The Effect Of Astragaloside IV Combined With Trimetazidine On Energy Metabolism In Dogs With Heart Failure

Purpose:In this study,we used molecular biology technology to observe the effects of astragaloside IV and trimetazidine on the general state,cardiac structure,cardiac function and myocardial energy metabolism related indicators of heart failure dog in vitro.Determination of drugs before and after treatment in the myocardial tissue of adenosine triphosphate?ATP?content of myocardial sarcoplasmic reticulum Ca²⁺-ATP enzyme fluorescence intensity of mRNA expression and intracellular Ca²⁺;Determination of myocardial tissue malondialdehyde?MDA?,superoxide dismutase?SOD?content,pyruvate dehydrogenase?PDH?and to observe the changes of expression of mRNA the pathomorphology and ultrastructure of myocardium in myocardial tissue.Objective To observe the changes of related indicators of astragaloside IV combined with trimetazidine before and after treatment,and to explore its mechanism of reducing myocardial injury and protecting myocardial cells in dogs with heart failure,so as to provide a new theoretical basis for the combination of Chinese and Western medicine in the treatment of heart failure,and guide clinical practice.Material and method:A heart failure model was established by rapid pacing in the right ventricle of dogs.16 dogs were randomly divided into 4 groups:sham operation group,model control group,trimetazidine group,astragaloside IV+trimetazidine group,4 dogs in each group.Sham-operation group only thoracotomy,no pacemaker;the other three groups were by rapid right ventricular pacing for 6 weeks to construct congestive heart failure model,were given 2 days before pacing in treatment of gastric perfusion simulation mode,trimetazidine group was given daily trimetazidine?5mg/kg/d?gavage;astragaloside IV+trimetazidine group daily with trimetazidine?5mg/kg/d?and astragaloside IV?1.5mg/kg/d?gavage;model control group normal saline gavage volume.The pacemaker is closed 6 weeks later.The living conditions of four groups of dogs before and after the model were observed,and the routine physical examination was performed daily.The left ventricular diameter,volume and function of the left ventricle were measured by Doppler ultrasound.The last time after intragastric administration,the 24h and fasting dogs in each group were sacrificed,thenhad tissue of left ventricular myocardium in dogs,determination of adenosine triphosphate by chemical Colorimetry?ATP?content;by reverse transcriptase polymerase chain reaction?RT-PCR?determination of myocardial sarcoplasmic reticulum Ca²⁺-ATP mRNA enzyme expression and pyruvate dehydrogenase?the expression of mRNA,PDH?to observe astragaloside IV and trimetazidine therapy on myocardial sarcoplasmic reticulum Ca²⁺-ATP mRNA enzyme pyruvate dehydrogenase?PDH?expression and mRNA expression;The changes of fluorescence intensity of Ca²⁺in cardiac myocytes were observed by laser scanning confocal microscopy with a calcium probe Fluo-3/AM staining.Determination of myocardial tissue MDA by thiobarbituric method?MDA?content;Determination of superoxide dismutase by xanthine oxidase?SOD?content;the application of H-E staining combined with semi quantitative method,observe astragaloside IV and trimetazidine treatment effect on myocardial tissue pathological morphology,determination of dissolved muscle area and ratio;application of uranyl acetate and lead citrate double staining method to observe astragaloside IV and trimetazidine treatment effect on ultrastructure of myocardium.Results:The influence of the general state:the sham-operation group is smooth,dense,sensitive,active,good appetite,and urine as usual.Compared with the model control group of dog,dog fur thin,dull,listlessness,loss of appetite,abdominal distension,urine volume significantly decreased,after an extreme loss of endurance,shortness of breath,calm,shortness of breath after activity increased,the lungs could be heard and rales.The drug therapy group compared with the model control group improved,dogs showed activity tolerance after decreasing,calm breathing smooth movement,shortness of activity,slightly slow reaction,mild abdominal distension,decreased urine output,I can hear a few rales of lung.However,the improvement of the state of astragaloside IV and trimetazidine in the drug treatment group was better than that of the trimetazidine group.1.The results of echocardiographyThe four groups of dogs were to take a leftlie,skinpreparation two-dimensional Doppler echocardiography,determination of diastolic diameter of 6 cardiac cycles in canine left ventricular?LVEDD?,left ventricular end diastolic volume?LVEDV?,end systolic volume?LVESV?and left ventricular ejection