRA、ETRB、α-SMA and PCNA and HE staining shows that the vascular structures of each group have no abnormal changes.4 Use high resolution ultrasonography to evaluate the AS model we find that intima-media thickness and end diastolic velocity in model group are increased remarkably than sham-operated group(p<0.01,p<0.05)and there are local scattered plaques informed.According to HE staining,we find that the Pathological features of this model are intimal and adventitia thickening,a large amount of foam cell deposition in the middle membrane but no significant stenosis is caused[stenosis rate(25.59±10.11)%].This model conforms to the pathological features of early atherosclerosis and it is suitable for the interventional study of early AS vascular remodeling.5 To count the daily intake of high-fat feed in the model,huotanjiedutongluo and atorvastatin group,we find that there is no statistically significant difference in average daily intake(p>0.05).The survival rate of the model and atorvastatin group is 80%and 46.7%,while the survival rate is 81.8%and 85.7%in huotanjiedutongluo and sham-operated group respectively.The positive area of aortic oil red staining is significantly increased in model group(Comparison with sham-operated group,p=0.002).Compared with model group,the positive area of aortic oil red staining is significantly decreased in huotanjiedutongluo group(p=0.048).The atorvastatin group has a tendency to decrease lipid deposition,but the difference is not statistically significant(p=0.172).Compared with model group,the lipid adhesion of liver in huotanjiedutongluo and atorvastatin group is significantly reduced,the hardness decreased,and the coloris more normal.TC,TG,HDL,LDL and OX-LDL in model group are evidently higher than that in sham-operated group(p<0.01).Huotanjiedutongluo decoction can significantly reduce the serum TC and OX-LDL level in rabbits(p<0.05).Compared with sham-operated group,the intimal thickness,intimal area,stenosis rate and adventitial thickness are all significantly increased in model group(p=0.008,p=0.009,p=0.008,p=0.022)while the huotanjiedutongluo decoction can reduce all of them(p=0.037,p=0.048,p=0.042,p=0.021).The CD 11 a+LMP of plasma in model group is higher than in sham-operated group(p<0.01)and Huotanjiedutongluo decoction atorvastatin can lower the level of it(p<0.05).Atorvastatin decoction has a tendency to decrease it but the difference is not statistically significant(p=0.147).Comparison with sham-operated group,eNOS dimer/monomer decreased evidently in model group(p<0.01)and Huotanjiedutongluo and atorvastatin has a tendency to increase it but the difference is not statistically significant(p=0.084,p=0.106).Comparison with sham-operated group,ROS increased evidently in model group(p=0.007)and Huotanjiedutongluo decoction can decrease it in early AS model(p=0.043).Discussion1 LMP CD45+MP/μl,CD11a+ MP/μl,CDlla+/CD45+MP/μl and PMP CD31+/CD42b+MP/μl have potential to predict the development of carotid atherosclerosis as new biomarkers,which have good sensitivity and specificity.2 BH4 related eNOS decoupling may occur in the circulation of early CAS patients.The white blood cell count,platelet count,total cholesterol,triglyceride and low density lipoprotein in group CAS were significantly higher than those in healthy people.The correlation between eNOS level and blood lipids and blood cell counts showed that the increase of serum lipids,inflammation and thrombocytosis were closely related to BH4 related eNOS decoupling.3 BH4/eNOS uncoupling/ROS and iNOS/NO/ROS.Compared with healthy people,recycling particles of early CAS patients result in greater degree of eNOS uncoupling,which is one of the causes of endothelial dysfunction in early AS pathological process.4 Huotanjiedutongluo decoction reduce the level of ox-LDL,area of lipid deposition,intimal and adventitial thickness and stenosis rate of lumen in the early model of rabbit AS.In terms of the mechanism,it has the tendency to reduce the expression of CD 11 a+ LMP,and can improve the eNOS uncoupling in the model of rabbit.