超微和普通生脉散对大鼠心肌缺血再灌注损伤的保护作用和机制的比较研究
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摘要
背景
心肌细胞发生缺血再灌注损伤(RI)时,细胞的钙调控发生改变,肌浆网的钙摄取功能和肌膜钙ATP酶受到抑制,钙离子在细胞内蓄积,形成钙超载;细胞内过多的钙离子可激活胞内的磷脂酶A2,使细胞膜磷脂分解,由黄嘌呤氧化酶途径生成大量的氧自由基,后者直接造成心肌细胞损伤,所以细胞内的钙超载和氧自由基产生增多是造成损伤的重要原因。
心肌缺血再灌注损伤最常见的表现包括心肌酶谱的变化、各种心律失常和心肌顿抑。西医缺少积极的防治办法,中医治疗能起到一定的疗效。生脉散及其注射剂是临床广为应用的益气补阴固脱的方剂,其抗心肌缺血再灌注损伤(RI)的作用备受关注。超微饮片是近年来新研制的一种中药饮片,目前许多研究表明超微饮片不仅可以替代传统饮片用于临床,而且可以加速药物成分的吸收,减少药物的剂量。关于超微生脉散在抗心肌缺血再灌注损伤(RI)的作用目前缺少研究。
目的
1.通过对超微生脉散和普通生脉散抗心肌RI的对比研究,为超微饮片的进一步研究和开发应用提供理论依据。
2.探索生脉散在抗RI过程中的抗细胞内钙超载作用的调控机制,研究超微生脉散和普通生脉散对心肌RI模型细胞内钙离子浓度的影响和对心肌细胞钙调节蛋白SERCA2、RYR2的mRNA表达的影响。
方法
1.用结扎30min/松开大鼠左冠状动脉前降支的方法造成再灌注损伤模型。在实验前60min,分别给予生理盐水和药物,分组为:正常组(C)、模型组(M),普通生脉散低剂量治疗组(T1)、普通生脉散中剂量治疗组(T2)、普通生脉散高剂量治疗组(T3)、超微生脉散低剂量治疗组(F1)、超微生脉散中剂量治疗组(F2)、超微生脉散高剂量治疗组(F3)。实验中观察心律失常的发生情况。再灌注180分钟后,取血分离血清,检测LDH、CK-MB、MDA、SOD。摘取心脏,免疫组化测Bax、Bcl-2和caspase-3的表达。
2.培养SD乳鼠心肌细胞,用缺氧/复氧法造成培养心肌细胞损伤模型,复氧同时分别给予空白血清冻干粉或含药血清冻干粉,细胞分组同前。分组处理后,正常培养24h,各组检测心肌细胞活力、细胞内钙离子浓度,继而用RT-PCR方法检测钙调节蛋白SERCA2、RyR2的mRNA表达。
结果
1.生脉散对大鼠急性心肌缺血再灌注性心律失常的影响:再灌注180min内,模型组大鼠再灌注性心律失常的总发生率为83.3%(10/12),各种心律失常的发生率较缺血阶段高,并出现了室性颤扑,死亡率25.0%。普通生脉散低剂量组死亡率11.1%,其它各组无死亡。各生脉散治疗组各种再灌注性心律失常的发生率均有降低,以超微生脉散中、高剂量组和普通生脉散高剂量组尤为明显,降至44.4%(4/9),二者与模型组比较,差异有非常显著性意义(p＜0.01)。
2.生脉散对缺血再灌注大鼠LDH、CK-MB的影响:模型组心肌酶LDH、CK-MB均较假手术组升高,分别为1615±176、665.4±39.8u/L和742±153、289.2±42.5u/L,两组比较差异有非常显著性意义(p＜0.01)。各治疗组的LDH、CK-MB也均高于假手术组,但低于模型组,与模型组比较,差异有非常显著性意义(p＜0.01)。同等剂量的超微生脉散和普通生脉散相比,超微生脉散降低缺血再灌注损伤所致LDH、CK-MB升高的作用更为明显,差异有非常显著性意义(p＜0.01)。
3.生脉散对缺血再灌注大鼠MDA、SOD的影响:模型组MDA均较假手术组升高,分别为6.31±0.52和0.32±0.14nmol/ml,各治疗组MDA也较假手术组升高,它们和假手术组比较,差异均有非常显著性意义(p＜0.01)。各生脉散治疗组MDA均低于模型组,与模型组比较差异有非常显著性意义(p＜0.01)。同等剂量的超微生脉散和普通生脉散相比,各超微生脉散组的MDA值均低于同等剂量的普通生脉散组的MDA值,两者差异有显著性(p＜0.05)。
与MDA的变化相反,模型组SOD较假手术组降低,分别为34.5±15.4和89.6±11.5umol/ml,和假手术组比较差异有非常显著性意义(p＜0.01)。各生脉散治疗组SOD均高于模型组、低于假手术组,和假手术组及模型组比较差异均有非常显著性意义(p＜0.01)。同等剂量的超微生脉散和普通生脉散相比,超微生脉散升高SOD的作用更为明显,两者差异有显著性(P＜0.05),其中超微生脉散高、中剂量组和普通生脉散高剂量组的SOD值略低于假手术组,但与假手术组比较无显著性差异(P＞0.05)。
4.生脉散对Bcl-2表达的影响:模型组及各生脉散组Bcl-2的表达较假手术组明显降低,与假手术组比较差异有非常显著性意义(p＜0.01);同时,各生脉散组Bcl-2表达均高于模型组,与模型组比较差异有非常显著性意义(p＜0.01);同剂量的超微生脉散组与普通生脉散组相比,超微生脉散组Bcl-2表达均高于普通生脉散组(P＜0.05)。
