| Objective:to establish heart failure rat model by ligation of anterior descending branch of coronary artery,and to evaluate the effect of optimization new Shengmaisan Recipe on heart function of heart failure rats by using echocardiography and exhaustive swimming Blood and other technologies were used to detect the apoptosis of cardiac myocytes and the expression of related regulatory proteins.From the perspective of the overall effect of multi-target of traditional Chinese medicine,the molecular biological mechanism of it formula to improve the heart function of rats with heart failure was revealed,providing scientific basis for optimization new Shengmaisan formula to prevent and treat heart failure Research methods100 normal and healthy Wistar rats(male)weighing about 200-240g were selected to establish the model of post myocardial infarction heart failure by ligation of anterior descending branch of coronary artery.Six weeks after the operation,vevo2100 echocardiography(ms-250)was used to evaluate the formation of the model,that is,left ventricular ejection fraction(LVEF)was less than 45%.The heart failure rats were randomly divided into model group,western medicine group,traditional Chinese medicine middle dose group and traditional Chinese medicine high dose group.In addition,the normal rats were given the anterior descending branch of the coronary artery without ligation.The western medicine group was intervened by Benazepril Hydrochloride(0.9mg/kg),the traditional Chinese medicine group was intervened by optimized xinmaisan formula(medium dose 8.1g/kg,high dose 16.2g/kg),the model group and the sham operation group were given the same dose of blank solvent(0.5%CMC Na),each group was given by gavage for 4 weeks.Before and 12 hours after the last administration,vevo2100 echocardiography was used to measure LVEF,left ventricular shortening(LVFS),and multi-channel physiological instrument was used to measure the maximum rate of left ventricular pressure rise or fall(ądp/dt max)and other indexes were used to evaluate the heart function;the rats in each group were weighed,and the lead strips(5%body weight)were rolled into small bundles at the root of the tail of the rats in a plastic water tank filled with water,the water depth was three times the length of the tail of the rats,the water temperature was constant at(25-29)?,and the rats began to count after entering the water tank until the head of the rats sank into the water for 10 seconds and could not surface(in physical consumption)The duration of swimming was used as the evaluation index of heart function.Rats were killed and myocardial tissue was fixed in formaldehyde solution and preserved in liquid nitrogen.TUNEL staining was used to observe the cells on the tissue sections of each group under microscope.The staining showed that green cells were positive apoptotic cells,and the cells with blue nuclei were normal cells.Immunofluorescence was used to measure caspase-3 proteasome activity,that is,the cells with blue nuclei were stimulated by UV,and the positive expression was red light labeled with corresponding fluorescein The expression of apoptosis related regulatory proteins such as PI3K,Akt,JNK,Bcl-2,Bax and caspase-3 were detected by blotting.To evaluate the effect of optimizing xinmaisan Recipe on heart function in rats with heart failure and analyze its mechanismResults:Effect of optimization new Shengmaisan on heart function of rats with heart failure(1)Echocardiographic indexCompared with the sham operation group,the LVEF and FS of the model group,the middle dose group of traditional Chinese medicine,the high dose group of traditional Chinese medicine and the western medicine group decreased significantly(P<0.01)6 weeks after operation;compared with the sham operation group,the LVEF and FS of the model group decreased significantly(P<0.01)4 weeks after operation;compared with the model group,the LVEF and FS of the western medicine group,the middle dose group of traditional Chinese medicine and the high dose group of traditional Chinese medicine increased significantly(P<0.01)There was no significant difference among Western medicine group,middle dose group and high dose group(P>0.05)(2)Hemodynamic indexesCompared with the sham operation group,the ądp/dt max(mmHg/s)of the model group was significantly lower(P<0.01),which was statistically significant;after drug intervention,the ądp/dt max(mmHg/s)of the Chinese medicine group,the high dose group and the western medicine group was significantly higher than that of the model group(P<0.01).There was no significant difference between the two