Shenfu Injection On Acute Pressure Overload And Neonatal Rat Myocardial Cells Of Oxygen And Glucose Deprivation

Objective: The acute pressure-overloaded rat models were made by banding abdominal aorta. We studied the change of hemodynamics at different points of time and the pathological change of myocardial tissues after the acute pressure-overloaded rats were treated with Shen-Fu Injection or were pretreated with Shen-Fu Injection. It was also observed how Shen-Fu Injection had influenced on the expression level of ANP\Na⁺-K⁺ ATPase and Ca²⁺-ATPase of myocardial tissues. We also studied the influence of Shen-Fu Injection on the apoptosis rate of myocardial cells and the protein expression of apoptosis rate of myocardial cells and the protein expression of apoptosis-related genes BCL-2 and BAX. At the same time, by means of cultivating myocardial cells in vitro culture. glucose-deficiency-and-oxygen-deficiency-led injury models were made for the study of the influence of Shen-Fu Injection on the myocardial cells injured by glucose deficiency and oxygen deficiency.Methods: The acute pressure-overloaded rat models were made according to relative references, which were divided into: (1) Medicine groups: From 50 male Wistar rats, 8 of them were operated on by means of banding abdominal aorta, among which 10 rats died during operation and 32 rats survived. The 32 survivors were divided into the following four groups at random: the model group. The Shen-Fu Injection group, Cedilanid and Dobutamine, with 8 rats in each group. In the sham operation group, the abdominal aorta were only separated instead of being banded. In other groups, two hours after operation of model-making, the left jugularis external veins of the rats were separated, into which the following medicines were injected:â‘ in the Shen-Fu Injection group, 0.5ml of Shen-Fu Injection (Containing Jingsheng 3.5mg/ml)â‘¡in the Cedilanid group, 0.5ml of Cedilanid(0.036mg/ml)â‘¢in the Dobutamine group, 0.5ml of Dobutamine(1.8mg/ml). (2) medicine pretreatment groups: 65 male Wistar rats were divided into 5 groups at random: the sham operation group, the model group, the high-dosed Shen-Fu Injection(HDSFI) group, the low-dosed Shen-Fu Injection(LDSFI)group and the Enalapril group. There were 5 rats in the HDSFI group and the LDSFI group were injected with Shen-Fu Injection through abdominal cavity at a dose of 8mg/kg and 4ml/kg respectively, at the same time, normal saline was poured into the stomachs of the rats at a dose of 10 ml/kg; In the Enalapril group, Enabpril solution was poured into the stomachs of the rats at a dose of 10ml/kg(0.5mg/ml), at the same time, normal saline was injected into the abdominal cavities at a dose of 8ml/kg. These administrations were given once a day and after successive administrations of 7 days the abdominal aorta were banded; In the sham operation group,normal saline was admini-stered turough both abdominal injection(8ml/kg) and stomach-pouring (10ml/kg),7days later the abdominal aorta of the rats in this group were obtusely separated without being banded. (3)Hemodynamics parameters:â‘ Medicine treatment groups: two hours after operation, a conduit was inserted into the left venticle and the prdssure curve was recorded with the Eight Tracks Physiological Record Instrument. These are the left ventricular contracticity parameters to be calculated: Left Ventricular Systole Prdssure (LVSP). Identical Volumed Ventricular end systolic pressure's maximum ascending speed(+dp/dtmax) and these are the left ventricular diastolic function parameters: Left Ventricular End-Diastolic pressure(LVEDP). Left Ventricular end diastolic pressure's maximum descending speed(-de/dtmax). After an interval of 5 minutes, Shen-Fu Injection, Cedilanid and Dobutamine were respectively injected into the veins of the rats in the corresponding groups. At an interval of 10 minutes, the hemodynamics parameters of the rats in the SFI group, the cedilanid group and the Dobutamine group lvere recorded again.â‘¡Medicine. Pretreatment groups: Respectively 1/2 hour, 1 hour, 2 hour after the abdominal aorta banding operations, from the model group,the high-dosed Shen Fu Injection(HDSFI) group,the low-dosed Shen Fu Injection(LDSFI) group and the Enalapril group.There were 5 rats in the sham operation group,in every other group,there were 15 rats.The rats in the HDSFI group and the LDSFI group were injected with Shen Fu Injection through abdominal cavity at a dose of 8mg/kg and 4ml/kg respectively, at the same time, normal saline was poured into the stomachs of the rats at a dose of 10ml/kg;In the Enalapril group,Enalapril solution was poured into the stomachs of the rats at a dose of 10ml/kg(0.5mg/ml),at the same time,normal saline was injected into the abdominal cavities at a dose of 8ml/kg.These