生脉散对Bax和caspase-3的表达的影响:模型组及各生脉散组Bax和caspase-3的表达较假手术组明显增加,与假手术组比较差异有非常显著性意义(p＜0.01);同时,各生脉散组Bax和caspase-3的表达均低于模型组,与模型组比较差异有非常显著性意义(p＜0.01);同剂量的超微生脉散组与普通生脉散组相比,超微生脉散高、中剂量组caspase-3的表达均低于同剂量普通生脉散组,两者比较差异有显著性意义(P＜0.05)。
5.超微和普通生脉散对缺氧复氧心肌细胞活力的影响:模型组心肌细胞严重受损,心肌细胞活力较正常对照组降低,与对照组相比差异有非常显著性意义(p＜0.01)。预先加入生脉散血清,可见(?)治疗组细胞存活率高于模型组,差异有非常显著性意义(p＜0.01)。说明生脉散血清可以提高RI致心肌细胞损伤时细胞的活力。将超微生脉散和普通生脉散相比较,可以发现,同等剂量超微生脉散血清作用强于普通生脉散。
6.超微和普通生脉散对RI心肌细胞内钙的影响:经缺氧复氧处理后,模型组心肌细胞内的钙(112.57±10.97)比正常对照组(75.15±4.87)显著升高,各生脉散治疗组的心肌细胞内钙也较正常对照组有较大幅度升高,但较模型组有不同程度下降,与正常对照组和模型组比较,差异均有非常显著性意义(p＜0.01),提示生脉散血清能抑制缺氧复氧造成的心肌细胞内钙超载。
将超微生脉散和普通生脉散血清作用相比较,可以发现,无论是在提高缺氧复氧损伤后所致的心肌细胞活力下降方面,还是在降低缺氧复氧损伤后所致的心肌细胞内钙超载方面,同剂量超微生脉散血清作用均强于普通生脉散血清,两者比较差异均有显著性(P＜0.05),且在实验所用的三种剂量情况下,剂量较高者作用相对较强,但超微生脉散高、中浓度血清治疗组组间差异并不显著(P＞0.05),提示在提高心肌细胞活力及抑制心肌细胞内钙超载方面,超微生脉散中等剂量水平即基本上能达到高剂量水平的效果。
7.超微和普通生脉散对RI心肌细胞SERCA2、RyR2的mRNA表达的影响:模型组心肌细胞SERCA2、RyR2的mRNA表达均较正常对照组降低,其相对表达量分别为0.48±0.11、0.47±0.09和0.79±0.09、0.80±0.11,两者比较差异有非常显著性意义(p＜0.01)。各生脉散组SERCA2、RyR2的mRNA表达量也较正常对照组低,但高于模型组,与两者比较,差异均有非常显著性意义(p＜0.01);将同剂量超微生脉散组和普通生脉散组比较,可以发现前者提高SERCA2、RvR2的mRNA的表达作用强于普通生脉散,两者比较差异均有显著性(P＜0.05),且在实验所用的高、中、低三种剂量情况下,剂量较高者作用相对较强,但超微生脉散高、中浓度血清治疗组组间差异并不显著(P＞0.05)。
结论
1.本研究的结果表明,超微生脉散不仅可以替代普通生脉散发挥抗RI的作用而且在同等剂量下,其作用更强。
2.生脉散抗心肌RI的作用,与其抗心肌细胞内钙超载密切相关。本研究首次表明,生脉散抗心肌细胞内钙超载的作用,可能与上调心肌细胞SERCA2、RyR2 mRNA的表达密切相关。这为临床使用超微生脉散治疗心肌RI提供了依据。
After myocardial ischemic/reperfusion injury(RI), there are a series of functional and morphological changes that are caused by intracellular calcium overloading, oxygen free radicals and so on. During the process of RI, myocardial calcium regulation function changes,which results in the depressing of myocardial cell membrane calcium ATP-ase and sarcoplasmic reticulum (SR) calcium intaking function. Intracellular redundant calcium ion activates intracellular phospholipaseA2 that can decompose cellular membrane phospholipid,which can product a devil of oxyradical by the way of xanthinoxidase(XO) which results in myocardial cell damage.
