| ObjectiveAt present,the incidence of coronary heart disease has been increasing.With the benefit from percutaneous coronary intervention,acute thrombolytic therapy,antiplatelet aggregation treatment and other drug treatments in the acute phase,the mortality of acute myocardial infarction has significantly decreased.But The incidence of heart failure after myocardial infarction has increased year by year.The reperfusion treatment has also brought about secondary damage to the heart,namely cardiac ischemia-reperfusion injury.Nowdays,the treatment of myocardial infarction is still mainly based on antiplatelet aggregation and inhibition of sympathetic nerves system.but the treatments with western medicine have some limitations and can cause side effects such as gastrointestinal bleeding,bradycardia and so on.Multi-target effects of traditional Chinese medicine have advantages in the treatment of myocardial infarction and can make up for the shortage of western medicine.Tongguan capsule,which is a Chinese patent drug developed by our own hospital,is widely used for the treat of myocardial infarction.In this study,we aim to explore the therapeutic effect and potential mechanism of Tongguan Capsule in the treatment of heart failure after myocardial infarction at the animal and cellular level.MethodsFirstly,we established a stable rat model of heart failure after myocardial infarction by subcutaneous injection of isoproterenol.The experimental animals were divided into four groups:blank control group,ISO heart failure model group,positive drug group treated with metoprolol and the group treat with Tongguan capsule.Seven days after injection with ISO and treatment with different drugs or saline as control,cardiac function was measured by cardiac echo for small animals.After anesthesia and sacrifice,the heart and lung tissues were weighed and the length of the tibia was measured.Hematoxylin-eosin staining and Sirius red Staining was used to detect the cardiac pathological changes.Western blot and immunohistochemical staining were used to detect protein expression and localization in the heart tissue.Previous research by our group confirmed that the main component of Tongguan Capsule is salvianolic acid by high performance liquid chromatography.This study also aimed to explore whether the cardioprotective effect of Tongguan Capsule based on the role of salvianolic B on this experimental model of heart failure.Animals were divided into four groups:the blank control group,ISO model group,positive drug gr oup treated with metoprolol,and the group treated with salvianolic acid B.Seven days after modeling and administration,cardiac function was detected by cardiac echo for small animals.After anesthesia and sacrifice,the heart and lung tissue were weighed and the length of the tibia was measured.Heart pathological changes were detected by hematoxylin-eosin staining and Sirius red staining.Western blot was used to detect protein expression in cardiac tissue.Tissue immunofluorescence was used to detect cell apoptosis in the hearts.At the cellular level,the same model of cardiomyocyte apoptosis was established with ISO in H9C2 cell line,which originated from rat ventricular cell.Cell viability was detected by MTT test.The effect of salvianolic acid B on calcium overload in cardiomyocytes was detected with calcium sensitive fluorescence staining Fluo4-AM.Western blot was used to detect the expression level of calcium regulating proteins and apoptosis-related protein.ResulcsTongguan capsule significantly increased the survival rate of rats after ISO injection,improve cardiac function damaged by ISO,as well as inhibited myocardial cell hypertrophy and cardiac collagen deposition after myocardial infarction.Tongguan capsule downregulated the overexpression of CaMKII caused by ISO,inhibited the translocation of transcription repression factor HDAC4 and myocyte enhancer factor(MEF2)expression,all of which inhibited ISO-induced cardiomyocytes hypertrophy and heart failure.At the same time,the mitogen-activated protein kinase(MAPKs)pathway,which is a classic cardiomyocytes hypertrophy signal pathway,also participated in ISO-induced heart failure.Tongguan Capsule protected the cardiac function by inhibiting the activation of MAPKs and the expression of downstream transcription factor GATA-4,therefore inhibited the expression of ANP which is a protein promoting cardiac hypertrophy.Salvianolic acid B,the main active ingredient of Tongguan Capsule,also improved cardiac function in rats with heart failure after myocardial infarction.It also inhibited cardiac hypertrophy and cardiac collagen deposition.We found that salvianolic acid B down-regulated ISO-induced overexpression of Bax,a mitochondrial dependent apoptosis-associated protein,promoted the expression of the anti-apoptotic protein Bcl-2,and inhibited the cleavage of Caspase-3 and the production of apoptotic bodies.At the cellular level,we found that salvianolic acid B can improve ISO induced cell death,apoptosis and intracellular calcium overload.Salvianolic acid B inhibited CaMKII activation and upregulated SERCA expression,and therefore alleviated intracellular calcium overload.Moreover,salvianolic acid B inhibited the overexpression of apoptosis-related proteins Bax and cleaved caspase-3 caused by ISO,as well as promoted the expression of anti-apoptotic protein Bcl-2,which eventually inhibited the formation of apoptotic body.ConclusionTongguan capsule improved cardiac function and survival rate of rats after myocardial infarction by inhibiting cardiac hypertrophy and fibrosis.Tongguan Capsule may exert its cardioprotective effect by inhibiting CaMKII/HDAC4/MEF2 and MAPKs/GATA-4 signal pathway.Salvianolic acid B,as the main active ingredient of Tongguan Capsule,also protected against cardiac hypertrophy in this experimental model of heart failure.It improved cardiac function and survival rate of rats during heart failure after myocardial infarction.Salvianolic acid B protected cardiac myocytes from apoptosis,where Ca2+/CaMKII signal pathway and SERCA may mediate the protective effect of salvianolic acid B against mitochondrial dependent apoptosis. |
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