administrations were given once a day and after successive administrations of 7days, the abdominal aorta were banded;In the sham operation group,normal saline was administered hrough both abdominal injection(8ml/kg) and stomach-pouring(10ml/kg),7 days later the abdominal aorta of the rats in this group were obtusely separated without being banded.3 Hemodynamics Parameters(1)medicine treatment groups:tow hours after operation,a conduit was inserted into the left ventricle and the pressure curve was recorded with the Eight Tracks Physiological Record Instrument.These are the left ventricular contractility parameters to be calculated:Left Ventricular Systole Pressure(LVSP),Identical Volumed end systolic pressure's maximum ascendin speed(+dp/dtmax)and these are the left ventricular diastolic function parameters:Left Ventricular End-Diastolic pressure(LVEDP),Left Ventricular end diastolic pressure's maximum descending speed(-dp/dtmax).After an interval of 5 minutes,Shen Fu Injection cedilanid and dobutamine were respectively injected into the veins of the rats in the corresponding group.At an interval of 10 minutes,the hemodynamics parameters ofthe rats in the SFI group,the cedilanid group and the dobutamine group were recorded again.(2)Medicine pretreatment group:Respectively 1/2hour, 1hour, 2hours after the abdominal aorta banding operations,from the model group,the HDSFI group,the LDSFI group and the Enalapril group 5 rats were respectively taken for hemodynamics examination and other index checks.So it was with the rats in the same operation group.The ANP expression of myocardial tissues was examined by radiommunoassay(RIA),thecontent of Na+/K+ATPase and Ca+ATPase in the myocardial tissues was examined through spectrophotometryThe apoptosis of myocardial cells was examined through the method of TUNEL,and the proteins Bcl-2 and Bax of myocardial cells were examined through immunoassay.2-3 days old newborn clean-level suckling Wister rats were prepared for the separation,purification and cultivation of myocardial cells. In our experiment glucose-deficiency solution Hanks was saturated with high-purity nitrogen gas (99.999%) for 15 minutes.After that,nitrogen gas was put into the culture bottle at the speed of 1 liter/minute for 30 minutes to expel the air inside the bottle.In the culture medium of the control group there was no nitrogen gas.The cultivated myocardial cells were taken and divided at random into the following group:â‘ the control groupâ‘¡the glvcose-deficiency and oxygen-deficiency(GDOD)groupâ‘¢the GDOD plus HDSFI group(containing ginseng 1000ug/ml)â‘£the GDOD plus MDSFI group(containing ginseng 500ug/ml)⑤the GDOD plus LDSFI group(containing ginseng 250ug/ml)â‘¥the GDOD plus Enalapril(500ug/ml) group.The growth situation and the pulsng frequency were observed under microscope.The vitality and DNA content of myocardial cells and cellular cycle and apoptosis rate of myocardial cells were examined through different ways.Result: After the acute pressure-overloaded rat models were made,it was observed that the heart rate(HR) of the rats in the SFI group and the Cedilanid group declined significantly after the medicine treatment,compared with the HR of the rats in the same groups before the medicine treatment(p<0.01). But the HR of the rats in the Dobutamine group increased significantly(p<0.01).The LVSP and +dp/dtmax in the SFI group,the Cedilanid group and the Dobutamine group increased significantly after the medicine treatment(p<0.01) compared with those in the same groups before the medicine treatment.The -dp/dtmax in the SFI group increased significantly after the medicine treatment compared with that before the treatment,but in the Cedilanid group and the Dobutamine group it decreased significantly(p<0.01).After the medicine treatment,the LVEDP in the SFI group declined significantly, but in the Cedilanid group and the Dobutamine group it increased significantly (p<0.01).At the same time,with the pretreatment of Shen Fu Injection or Enalapril,the hemodynamics parameters of the acute pressure-overloded rats showed siginificant change at different time points(p<0.01):For example,the LVSP and -dp/dtmax of the rats in the LDSFI group declined significantly compared with those indexes of the rats in the HDSFI group after the pretreatment(p<0.05);the LVSP and +dp/dtmax of the rats in the Enalapril group declined significantly compared with those indexed of the rats in the SFI grou(p<0.01).In the experiment we discovered that 0.5hour, 1hour,2hours after the abdominal aorta banding operation,the ANP level of the myocardial tissuses of the rats went up gradually, and that 2hours after the abdominal aorta banding operation,the ANP level of the myocardial tissues of the rats was 5.45 times higher than that in the sham operation group,and at