RI often manifests the change of the myocardial zymogram , arrhythmia that may be tachy- or brady-arrhythmia and myocardial stunning that may display persistent depression of ventricular systolic function. There is no possitive prevention and cure measure in modern medicine while traditional Chinese medicine treatment can have better curative effect.
ShengMai San(SMS, Powder for Restoring Pulse Beat)and its injectable preparation have been paid close attention to because some studies indicated that SMS could alleviate myocardial ischemia/reperfusion injury.
Ultrafine particle herb is a new Chinese medicinal material. At present, many investigations have indicated that it can not only substitute traditional cut crudedrug, but also speed up the absorption of medicine ingredient and reduce intaking dosage.
Purpose
1. The comparative studies on the effect of SMS traditional decoction and SMS ultrafine particle leachate on myocardium RI were designed to provide theoretical evidences for the ulterior investigation and development and application of ultrafine particle herbs.
2. This experiment was designed to research the intracellular Ca~(2+) regulation mechanism of SMS on decreasing calcium overload in the process of myocardium RI and investigate the effects of SMS traditional decoction and SMS ultrafine particle leachate on intracellular Ca~(2+) in RI model and their effects on the mRNA expression of myocardial cell calmodulin (CaM) SERCA2、RyR2.
Methods
1. In this experiment, the model foundation in vivo was made up of two main steps:ligating the left-anterior coronary artery for 30 minutes and then loosing it for 180 minutes.60 minutes before this experiment, intragastric administration was implemented. During the process of the experiment, the electrocardiogram was recorded continuously. SD rats were devided into 8 groups:control group (C), model group(M), SMS traditional decoction low dose group(Tl), SMS traditional decoction middle dose group(T2),SMS traditional decoction high dose group(T3),SMS ultrafine particle leachate low dose group(F1),SMS ultrafine particle leachate middle dose group(F2), and SMS ultrafine particle leachate high dose group(F3). We observed the incidence of arrhythmia caused by reperfusion in different groups.After 180minutes' reperfusion,we collected these rats' blood to demesh their serum and measure the contents of LDH、CK-MB、MDA and SOD, and picked up their hearts to survey the expression of Bax、Bcl-2 and caspase-3 bymeans of immunohistochemistry.
2.Cultured neonatal rat cardiomyocytes RI model in vitro were made by Hypoxia/Reoxygenation.When oxygen was reprovided , serum freeze drying powder without SMS constituent and that with SMS constituent was added to the culture fluid respectively at the same time.Cultured myocardial cells were devided into 8 different groups as before . Cadiocyte vitality and intracellular free Ca~(2+) of all the groups were measured .The mRNA expressing of calmodulin(CaM) sarcoplasmic reticular Ca~(2+)-ATPase-2 (SERCA2) and ryanodine receptor-2(RyR2) of all the groups were measured by reverse transcription-polymerase chain reaction(RT-PCR) and normalized to the mRNA levels ofβ-actin.
Results
1.The incidence of arrhythmia caused by RI in the model group was 83.3%. SMS decreased the arrhythmia incidence to the scale between 66.7% and 44.4%.There was remarkable difference between the SMS groups and the model group (P<0.01).
