乌附经方预处理对大鼠缺血再灌注心肌细胞凋亡的影响

目的观察含乌头、附子、半夏、栝楼的乌附经方对大鼠心肌缺血再灌注损伤的作用，探讨含有十八反药物的方剂用于特定病理状态的可行性。方法雄性Wistar大鼠50只随机分为5组，即：假手术对照组（SH组）、心肌缺血再灌注模型对照组（I/R组）、乌附经方低剂量组（I/R+JFL组）、乌附经方中剂量组（I/R+JFM组）及乌附经方高剂量组（I/R+JFH组），每组10只。乌附经方低、中、高剂量组分别以0.75、1.50、3.00 mL/100 g体质量的剂量灌胃14天，浓度500 g/L；除SH组外，其他各组结扎冠状动脉左前降支，使心肌缺血30 min，再灌注120 min。透射电镜观察心肌线粒体的超微结构改变；一步法脱氧核糖核苷酸末端转移酶介导的缺口末端标记法（terminal-deoxynucleotidyl transferase mediated nick end labeling，TUNEL）检测各组缺血心肌细胞凋亡；RT-PCR法测B细胞白血病/淋巴瘤－2基因（B-cell leukemia/lymphoma 2，Bcl-2）、与Bcl-2相关的x蛋白（Bcl-2 associated x protein，Bax）的mRNA表达；ELISA检测血清乳酸脱氢酶（lactic dehydrogenase，LDH）、肌酸激酶（creatine kinase，CK）活性，并测定心肌梗死面积的大小。结果与I/R组比较，乌附经方各组均能降低血清中LDH、CK的释放量（且随着剂量的增加LDH、CK的释放量递减），减小心肌梗死面积、降低凋亡指数（P<0.05）；经方组凋亡基因Bcl-2表达水平上升，Bax表达水平下降，Bcl-2/Bax比值升高。结论乌附经方预处理可能通过减少细胞凋亡，对心肌缺血再灌注损伤产生保护作用。说明“十八反”不是绝对的，而是相对的，含有相反中药的复方制剂可用于缺血性心肌病等特定的病理状态。

英文摘要:

ObjectiveTo observe the protective effect of Wufu Jingfang （WJ， containing Aconitum carmichaeli Debx， Radix Aconiti Lateralis Preparatae，Rhizoma Pinelliae， and snakegourd fruit） on myocardial ischemia-reperfusion injury （I/R） of rats， thus exploring the feasibility of recipes containing eighteen imcompatible pairs for specific pathological conditions. MethodsFifty male Wistar rats were randomly divided into five groups， i.e.， the sham-operative control group （the SH group）， the I/R group， the low dose WJ I/R group （the I/R+JFL group）， the middle dose WJ I/R group （the I/R+JFM group）， the high dose WJ I/R group （the I/R+JFH group）， 10 in each group. Rats in the latter three groups were administered with WJ at 0.75 mL/100 g， 1.50 mL/100 g， and 3.00 mL/100 g body weight for 14 consecutive days by gastrogavage. All groups except the SH group received ligation of left anterior descending branch of coronary artery for 30-min ischemia followed by 120-min reperfusion. The micro-structural changes of myocardial mitochondria were observed by transmission electron microscope. The ischemic cardiomyocyte apoptosis was detected in each group using one-step terminal-deoxynucleotidyl transferase mediated nick end labeling （TUNEL）. The mRNA expressions of B-cell leukemia/lymphoma 2 （Bcl-2） and Bcl-2 associated x protein （Bax） were detected by RT-PCR. The activities of lactic dehydrogenase （LDH） and creatine kinase （CK） were detected using ELISA. The myocardial infarct size was detected. ResultsCompared with the I/R group，WJ pretreatment significantly suppressed the release of LDH and CK （Besides， the release of LDH and CK reduced along with increased dose.）， reduced the myocardial infarct size， and lowered myocardial apoptosis index （P<0.05）. WJ pretreatment also modulated Bcl-2/Bax ratio by up-regulating Bcl-2 expression level while decreasing Bax expression level. ConclusionsWJ pretreatment might protect the heart from I/R injury via decreasing myocardial cell apoptosis. The results suggested that eighteen incompatible pairs is not absolute， but relative. Chinese medical preparation containing opposite Chinese herbs could be used in specific pathological states such as ischemic cardiomyopathy.

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