| Purpose:The experiment study through the establishment of the heart failure after myocardial infarction rats models,observed the Chinese drug formulae of invigorating Qi and promote blood circulation for the effect of of ventricular remodeling and its relevant factors in the heart failure rats after myocardial infarction,matrix metalloprotein ases-3(MMP-3),tissue inhibitor of metalloproteinase-3(TIMP-3),osteopontin(OPN),transforming growth factor-?1(TGF-?1)and connective tissue growth factor(CTGF).To study the mechanism and target of formulae of invigorating Qi and promote blood circulation from the aspects of tissue morphology,protein and gene,to develop new ideas and provide new strategies for the prevention and treatment of heart failure in traditional Chinese medicine.Material and method: 1.TO choose weight of 230?260 grams,and the SPF 12 male healthy one hundred SD rats.Randomly selected the 10 rats that were not treated with any treatment were blank control group.The 90 rats were treated with adaptive feeding for 1 week,and were injected with 10% Chloral hydrate 0.3?0.4ml/100 g intraperitoneal injection anes thesia.Supine fixed,used LED tongue depressors,pressed down on the root of the ton gue,a glottis of the respiratory rhythm can be seen.When the glottis opened,used 12 rat gavage needle inserted the airway,and connected the small animal ventilator for artificial respiration(the frequency was 70 times/min,the tidal volume was 6?8ml,the breathing time ratio was 1:1).After chest preparation,the needle electrode was injected into the limbs and chest wall of the rat,and the small animals electrocardiogram machine was connected to record the electrocardiogram.On the leftrib between 4 open chest,blunt separation chest muscles,exposed the ribs,blunt separation muscles between the ribs,the thymus,broke the pericardium and the starting point in the left an terior descending coronary artery in 2mm with No.6 suture line was the anterior desce nding coronary artery ligation.The heart surface was given to lidocaine injection 0.1m l to prevent ventricular arrhythmias,resetting the heart to the chest and suturing the chest wall.To recorded the postoperative electrocardiogram.The postoperative electrocar diogram showed the clearly st-segment elevation,which was determined as a myocardial infarction.Continuous injection of penicillin sodium 40000U/rat,continuous 3d,to p revent infection.After regular feeding for 1 week,the feed was cut in half,and daily swam to its exhaustion(The rats in the pool of the 1.5m long,1.0m wide,about 0.5m water depth,water temperature 32?34?).The rat's head sunk completely into the water andt he limbs did not swing clearly,judgment for exhaustion),1 time/d for 4 weeks.Echocardiography was performed to detect the ejection fraction(EF)of rats,and the cardiac index(CI)was calculated.The criteria for the successful model of heart failure were EF?45% or CI?180ml·min-1·kg-1.In the process of building model to the 90 rats,because of anesthetic deaths to the 3 rats,the 10 rats deaths in the oper ation,the 17 rats deaths after the operation,the 8 rats deaths for exhausted swimming correlation.2.According to the random number table method,the successful rats were divided into the model group 18 rats,formulae of invigorating Qi and promote blood circulation group 17 rats,the lisinopli group 17 rats(at the end of the experimen,ten rats were killed in each group).The blank control group and the model group rats were given with distilled water to saline irrigation,3m L/time,1 time/d.The dose formula of the human dose was converted into the dose formula of the rat,in the all groups.The lisinopril group was given to lisinopril 1.5mg/Kg/d,formulae of invigorating Qi and pro mote blood circulation group was given 9.2g raw Chinese drug/Kg/d.The two groups drug were dissolved in 3m L distilled water to gavage stomach with medicine 1 time/d.The four groups were to gavage stomach with 4 weeks.During the treatment,to clo sely observe general situation of the rats in all groups,including: hair color,skin,mental state,appetite,feces,urine,edema and tongue color,etc.During the treatment,th ree rats died in the model group,two rats died in the lisinopril group,two rats died in ormulae of invigorating Qi and promote blood circulation group.During the experiment,there was no death in the blank control group.General situation,the blank control group rats showed good mental state,lively and active,the appetite was exuberant,the hair was bright,the skin was pliable and elastic,the limbs were not edema,urine output and feces were normal.The model group rats showed spirits drooping,slowed response,loss of appetite,hair dry draft,skin flabby inelastic,limb edema,urine volu me decreased significantly,defecate rare pond,tongue color dark reddish purple,with ecchymosis,petechiae,etc.Two drug groups rats showed not inactive,less sensitive,slightly reduced appetite,hair no luster,less skin flabby,limb mild edema,slightly reduce of urine,stool sticky soft material,tongue color dark reddish purple,no ecchymos is,petechiae,etc.3.End of the treatment,the cardiac structure and cardiac functional changes of rats were detected by echocardiography(ten rats were randomly selected from the model group,the lisinopril group and invigorating Qi and promote blood circulation group,and comparative analysis was conducted with the blank control group),including: left ven tricular end-diastolic systolic(LVIDs),left ventricular end-Diastolic dimension(LVIDd),ejection franction(EF),fractional shortening(FS),indicators such as.Then all the rat s were killed and their heart muscle tissue was taken.4.The myocardial tissue of the rats was observed by electron microscopy.5.The expression of MMP-3,TIMP-3 and OPN in the myocardial tissues of the rats