参附注射液对大鼠急性压力超负荷及乳鼠心肌细胞缺糖缺氧损伤的影响

【作者】 徐伟建；

【导师】 凃晋文；

【作者基本信息】 湖北中医学院 ， 中医内科学， 2006， 博士

【摘要】 目的：采用腹主动脉结扎的方法制作急性压力超负荷大鼠模型，研究参附注射液对其进行治疗后，以及参附注射液提前干预后大鼠急性压力超负荷在不同时间点的血流动力学及心肌组织病理学变化，观察参附注射液对急性压力超负荷大鼠心肌组织ANP表达水平，及对急性压力超负荷大鼠心肌组织Na⁺-K⁺ATPase与Ca²⁺-ATPase表达水平的影响，并对腹主动脉结扎致压力超负荷大鼠心肌细胞凋亡率及凋亡相关基因Bcl-2、Bax蛋白表达的影响进行研究，同时采用体外培养心肌细胞的方法，并造成缺糖缺氧损伤模型，探讨参附注射液对急性压力超负荷大鼠的干预及治疗作用及缺糖缺氧损伤的心肌细胞的作用。方法：参照相关文献制作急性压力超负荷大鼠模型。实验分为：（1）药物治疗组：雄性Wistar大鼠50只，随机取8只作为假手术（Sham）组，其余42只行腹主动脉结扎术。其中10只大鼠于腹主动脉结扎术中死亡，32只存活大鼠随机分为模型组、参附组、西地兰组、多巴酚丁胺组，每组8只。假手术组仅分离腹主动脉而不结扎。术后2小时，分离大鼠左侧颈外静脉，分别推注①参附组：参附注射液0.5ml（人参皂甙3.5mg/1ml）；②西地兰组：西地兰0.5ml（0.036mg/1ml）；③多巴酚丁胺组：多巴酚丁胺0.5ml（1.8mg/1ml）。（2）药物提前干预组：65只雄性Wistar大鼠随机分为5组。假手术组，模型组、参附高剂量组、参附低剂量组及依那普利组，其中假手术组为5只大鼠，其余四组各15只大鼠。参附高剂量组与低剂量组大鼠分别以8ml/kg及4ml/kg腹腔注射参附注射液，同时以生理盐水10ml/kg灌服；依那普利组大鼠灌服依那普利溶液10ml/kg（0.5mg/ml），同时腹腔注射生理盐水（8ml/kg），每日一次，连用7日后行腹主动脉结扎术；假手术组同时进行腹腔注射（8ml/kg）及灌服生理盐水（10ml/kg），7日后作腹部正中切口2cm后钝性分离腹主动脉但不结扎腹主动脉。血流动力学参数（1）药物治疗组：术后2小时，插入左室导管，八道生理记录仪记录压力曲线，计算左室收缩功能参数：左室内压峰值（LVSP）、左室压力上升最大速度(+dp/dt_max)及左室舒张功能参数：左室舒张末内压（LVEDP）、左室压力下降最大速度(-dp/dt_max)。间隔5分钟后，分别按不同组别从静脉推注参附注射液、西地兰及多巴酚丁胺，间隔10分钟后再次记录参附组、西地兰组、多巴酚丁胺组大鼠的血流动力学参数。（2）药物干预组：腹主动脉结扎术后1/2小时、1小时及2小时，于模型组、参附高剂量组、参附低剂量组及依那普利组中分别取5只大鼠行血流动力学检测及其他指标检测，同时测定假手术组上述3个时段血流动力学参数及其他指标采用放射免疫（RIA）法检测心肌组织ANP表达，分光光度法测定心肌组织中Na⁺/K⁺ATPase,Ca²⁺-ATPase含量。具体操作步骤按说明书进行采用脱氧核糖核苷酸末端转移酶介导的缺口末端标记法（TUNEL）检测心肌细胞凋亡，采用免疫组织化学染色检测心肌细胞Bcl-2与Bax蛋白。采用全自动图像分析仪于HPIAS-2000图像分析系统分析棕黄色阳性颗粒的平均吸光度（A）用2-3天Wistar乳鼠进行心肌细胞的分离、纯化、培养，实验时用缺糖Hanks液代替正常培养基。培养瓶内立即充入氮气（1升/分流量）30秒，造成缺糖缺氧心肌细胞损伤模型。