Effect Of Tongxinluo On Microvascular Neovascularization And Fibrosis In Ischemic Myocardium Of Rats

PurposeMyocardial infarction is a severe disease threatening human health.It is caused by the formation of thrombus on the basis of rupture and erosion of unstable coronary plaque,then complete occlusion generated in coronary arteries.That followed with a series of pathological changes including anoxia and ischemia,necrosis and inflammation.The morbidity and mortality of myocardial infarction in China are rising year by year,and it has become a severe threat to human health.Treatment of effective angiogenesis and reducing and reversing myocardial fibrosis,is the important goal in curing myocardial infarction,and the key link in the process of ventricular remodeling and heart failure.In recent years,with the development of thrombolysis,nterventional operation and surgical treatment,the survival rate and prognosis of patients has a obvious improvement.But many patients’ condition will deteriorate because they can’t have revascularization due to a variety of factors.So angiogenesis is a new treatment to improve the supply of blood.Myocardial fibrosis is the inevitable stage in the process of ventricular remodeling and heart failure.It is significant to control fibrosis progress. The disease belongs to the category of "chest depression","true heart pain" and "precordial pain" in Traditional Chinese Medicine.The treatment mainly includes activating yang by removing obstruction of qi and blood circulation,benefing heart qi,promoting blood circulation to remove meridian obstruction,and resolve tetany and relieve pain.A variety of traditional Chinese medicine monomer and compound had obvious curative effect.On the basis of collaterals theory,Tongxinluo can tonify qi,activate meridians to stop pain and promote circulation and remove stasis.It can improve myocardial ischemia,inhibit ventricular remodeling,and reduce the cardiovascular events,which performs the characteristics of multi-channel and multi-target.Neovascularization and fibrosis are the hotspot issues in current research,while,the function and specific regulatory mechanism of Tongxinluo on angiogenesis of myocardial infarction and myocardial fibrosis is obviously insufficient.It still need to be further discussed.This study establishedAMIrat model,then observed the injury situation of cardiac muscle,the angiogenesis of ischemia area,the degree of myocardial fibrosis.Explore the effect and regulatory mechanismof Tongxinluo,and provide a new basis for the prevention of myocardial infarction.MethodsSD male rats were randomly divided into model group,Tongxinluo group,perindopril group,Tongxinluo+perindopril group,with 10 rats respectively.There were 9 rats in the sham group.Prepare myocardial infarction model by the method of ligation anterior descending branch of heart.The sham group thread only without ligation.Drugs were dissolved in sodium carboxymethyl cellulose solution to make suspension.The Tongxinluo group,perindopril group,Tongxinluo+perindopril group were respectively given Tongxinluo,perindopril, Tongxinluo combined with perindopril,and the model group and sham group were given sodium carboxymethyl cellulose solution.2 weeks later, detect the electrocardiogram,blood routine.Use the relative kits to test TUNEL apoptosis and SOD, MDA, NO, ET-1 level.The myocardial tissue are staining with HE and Masson method.Then observe the pathological characteristics,the structure of the left ventricle,collagen volume fraction.Immunohistoche-mical staining to detect myocardial CD31,alpha SMA,Notchl and Jagged1,VEGF,Hif-1 alpha, TGF-beta 1,Smad2,CTGF expression.Results1. Effects of Tongxinluo on damage degree of myocardial infarction of ratsElectrocardiogram:Compared with model group,Tongxinluo group,perindopril group, Tongxinluo+perindopril group reduced heart rate and incidence of arrhythmia,and the ST segment fell back (p<0.05).Blood RT:Platelet of Tongxinluo group was less than model group (p<0.05). Oxidative stress and endothelial function:Compared with model group,the MDA, ET-1 of drug groups were lower and SOD,NO were higher(p<0.05).Microstructure: Myocardial cell enlargement,elongate,fracture,disordered arrangement,clearance increases, neutral cells and mast cells gathered in model group. While the drug groups were improved. Left ventricular structure:Compared with model group,infarction area of drug groups was reduced;the minimum thickness of infarction area,the ratio of minimum thickness and interventricular septum thickness increased (p<0.05).Apoptosis index:That of drug groups was decreased than model group (p<0.05).2. Effectsof Tongxinluo on angiogenesis and Notch signaling pathwayPathological features:Myocardial cells and capillaries in survival edge area of Tongxinluo group,perindopril group, Tongxinluo+perindopril group were increased compared with model group,and the structure was more complete.CD31 positive cell count, CD31+alpha SMA positive cell count:Drug groups increased significantly than model group (p<0.05).Notch signaling pathways:Notchl,Jagged1,VEGF expression of drug groups were higer than model group (p<0.05).Correlation between angiogenesis and Notch signaling pathway:Microvascular density were positively correlated with Jagged1 and VEGF (p<0.01).3. Effects of Tongxinluo on myocardial fibrosis and TGF-beta 1 signaling pathwayPathological features:Compared with model group,collagen deposition in Tongxinluo group,perindopril group,Tongxinluo+perindopril group decreased,remnants of the myocardial cells and collagen fiber staggered.Collagen volume fraction:Drug groups were less than model group(p<0.01).TGF-beta 1/Smads signaling pathway:Hif-1 alpha,TGF-beta 1,Smad2 and CTGF expression in drug groups were reduced,compared with model group(p<0.05). Correlation between fibrosis and TGF-beta 1 signaling pathway:Collagen volume fraction were positively correlated with Hif-1 alpha,TGF-beta 1,Smad2 and CTGF (p<0.01).4.Influence Mechanism of Tongxinluo on endothelial-to-mesenchymal transitionCD31:Compared with model group,Tongxinluo group,Tongxinluo+perindopril group increased(p<0.05). Alpha SMA:Drug groups were less than model group(p<0.01). Notch and TGF-beta 1/Smads signaling path way:The relevance of the cluster is divided into two groups of Notch1,Jaggedl and Hif-1 alpha,VEGF,Smad2,CTGF.The indicators of 2 combinazation related to TGF-beta 1 at the same time.Correlation between endothelial-to-mesenchymal transition and signaling pathways:CD31 were positively correlated with Jaggedl,VEGF of Notch pathway;CD31 were negatively correlated with CTGF of TGF-beta 1 pathway(p<0.05).Alpha SMA were positively correlated with TGF-beta 1 pathway(p<0.05), and there is no correlation between Alpha SMA and Notch pathway.Conclusion1. Tongxinluo could maintainthe heart rate,reduce the incidence of arrhythmia,improve ischemiaper formance in ECG,resist oxidative stress,improve endothelial function,and reduce the apoptosis.2. Tongxinluo could activate Notch1/Jagged1/VEGF signaling pathway,then promote angiogenesis in edge area of infarction,and increase the maturing rate of microvasculars, which effectively improve myocardial blood supply.It also could inhibit Hif-1 alpha/TGF-beta 1/Smad2/CTGF signaling pathway to reduce the degree of myocardial fibrosis,then prevent ventricular remodeling and improve prognosis.3. Tongxinluo could inhibit endothelial-to-mesenchymal transition by regulating the above two signaling pathways.That protected vascular endothelial cells and reduced the stromal cells,thereby palyed the role of promoting angiogenesis and inhibiting myocardial fibrosis.4. Tongxinluo combined with perindopril had a better effect than Tongxinluo or perindopril alone.The function of Tongxinluo and perindopril was similar,but Tongxinluo was better than perindopril in reducing the platelet and promoting angiogenesis.