The contents of LDH and CK-MB in the model group were 1615±176 and 665.4±39.8u/L respectively,while in SMS groups they were from 1488±143 to 971±168 and from 482.76±40.3 to 314±53.2 u/L respectively.Compared with the model group, the contents of LDH and CK-MB in the SMS groups decreased markedly (P<0.01).
The content of MDA in the model group was 6.31±0.52nmol/ml,while in the SMS groups it was from 4.68±0.76 to 2.55±0.30 nmol/ml. Compared with the model group, the content of MDA in SMS groups decreased markedly (P<0.01).The content of SOD in the model group was 34.5±15.4 umol/ml, while in SMS groups it was from 51.7±14.6 to 86.2±13.2 umol/ml.There is remarkable difference between the SMS groups and the model group, that is, the contents of SOD in the SMS groups raised markedly (P<0.01).
Compared with the model group, the expression of Bcl-2 in the SMS groups raised markedly(P<0.01),but that of Bax and caspase-3 decreased markedly (P<0.01).
2.The experiment in vitro manifested that the cadiocyte vitality in the model group was impaired seriously. Compared with the model group, the cadiocyte vitality in the SMS groups raised markedly (P<0.01).
The cadiocyte intracellular Ca~(2+) in the model group raised seriously. Compared with the model group, the cadiocyte intracellular Ca~(2+) in the SMS groups decreased markedly (P<0.01)..
Compared with the control group, the mRNA expression of cadiocyte SERCA2、RyR2 in the model group decreased markedly (P<0.01). Compared with the model group, the mRNA expression of cadiocyte SERCA2、RyR2 in the SMS groups raised markedly (P<0.01).
In all the experiments,SMS traditional decoction and ultrafine particle leachate had the similar effect, but the effect of the latter was more effective.
Conclusion
1. The studies indicated that SMS ultrafine particle herbs can substitute SMS traditional Chinese cut crude drugs, furthermore, the effect of isodose SMS ultrafine particle herbs is more effectual.
2. The effect of SMS on resisting myocardium RI was correlated to decreasing cardiocyte intracellular calcium overload through reinforcing the mRNA expression of SERCA2、RyR2.The studies provided substantial evidence for clinical application of SMS ultrafine particle herbs to treat myocardial ischemic/reperfusion injury(RI).
心肌细胞发生缺血再灌注损伤(RI)时,细胞的钙调控发生改变,肌浆网的钙摄取功能和肌膜钙ATP酶受到抑制,钙离子在细胞内蓄积,形成钙超载;细胞内过多的钙离子可激活胞内的磷脂酶A2,使细胞膜磷脂分解,由黄嘌呤氧化酶途径生成大量的氧自由基,后者直接造成心肌细胞损伤,所以细胞内的钙超载和氧自由基产生增多是造成损伤的重要原因。
心肌缺血再灌注损伤最常见的表现包括心肌酶谱的变化、各种心律失常和心肌顿抑。西医缺少积极的防治办法,中医治疗能起到一定的疗效。生脉散及其注射剂是临床广为应用的益气补阴固脱的方剂,其抗心肌缺血再灌注损伤(RI)的作用备受关注。超微饮片是近年来新研制的一种中药饮片,目前许多研究表明超微饮片不仅可以替代传统饮片用于临床,而且可以加速药物成分的吸收,减少药物的剂量。关于超微生脉散在抗心肌缺血再灌注损伤(RI)的作用目前缺少研究。