that time the application of Shen Fu Injection could effectively bring down the ANP level of the myocardial tissues of the actute pressure-overloaded rats; It was also found that the application of Shen Fu Injection before banding the abdominal aorta of the rats,no matter high-dosed,could effectively bring down the ANP-level of the myocardial tissues of the rats after the abdominal aorta banding operation,which indicated that under the acute pressure-overloaded situation the ANP secretion of the atria went up,but Shen Fu Injection could decrease the secretive action,and the tyeatment effect of Shen Fu Injection was better than that of Cedilanid and Dobutamine,which indicated that Shen Fu Injection has a rather good eary pretreatment effect on the ANP expression of the rat hearts under the acute pressure-overloaded situation.The experiment also showed that Shen Fu Injection,Cedilanid and Dobutamine could effectively increase the expression level of Na+/K+ATPase and Ca2+ATPase of the myocardial tissues of the rats.It was also found that after the pretrentment of Shen Fu Injection,the Na+/K+ATPase and Ca2+ATPase expression level of the myocardial tissues of the rats in the HDSFI group,the LDSFI group and the Enalapril group increased significantly compared with the Na+/K+ATPase and Ca2+ATPase expression level in the control group at different time points. It was also discovered that 0.5hour, 1hour and 2hours after the abdominal aorta banding operation,the apoptosis rate of the myocardial cells of the rats in the model group was significantly higher than that in the sham operation group.The apoptosis rate of the myocardial cells of the rats in the model group 2hours after the abdominal aorta banding operation was 9.13 times higher than that in the sham operation group at the corresponding time,and the apoptosis rate of the myocardial cells of the acute pressure-overloaded rats tended to go up with the moving of time.And the application of high-or-low-dosed Shen Fu Injection and Enalapril in advance could obviously bring down the apoptosis rate of the myocardial cells of the rats at different time points after the abdominal aorta banding operation.It was also found that the application of Shen Fu Injection 2hours after the abdominal aorta banding operation could also decrease the apoptosis rate of the myocardial cells of the rats.The in vitro experiment showed that the apoptosis rate of the normal myocardial cells was (1.08±0.84)%; 3hours after the glucose-deficiency and oxygen-deficiency the apoptosis rate of the myocardial cells was (36.02±0.48)%; 3hours after the glucose-deficiency and oxygen-deficiency culture combined with the pretreatment of the high-dosed Shen Fu Injection,the apoptosis rate of the myocardial cells was (2.23±0.89)%,which was not signicantly different from that of the control group.For the rats in the MDSFI group and LDSFI group,3hours after the cultivation in glucose-deficiency and oxygen-deficiciency, the apoptsis rates of the myocardial cells were respectively(4.30±0.47)% and (5.7±0.56)%,there was significant difference when then the apoptosis rates in the two groups were compared with the apoptosis rate in the control group(p<0.05).For the rats in the GDOD plus Enalapril group,3hours after the cultivation the apoptosis rate of the myocardial cells was(11.23±0.34)%,compared with that in the GDOD group,there was signicant difference(p<0.05).As far as the apoptosis rate is concerned,when the Shen Fu Injection of different doses were compared with Enalapril,there was significant difference(p<0.05).The results of the experiment showed that 3hours after GDOD culture, the ANP expression in the GDOD group increased significantly, compared with that in the control group(p<0.01).The pretreatment of Shen Fu Injection could obviously restrain the GDOD-induced synthesis of the ANP of the myocardial cells(p<0.05),but when Enalapril was compared with Shen Fu Injection of different doses,there was significant different(p<0.05).Conclusion: Shen Fu Injection could restrain the ANP expression of the myocardial cells which was induced by acute overloaded pressure or GDOD condition.It could effectively enhance the expression level of Na+/K+ATPase and Ca2+ATPase of the myocardial cells and improve the abnormal absorption and release of Ca2+ of the myocardial cells under acute overloaded pressure.It could also increase the expression of protein Bcl-2 and the ratio of Bcl-2/Bax.Thus,it could effectively prevent and bring down the occurance of apoptsis of the myocardial cells induced by acute overloaded pressure and the culture of GDOD,and it could reduce the loss of the number of the myocardial cells.In this way, it could improve the myocardial contractility and diastolic capacity of the heart when HF happened and sustain the function of the heart.