fraction?LVEF?.Compared with the sham-operation group,the heart of the remaining 3 groups was enlarged in varying degrees and the ejection fraction was decreased.The model of dogs in the control group were significantly lower LVEF,LVEDD,LVEDV and LVESV increased significantly?P<0.05?;compared with the model group,the two groups after treatment,the results of echocardiography changes,trimetazidine group,astragaloside IV+trimetazidine group LVEF dogs was significantly higher than the model group,there were significant differences?P<0.05?,astragaloside IV+trimetazidine group LVEF was significantly higher than that of trimetazidine?P<0.05?,there was significant difference.And LVEDD,LVEDV,LVESV have no significant difference?p>0.05?,but the value is decreased compared with the model group.2.Determination of ATP content in myocardial tissueCompared with the sham-operation group,model control dogs,myocardial ATP content group were significantly lower than the sham-group,there was significant difference?P<0.05?;compared with the model group,trimetazidine group,astragaloside IV+trimetazidine group canine myocardial ATP content was significantly higher than that of the model control group,there were significant differences?P<0.05?;astragaloside IV+trimetazidine group myocardial tissue ATP content was significantly higher than that of trimetazidine group canine myocardial ATP content,but still lower than the sham group,there were significant differences?P<0.05?.3.Detection results of RT-PCR methodRelative expression of Ca²⁺-ATP enzyme mRNA in sarcoplasmic reticulum of canine ventricular muscle.Using 2000bp DNA Ladder Marker as a marker,amplification products were found at477bp and 379bp,and sequenced as Ca²⁺-ATP gene fragments and Ca²⁺-ATP-actin gene fragments.Compared with the sham group,the myocardial sarcoplasmic reticulum Ca²⁺-ATP enzyme mRNA expression in the model control group was significantly lower than that in the sham operation group?P<0.05?.Compared with the model control group,the expression of myocardial sarcoplasmic reticulum Ca²⁺-ATP enzyme mRNA in the triazetazine group,astragaloside and trimetazidine group was significantly higher than that of the model control group,with a statistically significant difference.?P<0.05?;the expression of Ca²⁺-ATP enzyme mRNA in myocardial sarcoplasmic reticulum of astragaloside IV+trimetazidine group was significantly higher than that of Ca²⁺-ATP enzyme mRNA expression in the myocardial sarcoplasmic reticulum of trimetazidine group,but it was still lower than that of sham operation group?P<0.05?.Relative expression of PDH mRNA in the canine ventricular muscle.The amplified products were visible at 205bp and 379bp at 2000 bp DNA Ladder Marker,which were confirmed by sequencing to be the encoding gene fragment of PDH mRNA and?-actin.Compared with the sham-operation group,model control group dogs,canine ventricular muscle PDH mRNA expression was significantly lower than the sham group,the difference was statistically significant?P<0.05?;compared with the model group,trimetazidine group,astragaloside IV+trimetazidine group canine ventricular muscle PDH mRNA expression was significantly higher than the model control group,there are statistical difference?P<0.05?,astragaloside IV+trimetazidine group ventricular PDH mRNA expression was significantly higher than that of trimetazidine group trimetazidine ventricular PDH mRNA expression,but still lower than the sham group,there was statistical difference?P<0.05?.4.Determination of calcium ion fluorescence intensity in cardiac myocytes.Compared with sham-operation group,model control group,the mean fluorescence intensity of canine myocardial cells was significantly higher than the sham group,there was statistical the difference?P<0.05?;compared with the model group,trimetazidine group,astragaloside IV+trimetazidine group of canine myocardial cells in the average fluorescence intensity was significantly lower than the model control group,there was statistical the difference?P<0.05?;astragaloside IV+trimetazidine group of canine myocardial cells in the average fluorescence intensity was significantly lower than trimetazidine group,but still higher than the sham group,there were statistical the differences?P<0.05?.5.Content of MDA and SOD in canine myocardiumCompared with sham-operation group,the MDA content in ventricular muscle tissue of model control group was significantly higher than that of sham-operation group,and SOD content was significantly lower than that of sham-operation group,there were statistical the differences?P<0.05?.Compared with model control group,the content of MDA in trimetazidine group,astragaloside IV+trimetazidine group was significantly lower than that in model control