目的
1.通过对超微生脉散和普通生脉散抗心肌RI的对比研究,为超微饮片的进一步研究和开发应用提供理论依据。
2.探索生脉散在抗RI过程中的抗细胞内钙超载作用的调控机制,研究超微生脉散和普通生脉散对心肌RI模型细胞内钙离子浓度的影响和对心肌细胞钙调节蛋白SERCA2、RYR2的mRNA表达的影响。
方法
1.用结扎30min/松开大鼠左冠状动脉前降支的方法造成再灌注损伤模型。在实验前60min,分别给予生理盐水和药物,分组为:正常组(C)、模型组(M),普通生脉散低剂量治疗组(T1)、普通生脉散中剂量治疗组(T2)、普通生脉散高剂量治疗组(T3)、超微生脉散低剂量治疗组(F1)、超微生脉散中剂量治疗组(F2)、超微生脉散高剂量治疗组(F3)。实验中观察心律失常的发生情况。再灌注180分钟后,取血分离血清,检测LDH、CK-MB、MDA、SOD。摘取心脏,免疫组化测Bax、Bcl-2和caspase-3的表达。
2.培养SD乳鼠心肌细胞,用缺氧/复氧法造成培养心肌细胞损伤模型,复氧同时分别给予空白血清冻干粉或含药血清冻干粉,细胞分组同前。分组处理后,正常培养24h,各组检测心肌细胞活力、细胞内钙离子浓度,继而用RT-PCR方法检测钙调节蛋白SERCA2、RyR2的mRNA表达。
结果
1.生脉散对大鼠急性心肌缺血再灌注性心律失常的影响:再灌注180min内,模型组大鼠再灌注性心律失常的总发生率为83.3%(10/12),各种心律失常的发生率较缺血阶段高,并出现了室性颤扑,死亡率25.0%。普通生脉散低剂量组死亡率11.1%,其它各组无死亡。各生脉散治疗组各种再灌注性心律失常的发生率均有降低,以超微生脉散中、高剂量组和普通生脉散高剂量组尤为明显,降至44.4%(4/9),二者与模型组比较,差异有非常显著性意义(p＜0.01)。
2.生脉散对缺血再灌注大鼠LDH、CK-MB的影响:模型组心肌酶LDH、CK-MB均较假手术组升高,分别为1615±176、665.4±39.8u/L和742±153、289.2±42.5u/L,两组比较差异有非常显著性意义(p＜0.01)。各治疗组的LDH、CK-MB也均高于假手术组,但低于模型组,与模型组比较,差异有非常显著性意义(p＜0.01)。同等剂量的超微生脉散和普通生脉散相比,超微生脉散降低缺血再灌注损伤所致LDH、CK-MB升高的作用更为明显,差异有非常显著性意义(p＜0.01)。
3.生脉散对缺血再灌注大鼠MDA、SOD的影响:模型组MDA均较假手术组升高,分别为6.31±0.52和0.32±0.14nmol/ml,各治疗组MDA也较假手术组升高,它们和假手术组比较,差异均有非常显著性意义(p＜0.01)。各生脉散治疗组MDA均低于模型组,与模型组比较差异有非常显著性意义(p＜0.01)。同等剂量的超微生脉散和普通生脉散相比,各超微生脉散组的MDA值均低于同等剂量的普通生脉散组的MDA值,两者差异有显著性(p＜0.05)。
与MDA的变化相反,模型组SOD较假手术组降低,分别为34.5±15.4和89.6±11.5umol/ml,和假手术组比较差异有非常显著性意义(p＜0.01)。各生脉散治疗组SOD均高于模型组、低于假手术组,和假手术组及模型组比较差异均有非常显著性意义(p＜0.01)。同等剂量的超微生脉散和普通生脉散相比,超微生脉散升高SOD的作用更为明显,两者差异有显著性(P＜0.05),其中超微生脉散高、中剂量组和普通生脉散高剂量组的SOD值略低于假手术组,但与假手术组比较无显著性差异(P＞0.05)。
4.生脉散对Bcl-2表达的影响:模型组及各生脉散组Bcl-2的表达较假手术组明显降低,与假手术组比较差异有非常显著性意义(p＜0.01);同时,各生脉散组Bcl-2表达均高于模型组,与模型组比较差异有非常显著性意义(p＜0.01);同剂量的超微生脉散组与普通生脉散组相比,超微生脉散组Bcl-2表达均高于普通生脉散组(P＜0.05)。
生脉散对Bax和caspase-3的表达的影响:模型组及各生脉散组Bax和caspase-3的表达较假手术组明显增加,与假手术组比较差异有非常显著性意义(p＜0.01);同时,各生脉散组Bax和caspase-3的表达均低于模型组,与模型组比较差异有非常显著性意义(p＜0.01);同剂量的超微生脉散组与普通生脉散组相比,超微生脉散高、中剂量组caspase-3的表达均低于同剂量普通生脉散组,两者比较差异有显著性意义(P＜0.05)。
5.超微和普通生脉散对缺氧复氧心肌细胞活力的影响:模型组心肌细胞严重受损,心肌细胞活力较正常对照组降低,与对照组相比差异有非常显著性意义(p＜0.01)。预先加入生脉散血清,可见(?)治疗组细胞存活率高于模型组,差异有非常显著性意义(p＜0.01)。说明生脉散血清可以提高RI致心肌细胞损伤时细胞的活力。将超微生脉散和普通生脉散相比较,可以发现,同等剂量超微生脉散血清作用强于普通生脉散。
6.超微和普通生脉散对RI心肌细胞内钙的影响:经缺氧复氧处理后,模型组心肌细胞内的钙(112.57±10.97)比正常对照组(75.15±4.87)显著升高,各生脉散治疗组的心肌细胞内钙也较正常对照组有较大幅度升高,但较模型组有不同程度下降,与正常对照组和模型组比较,差异均有非常显著性意义(p＜0.01),提示生脉散血清能抑制缺氧复氧造成的心肌细胞内钙超载。
将超微生脉散和普通生脉散血清作用相比较,可以发现,无论是在提高缺氧复氧损伤后所致的心肌细胞活力下降方面,还是在降低缺氧复氧损伤后所致的心肌细胞内钙超载方面,同剂量超微生脉散血清作用均强于普通生脉散血清,两者比较差异均有显著性(P＜0.05),且在实验所用的三种剂量情况下,剂量较高者作用相对较强,但超微生脉散高、中浓度血清治疗组组间差异并不显著(P＞0.05),提示在提高心肌细胞活力及抑制心肌细胞内钙超载方面,超微生脉散中等剂量水平即基本上能达到高剂量水平的效果。
