Establishment And Evaluation Of Coronary Heart Disease Model Of Qi-deficiency And Blood-stasis Syndrome Type In Rats

Research BackgroundCoronary Heart Disease(CHD)is the coronary atherosclerotic heart disease’s abbreviation, also known as ischemic heart disease.Cardiovascular disease is the most serious disease which causes of human death and disability. The heart disease develops when the coronary functional changes (cramps) or systemic atherosclerosis involving the coronary arteries, leading to stenosis or obstruction.The disease were describedwithmanytermssuchasthoracicobstruction,cardialgia,chestcomplaint,cardia construction and etc. TCM syndrome type of coronary heart disease can be classified in three major categories:deficiency in origin-excess in superficiality,deficiency in origin, excess in superficiality.The deficiency in origin-excess in superficiality is a common syndrome type. In recent years, changes in the way of Chinese people life lead to a lot of deficiency of Qi and blood stasis syndrome. The proportion of this two types of syndrome elements in coronary heart disease is gradually increasing.There have some progress about animal model for the study of Coronary heart disease (CHD) deficiency of blood stasis syndrome at present, but it still not mature enough in the model methods and model evaluation system, there have no standardized model for the establishment of recognized standards and evaluation methods. It is urgent to establish a highly stable, reproducible animal model,so that we can study the pathogenesis of Coronary Heart Qi and Blood and its material basis syndrome. Therefore,on the basis of previous studies, this study explore qi deficiency blood stasis syndrome animal model of coronary artery disease to establish and build the evaluation system. This study has important significance in theory and application for revealing syndromes material base, the development of new drugs and effective traditional Chinese medicine treatment of the coronary heart disease screening, clarify mechanism of traditional Chinese medicine compound function.Part 1 Exploration on the establishment of Coronary Heart Disease model of Qi-deficiency and blood-stasis syndrome type in rats1.ObjectiveTo establish a method for coronary heart disease in rats with qi deficiency and blood stasis of the stable by comparing different model establishment methods.2.Materials and methodsMale WISTAR rats were randomly divided into six groups:A control group (Con):Normal food and water n=8; B. Sham operation group(Sham):thoracotomy, threading but without ligation, normal food and water n=8; C. ligation of coronary artery group (L):normal feeding two weeks and then ligation the left anterior descending coronary artery n=8; D. Multifactor composite model group:dl:Exercise fatigue combine ligation of coronary artery group (EL):through two weeks of treadmill running built sports fatigue model in rats+coronary ligation surgery,every other day continue running after 3 days recovery n=8; d2:Diet combine ligation of coronary artery group,(DL):by 2 weeks diet caused qi deficiency of spleen and stomach deficiency of general model in rats+coronary artery ligation surgery continues to control diet after 3 days recovery n=7; d3:Diet, exercise fatigue combine ligation of coronary artery group,(DEL):through two weeks control diet, treadmill exercise cause systemic blood stasis model in rats+coronary artery ligation surgery control diet and every other day continue running after 3 days recovery n=5.After 6 weeks control,macroscopic signs of rats through collecting echocardiographic detection index (ejection fraction, fractional shortening),breathing extent to evaluate the rat qi deficiency blood stasis primary symptom of CHD;collecting empty field experiments related indicators (movement distance, average speed, exercise time), exhaustive exercise time, foot on the image color saturation to evalate accompanied symptom;gathering the lingual color saturation to evaluate the tongue;gathering pulsation amplitude of the pulse to evaluate pulse condition.At the same time detection the structure and function of the heart, vascular endothelial function, coagulation, energy metabolism, the heart muscle tissue and other related indicators.3.Results3.1.1 State of the body:Compared with SHAM group,the DL, EL> DELs’weight decreased 38.3%,10.6%,45.3%(P<0.01), L group rats compared with SHAM group rats the weight decreased 2.6%(P>0.05);compared with L group,the DL, EL DELs’weight decreased 