基于HPLC观察电针预处理对心肌缺血再灌注损伤大鼠心肌组织中腺苷酸的影响

Effect of electroacupuncture pretreatment on adenine nucleotides in myocardial tissues of rats with myocardial ischemia-reperfusion injury detected by high performance liquid chromatography(HPLC)

目的:观察电针(EA)预处理对心肌缺血再灌注损伤(MIRI)大鼠心肌组织中腺苷酸的影响,探讨EA预处理对MIRI大鼠心肌预防保护的作用机制。方法:将40只SPF级雄性Sprague-Dawley(SD)大鼠随机分为5组,空白组、假手术组、模型组、EA内关组和EA合谷组,每组8只。空白组仅用鼠板束縛大鼠,每次30 min,每天1次,连续7 d;假手术组于第7天在左冠状动脉前降支穿线不结扎,穿线40 min后取线,静置60 min后取材;模型组于第7天在左冠状动脉前降支结扎,40 min后恢复血流,60 min后取材;EA内关组和EA合谷组每天EA内关或合谷30 min,每天1次,连续7 d,于第7天进行造模和取材,方法同模型组。收集5组大鼠的左冠状动脉前降支下段左心室心肌组织。采用苏木精-伊红染色法(HE)及透射电子显微镜(TEM)检测心肌病理形态学的变化,用高效液相色谱法(HPLC)测定心肌组织中腺苷酸的含量变化。结果:在心肌HE染色及超微结构方面,与假手术组比较,模型组心肌损伤严重;与模型组比较,EA内关组心肌损伤较轻微,EA合谷组心肌损伤亦较轻微,且趋于正常。在腺苷酸含量方面,与空白组比较,假手术组和模型组腺苷酸含量均降低(均P<0.05);与假手术组比较,模型组腺苷酸含量亦降低,但差异无统计学意义(P>0.05);与模型组比较,EA内关组腺苷酸含量升高(P<0.05),EA合谷组腺苷酸含量有明显升高(P<0.01);与Ea A关组比较,EA合谷组腺苷酸含量高于EA内关组,但差异无统计学意义(P>0.05);与EA内关组比较,EA合谷组中三磷酸腺苷(ATP)、二磷酸腺苷(ADP)和一磷酸腺苷(AMP)含量明显升高(均P<0.01)。结论:EA内关和合谷均能减轻MIRI大鼠心肌的病理损伤,升高心肌组织中腺苷酸含量,对M IRI大鼠具有预防保护作用,且EA合谷比EA内关的预防保护效果更佳。

Objective:To observe the effect of electroacupuncture(EA)pretreatment on adenine nucleotides in the myocardial tissues of the myocardial ischemia-reperfusion injury(MIRI)rats,and to explore the mechanism of EA pretreatment on myocardial prevention and protection in MIRI rats.Methods:Forty SPF male Sprague-Dawley(SD)rats were randomly divided into 5 groups:a blank group,a sham operation group,a model group,an EA at Neiguan(PC 6)group and an EA at Hegu(LI 4)group,with 8 rats in each group.Rats in the blank group only received binding to the rat plate,30 min/time,once a day for 7 d;on the 7th day,rats in the sham operation group were subjected to threading for 40 min at the left anterior descending coronary artery without ligation,and then the rats were allowed to stand for 60 min before collection of the specimens;on the 7th day,rats in the model group were subjected to threading at the left anterior descending coronary artery with ligation,for 40 min before the blood flow was restored,and then the rats were allowed to stand for 60 min before collection of the specimens;on the 7th day of pretreatment with EA at Neiguan(PC 6)or Hegu(LI 4)for 30 min per day(once a day for 7 d),rats in the EA at Neiguan(PC 6)group and EA at Hegu(LI 4)group were subjected to modeling and sample collection same as in the model group.The left ventricular myocardium of the lower left anterior descending coronary artery was collected from rats in all 5 groups.Hematoxylin-eosin(HE)staining and transmission electron microscope(TEM)were used to observe the changes in myocardial pathological morphology.The change in the adenine nucleotide level of myocardial tissue was measured by high performance liquid chromatography(HPLC).Results:The HE staining and ultrastructure showed that the myocardial injury was severer in the model group compared with the sham operation group.Compared with the model group,the myocardial injury in the EA at Neiguan(PC 6)and the EA at Hegu(LI 4)groups was mild or hardly any.The adenine nucleotide levels in the sham operation group and the model group were all decreased compared with the blank group(all P<0.05);compared with the sham operation group,the adenine nucleotide level of the model group was also decreased,but the difference was not statistically significant(P>0.05);compared with the model group,the adenine nucleotide level in the EA at Neiguan(PC 6)group was increased(P<0.05),and the adenine nucleotide level in the EA at Hegu(LI 4)group was significantly increased(P<0.01).The adenine nucleotide level in the EA at Hegu(LI 4)group was higher than that in the EA at Neiguan(PC 6)group,but the difference was not statistically significant(P>0.05).Compared with the EA at Neiguan(PC 6)group,the levels of adenosine triphosphate(ATP),adenosine diphosphate(ADP)and adenosine monophosphate(AMP)in the EA at Hegu(LI 4)group were significantly increased(all P<0.01).Conclusion:Both EA at Neiguan(PC 6)and Hegu(LI 4)can alleviate the pathological damage to myocardium in MIRI rats,and increase the adenine nucleotide level in myocardial tissues,and thus protect MIRI rats.EA at Hegu(LI 4)has a better protective effect than Neiguan(PC 6).