7.超微和普通生脉散对RI心肌细胞SERCA2、RyR2的mRNA表达的影响:模型组心肌细胞SERCA2、RyR2的mRNA表达均较正常对照组降低,其相对表达量分别为0.48±0.11、0.47±0.09和0.79±0.09、0.80±0.11,两者比较差异有非常显著性意义(p＜0.01)。各生脉散组SERCA2、RyR2的mRNA表达量也较正常对照组低,但高于模型组,与两者比较,差异均有非常显著性意义(p＜0.01);将同剂量超微生脉散组和普通生脉散组比较,可以发现前者提高SERCA2、RvR2的mRNA的表达作用强于普通生脉散,两者比较差异均有显著性(P＜0.05),且在实验所用的高、中、低三种剂量情况下,剂量较高者作用相对较强,但超微生脉散高、中浓度血清治疗组组间差异并不显著(P＞0.05)。
结论
1.本研究的结果表明,超微生脉散不仅可以替代普通生脉散发挥抗RI的作用而且在同等剂量下,其作用更强。
2.生脉散抗心肌RI的作用,与其抗心肌细胞内钙超载密切相关。本研究首次表明,生脉散抗心肌细胞内钙超载的作用,可能与上调心肌细胞SERCA2、RyR2 mRNA的表达密切相关。这为临床使用超微生脉散治疗心肌RI提供了依据。
After myocardial ischemic/reperfusion injury(RI), there are a series of functional and morphological changes that are caused by intracellular calcium overloading, oxygen free radicals and so on. During the process of RI, myocardial calcium regulation function changes,which results in the depressing of myocardial cell membrane calcium ATP-ase and sarcoplasmic reticulum (SR) calcium intaking function. Intracellular redundant calcium ion activates intracellular phospholipaseA2 that can decompose cellular membrane phospholipid,which can product a devil of oxyradical by the way of xanthinoxidase(XO) which results in myocardial cell damage.
RI often manifests the change of the myocardial zymogram , arrhythmia that may be tachy- or brady-arrhythmia and myocardial stunning that may display persistent depression of ventricular systolic function. There is no possitive prevention and cure measure in modern medicine while traditional Chinese medicine treatment can have better curative effect.
ShengMai San(SMS, Powder for Restoring Pulse Beat)and its injectable preparation have been paid close attention to because some studies indicated that SMS could alleviate myocardial ischemia/reperfusion injury.
Ultrafine particle herb is a new Chinese medicinal material. At present, many investigations have indicated that it can not only substitute traditional cut crudedrug, but also speed up the absorption of medicine ingredient and reduce intaking dosage.
Purpose
1. The comparative studies on the effect of SMS traditional decoction and SMS ultrafine particle leachate on myocardium RI were designed to provide theoretical evidences for the ulterior investigation and development and application of ultrafine particle herbs.
2. This experiment was designed to research the intracellular Ca~(2+) regulation mechanism of SMS on decreasing calcium overload in the process of myocardium RI and investigate the effects of SMS traditional decoction and SMS ultrafine particle leachate on intracellular Ca~(2+) in RI model and their effects on the mRNA expression of myocardial cell calmodulin (CaM) SERCA2、RyR2.