对照组用正常培养基不含氮气取培养心肌细胞，随机分为：1组：正常对照组；2组：缺糖缺氧组；3组：缺糖缺氧+参附液高剂量组（含人参皂甙1000μg/ml）；4组：缺糖缺氧+参附液中剂量组（含人参皂甙500μg/ml）；5组：缺糖缺氧+参附液低剂量组（含人参皂甙250μg/ml）；6组：缺糖缺氧+依那普利组（500μg/ml），于倒置显微镜下观察心肌细胞生长情况和搏动频率，MTT比色法检测细胞活力，流式细胞仪检测心肌细胞DNA含量、细胞周期及凋亡率结果：在腹主动脉结扎后，大鼠出现呼吸浅而快，少动，唇舌紫绀等症状，符合中医心肾阳虚病机范畴，在结扎大鼠腹主动脉导致急性压力超负荷后，心率在参附组与西地兰组药物治疗后较治疗前显著降低（P＜0.01），而多巴酚丁胺组用药后HR显著增加（P＜0.01）；参附组、西地兰组、多巴酚丁胺组药物治疗后与治疗前相比，LVSP、+dp/dt_max在治疗后均有非常明显的升高（P＜0.01）；参附组药物治疗后-dp/dt_max较治疗前明显上升，而西地兰组和多巴胺组则显著下降（P＜0.01）；药物治疗后，参附组LVEDP有非常显著性的下降，而西地兰组与多巴酚丁胺组则明显上升（P＜0.01）。同时，提前使用参附注射液或依那普利大鼠各项血流动力学参数在急性压力超负荷后各时间点均出现非常明显的改变（P＜0.01）：在参附注射液提前干预组中，与模型组相比，高、低剂量组参附注射液各项血流动力学参数均有非常显著的差异（P＜0.01），其中提前使用参附低剂量组大鼠LVSP与-dp/dt_max较高剂量组明显降低（P＜0.05）；依那普利组大鼠LVSP及+dp/dt_max较参附组明显降低（P＜0.01）。实验中我们发现腹主动脉结扎后0.5小时、1小时、2小时，大鼠心肌组织ANP水平随时间逐渐升高，腹主动脉结扎2小时后大鼠心肌组织ANP水平较假手术组升高了5.45倍，而此时应用参附注射液即能有效地降低急性压力超负荷大鼠心肌组织ANP水平；同时，在结扎大鼠腹主动脉前使用参附注射液，无论高剂量或是低剂量，其均能有效地降低腹主动脉结扎后大鼠心肌组织ANP水平，显示在急性压力超负荷时心房ANP分泌增加，而参附注射液有减少其分泌的作用，且疗效较西地兰和多巴酚丁胺为佳，提示参附注射液对急性压力超负荷状态下的心脏ANP表达具有良好的早期干预作用。同时我们发现急性压力超负荷大鼠在腹主动脉结扎后0.5小时，心肌组织Na⁺/K⁺ATPase表达水平较假手术组有显著性的升高，但在随后的时间点（1小时和2小时），模型组大鼠心肌组织Na⁺/K⁺ATPase表达水平较假手术组有非常显著性的降低（P＜0.01），且心肌组织Na⁺/K⁺ATPase表达水平随时间的推移有降低的趋势，腹主动脉结扎2小时后，大鼠心肌组织Na⁺/K⁺ATPase与Ca²⁺-ATPase表达水平均较假手术组有非常显著性的降低（P＜0.01），同时，我们在实验中亦发现，Na⁺/K⁺ATPase与Ca²⁺-ATPase表达水平与心肌组织ANP蛋白表达水平呈负相关。实验结果显示，参附注射液、西地兰及多巴酚丁胺均能有效地增加大鼠心肌组织Na⁺-K⁺ATPase表达水平以及Ca²⁺ATPase表达水平；而提前使用参附注射液干预后，与不同时间点模型组大鼠心肌组织Na⁺-K⁺ATPase表达水平相比，高、低剂量参附注射液及依那普利均能显著增加Na⁺/K⁺ATPase及Ca²⁺ATPase表达水平。实验中我们发现在腹主动脉结扎后0.5小时，1小时和2小时，模型组大鼠心肌细胞凋亡率较假手术组明显升高，腹主动脉结扎后2小时模型组大鼠心肌细胞凋亡率较假手术组升高9.13倍，且急性压力超负荷大鼠心肌细胞凋亡率随着时间的推移有上升趋势。而预先使用高、低剂量参附注射液及依拉普利均能明显降低腹主动脉结扎后在不同的时间点的大鼠心肌细胞凋亡率，同时在腹主动脉结扎后2小时使用参附注射液能降低大鼠心肌细胞凋亡率。我们在实验中采用流式细胞术对使用不同剂量参附注射液缺糖缺氧培养的心肌细胞的细胞周期及心肌细胞凋亡率进行了监测。结果显示，在培养的正常心肌细胞在空白对照组心肌细胞G₁期之前，存在亚二倍体峰，提示处于正常条件培养的乳鼠心肌细胞即有细胞凋亡的存在，凋亡率为（1.08±0.84）％；参附注射液高剂量组+缺糖缺氧培养3小时后心肌细胞凋亡率为（2.23±0.89）％，与正常对照组相比无显著性差异。