| BACKGROUNDHeart failure is the final outcome of the development of a variety of cardiovascular diseases,with high morbidity and mortality.It is an urgent problem to find effective treatment to improve heart failure.Aconite has the function of restoring yang and saving adversity,and it is the first choice of traditional Chinese medicine in the clinical treatment of heart failure,but its clinical application is greatly limited because it is easy to cause side effects such as arrhythmia.Aconitine is not only the main active component of aconitine,but also its main toxic component,so it is of great significance to.find a way to increase the efficacy and reduce toxicity of aconitine,which can not only ensure the efficacy of aconitine,but also improve the safety of aconitine.A large number of previous studies had shown that the abnormal calcium regulation of sarcoplasmic reticulum in cardiomyocytes was the main pathological mechanism of heart failure,and the effects of aconitine on heart failure and its toxic and side effects were also closely related to the regulation of intracellular calcium in cardiomyocytes.On the other hand,a large number of existing research data and previous research work in our laboratory showed that acupuncture could reduce intracellular calcium overload and enhance myocardial contractility by regulating the expression of key proteins regulated by calcium in cardiomyocytes.and it could also improve ischemic arrhythmia and had a good protective effect on myocardium.According to the above research results,it was obvious that the occurrence and development of heart failure,the pharmacological and toxicological mechanism of aconitine and the effect of acupuncture on improving cardiac function were inseparable from the regulation mechanism of intracellular calcium in cardiomyocytes.In view of the fact that clinical electroacupuncture combined with conventional drugs for the treatment of heart failure could enhance the curative effect and complement each other’s advantages,and combining the our previous research results of electroacupuncture on the synergistic/attenuating effect and mechanism of digitalis drugs,our assumption is that the combination of electroacupuncture and aconitine should be more effective in improving heart failure more,and the key factors related to intracellular calcium regulation may be involved in mediating the synergistic and attenuating mechanism of electroacupuncture on aconitine.OBJECTIVEBased on the mechanism of calcium regulation in myocardial cells,the pathological mechanism of heart failure,the pharmacological and toxicological effect of aconitine,and the relationship between the effect of electroacupuncture combined with aconitine and the mechanism of calcium regulation in cardiomyocytes were analyzed.Firstly,it was determined that electroacupuncture combined with aconitine could improve myocardial function and reduce toxic and side effects in rats with heart failure,and from the point of view of the expression of calcium regulation related proteins in cardiomyocytes,the scientific mechanism of electroacupuncture combined with aconitine in improving the synergistic and attenuating effect of heart failure was further analyzed and explored.The above study will open up an effective and safe way for the combination of electroacupuncture and aconitine to improve heart failure.At the same time,and provide scientific evidence for the combination of acupuncture and medicine in the treatment of other diseases.METHODSIn this study,the rat model of acute heart failure was established by continuous infusion of propranolol hydrochloride into the femoral vein,and the successful model of acute heart failure was established by the decrease of+dp/dtmax to less than 60%of the normal(baseline).Fifty-four healthy male SD rats weighing 280g+30g were randomly divided into normal control group(NC),model group(M),electroacupuncture group(EA),aconitine(ACO)low(5μg/kg),medium(10μg/kg),high(20μg/kg)dose groups(ACO5,ACO10,ACO20),low,medium and high dose aconitine plus electroacupuncture groups(ACO5+EA,ACO10+EA,ACO20+EA),with 6 rats in each group.NC group received no medication,no electroacupuncture,only the same volume of normal saline injection;other groups received propranolol hydrochloride to establish acute heart failure model.Group M did not give electroacupuncture after successful modeling,but only injected normal saline through the femoral vein;after successful modeling of aconitine alone,each group did not receive electroacupuncture,only injected the corresponding concentration of aconitine saline solution into the femoral vein.EA group and aconitine combined with aconitine group were given bilateral Neiguan acupoint electroacupuncture to stimulate 30min after successful modeling(EA parameters:intensity 3mA,frequency 2/15 Hz),EA and immediately injected normal saline or aconitine saline solution of corresponding concentration into the femoral vein.This present study was divided into the following two parts:The first part of the experiment mainly observed and determined the synergistic/attenuating effect of electroacupuncture combined with aconitine on acute heart failure rats.The hemodynamic indexes such as LVSP,HR,+dp/dtmax and-dp/dtmax were detected by carotid artery intubation technique,and the cardiac function indexes such as LVEF and LVFS were detected by small