group,and the content of SOD was significantly higher than that in model control group,there were statistical the differences?P<0.05?.The content of MDA in myocardial tissue of astragaloside IV+trimetazidine group was significantly lower than that in trimetazidine group,and the content of SOD was significantly higher than that in trimetazidine group,there were statistical the differences?P<0.05?.6.Histopathological changes of ventricular myocardium in dogsThe sham-operation group:the myocardial cells were arranged in order and the structure was normal.Model control group:myocardial cells lose normal and orderly arrangement,extremely disorder,appear compression atrophy or compensatory hypertrophy,nuclear malformation is different,myocardial fibrinolysis,severe myocardial dissection in some areas,fibrous tissue hyperplasia in myocardial interstitium.In the drug treatment group,the arrangement of myocardial fibers was irregular,myolysis occurred in a few regions,some of the membrane was not clear,and the interstitium of the myocardium was scattered in a small amount of fibrous tissue.Compared with the model control group,the myocardial histopathology injury in the Trimetazine group,astragaloside IV and trimetazidine group was less than that in the model control group.astragaloside IV+trimetazidine group were significantly less histopathological than that of the trimetazine group.7.Ultrastructural changes of ventricular myocardium in dogs.Sham-operation group:myocardial ultrastructure basically intact;sarcomere structure is complete,each with clear nucleus;normal development,prominent nucleoli,abundant mitochondria,cristae;intercalated disc is well developed.Model control group:myocardial fiber dissolution,deletion,karyopyknosis are sparse;very obvious;perinuclear organelles disappeared;intercellular junctions formed high electron dense plaque;mitochondria swelling,cristae decreased,size,shorter,and even dissolved,visible megamitochondria;interstitial edema,visible and phagocytic cells lymphocytes,phagocytic cells seen in lysosomes;individual muscle cells completely dissolved;interstitial connective tissue cells.In the drug treatment group,some of the muscle fibers were dissolved and absent,and arranged in disorder.Visible nuclear is condensation.The number of mitochondria and organelles decreased,the lysis phenomenon appeared,glycogen granules increased,lysosomes appeared,and mitochondria floc changed.Among,astragaloside IV+trimetazidine group,trimetazidine group and model control group compared with the model control group,the myocardium ultrastructural changes of dogs were reduced.The changes of myocardial ultrastructure of the group of astragaloside IV+trimetazidine were significantly lower than that of the trimetazine group.Conclusion:1.Combined application of astragaloside IV and trimetazidine can significantly increase the heart failure dog LVEF,reduce the increase of LVEDD,LVEDV and LVESV,improve cardiac function,and reduce the change of cardiac structure and function.2.The combination of astragaloside IV and trimetazidine can significantly increased the content of ATP in myocardium,significantly increased the expression of Ca²⁺-ATP mRNA in cardiac sarcoplasmic reticulum,and reduced the calcium fluorescence intensity in cardiac myocytes.Two kinds of drugs may be rectified by heart failure myocardial cells of ATP metabolism,energy metabolism regulation of myocardial cell abnormalities,thereby regulating Ca²⁺-ATP mRNA expression,reduce heart failure myocardial cells with the concentration of Ca²⁺,reduce myocardial Ca²⁺overload,protect myocardial cells,improve myocardial cell function.3.The combined use of astragaloside IV and trimetazidine can significantly reduce the content of MDA in the myocardium and significantly increase the content of SOD in the myocardium.The degree of myocardial histopathology injury in heart failure dogs was improved and the changes of myocardial ultrastructure were alleviated.The combination of the two drugs may increase glucose oxidation,inhibit lipid peroxidation and improve myocardial energy metabolism,thereby improving the oxidative stress state of myocardial cells,reducing myocardial cell injury and maintaining the structure and function of cardiac myocytes in dogs with heart failure.4.Combined application of astragaloside IV and trimetazidine can significantly increase the expression of PDH mRNA in canine ventricular myocardium,indicating that combination therapy increases the glucose oxidation efficiency in cardiac myocytes,ATP is relatively increased,myocardial energy metabolism is optimized,NADH/NADPH system function is improved,NADH accumulation is reduced,thereby reducing the inhibition of NADH on PDH and reducing ROS damage.