36.6%,8.2%,43.9%(P<0.05 P<0.01)3.1.2 Cardiac function:Compared with SHAM ejection fraction,the DL,EL,DEL and L group decreased 71.4%,62.4%,71.6%,49.4%(P<0.01) fractional shortening decreased 77.2%,69.2%,77.5%,57.9%(P<0.01),left ventricular end-diastolic diameter expanded 123.9%,122.6%,124.4%,83.6%(P<0.01),left ventricular end-systolic diameter expanded 76.7%,99.3%,83.7%,50.7%(P<0.01),left ventricular end-diastolic volume expanded 525.7%,515.9%,522.6%,303.8% (P<0.01),at the end of the left ventricular contraction volume expanded 525.7%, 515.9%,522.6%and 303.8%(P<0.01);compared with L group rats shortening fractional DL and DEL group decreased 45.9%,46.6%(P<0.01), EL group decreased 26.9%(P>0.05),DL, EL, DEL group compared with L group ejection fraction decreased 43.4%,25.6%,43.9%(P<0.05 P<0.01), left ventricular end-diastolic diameter expanded 7.8%,14%,9.9%(P<0.05 P<0.01), left ventricular end systolic diameter expand 22%,21.2%,22.2%(P<0.01),left ventricular end diastolic volume expanded 17.3%,32.3%,21.9%(P<0.05 P<0.01),left ventricular end-systolic volume expanded 55%,52.5%,54.2%(P>0.05)3.1.3 Biological indicators:Compared with SHAM group,other four model groups’content of BNP、NT-proBNP、ET、ATP synthase increased obviously (P<0.05 P<0.01),the content of NO decreased obviously (P<0.01).compared with SHAM group,the 6Keto-PGF1α/TXB2 of DL、EL、DEL decreased obviously (P<0.05 P<0.01),the L’s decreased (P>0.05).Compared with L group,the EL、DL、DEL’s content of BNP、ET、ATP synthase increased obviously (P<0.01),the content of NO and 6Keto-PGFla/TXB2 decreased obviously (P<0.01)3.1.4 Myocardial histopathologic observation:By histological staining and electron microscopy, Con, Sham have normal myocardial tissue, the rest of the model have different degrees of pathological changes.3.1.5 Sympotoms:Compared with SHAM group,the primary symptom、 accompanied symptom、tongue picture and pulse of the other four model groups performanced in different level.EL、DL、DEL performanced more obviously than L.The index related the primary symptom、accompanied symptom、tongue picture and pulse had changed (P<0.05 P<0.01)4.Conclusion(1)Both the method of L(ligation of coronary artery)and DL, EL, DEL(multi-originated information complex)can be successfully established Coronary Heart Disease model of Qi-deficiency and blood-stasis syndrome type in rats.(2) Model set up by using the compound factors (DL, EL, DEL), and through simple ligation of coronary artery to build model (L) to compare the clinical etiology and linked,DL, EL and DEL are more closely with relevant theoretical system of TCM and have a certain advantage in complete reflect the characteristics of TCM syndrome.(3) Among three kinds of compound factors model, EL(Exercise fatigue combine ligation of coronary artery) model compared with other two model DL,and DEL is more consistent with clinical practical reasons and characteristics of the disease.Through EL the CHD deficiency of blood stasis rat model of combined disease can be controlled and good repeated.Part 2.Research the development process of coronary heart disease of qi deficiency and blood stasis and the intervention effect of drugs for supplementing qi and activating blood circulation1.ObjectiveTo observe the dynamic model is established for three different stages of disease development process and use drugs counterevidence approach to evaluate the rat model, further to discuss the modeling method of reliability and stability.2.Materials and methodsMale Wistar rats were randomly divided into 11 groups, the first stage is 2 weeks after intervention, the rats were divided into three groups:A.control group2(Con2) n=8;B.model group2(M2) n=7;C.drug intervention group2(D2):exercise fatigue model based on intragastric administration of SSTG 22.5mg/kg per day 1 times, lasting 2 weeks n=8. The second stage is 4 weeks after intervention, rats were divided into 4 groups,n=8:A.control group4(Con4);B.Sham operation group4 (Sham4);C. Model group4(M4); D.drug intervention group4(D4):coronary artery ligation surgery 24 hours after intragastric administration of SSTG 22.5mg/kg,1 times a day, for 2 weeks. The third stage is 6 weeks after the intervention, the rats were divided into 4 groups,n=8:A.control group6(Con6); B.sham operation group6(Sham6);C.model group6(M6);D.drug intervention group6(D6): coronary artery ligation surgery 24 hours after intragastric administration of SSTG 22.5mg /kg,1 times a day, for 4 weeks.After the end of each phase, the test method of the index was the same as the