animal ultrasound imaging technique to evaluate the synergistic effect of electroacupuncture combined with aconitine in improving heart failure.On the other hand,the type of arrhythmia was analyzed and scored by ECG changes to determine the attenuated effect of electroacupuncture on aconitine.The second part of the experiment mainly explored the calcium regulation mechanism of electroacupuncture combined with aconitine in improving heart failure.The expression of SECAR2a,PLB and NCX1 protein in left ventricular myocardial tissue of rats in each group was detected by fluorescent western blotting,and the content of cTnT in serum of rats in each group were detected by enzyme-linked immunosorbent assay to explore whether electroacupuncture could mediate the synergistic and attenuating effect of aconitine on heart failure by affecting the key proteins of calcium regulation mechanism in cardiomyocytes.RESULTS1 Observation and determination of the synergistic and attenuating effect of electroacupuncture combined with aconitine on heart failure.1.1 Screening of the optimal dose of aconitine.1.1.1 Changes of hemodynamic parameters.In NC group,LVSP,HR,+dp/dtmax,-dp/dtamax,LVEF and LVFS were at a high level and remained relatively stable throughout the experiment.The above-mentioned hemodynamic indexes in group M maintained a relatively low level compared with those in group NC at each time point after successful establishment of the model(P<0.01).The levels of +dp/dtmax and+dp/dtmax in ACO5 group were slightly higher than those in M group,LVSP and+dp/dtmax in ACO5+EA group were significantly higher than those in M group(P<0.05 or P<0.01),the level of+dp/dtmax in ACO10 group was significantly higher than that in M group(P<0.01),and there was a tendency to increase LVSP in heart failure rats.The levels of HR and-dp/dtmax,in rats with heart failure in ACO10+EA group were significantly higher than those in ACO10 group(P<0.05 or P<0.01 respectively).Compared with group M,+dp/dtmax increased significantly in rats with heart failure in ACO20 group,and there was a tendency to increase LVSP in rats with heart failure,while the levels of HR and-dp/dtmax in group M were significantly lower than those in group M(P<0.05).The levels of LVSP and+dp/dtmax in ACO20+EA group were significantly higher than those in M group(P<0.01).The levels of LVSP and+dp/dtmax in M group were significantly higher than those in M group.The levels of LVEF and LVFS in different intervention groups were significantly higher than those in group M(P<0.05 or P<0.01).Among them,the improvement of LVEF and LVFS in ACO10+EA group and ACO20+EA group was more significant,which was similar to that in NC group(P<0.05).1.1.2 Changes in arrhythmia score.In the 60min of rats after intravenous administration,different degrees of arrhythmias were found in each group,especially in the ACO20 group.100%of the rats in the same group had frequent ventricular extrasystole,and the arrhythmia score was significantly higher than that in the ACO5 group and the ACO10 group(P<0.05or P<0.01).However,the incidence and score of arrhythmia in aconitine combined with electroacupuncture group decreased in varying degrees,especially in ACO10+EA group,and the arrhythmia score in aconitine combined with electroacupuncture group was significantly lower than that in ACO10 group,ACO20 group and ACO20+EA group(all P<0.01).In addition,although the incidence and score of arrhythmia in the ACO20+EA group were lower than those in the ACO20 group,there was no significant difference in the arrhythmia score between the ACO20+EA group and the ACO20 group.In the 60min after electroacupuncture and administration,the ventricular extrasystole still occurred frequently in the ACO20+EA group.Based on the above results,we took electroacupuncture combined with intravenous injection of 10μg/kg aconitine as the main intervention to further observe the effect of electroacupuncture combined with aconitine on heart failure in rats and its related calcium regulation mechanism.1.2 Observation and determination of the synergistic/attenuating effect of electroacupuncture combined with aconitine on acute heart failure in rats.1.2.1 Changes of hemodynamic indexes.In NC group,LVSP,HR,+dp/dtmax,-dp/dtmax,LVEF and LVFS were at a high level and remained relatively stable throughout the experiment.The above-mentioned hemodynamic indexes in group M maintained a relatively low level compared with those in group NC at each time point after successful establishment of the model(P<0.01).Electroacupuncture could significantly increase+dp/dtmax in heart failure rats immediately,but then decreased gradually.Aconitine alone and electroacupuncture combined with aconitine could significantly increase+dp/dtmax,in heart failure rats,and LVSP and-dp/dtmax in ACO10+EA group were also significantly higher than those in EA group and ACO10 group at the corresponding time point(P<0.05 or P<0.01),and at the corresponding time point,it was also significantly higher than that in EA group and ACO10 group(P<0.05 or P<0.01),and at the corresponding time point,it was also significantly higher than that in EA group and ACO10 group(P<0.05 or P<0.01).It also has a tendency to increase the HR of rats with heart failure.On the other hand,the LVEF and LVFS of rats in different intervention groups were increased in different degrees,which were