groups(P>0.05)(3)Experimental index of exhausted swimmingCompared with the sham operation group,the swimming time in the model group decreased significantly(P<0.01);compared with the model group,the swimming time in the traditional Chinese medicine group,the high dose group and the western medicine group increased significantly(P<0.01);there was no statistical difference between the traditional Chinese medicine group and the western medicine group(P>0.05)2.Study on the molecular biological mechanism of improving Heart function of heart failure rats by optimization new Shengmaisan recipe(1)The condition of myocardium of rats in TUNEL staining groupAfter TUNEL staining,there were a small number of green fluorescent cells in the sham operation group,but compared with the sham operation group,there were a large number of green fluorescent cells in the model group,while there were fewer green fluorescent cells in the middle dose group,high dose group and Western medicine groupCompared with the sham operation group,the apoptotic rate of cardiomyocytes in the model group increased significantly(P<0.01);after drug intervention,compared with the model group,the apoptotic rate of cardiomyocytes in the western medicine group and the middle dose group and the high dose group of traditional Chinese medicine decreased significantly(P<0.01);there was no significant difference between the Chinese medicine group and the western medicine group(P>0.05)(2)Detection of caspase-3 activity in myocardium of heart failure rats by fluorescence colorimetryCompared with the sham operation group,the model group showed more cells with red fluorescence;compared with the model group,the traditional Chinese medicine group showed less cells with red fluorescence;while the western medicine group and the traditional Chinese medicine high dose group also observed a small number of cells with red fluorescenceCompared with the sham operation group,the caspase-3 protein in the model group increased significantly(P<0.01);compared with model group,caspase-3 protein in western medicine group,middle dose group of traditional Chinese medicine and high dose group of traditional Chinese medicine decreased significantly(P<0.01);there was no statistical difference between middle dose group,high dose group and Western medicine group(P>0.05)(3)PI3K,Akt protein and p-PI3K,p-Akt phosphorylation level expression:there was no statistical difference in the protein level of PI3K and Akt in each group(P>0.05);compared with the sham operation group,the phosphorylation level of p-PI3K and p-Akt in the model group decreased(P<0.05);the phosphorylation level of p-PI3K and p-Akt in the western medicine group,the middle dose group of traditional Chinese medicine and the high dose group of traditional Chinese medicine increased(P<0.05);there was no statistical difference between the western medicine group and the traditional Chinese medicine group(P>0.05)(4)The expression of JNK protein in heart failure rats in each group:compared with sham operation group,the expression of JNK protein in model group increased(P<0.05);the expression of JNK protein in western medicine group and traditional Chinese medicine group decreased significantly(P<0.05);there was no statistical difference between Western Medicine group and traditional Chinese medicine group(P>0.05)(5)The expression of Bcl-2,Bax and caspase-3 protein in heart failure rats in each group compared with sham operation group,the expression of Bax and Caspase-3 in heart failure rats in model group increased,while that of Bcl-2 decreased(P<0.05 orP<0.01);compared with model group,the expression of Bax and caspase-3 protein in heart failure rats in western medicine group and traditional Chinese medicine group decreased significantly,while that of Bcl-2 increased(P<0.05 orP<0.01);there was no statistical difference between western medicine group and traditional Chinese medicine group difference(P>0.05)Conclusion1.optimization new Shengmaisan can improve the heart function of heart failure rats after myocardial infarction2.The optimization new Shengmaisan has obvious inhibition on cardiomyocyte apoptosis Its mechanism may be inhibition of PI3K/Akt signal pathway,inhibition of target JNK signal pathway activation,JNK pathway deactivation and inhibition of downstream gene Bax and Caspase-3 expression,inhibition of cardiomyocyte apoptosis,at the same time enhance Bcl-2 to resist apoptosis.It is suggested that the optimized xinmaisan can inhibit the apoptosis of myocardial cells in heart failure rats through PI3K/Akt pathway. |
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