Methods
1. In this experiment, the model foundation in vivo was made up of two main steps:ligating the left-anterior coronary artery for 30 minutes and then loosing it for 180 minutes.60 minutes before this experiment, intragastric administration was implemented. During the process of the experiment, the electrocardiogram was recorded continuously. SD rats were devided into 8 groups:control group (C), model group(M), SMS traditional decoction low dose group(Tl), SMS traditional decoction middle dose group(T2),SMS traditional decoction high dose group(T3),SMS ultrafine particle leachate low dose group(F1),SMS ultrafine particle leachate middle dose group(F2), and SMS ultrafine particle leachate high dose group(F3). We observed the incidence of arrhythmia caused by reperfusion in different groups.After 180minutes' reperfusion,we collected these rats' blood to demesh their serum and measure the contents of LDH、CK-MB、MDA and SOD, and picked up their hearts to survey the expression of Bax、Bcl-2 and caspase-3 bymeans of immunohistochemistry.
2.Cultured neonatal rat cardiomyocytes RI model in vitro were made by Hypoxia/Reoxygenation.When oxygen was reprovided , serum freeze drying powder without SMS constituent and that with SMS constituent was added to the culture fluid respectively at the same time.Cultured myocardial cells were devided into 8 different groups as before . Cadiocyte vitality and intracellular free Ca~(2+) of all the groups were measured .The mRNA expressing of calmodulin(CaM) sarcoplasmic reticular Ca~(2+)-ATPase-2 (SERCA2) and ryanodine receptor-2(RyR2) of all the groups were measured by reverse transcription-polymerase chain reaction(RT-PCR) and normalized to the mRNA levels ofβ-actin.
Results
1.The incidence of arrhythmia caused by RI in the model group was 83.3%. SMS decreased the arrhythmia incidence to the scale between 66.7% and 44.4%.There was remarkable difference between the SMS groups and the model group (P<0.01).
The contents of LDH and CK-MB in the model group were 1615±176 and 665.4±39.8u/L respectively,while in SMS groups they were from 1488±143 to 971±168 and from 482.76±40.3 to 314±53.2 u/L respectively.Compared with the model group, the contents of LDH and CK-MB in the SMS groups decreased markedly (P<0.01).
The content of MDA in the model group was 6.31±0.52nmol/ml,while in the SMS groups it was from 4.68±0.76 to 2.55±0.30 nmol/ml. Compared with the model group, the content of MDA in SMS groups decreased markedly (P<0.01).The content of SOD in the model group was 34.5±15.4 umol/ml, while in SMS groups it was from 51.7±14.6 to 86.2±13.2 umol/ml.There is remarkable difference between the SMS groups and the model group, that is, the contents of SOD in the SMS groups raised markedly (P<0.01).
Compared with the model group, the expression of Bcl-2 in the SMS groups raised markedly(P<0.01),but that of Bax and caspase-3 decreased markedly (P<0.01).
2.The experiment in vitro manifested that the cadiocyte vitality in the model group was impaired seriously. Compared with the model group, the cadiocyte vitality in the SMS groups raised markedly (P<0.01).
The cadiocyte intracellular Ca~(2+) in the model group raised seriously. Compared with the model group, the cadiocyte intracellular Ca~(2+) in the SMS groups decreased markedly (P<0.01)..
Compared with the control group, the mRNA expression of cadiocyte SERCA2、RyR2 in the model group decreased markedly (P<0.01). Compared with the model group, the mRNA expression of cadiocyte SERCA2、RyR2 in the SMS groups raised markedly (P<0.01).
In all the experiments,SMS traditional decoction and ultrafine particle leachate had the similar effect, but the effect of the latter was more effective.
Conclusion
1. The studies indicated that SMS ultrafine particle herbs can substitute SMS traditional Chinese cut crude drugs, furthermore, the effect of isodose SMS ultrafine particle herbs is more effectual.
2. The effect of SMS on resisting myocardium RI was correlated to decreasing cardiocyte intracellular calcium overload through reinforcing the mRNA expression of SERCA2、RyR2.The studies provided substantial evidence for clinical application of SMS ultrafine particle herbs to treat myocardial ischemic/reperfusion injury(RI).
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