was detected by immunohistochemistry.The expression levels of MMP-3,TIMP-3,OP N,TGF-?1 and CTGF in the myocardial tissues of the rats were determined by realtime quantitative fluorescence PCR and Western blot.6.The statistical methods: SPSS 18.0 statistical software was used for data analysis,the measurement data was expressed in(X—±S),univariate analysis of variance was used in multiple groups,SNK-q test was used for comparison between groups,P<0.05 was considered to be statistically significant.Results: 1.Heart structure and cardiac function of the rats in the groups Compared with the blank control group,LVIDs and LVIDd in the model group and the two treatment groups(formulae of invigorating Qi and promote blood circulation group,lisinopril grou p)increased significantly,and the level of EF and FS significantly decreased,P<0.05,which was statistically significant.After the drugs intervention,LVIDs and LVIDd of the two treatment groups(formulae of invigorating Qi and promote blood circulation group,lisinopril group)were significantly lower than the model group,but EF and FS were significantly increased,P<0.05,which was statistically significant.2.Under electron microscope to observe myocardial tissue ultrastructure of the rats in the groups The blank control group: the myocardial fibers arranged neatly,the myofilament was orderly arrangement,the myocardial horizontal lines were clear,the mitochondrial morphology was normal,structural integrity,the mitochondrial membrane structure was clear,the internal mitochondria crest was very tight,invisible swelling and cavitation.The model group: the myocardium was severely damaged,the myofilament was loose and fractured,the myocardial joint was incomplete,the myocardial horizontal line s were not clear,some of the mitochondria were swollen and broken,visible double membrane fusion,blurred and visible cavitation,the mitochondrial crest fracture and fusion,partial disappearance.Formulae of invigorating Qi and promote blood circulation group:myofilament wa s roughly Orderly arrangeme,the partially myofilaments was disorganized,the myocard ial joint was roughly complete,the horizontal lines were slightly blurred,the mitochon dria was closely spaced,the swelling was significantly reduced compared with the mo del group,the cavitation was significantly reduced compared with the model group,the crest structure was clear,occasionally,the mitochondria dissolved and disappeared.The lisinopril group: the myofilament was roughly orderly arrangement,the partially myofilaments was disorganized and loose,the myocardial joint was roughly complet e,the horizontal grain was not clear,the mitochondria was closely spaced,the swellin g was significantly reduced compared with the model group,the cavitation was signifi cantly reduced compared with the model group,the mitochondrial crest structure was clear,individual mitochondria dissolved and disappeared.3.The expression of the rats myocardial tissue MMP-3 and TIMP-3 in the groups 3.1 Protein and mRNA expression of MMP-3 and TIMP-3 of the rats myocardial tissue in the groups: compared with the blank control group,MMP-3 mRNA and protein expression in the model group and the two treatment groups(formulae of invigorating Qi and promote blood circulation group,lisinopril group)increased significantly,P<0.05,which was statistically significant.After the drugs intervention,MMP-3 m RNA an d protein expression of the two treatment groups(formulae of invigorating Qi and promote blood circulation group,lisinopril group)were significantly lower than the model group,and P<0.05 was statistically significant.Compared with the blank control gro up,the expression of TIMP-3 mRNA and protein in the model group and the two tre atment groups(formulae of invigorating Qi and promote blood circulation group,lisino pril group)significantly decreased,and P<0.05 was statistically significant.After drug s intervention,the expression of TIMP-3 m RNA and protein expression the two treatment groups(formulae of invigorating Qi and promote blood circulation group,lisinopril group)were significantly higher than the model group,and P<0.05 was considered s tatistically significant.3.2 Immunohistochemical expression of the rats myocardial tissue MMP-3 and TIMP-3 in the groups: compared with the blank control group,the positive expression of M MP-3 in the model group and the two treatment groups(formulae of invigorating Qi and promote blood circulation group,lisinopril group)significantly increased.Compared with the model group,the positive expression of MMP-3 of the two treatment grou ps(formulae of invigorating Qi and promote blood circulation group,lisinopril group)significantly decreased.Compared with the blank control group,the positive expressio n of TIMP-3 in the model group and the two treatment groups(formulae of invigorati ng Qi and promote blood circulation group,lisinopril group)significantly decreased.C ompared with the model group,the positive expression of TIMP-3 of the two treatment groups(formulae of invigorating Qi and promote blood circulation group,lisinopril group)significantly increased.The average grayscale value of the MMP-3 immunohistochemical method in the model group was 161.23±8.02 after software analysis,its expression was significantly higher than that of the blank control group 91.76±4.34,P<0.05,which was statistic ally significant.After the drug intervention,the average grayscale value of formulae of invigorating Qi and promote blood circulation group was 126.58±6.43,Lisinopril group was 127.24±6.49,the expression levels of the two treatment groups were significa ntly lower than the model group,P<0.05,which was statistically significant.Compare d with the blank control group,the immunohistochemical expression of MMP-3 was significantly