first part of the experiment.3.Results3.1.1 State of the body:2weeks,compared with CON2 group,M2’s weight decreasd 12.5%(P<0.01);compared with M2,D2’s weight increased 10.7% (P<0.05).4weeks,compared with SHAM4 group,M4’s weight decreasd 14.4% (P<0.05);compared with M4,D4’s weight increased 11% (P>0.05).6weeks,compared with SHAM6 group,M6’s weight decreasd 10.6% (P<0.01); compared with M6,D6’s weight increased 8.3%(P<0.05)3.1.2 Cardiac function:2weeks,compared with CON2 group,M2’s ejection fraction^ fractional shortening decreased 7.6%、10.9%(P>0.05),left ventricular end-diastolic diameter decreased 6.2%(F<0.01),left ventricular end-diastolic volume decreased 13.6%(P<0.01);compared with M2,D2’s ejection fractions fractional shortenings left ventricular end-diastolic diameters left ventricular end-diastolic contraction volume increased 5.6%、8.6%、3.9%、9%(P>0.05)4weeks,compared with SHAM4 group,M4’s ejection fraction-, fractional shortening decreased 46.1%、54%(P<0.0\), left ventricular end-diastolic diameters left ventricular end-systolic diameter increased 27.4%、78.2%(P<0.01), left ventricular end-diastolic volume、left ventricular end-systolic volume increased 71.2%.267.2%(P<0.01); compared with M4,D4’s ejection fraction, fractional shortening increased 6.9%.8.2%(P>0.05),left ventricular end-diastolic diameter decreased 6.2%(P<0.05),left ventricular end-systolic diameter decreased 7.1% (P>0.05),left ventricular end-diastolic volume. left ventricular end-systolic volume decreased 12.7%.14%(P>0.05).6weeks, compared with SHAM6 group,M6’s ejection fraction n fractional shortening decreased 54,5%、60.1%(P<0.01), left ventricular end-diastolic diameter left ventricular end-systolic diameter increased 20.9%.70.1%(P<0.01), left ventricular end-diastolic volume. left ventricular end-systolic volume increased 53%、 243.5%(P<0.05 P<0.01); compared with M6,D6’s ejection fraction, fractional shortening increased 28.9%、29.4%(P>0.05), left ventricular end-diastolic diameter. left ventricular end-systolic diameter decreased 0.6%.7%(P>0.05), left ventricular end-diastolic volume、left ventricular end-systolic volume decreased 1.4%、17.4% (P>0.05).3.1.3Biological indicators:After 2weeks.4weeks.6weeks, compared with control group,M group’s content of BNP、NT-proBNP、ET、ATP synthase increased obviously (P<0.01),the content of NO decreased obviously (P<0.01).Compared with M group,D’s content of ET. BNP. NT-proBNP. ATP synthase decreased obviously (P<0.01),the content of NO increased obviously (P<0.01)3.1.4 Myocardial histopathologic observation:After tissue staining and electron microscope observation, the three stages of Con, Sham group of normal myocardial tissue, M group of myocardial pathological changes in different degrees. Group D myocardial changes between Con group and M group, the degree of pathological changes to reduce.3.1.5 Sympotoms:With the time of the model estblshment extended,the model group’s primary symptom. accompanied symptom. tongue picture and pulse performanced more and more obviously.SSTG could improve the representation of primary symptom, accompanied symptom and pulse.4.Conclusion(1) Starting modeling 2 weeks later, the way of running to establish the model of exercise induced fatigue began to perform Qi deficiency and blood stasis syndrome and related symptoms. After 4 weeks, coronary heart disease (CHD) rat model of qi deficiency and blood stasis syndrome is more obvious; after 6 weeks, rat model of coronary heart disease can discriminate syndrome of qi deficiency and blood stasis syndrome.(2) The development of the disease affected abnormal energy metabolism and then expanded to myocardial cell injury; coronary artery injury resulting in the destruction of the endothelial cells had the adverse effects on normal vascular function maintenance; with the extension of duration of diseases, further aggravating the myocardial damage, resulting in the occurrence of myocardial infarction and myocardium showed serious lesions affecting cardiac systolic function, coagulation function with obstacles.(3) SSTG can make a good improvement in exercise tolerance, endothelial function, energy metabolism of the CHD of Qi-deficiency and blood stasis in rats.In conlusion,on the basis of comparing the four kinds of different modeling metods of CHD of Qi-deficiency and blood stasis in rats and we got a reliable and stable method.Dynamic observe the disease pathology evolution process,we used SSTG to intervene the model rats,verified the model reliability and the efficacy of SSTG.