摘要
活络效灵丹是活血化瘀的经典方剂,出自清代名医张锡纯的《医学衷中参西录》,具有活血祛瘀、通络止痛的作用,是活血止痛的常用方剂。近年来,活络效灵丹在临床上也开始应用于治疗各类冠心病,但对其治疗心肌缺血再灌注的研究尚少。
本研究运用血清药理学方法,在原代大鼠乳鼠心肌细胞上建立缺氧/复氧(Hypoxia/Reoxygenation, H/R)模型,模拟在体心肌缺血/再灌注(Ischemia/Reperfusion,I/R)损伤,并采用冠心病常用药单硝酸异山梨酯(Isosorbide Mononitrate)作为阳性对照药物,观察活络效灵丹及其系列组方对缺氧/复氧损伤心肌细胞的保护作用。主要研究结果如下：
1.活络效灵丹对缺氧/复氧损伤心肌细胞的保护作用的评价。采用MTT法,比色法和流式细胞术,考察其对H/R损伤心肌细胞的存活率、LDH漏出量、MDA含量、SOD活性以及心肌细胞凋亡率的影响。结果提示,活络效灵丹对缺氧/复氧损伤心肌细胞具有良好的保护作用。
2.活络效灵丹系列组方对缺氧/复氧损伤心肌细胞保护作用的初步筛选。采用MTT法和比色法,考察其对H/R损伤心肌细胞的存活率、LDH漏出量的影响。结果提示,活络效灵丹组、丹参当归组、丹参乳香组、丹参当归乳香组在保护缺氧/复氧损伤心肌细胞方面疗效更为显著,用来进行进一步研究。
3.活络效灵丹系列优化组方对H/R损伤心肌细胞保护作用相关机制的评价。采用比色法和PCR法,考察其对H/R损伤心肌细胞的MDA含量、SOD活性以及心肌细胞凋亡基因Bcl-2和Bax mRNA的影响。结果提示,活络效灵丹优化组方在保护H/R损伤心肌细胞的作用机制可能与其对抗自由基损伤、上调Bcl-2基因表达和下调Bax基因表达有关。
4.活络效灵丹方中的丹参和当归在保护心肌细胞抗氧化损伤及调控细胞凋亡基因表达方面起主导作用。
综上研究表明,活络效灵丹及其系列组方对H/R损伤心肌细胞具有良好的保护作用,在冠心病尤其是心肌缺血再灌注疾病的治疗中具有潜在的应用价值。
Huoluoxiaolingdan is a classic decoction, which has powerful curative effects on blood circulation, dredging collaterals, relieving pain. In recent years, Huoluoxiaolingdan has already have a clinical use on coronary heart disease. But to date the study of myocardial ischemia and reperfusion is still less.
To study the protective effects of medicated serum with Huoluoxiaolingdan and its series on hypoxia/reoxygenation (H/R) injury in neonatal rat cardiomyocytes, myocardial H/R model was established on cultured primary myocardial cells of neonatal rat. The isosorbide mononitrate has been used as positive control. The key results are shown as below:
1. Evaluating the protective effects of Huoluoxiaolingdan against H/R injury in neonatal rat cardiomyocytes. The protective role of Huoluoxiaolingdan in cardiocytes survival rate was detected with MTT colorimetric method after H/R injury. The cell apoptosis was determined by flow cytometry. Moreover, the lactate dehydrogenase (LDH) activity in the supernatant, the malondialchehyche (MDA) content and the superoxide dismutase (SOD) activity in the supernatant were measured. Then the cell apoptosis was determined by flow cytometry. The results showed that Huoluoxiaolingdan has a significant protective effects on H/R injury cardiomyocytes.
2. Preliminaryly screening the series of Huoluoxiaolingdan by the protective effects on H/R injury cardiomyocytes. The protective role of Huoluoxiaolingdan in cardiocytes survival rate was detected with MTT colorimetric method after H/R injury. Meanwhile, the lactate dehydrogenase (LDH) activity in the supernatant was measured. The results showed that the medicated serum of Huoluoxiaolingdan, danshendanggui, danshenruxiang and danshendangguiruxiang have significant protective effects.
3. Investigating the action mechanism of protective effects on H/R injury cardiomyocyte. The malondialchehyche (MDA) content and the superoxide dismutase (SOD) activity in the supernatant were measured by colorimetric method. And RT-PCR was used to test the expression of gene-related apoptosis Bcl-2 mRNA and Bax mRNA. The results showed that the reducing of oxygen free radical and inhibiting myocardial apoptosis may be the possible mechanisms.
4. Angelica Sinensis and Salvia Miltiorrhiza in Huoluoxiaolingdan play an leading role on the protection of myocardial cells in antioxidant injury and inhibiting myocardial apoptosis gene expression.
In summary, Huoluoxiaolingdan and its series can protect cardiomyocytes from H/R-induced injure and exhibit a potentional application value on the treatment ofmyocardial ischemia and reperfusion.
引文
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