参附注射液中与低剂量组+缺糖缺氧培养3小时后心肌细胞凋亡率为分别为（4.30±0.47）％与（5.7±0.56）％，二组心肌细胞凋亡率与对照组相比有显著性差异（p＜0.05），依那普利+缺糖缺氧培养3小时后心肌细胞凋亡率为（11.23±0.34）％，与缺糖缺氧相比有显著性差异（p＜0.05），不同浓度的参附注射液与依那普利相比，亦有显著性的差异（p＜0.05）。本实验结果显示：缺糖缺氧培养3小时后，与正常对照组ANP表达量相比，缺糖缺氧组ANP表达明显增高（P＜0.01）。参附注射液干预后能明显降低缺糖缺氧诱导心肌细胞ANP的合成（P＜0.01），依那普利亦能明显降低缺糖缺氧诱导心肌细胞ANP的合成（P＜0.05），但与不同浓度的参附注射液相比，仍有显著性的差异（P＜0.05）。结论：总结以上实验结果，我们认为急性压力超负荷导致心肌射血时阻力增加，心脏做功增加的同时出现结构性的变化，以适应这种后负荷的改变，即心肌重塑，在此过程中心肌的耗氧耗能急剧增加。但机体储备有限，因此心肌细胞供氧供能相对不足，结果引起机体神经内分泌的激活如ANP表达增多，细胞因子分泌增多，进一步引起心肌细胞的重塑，最终形成心室扩张，肥厚；另一方面压力牵张可直接导致心肌细胞的结构及功能改变，引起相关基因的表达异常，Na⁺-K⁺-ATP酶、Ca⁺⁺-ATP酶表达的异常，并引发心肌细胞离子流障碍，同时由于能量及氧代谢异常可引起心肌细胞凋亡，心肌细胞数目减少，这些因素共同形成心脏舒缩功能障碍，心功能衰竭，临床上出现心慌、气喘、乏力、肢冷、水肿等症状，这些与中医之心气不足、心肾阳虚证候极为相似，故推测作为心衰根本病因的心肾阳虚之生物学基础可能在于心肌能量与氧代谢障碍。因此参附注射液益气温阳作用机制可能在于改善了急性压力超负荷状态下心肌异常的能量及氧代谢，减少了心肌ANP表达，有效提高心肌细胞的Na⁺/K⁺ATPase及Ca²⁺ATPase的表达水平，改善急性压力超负荷状态下心肌细胞异常的Ca²⁺摄取和释放，有效的预防和治疗急性压力超负荷状态下和缺糖缺氧条件下培养心肌细胞凋亡的发生，减少心肌细胞数量的丧失，从而改善心力衰竭时心脏收缩力减弱和舒张异常延迟的状态，维持心脏的功能。更多还原

【Abstract】 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:①in the Shen-Fu Injection group, 0.5ml of Shen-Fu Injection （Containing Jingsheng 3.5mg/ml）②in the Cedilanid group, 0.5ml of Cedilanid（0.036mg/ml）③in 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:①Medicine 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.②Medicine. 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:①the control group②the glvcose-deficiency and oxygen-deficiency（GDOD）group③the GDOD plus HDSFI group（containing ginseng 1000ug/ml）④the GDOD plus MDSFI group（containing ginseng 500ug/ml）⑤the GDOD plus LDSFI group（containing ginseng 250ug/ml）⑥the 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.更多还原