increased in the two treatment groups,P<0.05,which was statistically sign ificant.The average grayscale value of the TIMP-3 immunohistochemical method of the model group was 73.54±7.14,the expression was significantly lower than the blank control group 152.72±10.24,P<0.05,which was statistically significant.After the inter vention of the drug,the average grayscale value of formulae of invigorating Qi and p romote blood circulation group was 117.15±8.35,lisinopril group was 116.62±8.37,t he expression level of the two treatment groups were significantly higher than the model group,P<0.05,which was statistically significant.Compared with the blank control group,the immunohistochemical expression of TIMP-3 in the two treatment groups decreased significantly.4.OPN expression of the rats myocardial tissue in the groups 4.1 The expression of OPN m RNA and protein of the rats myocardial tissue in the gr oups: compared with the blank control group,the expression of OPN mRNA and prot ein in the model group and the two treatment groups(formulae of invigorating Qi and promote blood circulation group,lisinopril group)increased significantly,P<0.05,whi ch was statistically significant.After the drugs intervention,the expression of OPN m RNA and protein in the two treatment groups(formulae of invigorating Qi and promo te blood circulation group,lisinopril group)were significantly lower than the model gr oup,P<0.05,which was statistically significant.4.2 The OPN immunohistochemistry expression of the rats myocardial tissue in the groups: compared with the blank control group,the positive expression of OPN in the model group and the two treatment groups(formulae of invigorating Qi and promote blood circulation group,lisinopril group)significantly increased.Compared with the mo del group,the positive expression of OPN of the two treatment groups(formulae of invigorating Qi and promote blood circulation group,lisinopril group)significantly decreased.After software analysis,the average grayscale value of OPN immunohistochemical method in the model group was 113.36±11.73,the expression was significantly higher than that in the blank control group 40.64±4.47,P<0.05,which was statistically significant.After the drugs intervention,the average grayscale value of the formulae of invigorating Qi and promote blood circulation group was 73.31±7.17,the lisinopril g roup was 72.86±7.85,the expression levels of the two treatment groups were signific antly lower than the model group,P<0.05,which was statistically significant.Compar ed with the blank control group,the expression of OPN immunohistochemistry was significantly increased in the two treatment groups,P<0.05,which was statistically sign ificant.5.The mRNA and protein expression of the rats myocardial tissue TGF-?1 and CTGF in the groups 5.1 The expression of TGF-?1 m RNA and protein of the rats myocardial tissue in th e groups: compared with the blank control group,the expression of TGF-?1 mRNA and protein in the model group and the two treatment groups(formulae of invigorating Qi and promote blood circulation group,lisinopril group)increased significantly,P<0.05,which was statistically significant.After the drugs intervention,the mRNA and protein expression of TGF-?1 in the two treatment groups(formulae of invigorating Qi and promote blood circulation group,lisinopril group)were significantly lower than the model group,P<0.05,which was statistically significant.5.2 The expression of CTGF m RNA and protein of the rats myocardial tissue in the groups: compared with the blank control group,the expression of CTGF m RNA and protein in the model group and the two treatment groups(formulae of invigorating Qi and promote blood circulation group,lisinopril group)increased significantly,P<0.05,which was statistically significant.After the drugs intervention,the mRNA and protein expression of CTGF in the two treatment groups(formulae of invigoratingormula e of invigorating Qi and promote blood circulation group,lisinopril group)were signif icantly lower than the model group,P<0.05,which was statistically significant.Conclusion: 1.By means of coronary artery ligation and combination of exhausted swimming to established the model of heart failure rats after myocardial infarction(Qi deficiency and blood stasis syndrome),through electrocardiogram,echocardiography and the rats symptoms manifestations can prove that the modeling method was feasible.Formulae of invigorating Qi and promote blood circulation can improve the symptoms manifestations of the heart failure rats after myocardial infarction.The methods of “disease and symptom-sign combined,prescription and symptom-sign corresponded,inspected symptom-sign with a prescription” conformed to the animal model characteristics of traditional Chinese medicine.2.Through echocardiography,the ultrastructure of myocardial tissue was observed by e lectron microscopy,formulae of invigorating Qi and promote blood circulation can improve myocardial structure,cardiac function and damaged myocardial tissue in the heart failure rats after myocardial infarction.3.The correlative factors of ventricular remodeling were detected by immunohistochemistry,real-time fluorescence quantitative PCR and Western blot,the expression of MM P-3,TIMP-3,OPN,TGF-1 and CTGF were found,formulae of invigorating Qi and pr omote blood circulation can reduce the expression levels of MMP-3,OPN,TGF-1 and CTGF in myocardium of the heart failure rats after myocardial infarction,and can en hance the expression of TIMP-3.It was indicated that formulae of invigorating Qi and promote blood circulation can regulate the expression of the above ventricular remode ling related factors and can play a role in the treatment of heart failure. |
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