significantly higher than those in M group(P<0.01).Among them,the increase of LVEF and LVFS in electroacupuncture combined with aconitine group was the most obvious,which was significantly higher than that in electroacupuncture group(P<0.05 or P<0.01),and had a tendency to be higher than that in aconitine group.1.2.2 Changes in arrhythmia score.All the rats in the aconitine group had arrhythmias in varying degrees,and the arrhythmia score was also higher.after aconitine combined with electroacupuncture,the incidence and degree of arrhythmia decreased significantly,and the arrhythmia score was also significantly decreased.The above results show that the obvious immediate effect of electroacupuncture combined with the rapid and sustained effect of intravenous injection of aconitine can significantly improve the myocardial contractile and diastolic function of rats with acute heart failure,and compared with electroacupuncture alone and aconitine alone,it does have the advantages of quick effect and wide time window,and electroacupuncture can enhance the improvement of myocardial contractile and contractile function in rats with acute heart failure.It can also significantly inhibit the side effects of arrhythmia caused by aconitine and reduce the toxicity to a certain extent.2 Study on calcium regulation mechanism of electroacupuncture combined with aconitine in improving heart failure.2.1 Involvement of SERCA2a in mediating the synergistic and attenuating effect of electroacupuncture on aconitine in improving heart failure.The relative expression of SERCA2a protein in NC group was higher than that in M group,while the expression of SERCA2a protein in M group was significantly lower than that in NC group(P<0.01).The expression of SERCA2a protein in EA group was significantly higher than that in M group(P<0.05).Although the expression of SERCA2a protein in ACO10 group was higher than that in M group,there was no significant difference between ACO10 group and M group.The expression level of SERCA2a protein in ACO10+EA group was the highest,which was significantly higher than that in M group,and also significantly higher than that in EA group and ACO10 group(P<0.05 or P<0.01).2.2 Involvment of PLB in mediating the synergistic and attenuating effect of electroacupuncture on aconitine in improving heart failure.The expression of PLB in NC group was relatively lower than that in NC group,while the expression of PLB in M group was significantly higher than that in NC group(P<0.01).The expression of PLB in EA group,ACO10 group and ACO10+EA group was significantly lower than that in M group(P<0.05 or P<0.01).Among them,the content of PLB in ACO10+EA group was the lowest,and its PLB expression was significantly lower than that in EA group and ACO10 group(all P<0.05).2.3 Involvement of NCX1 in mediating the synergistic and attenuating effect of electroacupuncture on aconitine in improving heart failure.The expression of NCX1 protein in NC group was relatively low,while the expression of NCX1 protein in M group was significantly higher than that in NC group,and the expression of NCX1 protein in ACO10 group was still higher than that in NC group,while the expression of NCX1 protein in EA group and ACO10+EA group was significantly lower than that in M group(P<0.01).2.4 Involvement of cTnT in mediating the synergistic and attenuating effect of electroacupuncture on aconitine in improving heart failure.The concentration of cTnT in serum of rats in NC group was lower,while the concentration of cTnT in group M was significantly higher than that in group NC(P<0.01).The content of cTnT in group),EA and ACO10+EA was significantly lower than that in group M,while the content of cTnT in group ACO10 was still at a high level,which was not significantly different from that in group M,and was significantly higher than that in group EA and group ACO10+EA(P<0.05 or P<0.01).CONCLUSIONS1.The expression of SERCA2a protein in cardiomyocytes of rats with acute heart failure was significantly decreased,while the expression of PLB protein and NCX1 protein was significantly increased.In addition,the concentration of troponin cTnT in peripheral blood was also abnormally increased.The disorder of calcium regulation mechanism in cardiomyocytes is an important factor in the weakening of cardiac contractile force in the process of heart failure.2.The cardiotonic effect and arrhythmogenic side effects of aconitine may be related to down-regulating the expression of PLB and increasing the expression of NCX1 in myocardium.3.Electroacupuncture can not only enhance the contractile and diastolic function of myocardium in rats with acute heart failure,but also significantly inhibit the side effects of arrhythmia caused by aconitine and reduce the toxicity to a certain extent.4.SERCA2a,PLB and NCX1 proteins in cardiomyocytes and cTnT in peripheral circulation may be involved in the protective effect of electroacupuncture and electroacupuncture combined with aconitine in improving heart failure,moreover,electroacupuncture may achieve the synergistic/attenuating effect of aconitine on heart failure by increasing the content of SERCA2a protein in myocardial tissue,down-regulating the expression of PLB and NCX1 protein in myocardial tissue and reducing the content of cTnT in peripheral circulation. |
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