The Comparative Studies Of The Protective Effect And Mechanisms Of Shengmai San Traditional Decoction And Ultrafine Particle Leachate On Myocardial Ischemia/Reperfusion Injury

After myocardial ischemic/reperfusion injury(RI), there are a series of functional and morphological changes that are caused by intracellular calcium overloading, oxygen free radicals and so on. During the process of RI, myocardial calcium regulation function changes,which results in the depressing of myocardial cell membrane calcium ATP-ase and sarcoplasmic reticulum (SR) calcium intaking function. Intracellular redundant calcium ion activates intracellular phospholipaseA2 that can decompose cellular membrane phospholipid,which can product a devil of oxyradical by the way of xanthinoxidase(XO) which results in myocardial cell damage.RI often manifests the change of the myocardial zymogram , arrhythmia that may be tachy- or brady-arrhythmia and myocardial stunning that may display persistent depression of ventricular systolic function. There is no possitive prevention and cure measure in modern medicine while traditional Chinese medicine treatment can have better curative effect.ShengMai San(SMS, Powder for Restoring Pulse Beat)and its injectable preparation have been paid close attention to because some studies indicated that SMS could alleviate myocardial ischemia/reperfusion injury.Ultrafine particle herb is a new Chinese medicinal material. At present, many investigations have indicated that it can not only substitute traditional cut crudedrug, but also speed up the absorption of medicine ingredient and reduce intaking dosage. Purpose1. The comparative studies on the effect of SMS traditional decoction and SMS ultrafine particle leachate on myocardium RI were designed to provide theoretical evidences for the ulterior investigation and development and application of ultrafine particle herbs.2. This experiment was designed to research the intracellular Ca²⁺ regulation mechanism of SMS on decreasing calcium overload in the process of myocardium RI and investigate the effects of SMS traditional decoction and SMS ultrafine particle leachate on intracellular Ca²⁺ in RI model and their effects on the mRNA expression of myocardial cell calmodulin (CaM) SERCA2,RyR2.Methods1. In this experiment, the model foundation in vivo was made up of two main steps:ligating the left-anterior coronary artery for 30 minutes and then loosing it for 180 minutes.60 minutes before this experiment, intragastric administration was implemented. During the process of the experiment, the electrocardiogram was recorded continuously. SD rats were devided into 8 groups:control group (C), model group(M), SMS traditional decoction low dose group(Tl), SMS traditional decoction middle dose group(T2),SMS traditional decoction high dose group(T3),SMS ultrafine particle leachate low dose group(F1),SMS ultrafine particle leachate middle dose group(F2), and SMS ultrafine particle leachate high dose group(F3). We observed the incidence of arrhythmia caused by reperfusion in different groups.After 180minutes' reperfusion,we collected these rats' blood to demesh their serum and measure the contents of LDH,CK-MB,MDA and SOD, and picked up their hearts to survey the expression of Bax,Bcl-2 and caspase-3 bymeans of immunohistochemistry.2.Cultured neonatal rat cardiomyocytes RI model in vitro were made by Hypoxia/Reoxygenation.When oxygen was reprovided , serum freeze drying powder without SMS constituent and that with SMS constituent was added to the culture fluid respectively at the same time.Cultured myocardial cells were devided into 8 different groups as before . Cadiocyte vitality and intracellular free Ca²⁺ of all the groups were measured .The mRNA expressing of calmodulin(CaM) sarcoplasmic reticular Ca²⁺-ATPase-2 (SERCA2) and ryanodine receptor-2(RyR2) of all the groups were measured by reverse transcription-polymerase chain reaction(RT-PCR) and normalized to the mRNA levels ofβ-actin.Results1.The incidence of arrhythmia caused by RI in the model group was 83.3%. SMS decreased the arrhythmia incidence to the scale between 66.7% and 44.4%.There was remarkable difference between the SMS groups and the model group (P<0.01).The contents of LDH and CK-MB in the model group were 1615±176 and 665.4±39.8u/L respectively,while in SMS groups they were from 1488±143 to 971±168 and from 482.76±40.3 to 314±53.2 u/L respectively.Compared with the model group, the contents of LDH and CK-MB in the SMS groups decreased markedly (P<0.01).The content of MDA in the model group was 6.31±0.52nmol/ml,while in the SMS groups it was from 4.68±0.76 to 2.55±0.30 nmol/ml. Compared with the model group, the content of MDA in SMS groups decreased markedly (P<0.01).The content of SOD in the model group was 34.5±15.4 umol/ml, while in SMS groups it was from 51.7±14.6 to 86.2±13.2 umol/ml.There is remarkable difference between the SMS groups and the model group, that is, the contents of SOD in the SMS groups raised markedly (P<0.01).Compared with the model group, the expression of Bcl-2 in the SMS groups raised markedly(P<0.01),but that of Bax and caspase-3 decreased markedly (P<0.01).2.The experiment in vitro manifested that the cadiocyte vitality in the model group was impaired seriously. Compared with the model group, the cadiocyte vitality in the SMS groups raised markedly (P<0.01).The cadiocyte intracellular Ca²⁺ in the model group raised seriously. Compared with the model group, the cadiocyte intracellular Ca²⁺ in the SMS groups decreased markedly (P<0.01)..Compared with the control group, the mRNA expression of cadiocyte SERCA2,RyR2 in the model group decreased markedly (P<0.01). Compared with the model group, the mRNA expression of cadiocyte SERCA2,RyR2 in the SMS groups raised markedly (P<0.01).In all the experiments,SMS traditional decoction and ultrafine particle leachate had the similar effect, but the effect of the latter was more effective.Conclusion1. The studies indicated that SMS ultrafine particle herbs can substitute SMS traditional Chinese cut crude drugs, furthermore, the effect of isodose SMS ultrafine particle herbs is more effectual.2. The effect of SMS on resisting myocardium RI was correlated to decreasing cardiocyte intracellular calcium overload through reinforcing the mRNA expression of SERCA2,RyR2.The studies provided substantial evidence for clinical application of SMS ultrafine particle herbs to treat myocardial ischemic/reperfusion injury(RI).